optimization, characterization and applications of glucose oxidase
TRANSCRIPT
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OPTIMIZATION, CHARACTERIZATION AND
APPLICATIONS OF GLUCOSE OXIDASE PRODUCED
FROM ASPERGILLUS AWAMORI MTCC 9645 FOR FOOD
PROCESSING AND PRESERVATION
A THESIS
Submitted by
P. SATHIYA MOORTHI, M.Sc.,
for the award of the degree
of
DOCTOR OF PHILOSOPHY
DEPARTMENT OF INDUSTRIAL BIOTECHNOLOGYDr. M.G.R.
EDUCATIONAL AND RESEARCH INSTITUTEUNIVERSITY
(Declared U/S 3 of the UGC Act 1956)
CHENNAI 600 095
AUGUST 2009
2
Dr. M.G.R.EDUCATIONAL AND RESEARCH INSTITUTE
UNIVERSITY(Declared U/S 3 of the UGC Act 1956)
CHENNAI 600 095
BONAFIDE CERTIFICATE
Certified that this thesis entitled “OPTIMIZATION,
CHARACTERIZATION AND APPLICATIONS OF GLUCOSE
OXIDASE PRODUCED FROM ASPERGILLUS AWAMORI MTCC
9645 FOR FOOD PROCESSING AND PRESERVATION” is the
bonafide work of Mr. P. SATHIYA MOORTHI, who carried out the
research under our supervision. Certified further that to the best of our
knowledge the work reported herein does not form part of any other
thesis or dissertation on the basis of which a degree or award was
conferred on an earlier occasion of this or any other candidate.
SIGNATUREDr. M. DEECARAMAN(CO-SUPERVISOR)Dean,Dept. of Industrial Biotechnology,Dr.M.G.R. Educational and Research Institute,Maduravoyal, Chennai-600 095.
SIGNATUREDr. P.T. KALAICHELVAN(SUPERVISOR)Professor,Centre for Advanced Studies in Botany,University of Madras, Guindy Campus,Chennai-600 025.
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DECLARATION
I declare that the thesis entitled “OPTIMIZATION,
CHARACTERIZATION AND APPLICATIONS OF GLUCOSE
OXIDASE PRODUCED FROM ASPERGILLUS AWAMORI MTCC
9645 FOR FOOD PROCESSING AND PRESERVATION” submitted
by me for the degree of Doctor of Philosophy is the record of work
carried out by me during the period from January 2006 to July 2009
under the supervision of Dr. P.T. Kalaichelvan, Professor, Centre for
Advanced Studies in Botany, University of Madras, Guindy Campus,
Chennai and the co-supervision of Dr. M. Deecaraman, Dean,
Department of Industrial Biotechnology, Dr. M.G.R. Educational and
Research Institute University, Maduravoyal, Chennai and has not formed
the basis for the award of any degree, diploma, associate-ship, fellowship
and titles in this or any other University or other similar institution of
Higher learning.
Signature of the candidate
(P. SATHIYA
MOORTHI)
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ACKNOWLEDGEMENT
I sincerely submit my respectful regards to our Chancellor Thiru
A.C. Chanmugam, BA., B.L., and Pro-Chancellor Er. A.C.S. Arunkumar
Dr.M.G.R. Educational and Research Institute University, Maduravoyal, Chennai
with whose blessing this work has been completed successfully.
I express my gratitude to our Vice-Chancellor Dr. K. Padmanabhan and
Vice-President (Academic) Dr. P.T. Manogaran, Dr.M.G.R. Educational and
Research Institute University, Maduravoyal, Chennai for their valuable advice.
I am extremely thankful and deeply indebted to my research supervisor
Dr. P.T. Kalaichelvan, Professor, Centre for Advanced Studies in Botany,
University of Madras, Chennai for his valuable suggestions, constructive criticism,
unstinted encouragement and care on me throughout my tenure.
I would like to place my deep sense of indebtedness to my co-supervisor
Dr. M. Deecaraman, Dean, Department of Industrial Biotechnology, Dr.M.G.R.
Educational and Research Institute University, Maduravoyal, Chennai for his
invaluable guidelines, suggestions and support through out this work.
I express my sincere gratitude to Dr. Rama vaidyanathan, HoD, Department
of Industrial Biotechnology, Dr.M.G.R. Educational and Research Institute
University, Maduravoyal, Chennai for providing laboratory facilities and her constant
encouragement to motivation of research.
I probably express my sincere thanks, Dr. M. Vijayalakshmi, Deputy HoD,
Department of Industrial Biotechnology, Dr.M.G.R. Educational and Research
Institute University, Maduravoyal, Chennai for her constant advice and motivation
thought my research.
I express my gratitude to Dr. P. Aravindan, Dean Research, Dr.M.G.R.
Educational and Research Institute University, Maduravoyal, Chennai for his constant
support and encouragement throughout the research.
I express my sincere thanks to Dr. S. Senthilvelan, Dean, Engineering and
Technology, Dr.M.G.R. Educational and Research Institute University, Maduravoyal,
Chennai for his valuable suggestions.
I would like to express my sincere thanks to Dr. N. Padmanaban, HoD,
Department of Chemical Engineering, Dr.M.G.R. Educational and Research Institute
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University, Maduravoyal, Chennai for his expert comments, valuable suggestions and
constant support throughout the research.
I would like to express my sincere thanks to Dr. V. Cyril Raj, HoD,
Department of Computer Science and Engineering, Dr.M.G.R. Educational and
Research Institute University, Maduravoyal, Chennai for his motivation and constant
support throughout the research.
I probably express my sincere thanks to Dr. R. Rengasamy, Professor and
Director, Centre for Advanced Studies in Botany, University of Madras, for his expert
commends and valuable suggestions throughout this research.
I express my sincere thanks to Dr. B.P.R. Vital, Professor, Centre for
Advanced Studies in Botany, University of Madras, for helping in fungal
identification and constant support throughout the research.
I would like to thank gratefully Er. Ap. Prabhaker, Managing Director,
Chinnu Exports, Chennai, for his constant support.
I would be failing in my duty if I forget the help rendered by my research lab
friends at Department of Industrial Biotechnology, Dr.M.G.R. Educational and
Research Institute University, Maduravoyal, Chennai.
I heartfelt thank to my research friends at Centre for Advanced Studies in
Botany, University of Madras, Chennai.
I also extend my thanks to the teaching and non-teaching staff of the
Department of Industrial Biotechnology, Dr.M.G.R. Educational and Research
Institute University, Maduravoyal, Chennai, for their kind co-operation.
My heartfelt thanks to all my friends, who have support and encourage me
during the research period.
Words are inadequate to thank my beloved father, mother and sister always
been with me in all my endeavors.
(P. SATHIYA MOORTHI)
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TABLE OF CONTENTS
CHAPTER No. TITLEPAGE
No.
ABSTRACT
LIST OF TABLES
LIST OF FIGURES
LIST OF SYMBOLS, ABBREVIATIONS AND
NOMENCLATURE
vi
viii
x
xiv
1. INTRODUCTION 1–4
2. LITERATURE SURVEY 5–52
2.1 Glucose oxidase production 9
2.2 Media optimization 10
2.3 Natural occurrence of GOx and its applications 10
2.4 Food processing additive 11
2.4.1 Bread making 11
2.4.2 Dry egg powder 12
2.4.3 Antioxidant/preservative (oxygen scavenger) 13
2.4.4 GOx in sea foods 14
2.4.5 Dairy and the lactoperoxidase system (LPS) 14
2.4.6 Reduced alcohol wine 16
2.4.7 Gluconic acid production 17
2.4.8 Glucose sensor/assay 18
2.4.9 Fuel cell 19
2.4.10 Other uses of GOx 20
2.5 Vegetable and food processing 22
2.5.1 Chlorination 22
2.5.2 Chlorine dioxide 22
2.5.3 Biodegradable Packaging 23
2.5.4 Edible coatings and films 24
2.5.4.1 Cellulose 26
7
2.5.4.2 Alginate 26
2.5.4.3 Zein 27
2.5.4.4 Banana powder 27
2.6 Antimicrobial agents in film coating 27
2.6.1 Ethylene diamine tetra acetic acid (EDTA) 28
2.6.2 Bacteriocin 29
2.6.3 Lysozyme 30
2.7 Microbial contaminates in vegetable products 31
2.8 Spices and edible oil in film making 32
2.9 Apple puree 32
2.9.1 Glucose oxidase-catalase system 33
2.9.2 Lactoperoxidase 34
2.10 Minimally processed fruits and vegetables 36
2.11 Reasons for quality changes in minimally processed produce 36
2.12 Methods to improve the shelf life and safety of minimally processed
produce
37
2.13 Microbial spoilage of vegetables and fruits 38
2.14 Pesticides as a source of microbial contamination of salad vegetables 43
2.15 Cut vegetable and fruit preservation techniques in practice 43
2.15.1 Combination preservation 45
2.15.2 Chemical preservative agents 46
2.16 Calcium ions on vegetables and fruits 47
2.16.1 Calcium sources to maintain the shelf-life of fresh vegetables
and fruits
48
2.17 Koruk juice 50
2.18 Effect of blanching 51
2.19 Cold storage 51
2.20 Economic loss 52
8
3. OBJECTIVE OF THE PRESENT WORK 53
4. MATERIALS AND METHODS 54–99
4.1 General 54
4.1.2 Sterilization 54
4.1.3 Chemicals 54
4.2 ISOLATION AND SCREENING OF GLUCOSE OXIDASE
PRODUCING FUNGI AND OPTIMIZATION OF MEDIUM
54
4.2.1 Isolation of GOx producing fungi 54
4.2.2 Screening of GOx producing fungi 55
4.2.3 Identification of fungi 56
4.2.4 Preparation of spore suspension 56
4.3 Analytical methods 57
4.3.1 Assay of GOx activity 57
4.3.2 Estimation of protein 59
4.3.3 Estimation of fungal biomass 60
4.3.4 Analysis of glucose 60
4.3.5 Analysis of gluconic acid 60
4.4 Culture condition for GOx production 61
4.4.1 Selection of suitable medium for GOx production 61
4.5 Media optimization 63
4.5.1 Single factor analysis (SFA) for GOx production 63
4.5.1.1 Effect of carbon source on GOx production 63
4.5.1.2 Effect of nitrogen sources on GOx production 63
4.5.1.3 Effect of Di-ammonium hydrogen phosphate,
potassium di-hydrogen phosphate and magnesium
sulphate on GOx production
63
4.5.1.4 Effect of calcium carbonate supplementation on GOx
production
64
4.5.1.5 Effect of pH and temperature on GOx production 64
4.5.1.6 Effect of fermentation time on GOx production 64
4.5.2 Statistical optimization by Response Surface Methodology 64
4.5.2.1 Experimental design of RSM for optimization of 65
9
media components
4.5.3 Modified composition of production medium GOxM 3 69
4.6 Production of GOx by laboratory batch fermentor 70
4.7 Morphological studies 70
4.7.1 Quantification and qualification of different types of
bioparticles
70
4.7.2 Large pellets (>3 mm diameter) 71
4.7.3 Small bioparticles (< 3 mm diameter) 71
4.7.4 Measurement and calculations 71
4.7.5 Time course study on cell growth, GOx production, substrate
utilization and acid formation during fermentation period
72
4.8 Purification and characterization of GOx 72
4.8.1 Preparation of enzyme for purification 72
4.8.2 Dialysis 72
4.8.3 Lyophilization 73
4.8.4 Ion exchange chromatography 73
4.8.5 Size exclusion chromatography 73
4.9 Polyacrylamide Gel Electrophoresis (PAGE) 74
4.9.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis 74
4.9.2 Native polyacrylamide gel electrophoresis 77
4.9.2.1 Zymogram analysis 79
4.9.3 Confirmation of enzyme activity using plate assay 79
4.10 Characterization of glucose oxidase 80
4.10.1 Kinetic charecterization 80
4.10.2 Effect of temperature and pH on GOx activity 80
4.10.3 Stability testing 80
4.10.4 Stability and inhibitory studies of GOx 81
4.10.4.1 Effect of metal ions on GOx activity 80
4.10.4.2 Effect of calcium ions on GOx activity 80
4.10.5 Preparation of carrier based enzyme 81
4.11. APPLICATION OF GLUCOSE OXIDASE IN FOOD
PROCESSING AND PRESERVATION
82
4.11.1 Enhancing the storage stability of vegetable by coating of 82
10
edible film incorporated with glucose oxidase,
lactoperoxidase and lysozyme
4.11.1.1 Microorganisms and culture conditions 82
4.11.1.2 Preparation and analysis of antimicrobial enzymes 82
4.11.1.3 Evaluation of antimicrobial activity of GOx, LPS
and lysozyme with EDTA
85
4.11.1.4 Film making 85
4.11.1.5 Antimicrobial film activity 86
4.11.1.6 Surface sterilization of carrots 86
4.11.1.7 Coating procedure 87
4.11.1.8 Determination of weight loss 87
4.11.1.9 Determination of soluble protein content 88
4.11.1.10 Microbial analysis by viable plate count method 88
4.11.1.11 Sensory analysis 88
4.11.2 Control of browning and enhancing shelf-life of apple
puree by applying glucose oxidase-catalase system with
lactoperoxidase
89
4.11.2.1 Preparation and analysis of antimicrobial enzymes 89
4.11.2.2 Determination antimicrobial activity of GOx, LPS
and catalase
90
4.11.2.3 Preparation of apple puree 90
4.11.2.4 Effect of GOx and ascorbic acid on dissolved
oxygen consumption
91
4.11.2.5 Experimental design 91
4.11.2.6 Evaluation of browning on apple puree 91
4.11.2.7 Examination of microbial populations 92
4.11.2.8 Sensory analysis 92
4.11.3 Studies on the effect of glucose oxidase-catalase with
calcium ions in stabilizing and improving the fruit salad
quality
93
4.11.3.1 Preparation of enzyme 93
4.11.3.2 Collection of fruits 93
4.11.3.3 Preparation of koruk juice 93
11
4.11.3.4 Analysis of antimicrobial activity of GOx, calcium
ions and koruk juice
93
4.11.3.5 Preparation of treatment solution 94
4.11.3.6 General fruit salad preparation procedure 94
4.11.3.7 Sensory analysis 95
4.11.3.8 Experimental design of RSM for optimization of
calcium ions
96
4.11.3.9 Effect of optimized calcium ions on fruit salad
preparation
98
4.11.3.10 Measurement of weight loss 98
4.11.3.11 Microbial analysis of fruit salad by viable plate
count method
98
4.12 Statistical analysis 99
5. RESULTS AND DISCUSSION 100–197
5.1 ISOLATION, MEDIA OPTIMIZATION AND PRODUCTION
OF GLUCOSE OXIDASE
100
5.1.1 Isolation of GOx producing fungi 100
5.2 Optimization of medium for glucose oxidase production 104
5.2.1 Single Factor Analysis (SFA) for GOx production 104
5.2.1.1 Effect of carbon source on GOx production 104
5.2.1.2 Effect of nitrogen sources on GOx production 106
5.2.1.3 Effect of di-ammonium hydrogen phosphate,
potassium di-hydrogen phosphate and magnesium
sulphate on GOx production
106
5.2.1.4 Effect of calcium carbonate on GOx production 109
5.2.1.5 Effect of pH on GOx production 109
5.2.1.6 Effect of temperature on GOx production 111
5.2.1.7 Effect of fermentation time on GOx production 111
5.2.2 Response surface methodology for GOx production 113
5.2.2.1 SET 1: Optimization of glucose, proteose peptone
and calcium carbonate for GOx production
113
5.2.2.2 SET 2: Optimization of di-ammonium hydrogen
phosphate, potassium di-hydrogen phosphate and
116
12
magnesium sulphate for GOx production
5.2.2.3 SET 3: Optimization of pH and temperature 126
5.3 Production of glucose oxidase by laboratory batch fermentor 130
5.3.1 Spore aggregation and pellet formation during the early
cultivation time
130
5.3.2 Morphological studies 133
5.3.3 Time course study on cell growth, substrate utilization, GOx
and gluconic acid production during fermentation period
133
5.4 Purification and characterization of glucose oxidase produced
from Aspergillus awamori MTCC 9645
136
5.4.1 DEAE-Cellulose column chromatography 136
5.4.2 Sephacryl S-200 column chromatography 136
5.4.3 Molecular mass determination of purified GOx by SDS-PAGE 140
5.4.4 Purified GOx activity on native-PAGE 140
5.4.5 Confirmation of enzyme activity using plate assay 140
5.4.6 Kinetic characterization of GOx 143
5.4.7 Effect of temperature and pH on GOx activity 143
5.4.8 Stability testing 143
5.4.9 Stability and inhibitory activity of GOx 148
5.4.9.1 Effect of metal ions on GOx activity 148
5.4.9.2 Effect of calcium ions on GOx activity (1mM) 149
5.4.9.3 Effect of calcium ions on pH stability of GOx 149
13
5.4.9.4 Effect of calcium ions on temperature stability of
GOx (1mM)
149
5.5 APPLICATION OF GLUCOSE OXIDASE IN FOOD
PROCESSING AND PRESERVATION
152
5.5.1 Edible films incorporated with glucose oxidase,
lactoperoxidase and lysozyme for carrot preservation
152
5.5.1.1 Assay of antimicrobial enzymes 152
5.5.1.2 Evaluation of antimicrobial activity of GOx, LPS and
lysozyme with EDTA
152
5.5.1.3 Antimicrobial activity of alginate film 154
5.5.1.4 Surface sterilization of carrot 156
5.5.1.5 Measurement of weight loss 156
5.5.1.6 Measurement of soluble protein content 160
5.5.1.7 Enumeration of bacterial population from treated and
control carrots
160
5.5.1.8 Sensory analysis 160
5.5.2 Control of browning and enhancing the shelf-life of apple
puree by glucose oxidase, catalase and lactoperoxidase
164
5.5.2.1 Assay of antimicrobial enzymes 164
5.5.2.2 Evaluation of antimicrobial activity of GOx, catalase
and LPS
164
5.5.2.3 Effect of GOx and ascorbic acid on removal of
dissolved oxygen from apple puree
166
5.5.2.4 Effect of GOx, catalase, LPS and ascorbic acid on
controlling of browning in apple puree
167
5.5.2.5 Examination of microbial populations 171
5.5.2.6 Sensory analysis 171
14
5.5.3 Studies on the effect of glucose oxidase-catalase with
calcium ions for the improvement of fruit salad quality
173
5.5.3.1 Evaluation of antimicrobial activity of GOx, catalase
calcium ions and koruk juice
173
5.5.3.2 Sensory analysis 173
5.5.3.3 Sensory analysis of GOx-catalase treated fruit salad 176
5.5.3.4 Sensory analysis of calcium chloride treated fruit salad 176
5.5.3.5 Sensory analysis of calcium propionate treated fruit
salad
176
5.5.3.6 Sensory analysis of calcium lactate treated fruit salad 178
5.5.3.7 Sensory analysis of koruk juice treated fruit salad 178
5.5.3.8 Optimization of calcium ions concentrations by
response surface methodology
182
5.5.3.9 Effect of RSM optimized combined of calcium ions,
GOx-catalase and koruk juice on fruit salad
194
5.5.3.10 Measurement of weight loss 197
5.5.3.11 Enumeration of bacterial population from treated
and control fruit salads
197
6. SUMMARY 199–202
7. CONCLUSIONS AND SCOPE FOR FUTURE WORK 203–204
REFERENCES i–xxviii
LIST OF PUBLICATIONS, PRESENTATIONS AND CONFERENCES xxix–xxxi
CURRICULUM VITAE xxxii
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ABSTRACT
The glucose oxidase (GOx) enzyme has considerable industrial
importance and used in different food products (dried egg, bread,
beverages and vegetables) for processing and preservation. Among the
different fungi isolated from various sugar rich products, Aspergillus
awamori MTCC 9645 was found to be produce high amount of GOx. The
compositions of production medium were optimized by Single Factor
Analysis (SFA) and a statistical tool Response Surface Methodology
(RSM). Glucose and proteose peptone were found to be a good carbon
and nitrogen source for maximum GOx production respectively. Addition
of di-ammonium hydrogen phosphate, potassium di-hydrogen phosphate
and magnesium sulphate supports the GOx production. Remarkably GOx
production was increased in the presence of 35–40 g/l calcium carbonate.
The pH 5.0–6.0 and the temperature between 30 and 35ºC were found to
be optimum for GOx production. The cell free extract was purified and
characterized. Enzyme stability studies were carried out with different
calcium ions in which calcium lactate was found to be effective for
stabilizing GOx activity. Metal ions such as mercuric chloride, copper
sulphate, and silver nitrate were significantly inhibit the activity of GOx.
The enzyme was subjected for the processing and preservation of food
products such as vegetable, apple puree and fruit salad. Alginate film
16
coated carrot with the formulation of GOx and lactoperoxidase (LPS),
lysozyme and EDTA exhibited good shelf-life. Apple puree was
processed with the combination of GOx-catalase with LPS exhibited
good antibrowning and antibacterial efficacy. The combination of GOx-
catalase with calcium ions showed antibacterial and antibrowning activity
in salad that increased the storage stability and also high sensory score.
The results from the present findings were clearly revealed that potential
of GOx would use as processing and preservation of food products.
Key words: Apple puree, Aspergillus awamori (MTCC 9645); Carrot;
Glucose oxidase; Optimization, Preservation; RSM; Salad;
Vegetable process.
17
LIST OF TABLES
TABLE No. TITLE OF TABLE
Table 2.1 Requirements for the commercial manufacture of pre-peeled and/or
sliced, grated or shredded fruit and vegetables
Table 4.1 Different production medium for GOx
Table 4.2 Set 1 Optimization of glucose, proteose peptone and calcium
carbonate for GOx production
Table 4.2.1 Design summary
Table 4.2.2 Experimendal design and of 23 factorial design
Table 4.3 Set 2 Optimization of (NH4)2HPO4, KH2PO4 and MgSO4 for GOx
production
Table 4.3.1 Design summary
Table 4.3.2 Experimendal design and of 23 factorial design
Table 4.4 Set 3 Optimization of pH and temperature for GOx production
Table 4.4.1 Design summary
Table 4.4.2 Experimendal design and of 22 factorial design
Table 4.5 Optimization of calcium ions for salad preparation
Table 4.5.1 Design summary
Tanle 4.5.2 Experimendal design and results of 23 factorial design
Table 5.1 Glucose oxidase production by different fungi
Table 5.2 Set 1 Optimization of glucose, proteose peptone and calcium
carbonate for the GOx production by CCD of response surface
methodology (23 factorial design)
Table 5.2.1 F-test analysis ( ANOVA for Response Surface Quadratic Model)
Table 5.2.2 Comparition of R2 predicted and estimated
Table 5.2.3 Model coefficient estimated by linear regression
Table 5.3 Set 2 Optimization of (NH4)2HPO4, KH2PO4 and MgSO4 for the GOx
production by CCD of response surface methodology
(23 factorial design)
Table 5.3.1 F-test analysis (ANOVA for Response Surface Quadratic Model)
Table 5.3.2 Comparition of R2 predicted and estimated
Table 5.3.3 Model coefficient estimated by linear regression
18
Table 5.4 Set 3 Optimization of pH and temperature for the GOx production by
CCD of response surface methodology (23 factorial design)
Table 5.4.1 F-test analysis (ANOVA for Response Surface Quadratic Model)
Table 5.4.2 Comparition of R2 predicted and estimated
Table 5.4.3 Model coefficient estimated by linear regression
Table 5.5 Spore aggregation and pellet formation during the early cultivation
time in the laboratory batch fermentor
Table 5.6 Summary of purification of GOx from A. awamori MTCC 9645
Table 5.7 Bacterial counts (CFU/ml± SD) on surface washing carrot treated
with chlorine dioxide at different concentration and time of exposure
Table 5.8 Soluble protein content (mg/g of dry wt ± SD) in alginate coated
(Formulation I) and uncoated control carrots.
Table 5.9 Optimization of calcium ions for salad preparation by CCD of
response surface methodology (23 factorial design)
Table 5.9.1 F-test analysis (ANOVA for Response Surface Quadratic Model)
Table 5.9.2 Comparition of R2 predicted and estimated
Table 5.9.3 F-test analysis (ANOVA for Response Surface Quadratic Model)
Table 5.9.4 Comparison of R2 predicted and estimated
Table 5.9.5 Model coefficient estimated by linear regression
19
LIST OF FIGURESFIGURE No. TITLE OF FIGURES
Figure 2.1 Enzymatic conversion of glucose to gluconic acid by GOx
Figure 2.2 3-Dimentional structure of glucose oxidase
Figure 5.3 Isolation, screening and identification of A. awamori (GOP-3)
Figure 5.4: Isolation, screening and identification of A. awamori (GOP-7) MTCC 9645
Figure 5.5 Effect of different media for GOx production
Figure 5.6 Effect of carbon sources on GOx production
Figure 5.7 Effect of different nitrogen sources for the GOx production
Figure 5.8 Effect of di-ammonium hydrogen phosphate on GOx production
Figure 5.9 Effect of potassium di-hydrogen phosphate and magnesium sulphate of
GOx production
Figure 5.10 Effect calcium carbonate on glucose oxidase production
Figure 5.11 Effect of pH on GOx production
Figure 5.12 Effect of temperature on GOx production
Figure 5.13 Effect of fermentation time on GOx production and cell growth
Figure 5.14 The contour and 3D response surface plot showing the effect of glucose and
calcium carbonate on GOx production
Figure 5.15 The contour and 3D response surface plot showing the effect of glucose and
peptone on GOx production
Figure 5.16 The contour and 3D response surface plot showing the effect of peptone and
calcium carbonate on GOx production
Figure 5.17 The contour and 3D response surface plot showing the effect of
(NH4)2HPO4 and KH2PO4 on GOx production
Figure 5.18 The contour and 3D response surface plot showing the effect of
(NH4)2HPO4 and MgSO4 on GOx production
Figure 5.19 The contour and 3D response surface plot showing the effect of KH2PO4
and MgSO4 on GOx production
Figure 5.20 The contour and 3D response surface plot showing the effect of pH and
temperature on GOx production
Figure 5.21 Production of GOx in A. awamori MTCC 9645 in laboratory bioreactor
Figure 5.22 Time course study on substrate utilization, production of gluconic acid and
biomass during the fermentation
20
Figure 5.23 Time course study on production of GOx and protein during the
fermentation
Figure 5.24 Flow chart for the purification and characterization of extracellular GOx of
A. awamori MTCC 9645
Figure 5.25 Purification of GOx from A. awamori MTCC 9645
Figure 5.25(a) Elution profile on DEAE column chromatography
Figure 5.25 (b) Elution profile on Sephacryl S-200 column chromatography
Figure 5.26 Molecular mass determination on SDS-PAGE (10%) of GOx from A.
awamori MTCC 9645
Figure 5.27(b) Zymogram of GOx (Iso enzyme patterning) on native-PAGE (8%)
developed with horse radishproxidase, o-dianisidine and glucose
Figure 5.27(b) Confirmation of GOx from A. awamori MTCC 9645 by plate assay
Figure 5.28 Kinetic parameters (apparent Km and Vmax) for purified GOx of A. awamori
MTCC 9645 by Lineweaver's Burk plot
Figure 5.29 Kinetic parameters (apparent Km and Vmax) for purified GOx of A. awamori
MTCC 9645 by Eadie-Hofstee plot
Figure 5.30 Kinetic parameters (apparent Km and Vmax) for purified GOx of A. awamori
MTCC 9645 by Hanes plot
Figure 5.31 Effect of temperature and pH on GOx activity
Figure 5.32 Effect of temperature stability of purified GOx
Figure 5.33 Effect of metal ions (1mM) on GOx activity
Figure 5.34 Effect of calcium ions (1mM) on GOx activity
Figure 5.35 Effect of calcium ions (1mM) on pH stability of GOx activity
Figure 5.36 Effect of calcium ions (1mM) on temperature stability of GOx
Figure 5.37 Effect of antimicrobial activity of GOx, LPS and lysozyme with EDTA
against E. coli
Figure 5.38 Effect of antimicrobial activity of GOx, LPS and lysozyme with EDTA
against S. aureus
21
Figure 5.39 Effect of antimicrobial alginate films incorporated with partially purified
enzymes of different formulations
Figure 5.40
(a&b)
Effect of GOx, LPS and lysozyme incorporated with alginate film coated
carrots stored at (a) Stored at ~26°C and (b) 6°C
Figure 5.41
(a&b)
Effect of alginate coated (Formulation VII) and uncoated carrots stored on
weight loss (%) during the storage period at (a) 6°C (b) and (b) ~26ºC
Figure 5.42 Showed the microbial population (10-6 dilution) the treated and control
carrots after the storage period (10 d) stored at 6ºC ~26ºC
Figure 5.43
(a&b)
Evaluation of the sensory profile of treated and control carrots after the
storage period (10 d) stored at (a) 6ºC and (b) ~26ºC
Figure 5.44
(a&b)
Effect of antimicrobial activity of GOx, catalase and LPS against (a) E. coli
and (b) S. aureus
Figure 5.45 Effect GOx and ascorbic acid on removal of dissolved oxygen from apple
puree
Figure 5.46 Effect GOx, catalase, LPS and ascorbic acid on controlling of browning in
apple puree
Figure 5.47
(a&b)
Effect GOx and ascorbic acid on controlling of browning in (a) Cut apple
and (b) Apple puree
Figure 5.48 Showed the microbial population of treated and control apple puree
Figure 5.49 Evaluation of the sensory profile of treated and control apple puree
Figure 5.50
(a&b)
Effect of antimicrobial activity of GOx, calcium ions and koruk juice
against (a) E. coli and (b) S. aureus
Figure 5.51
(A–G)
Effect of calcium ions, koruk juice and GOx on controlling browning in cut
(a) apple, (b) pomegranate and (c) guava
Figure 5.52
(a&b)
Evaluation of the sensory profile of different concentration of (a) GOx and
(b) calcium chloride treated and control fruit salad
Figure 5.53
(a&b)
Evaluation of the sensory profile of different concentration of (a) calcium
propionate and (b) calcium lactate treated and control fruit salad
Figure 5.54 Evaluation of the sensory profile of different concentration of koruk juice
treated and control fruit salad
Figure 5.55
(a&b)
Effect of GOx and koruk juice on fruit salad
22
Figure 5.56 The contour and 3D response surface plot showing the weight loss % of
calcium lactate and calcium propionate treatment on fruit salad
Figure 5.57 The contour and 3D response surface plot showing the weight loss % of
calcium propionate and calcium chloride treatment on fruit salad
Figure 5.58 The contour and 3D response surface plot showing the weight loss % of
calcium chloride and calcium lactate treatment on fruit salad
Figure 5.59 The contour and 3D response surface plot showing the overall sensory
acceptability profile of calcium propionate and calcium lactate treatment on
fruit salad
Figure 5.60 The contour and 3D response surface plot showing the overall sensory
acceptability profile of calcium propionate and calcium chloride treatment
on fruit salad
Figure 5.61 The contour and 3D response surface plot showing the overall sensory
acceptability profile of calcium lactate and calcium chloride treatment on
fruit salad
Figure 5.62 Effect of RSM optimized calcium ions on fruit salad
Figure 5.63 Evaluation of the sensory profile of combined calcium ions (obtained from
RSM) and GOx-catalase and koruk juice treated and control fruit salad
Figure 5.64
(a,b&c)
Effect of GOx-catalase, calcium ions and koruk juice on fruit salad
Figure 5.65 Effect of GOx, calcium ions and koruk juice for controlling weight loss on
fruit salad
Figure 5.66 Enumeration of microbial population of different treated and control fruit
salad
23
LIST OF SYMBOLS, ABBREVIATIONS AND NOMENCLATURE
$ Dollar
% Percentage
° Degree
°C Degree Celsius
°F Degree Fahrenheit
~ Tilde
< Less than
> Greater than
∆ Delta
µ Micro
µl Microlitre
A. awamori Aspergillus awamori
A. niger Aspergillus niger
ANOVA Analysis of variance
B. amyloliquefaciens Bacillus amyloliquefaciens
CBB Coomassie brilliant blue
CCD Central composite design
CFU Colony forming unit
cm Centimeter
CMC Carboxy methylcellulose
CT Cholera toxin
d Day
D Dolton
Df Dilution factor
dl Decilitre
DNA Deoxyribonucleic acid
DO Dissolved oxygen
E. coli Escherichia coli
E. faecium Enterococcus faecium
EAEC Enteroaggregative E. coli
24
EAEC Diffusely adherent E. coli
EAF EPEC Adherence factor
EDTA Ethylene diamine tetra acetic acid
EHEC Enterohemorrhagic E. coli
EIEC Enteroinvasive E. coli
EPEC Enteropathogenic E. coli
ETEC Enterotoxigenic E. coliFAD Flavine adenine dinucleotide
FDA Food and drug administration
g Gram
g Gravitational force
GOx Glucose oxidase
GP Gas permeability
GRAS Generally regarded as safe
h Hour
HOSCN Hypothiocyanous acid
HPMC Hydroxypropyl methylcellulose
IU International unit
kDa Kilo dalton
Kg Kilogram
Km Michaelis constant
L Litre
L. monocytogenes Listeria monocytogenes
LAB Lactic acid bacteria
LB Luria Bertani
LPS Lactoperoxidase
Ltd Limited
Lyz Lysozyme
M Molar
mA Milliamps
MAP Modified atmosphere packaging
mbar Millibar
25
MC Methylcellulose
MEA Malt extract agar
mg Milligram
min Minutes
ml Millilitre
mM Millimolar
mm Millimeter
MPV Minimally processed vegetables
MTCC Microbial type culture collection
N Normal
nm Nano meter
OP Oxygen permeability
OSCN- Hypothiocyante
P Probability
P. amagasakiens Penicillum amagasakiens
P. glaucum Penicillum glaucum
P. notatum Penicillum notatum
P. variabile Penicillum variabile
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffer solution
PCR Polymerase chain reaction
PDA Potato dextrose agar
PEF Pulsed electric fields
pH Potential hydrogen ion concentration
POD Peroxidase
ppm Parts per million
psi Pound per square inch
rpm Revolution per minute
RSM Response surface methodology
S. aureus Staphylococcus aureus
S. cerevisiae Saccharomyces cerevisiae
S. enteritidis Salmonella enteritidis
S. typhimurium Salmonella typhimurium
26
SCN− Thiocyanate
SDS Sodium dodecyl sulphate
SFA Single factor analysis
t Time
TDM Total dry mass
TEMED Tetramethyl ethylene diamine
TMB Tetramethylbenzidine
U Unit
UHP Ultra high pressure
US United states
UV Ultra violet
V. vinifera Vitis vinifera
v/v Volume per volume
Vmax Maximal limiting rate velocity
VP Vacuum packaging
W(t) Sample weight at time t
w/v Weight per volume
W0 Initial sample weight
WL Weight loss
WVP Water vapour permeability
X Magnification unit
α Alpha
β Beta
δ Delta
27
CHAPTER-1
INTRODUCTION
Enzymes possess very diverse specificity, reactivity and other
physiochemical, catalytic and biological properties highly desirable for various
industrial and medical applications. The use of enzymes, either alone or as part
of live cells, can be traced back to the dawn of civilization. Now, enzymes are
being rigorously and systematically developed as economically viable and
environment-friendly industrial biocatalysts, along with the fast advancement
and expansion of modern biotechnology (Feng Xu, 2005).
Current industrial enzyme sales are around $2 billion per year with >500
products for >50 major applications (Kirk et al., 2002). Although being a very
small part of the whole speciality chemicals market, the industrial enzyme field
is projected to grow fast, at a rate close to double digits annually for the near
future. Based on application, ~65% of the commercial enzymes are technical
enzyme applications such as detergent, textile and starch, ~25% are food
enzymes and ~10% are feed enzymes.
The majority of the commercial enzymes are hydrolases, while
oxidoreductase account for a miniscule share but it is in contrast to the high
occurrence of oxidoreductase in nature (Kirk et al., 2002; Burk, 2003). The gap
between vast natural oxidoreductase products creates the space and potential
for developing more oxidoreductase based biocatalysts. Currently, the
industrial-technical, food and environmental applications are the only markets
with a significant oxidoreductase commercialization although; it remains very
less in comparison to that of hydrolytic industrial enzymes. Further, improving
the cost competitiveness of existing oxidoreductase products and enhancing the
innovation effort in applying oxidoreductases to new field are vital for the
28
future growth of industrial oxidoreductase biocatalysts (Feng Xu, 2005).
Today, enzymes are used for an increasing range of food applications: bakery,
cheese making, starch processing and production of fruit juices and other
drinks. One such enzyme is glucose oxidase (GOx), which is used on food
industries, as an oxygen and glucose scavenger from foods and beverages
(Sathiya moorthi et al., 2007).
Glucose oxidase (EC 1.1.3.4) belongs to class oxidoreductase and
catalyzes the oxidation of β-D-glucose to gluconic acid, utilizing molecular
oxygen as an electron acceptor with the simultaneous production of hydrogen
peroxide. It is a dimeric protein composed of two identical subunits, each
subunit, or monomer, binds into domain: one domain binds to the substrate
glucose while the other domain binds non-covalently to a cofactor flavin
adenine dinucleotide (FDA).
The success of Aspergillus niger group for industrial production of
biotechnological products is largely due to the metabolic versatility of this
strain. A. niger is well known to produce a variety of enzymes, organic acids,
plant growth regulators, mycotoxins and antibiotics. The industrial importance
of A. niger groups are not limited on its >335 native products but also on
development and commercialization of the new products which are derived by
modern bioprocess and molecular biology techniques.
Glucose oxidase was first isolated from mycelia of A. niger and
Penicillium glaucum by Muller (1928) and later they were detected in different
sources (insects, honey, algae and micro fungi) and recently was obtained from
Penicillum amagasakiense and especially from A. niger. The most common
sources of GOx were A. niger, P. notatum, P. glaucum, P. amagasakiense,
P. purpurogenum, P. variabile and Alternaria alternate (Caridis et al., 1991).
29
Recent researches are focused on factors that regulate the production of
GOx. There are several media constituents like carbon, nitrogen, CaCO3 and
environmental factors such as temperature, pH and agitation involved in the
production of GOx. The above factors have to be optimized for the effective
production of GOx. There are few reports concerned in the optimization of
cultural conditions for the production of GOx from various fungi (Sandip et al.,
2008). Optimization of media composition and other factors by using statistical
method have few advantages over the single factor analysis at a time. It can be
used for easy determination of important parameter from a large number of
factors and the study of the interactions between the variables of the media
constituents.
Glucose oxidase has considerable industrial importance and used for the
removal of trace amounts of oxygen or glucose from different sources such as
dried egg, beer, wine and fruit juices (Reed and Underkoer, 1966). It has been
used to remove residual glucose and oxygen in foods and beverages in order to
prolong their shelf- life. The hydrogen peroxide produced by the enzyme acts
as a good bactericide and can be later removed using catalase which converts
hydrogen peroxide to oxygen and water. GOx can also be used to remove
oxygen from the top of bottled beverages before they are sealed. The GOx was
also found to be antagonistic potential against different food borne pathogens
like Salmonella infantis, Staphylococcus aureus, Clostridium perfringens,
Bacillus cereus, Campylobacter jejuni and Listeria monocytogenes (Tiina and
Sandhlm, 1989). In the glucose detection kits, GOx act as a basis of glucose
sensor (Degani and Heller, 1988).
Minimally processed vegetables (MPV) sold in ready-to-eat (salads) or
ready-to use forms have become a very important area of potential economic
growth for fresh-cut vegetables and fruit industry. Today MPV products have
gained popularity mainly because consumers perceive such products, besides
30
their well-known nutritional qualities, as fresh, healthy, convenient, tasty and
easy to use.
Current technologies for preservation and shelf-life extension of food
include chemical preservatives, heat processing, modified atmosphere
packaging, vacuum packaging or refrigeration. However, these steps do not
eliminate undesirable pathogens from these products or delay microbial
spoilage entirely.
Alternative preservation techniques such as novel non-thermal
technologies and naturally derived antimicrobial ingredients are under
investigation for their application to food products. Alginate-based edible films
and calcium ions have the ability to limit moisture loss and enhancing shelf-life
of vegetables has been studied by the incorporation of various natural
antimicrobial agents.
A number of naturally occurring antimicrobial agents have been
investigated that includes GOx, LPS, lysozyme, lactoferrin, avidin, various
plant extracts such as spices and their essential oils, sulfur and phenolic
compounds (Davidson et al., 2001). Combinations of preservation treatments
allow the required level of protection to be achieved while at the same time
retaining the natural qualities of the product such as, colour, flavour, texture
and nutritional value.
31
CHAPTER-2
LITREATURE SURVEY
Utilization of highly selective oxidizing biocatalysts such as oxygenases
and oxidases is an emerging field of white biotechnology, since, it may lead to
an environment-friendly production of enantiomerically pure compounds
serving as valuable chiral synthons for subsequent chemical syntheses of
biologically active substances (Stottmester et al., 2005).
Glucose oxidase a flavoprotein which catalyses the oxidation of β-D-
glucose to D-glucono-δ-lactone and hydrogen peroxide using molecular
oxygen as the electron acceptor. The reaction can be divided into a reductive
and an oxidative step. In the reductive-half reaction, GOx catalyses the
oxidation of β-D-glucose to D-glucono-δ-lactone which can in certain fungi
such as Aspergillus sp., be enzymatically or spontaneously hydrolyzed to
gluconic acid. Subsequently, the flavine adenine dinucleotide (FAD) ring of
GOx is reduced to FADH2 (Witt et al., 2000). In the oxidative half reaction the
reduced GOx is re-oxidised by oxygen to yield hydrogen peroxide (Figure 2.1).
The kinetics, mechanism of action, properties and molecular structure of
GOx were studied by many researchers (Swoboda and Massey, 1965; Tsuge et
al., 1975; Takegawa et al., 1991; Hecht et al., 1993). GOx from A. niger is a
homodimer with a molecular weight of 150–180 kDa. It contains two tightly
bound FAD molecules (Pazur and Kleppe, 1964). Dissociation of the sub-units
only occurs under denaturation conditions and is accompanied by the loss of
the cofactor FAD (Jones et al., 1982). The amino acid sequence for the 583
residues protein has been derived from the DNA sequence independently by
Kriechbaum et al. (1989) and Frederick et al. (1990).
32
Figure 2.1: Enzymatic conversion of glucose to gluconic acid by GOx
33
The enzyme is highly specific for β-D-glucose with other
monosaccharides, being oxidized at much lower rate (Adams et al., 1969).
GOx from A. niger is a highly glycosylated protein, the carbohydrate content is
ranged from 10% to 24% of its molecular weight (Pazur et al., 1965; Hayashi
and Nakamura, 1981). The glycosylated protein contains 190 mannose and 16
N-acetyl glucosamine residues. Several functions have been proposed for the
carbohydrate moiety of glycoproteins including correct targeting or proteins,
transport through membranes, biological function, immune response and
stabilization of the three dimensional structure (Figure 2.2) of the protein
(Kalisz et al., 1991). In case of GOx, the deglycosylation did not significantly
affect the three dimensional structure of the enzyme. Other properties such as
thermal stability, pH and temperature optimum of GOx activity and substrate
specificity were not affected. Thus, the carbohydrate moiety of GOx, like that
of other glycoproteins, does not appear to contribute significantly to the
biological properties of enzyme (Kalisz et al., 1991). On the other hand, the
carbohydrate-depleted GOx was more rapidly precipitated by the addition of
trichloroacetic acid and ammonium sulfate than the native enzyme. These
results show that the N-linked sugar chains of GOx contribute to the high
solubility of the enzyme in water (Takegawa et al., 1989).
Glucose oxidase was suitable in large-scale technological application
since, 1950’s, which includes the enzymatic determination of glucose with
biosensor technology (Vodopivec et al., 2000) for the production of gluconic
acid and as a food preservative. Implantable glucose sensors may find
significant application for monitoring of glucose in diabetics (Gerritsen, 2001).
Enhancement of the properties of GOx is still receiving more attention,
presumably due to the current and extensive applications of this enzyme.
34
Overall topology of GOx homoenzyme
Subunit structure of GOx showing FAD (red space fill)
Figure 2.2: 3-Dimentional structure of glucose oxidase
35
2.1 Glucose oxidase production
The carbon sources used for GOx production ranged from simple C3
such as glycerol up to a complex carbon source such as starch. Glucose was
mainly used in all cultivation media with a concentration ranging from 40 to 80
g/l. However, in the industrial scale, sucrose or corn steep liquor can be used in
case of bulk production of GOx in non-purified form which is utilized in
gluconic acid production, food preservation and other non-analytical purposes.
Also, different organic and inorganic nitrogen sources were added to the
cultivation medium to enhance production. Peptone or polypeptone with a
concentration ranging from 3 to 20 g/l or yeast extract were the main common
organic nitrogen sources. On the other hand, sodium nitrate was mainly used as
inorganic nitrogen source for GOx production followed by ammonium di-
hydrogen phosphate. The source of phosphate was in the form of either
potassium di-hydrogen phosphate or di-potassium hydrogen phosphate with a
concentration ranging from 0.2 to 1.0 g/l reported by Nakamatsu et al. (1975).
Inorganic salts for supplementation with Mg++, Fe++, Zn++ and K+
cations were also added in low quantity. Moreover, either magnesium
carbonate or calcium carbonate were used in some publications in case of
cultivation in shake flask to neutralize the acidity of the cultivation medium
due to gluconic acid production which was concomitant with GOx production
on using glucose as carbon source. Nakamatsu et al. (1975) studied the effect
of different complex carbon sources as well as different nitrogen sources on
GOx production.
Zetelaki and Vas (1968) investigated the effect of aeration and agitation
on the GOx production by A. niger in a 5 l stirred tank bioreactor. They found
that the maximum enzyme production was recorded at 700 rpm. Further
increases in the agitation speed resulted in neither a higher growth rate nor
higher activity.
36
2.2 Media optimization
There are some advantages of using statistical methods, over the one-
factor-at-a-time classical method for optimization of media. However,
statistical design enables easy selection of important parameters from a large
number of factors and explains the interactions between important variables. A
number of statistical experimental designs were used for optimizing the
fermentation variables. The Plackett–Burman design (Plackett and Burman,
1946) is a well known and widely used statistical technique for screening and
selection of most significant culture variables, while the central composite
design (CCD) provides an important information regarding the optimum level
of each variable along with its interactions with other variables and their
effects on product yield (Pardeep and Satyanarayana, 2006).
2.3 Natural occurrence of GOx and its applications
Glucose oxidase is naturally produced by various fungi and insects. The
main function of GOx is to act as antibacterial and antifungal agent through the
production of hydrogen peroxide. Permanent oxidative stress through the
maintenance of hydrogen peroxide at low concentration by GOx’s continued
catalytic activity was reported by many researchers (Tiina and Sandholm 1989;
Dobbenie et al., 1995) to be very effective against bacterial or fungal growth.
Breakdown of hydrogen peroxide by the microorganism’s intrinsic
catalase may protect it against hydrogen peroxide’s antibacterial or antifungal
effect. Presence of catalase at millimolar level of hydrogen peroxide was
required to inhibit the cell growth. Whereas, in the presence of GOx, the
micromolar level of hydrogen peroxide was constantly maintained by the
catalytic activity of GOx was already sufficient to inhibit the cell growth.
Interestingly, natural functions of GOx include assisting in plant infection,
lignin degradation, lowering pH of the environment, etc., (Chun Ming Wong et
al., 2008).
37
Detailed below are many industrial and commercial applications where
GOx may be used in. The usefulness of GOx in diverse fields had triggered the
search for new sources of GOx from other species of fungi (including A. niger;
Hatzinikolaou et al. 1996) and insects to satisfy the demand for improved
properties such as higher catalytic activity (Fiedurek and Gromada, 1997). The
following discussion will attempt to cover the major applications together with
the respective working principle utilized.
2.4 Food processing additive
Glucose oxidase has Generally Regarded As Safe (GRAS) status under
FDA classification (FDA/CFSAN 2002 a, b) and is available in bulk for use in
food industry as an additive in liquid or powder form. It is often classified with
antioxidant, preservative and stabilizer properties. There are many food
products in which GOx can be used. Some of them are elaborated below, and a
detailed list can be found in the database of Codex Alimentarius Commission
(2007a). The food grade GOx preparation used typically contains a mixture of
GOx and catalase because the two enzymes are found naturally together in the
mycelium cell wall (Witteveen et al., 1992). Separation of GOx from catalase
is costly and not essential in food grade preparations. Furthermore, catalase
assists in the breakdown of hydrogen peroxide produced by GOx, thereby
reducing inhibition and deactivation by hydrogen peroxide (Bao et al., 2001
and Bao et al., 2003).
2.4.1 Bread making
Maturing/oxidizing agents are an essential additive to flour. One of its
purposes is to strengthen gluten, thereby improve the bread’s final texture. This
is achieved through the oxidation of two proteins within flour, gliadin and
glutenen to allow more bonds to form when gluten develops. Gluten forms
when gliadin and glutenen are in contact with water and its maturation is
assisted by the actions of yeast (Corriher, 2001). Only a small amount of
38
maturing agents in the level of parts per million is needed in this process and
traditionally, potassium bromate was used (Figoni, 2003). However, it has
been recognized that bromate is carcinogenic, causing DNA damage invitro
and invivo that may contribute towards cancer (Moore and Chen, 2006). As a
result, most countries have prohibited the use of bromate in food, and an
alternative such as GOx is used in bakery (Enzyme Technical Association,
2001).
Glucose oxidase is an effective oxidant to produce bread with improved
texture and increased loaf volume (Vemulapalli et al., 1998; Rasiah et al.,
2005). The basis of oxidation by GOx has been validated to be a result of the
hydrogen peroxide produced which yields dough that is more elastic and
viscous than the control without GOx (Vemulapalli et al., 1998). In addition,
GOx also causes a drying effect on dough that is attributed to gel formation of
water soluble pentosans (Vemulapalli and Hoseney, 1998). As it is recognized
that potassium bromate does not cause this drying effect, it is postulated that
this effect is induced by GOx (Vemulapalli et al., 1998). Although, the exact
mechanisms by which hydrogen peroxide produced by GOx improves the
dough properties are not completely understood, more work are in progress,
and some theories have been proposed (Rasiah et al., 2005; Franziska Hanft
2006). Nevertheless, GOx is known to cause cross-linking of dough protein
(Rasiah et al., 2005) and exert effects such as reducing the sulfhydryl content
as well as increasing viscosity in the water soluble portion of dough
(Vemulapalli and Hoseney 1998).
2.4.2 Dry egg powder
Maillard non-enzymatic browning is a result of reaction between the
amino group of proteins or amino acid and reducing sugars. In the production
of dried egg powder this reaction causes undesirable browning and formation
of unwanted flavour. Therefore, the glucose present (~4 g/l) in liquid egg is
39
typically removed before spray drying (Sisak et al., 2006). Glucose removal
also has the added benefit of longer shelf-life and enhanced microbial
tolerance. Besides that, hydrogen peroxide produced in the reaction can also
kill or inhibit growth of microorganisms commonly present in liquid egg
(Dobbenie et al., 1995). One of the way to remove glucose from egg is by
adding GOx before spray drying takes place, as allowed under FDA
regulations. This is typically a batch process and contains the enzyme as an
impurity in the product. Through immobilization, continuous process is
possible, and the enzyme can also be retained and recycled instead as being an
impurity in the product. Hence, there were studies into the viability of reactors
for de-sugaring based on immobilized GOx (Sisak et al., 2006). It should be
noted that undesirable browning caused by Maillard reaction is not unique to
eggs. Potato products also suffer the same issue, and GOx can be employed to
reduce glucose content, thereby reducing browning (Low et al., 1989).
2.4.3 Antioxidant/preservative (oxygen scavenger)
The presence of oxygen is a problem in many food products. In high-fat
foods such as mayonnaise and salad dressing, lipid oxidation can cause
deterioration and rancid taste (Isaksen and Adler-Nissen, 1997). The same is
true for beverages such as wine and beer, keeping oxygen out of the drink
helps to maintain taste and flavour (McLeod and Ough, 1970; Labuza and
Breene, 1989). In canned/bottled/packaged food, oxygen also promotes
bacterial growth, hence, it is desirable to remove oxygen from the headspace to
maintain an anaerobic environment (Kirk et al., 2002). In puree processing,
oxygen contributes to maillard non-enzymatic browning, therefore appropriate
controls must be in place (Parpinello et al., 2002).
The overall reaction catalyzed by GOx involves the consumption of two
glucose molecules and one oxygen molecule to produce two gluconic acid
molecules. This reaction consumes oxygen, a trait that allows GOx to be used
40
as an active oxygen scavenger, antioxidant and preservative in various food
applications. Detailed analysis on using GOx as an oxygen scavenger can be
found in Labuza and Breene (1989). Moreover, the catalytic product, gluconic
acid, is safe for human consumption and WHO has not specified a limit on its
acceptable daily intake. This combined with the demand from consumers to
replace chemical antioxidant and oxygen scavenger with natural compounds
which makes GOx an ideal for food preservation.
2.4.4 Glucose oxidase in sea foods
As a preservative, GOx has been demonstrated to be useful to extend the
shelf-life of some sea food. Fillets or whole fish dipped with GOx/glucose
solution before being refrigerated could be stored for up to 21 versus 15 days
for control (Field et al., 1986), and similarly treated shrimp could also be
stored for up to 11 versus 6 days for control (Dondero et al., 1993). This
phenomenon is most likely due to growth inhibition of the spoilage bacteria
such as Pseudomonas fragi, which is commonly present in fish (Yoo and Rand
1995); Pseudomonas fluorescens, which is associated with shrimp (Kantt et al.,
1993); enterotoxic bacteria E. coli and Salmonella derby (Massa et al., 2001).
Other than the food industry, the “natural” advantage of GOx has also triggered
interests from the pharmaceutical company to pursue the use of GOx to replace
traditional antioxidant in their formulations (Uppoor et al., 2001).
2.4.5 Dairy and the lactoperoxidase system (LPS)
One of the most important applications of GOx in food processing
industry is food preservation. The LPS system, when used in conjunction with
GOx, is a very useful antimicrobial agent. LPS is part of the immune system’s
innate defense mechanism against foreign microorganisms and can be found in
mammalian secretions such as milk, tears and saliva. This system consists of
three components like LPS, thiocyanate and hydrogen peroxide. LPS activation
41
occurs only in the presence of thiocyanate and hydrogen peroxide. Catalysis by
LPS generates active intermediates, which has antimicrobial properties and is
completely safe to humans. The presence of GOx and its substrate (glucose)
allows hydrogen peroxide required by LPS to be continuously generated and
replenished (Seifu et al., 2005). Sathiya moorthi et al. (2008) state that, LPS
has been recognized as an effective antimicrobial agent since many years and
used extensively as an antibacteriostatic agent in reducing micro flora in milk
as well as while making cheese.
For the transportation and/or storage of raw milk, the use and the
activation of the LPS is effective against spoilage and it is recommended to be
used when refrigeration is unavailable or as a complement to refrigeration.
Experiments have demonstrated that the shelf-life of milk with active LPS
enzyme almost doubled the comparison to milk with inactivated LPS (Marks et
al., 2001). In addition, activation of the LPS is also suggested as a pre-
treatment for dairy products to enhance bacterial deactivation including
mastitis pathogens (Sandholm et al., 1988) and to allow lower temperature
treatments during pasteurization (Seifu et al., 2005).
The same LPS–GOx system mentioned previously can also be used in
cheese production. The hydrogen peroxide produced by GOx is utilized by the
LPS for cold, i.e. room temperature sterilization, while the gluconic acid
produced is used for direct acidification (Fox and Stepaniak, 1993). It should
be noted that this LPS-GOx antimicrobial system is not limited to food and has
been used in toothpaste (Biotene, 2006; National Library of Medicine, 2007a),
lotions (National Library of Medicine, 2007b), shampoos, cosmetics, meat
processing (Food Standards Australia New Zealand, 2002) and fish farming
(Seifu et al., 2005).
Further more, during incomplete reduction of molecular oxygen, the
super oxide radical, is generated hydrogen peroxide may lead together with the
42
super oxide radical and trace amounts of transition metal ions [eg., Fe(II)] in
the so called Fenton reaction to the formation of the extremely biocidal
hydroxyl radical (Luo et al., 1994). A wide range of both Gram-negative
bacteria (Wray and Mc Laren, 1987; Borch et al., 1989) and Gram-positive
bacteria (Oram and Reiper, 1966; Siragusa and Johnson, 1989) are inhibited by
LPS. However, studies have shown that Gram-negative bacteria were generally
found to be more sensitive to LPS mediated, (food) preservation than Gram-
positive species (Marshal and Reoter, 1980; De Wit and Van Hooydonk,
1996).
2.4.6 Reduced alcohol wine
Sugar is an important ingredient when it comes to alcohol production
through fermentation because it is the primary substrate used by
Saccharomyces cerevisiae to produce alcohol. Hence, reduction of glucose is
necessary to obtain the lower alcohol content. As there are demands for
reduced alcohol wines, partly driven by its lower tax and tariffs, there were
investigations on the feasibility of using various technologies (Gary, 2000).
One of them is to use GOx to reduce the amount of glucose available,
subsequently yielding lower alcohol content.
One of the easiest ways to do this is to add GOx to the must before
fermentation. GOx consumes some of the glucose present making them
unavailable for alcohol fermentation, thereby resulting in wine with reduced
alcohol (Pickering et al., 1998, 1999a, 1999b and 1999c). At the same time,
hydrogen peroxide generated may reduce the activity or growth of the
S. cerevisiae used for alcohol fermentation. Nevertheless, experiments had
shown that the must containing GOx completed fermentation in 10 days,
whereas the control required 12 days (Pickering et al., 1999a). Another
approach examined was to genetically engineer the S. cerevisiae used during
the fermentation process to express GOx. This approach was considered viable
43
but requires more work to be done before it can reach the market. One added
advantage in the use of GOx is that the hydrogen peroxide produced acts as a
bactericide, helping to act as preservative for the wine (Malherbe et al., 2003).
2.4.7 Gluconic acid production
Gluconic acid and its derivative salts are GRAS and can be used in a
wide range of industries (Ramachandran et al., 2006) including textile dying,
metal surface cleaning, food additives, detergents, concrete, cosmetics (Yu and
Scott, 1997) and pharmaceuticals (BACAS, 2004). As a food additive, it can be
used as an acidity regulator, raising agent, colour stabiliser, antioxidant and
chelating agent in bread, feed and beverage (Brookes et al., 2005; Codex
Alimentarius Commission, 2007b). Industrially, gluconic acid is mostly
produced from fermentation (Singh et al., 2005), with an estimated global
production of about 50,000–100,000 ton/year (BACAS, 2004; EuropaBio and
ESAB, 2005). Further information regarding the properties, applications and
microbial production of gluconic acid has been described by Ramachandran et
al. (2006).
As with all fermentation processes, there are some disadvantages.
Cultures require various added nutrients and at least a few days to grow and
perform bioconversion. In addition, culture solutions produce and contain
unwanted by-products, need downstream purifications and consume substrates
prohibiting high conversion efficiency. Hence, the use of enzyme-based
conversion is considered a viable method to reduce production cost and time
(Nakao et al., 1997). For example, during 1997 and 2003, there were patents
filed making claims of GOx based process that is capable of almost 100%
conversion efficiency, require less time than fermentation and do not contain
impurities (Vroemen and Beverini, 1999; Lantero and Shetty, 2004).
Bioreactor using immobilized GOx is one of the preferred setups being
investigated (Godjevargova and Turmanova, 2004). Immobilization allows the
44
enzymes to be recycled, reduces cost as well as permitting relatively easier
design and construction of reactor to produce and remove of the desired
product, gluconic acid continuously. Despite these potential advantages, the
lack of industrial adoption implies that there are major hurdles to overcome
before mass adoption of enzyme-based bioconversion process occurs. This is
evident, as there is patent covering industrial scale production of gluconic acid
from glucose using GOx dating decades old (Bergmeyer and Jaworek, 1976).
Nevertheless, electrodeionisation-based separative bioreactor (a technology
used to make deionised water; Arora et al., 2007) backed by the US
Department of Energy Biomass Program seems promising and may be adopted
by the industry in the near future.
2.4.8 Glucose sensor/assay
Recently estimated the world’s market value of biosensors to be about
$5 billion dollars, and 85% is attributed to glucose biosensors. Many glucose
sensors available in the market are based on immobilized GOx, and more
information about glucose sensors available in the market can be found in the
review article by Newman and Turner (2005).
Glucose oxidase is commonly used to construct amperometric
biosensors for medical (Wilkins and Atanasov, 1996; Newman and Turner,
2005) and food industry (Mello and Kubota, 2002). A constant electric
potential is applied between working and reference electrode, promoting the
catalytic reaction, which drive the current flow that is proportional to the
concentration of the target molecule (Terry et al., 2005). Such amperometric
glucose sensors based on GOx can be divided into three generations according
to its principle of operation and historical development (Wilkins and Atanasov,
1996; Newman and Turner, 2005; Park et al., 2006).
45
In the medical industry use of GOx in glucose sensors is not only
limited to the traditional “fingerpricking” blood glucose measurement devices
but has also been investigated to be used in continuous monitoring of glucose
in vivo (Wilson and Hu, 2000; Klonoff, 2005) such as fluorescent-based
glucose sensing (Pickup et al., 2005, Brown et al., 2006; Brown and McShane,
2006; Yang et al., 2006). Fluorescent-based glucose sensing has some
advantages over “fingerpricking” sensors, for e.g., extreme sensitivity and non-
invasive.
2.4.9 Fuel cell
The use of GOx in fuel cell is not a recent trend. Investigations on GOx
based fuel cells have been on-going since 1960s (Davis and Yarbrough, 1962;
Yahiro et al., 1964). Despite these efforts and ability to produce cells with near
100% current (faradic) efficiency (Weibel and Dodge, 1975), biofuel cells are
still not ready for applications outside the laboratory. As pointed out in many
litreatures (Calabrese Barton et al., 2004; Bullen et al., 2006; Davis and
Higson, 2006), two main hurdles of biofuel cells are the limited lifetime and
limited power output of the cells. Higher enzyme stability is needed to improve
lifetime of the cells from days/months to years, while higher enzyme catalytic
rate is needed to improve power output by several orders of magnitude.
One viable approach to tackle these problems is by using directed
evolution. For example, by using GOx detection assay which measures the
amount of NADPH produced from the downstream catalysis of GOx end-
products at 340 nm, Zhu et al., (2006) obtained GOx mutant E4, which
displays a higher catalytic activity. GOx are typically used in the anode of
biofuel cells to oxidise glucose, i.e., extract electrons and transfer it to the
anode electrode from which the electrons will flow through the load in the
circuit to the cathode at which the electron will be used to reduce molecules,
e.g., oxygen to water (Weibel and Dodge, 1975). At the same time, ions such
46
as proton, i.e. H+ will diffuse from the anode compartment through the
separating semi-permeable membrane to the cathode compartment to complete
the circuit. Typically, this semi-permeable membrane is necessary to prevent
mixing of materials in the two compartments, which may cause interference to
the operation of the cell.
2.4.10 Other uses of Glucose oxidase
Use of GOx is not limited to the applications described above. In the
textile industry, there are considerable interests to replace chemical bleaching
with environmentally friendly bio-bleaching processes. Chemical bleaching
requires around pH 10.5–11 and near boiling temperature, whereas bio-
bleaching can be conducted at lower temperature and around neutral pH. This
means significant cost saving in energy and effluent treatment. Research
conducted so far have shown promising results in using GOx to produce
hydrogen peroxide for bleaching, utilizing the glucose generated from the
upstream desizing and bioscouring processes (Buschle-Diller et al., 2002;
Tzanov et al., 2002). Although it was noted that the cost of the enzyme is too
expensive for textile processing (Hamlyn, 2000), a patent by Novozyme North
America, Inc. (Salmon et al., 2006) can be found, which covers the use of
carbohydrate oxidase, including GOx to bleach textiles. In other words, it
would be reasonable to speculate that the use of GOx in textile industry should
be economically viable in the near future.
While bio-bleaching can be performed in the factories, it is also possible
to do the same in the everyday laundry by adding GOx to laundry detergent
preparations (Pramod, 1999). In the laboratory, GOx also has diverse uses. For
example, GOx can be used in various immunoassays and/or staining
procedures as well as removal of excess glucose (Rathlev, 1983; Porter and
Porter, 1984; Pfreundschuh et al., 1988; Blais and Yamazaki, 1992; Dosch et
al., 1998; Megazyme, 2003). Whereas, in real-time fluorescent microscopy for
47
biological samples, GOx-catalase is often used for oxygen scavenging to
reduce photodamage (Desai et al., 1999). In geochemicalprospecting, heap
leaching, pollution studies, etc., GOx can be used to prepare mineral leaching
solutions as both hydrogen peroxide and gluconic acid produced facilitates
leaching (Clark, 1995; Clark, 1996).
In genetic engineering, expression of GOx in plants can provide
resistance to bacterial infection (Wu et al., 1995). Obviously, there is no
restriction to the number of potential applications that GOx may be employed
in. Other than the known uses mentioned in this article, there seems to be
plenty of room for new novel applications. For instance, when GOx is used as
preservative in packaged food, changes in pH due to glucose hydrolysis can
potentially be monitored using a pH strip visible outside the package. As the
GOx catalyzed reaction is oxygen limited, if the package is broken and air
leaks in, it will provide the necessary oxygen for GOx to hydrolyze glucose
into gluconic acid, causing pH to drop. Alternatively, a specially designed
container could permit some degree of air penetration, providing the necessary
oxygen for GOx to generate and maintain low level of hydrogen peroxide for
microbial inhibition.
Enzymes, including GOx are gaining importance and popularity in the
industry as an environmental friendly alternative to the traditional chemical
treatments, especially when it becomes more cost effective to produce. With
the enzyme market predicted to have annual growth rate at 7.6% per annum
and market value increase from $4.1 to $6 billion by 2011 (The Freedonia
Group, 2007), and they expecting more breakthroughs, investments as well as
innovations in the applications of GOx in the future.
48
2.5 Vegetable and food processing
2.5.1 Chlorination
Disinfection by chlorination has had many applications in the
propagation, production, harvest, post harvest handling, and marketing display
of fresh fruits and vegetables for many decades (Winston et al., 1953;
Endemann, 1969; Anon, 1970; Hough and Kellerman, 1971; Goodin, 1977;
Rabin 1986, Bartz and Lill, 1988). In the past, maintaining wash tank and
flume concentrations of 3,000 mg/ml for tomatoes and 6,000 mg/ml for citrus
were recommended to control decay (Winston et al., 1953). The primary uses
of chlorine have been to inactivate or destroy pathogenic bacteria, fungi,
viruses, cysts, and other propagates of microorganisms associated with seed,
cuttings, irrigation water, farm or horticultural implements and equipment,
contact surfaces, and human contact with fresh produce. Chlorination has been
routinely used to treat post harvest cooling water, in post harvest treatments
(i.e., calcium for firmness enhancement) and during rehydration at shipping
destinations. Chlorine, primarily as sodium or calcium hypochlorite, has been
an important part of a properly managed horticultural sanitation program for
several decades.
2.5.2 Chlorine dioxide
Chlorine dioxide (ClO2) has been recognized as a strong oxidizing agent
with a broad biocidal effectiveness due to the high oxidation capacity of about
2.5 times greater than chlorine. Many studies have demonstrated its
antimicrobial activity. Since its use was allowed in washing fruits and
vegetables by the efficacies of aqueous chlorine dioxide, ozonated water and
thyme essential oil alone or sequentially in killing mixed strains of E. coli
O157:H7 inoculated on alfalfa seeds. They found that the sequential washing
procedure (thyme oil followed by ozonated water and aqueous ClO2 was
significantly more effective in removal of E. coli O157:H7. However, the use
49
of the combination of these techniques may adversely affect the organoleptical
properties.
Chlorine dioxide has received a lot of attention in the last few years
because its effectiveness is less affected by pH and organic matter content than
that of chlorine. Another advantage is its high oxidative action, which has been
observed to be 2.5 times greater than chlorine. However there are some
disadvantages also. These include its poor stability, virus resistance, and its
tendency to explode at high concentrations. Chlorine dioxide decomposes at
temperatures above 30°C (86°F) and when it is exposed to light. Despite these
disadvantages, use of chlorine dioxide has been increasing because of new
technologies that permit shipment to areas of use instead of onsite generation.
Concentrations should not exceed 5 ppm for treating unpeeled fruits and
vegetables. Chlorine dioxide is approved as a wash treatment for uncut
produce, and is being reviewed for approval as a wash treatment for pre-cut
produce (Rabin, 1986).
2.5.3 Biodegradable packaging
The purposes of food packaging are to protect foods from outside
contamination during distribution, transmission and storage, to maintain the
correct moisture, oxygen or carbon dioxide content in a product or maintain a
desired atmosphere in the headspace around a product. Materials, especially
those used in the food and agriculture industries, have rapidly developed in last
few years. The availability in large quantities at low cost and favorable
functional characteristics enabled the broad application of petrochemical based
plastics such as polyolefins, polyesters, polyamides, and etc., However, their
non biodegradable characteristics lead to environmental pollution and
packaging material has been the target of environmental and consumer activist
groups as being a major contributor to the solid waste stream. Biodegradable
packaging materials received a great attention because of their functionality
50
and environmental-friendly attributes. Among them, edible coatings and films
show special advantages in increasing the shelf-life of product.
2.5.4 Edible coatings and films
Edible packaging and coatings must be free of toxic compounds and
should have a high biochemical, physico-chemical and microbiological
stability, before, during and after application (Risch, 2000). They also should
have good sensory qualities and good barrier and mechanical properties. The
main components of edible packaging that provide good film-forming
properties are polysaccharides, proteins and lipids. They are not only
biodegradable products from various food sources, but can also serve as
carriers for certain additives, such as antioxidants, preservatives, flavours, etc.,
(Day, 1998). Edible coatings could also provide a barrier against visible and/or
UV light which can modify the food characteristics via oxidation of lipids and
pigments (Risch, 2000).
Polysaccharide and protein based films have good mechanical properties
and present excellent barriers for gases, aromas, and lipids, but are highly
permeable to moisture (Krochta and Mulder-Johnson, 1997). Wu et al. (2001)
described that starch-alginate based edible films had the ability to limit
moisture loss and lipid oxidation of pre-cooked beef patties but the abilities
differed with the composition of films. Films made from high amylose starch
showed lower water vapour permeability (WVP) and gas permeability (GP)
than regular corn starch films while addition of oil decreased WVP of starch-
based films (Garcia et al., 2000).
Protective coatings based on zein are commercially available for use on
confectionery items, shelled nuts, and pharmaceutical tablets. As the other
protein films, zein films have high WVP. Wu et al. (2001) tested zein and
zein/lipid films and found that the addition of plasticizer and lipid to film
51
matrix lowered the WVP and increased the elasticity of the films. Studies
showed that, contrary to hydrophilic polysaccharide and protein based films,
hydrophobic lipid based films have poor mechanical properties but high
moisture resistance (Yang and Paulson, 2000). Therefore, significant efforts
have been directed towards the development of edible films by incorporation of
both hydrophilic and hydrophobic molecules into the film-forming matrix to
improve film’s physico-chemical properties. Yang and Paulson (2000) showed
that addition of lipids to gellan films significantly improved the WVP, but
lower the mechanical properties and caused the films to become opaque.
Garcia et al. (2000) found that the addition of sunflower oil to starch-based
film decreased the WVP and lowered the crystalline-amorphous ratio
compared to films without additives. The increase of wax content in lactic
acid-casein based edible films could significantly decrease WVP. Sebti et al.
(2002) introduced stearic acid in cellulose films what resulted in decreased
water vapor transmission rate, increased contact angle, decreased tensile
strength, and lowered air permeability of the films. Similarly, Ozdemir and
Floros (2003) reported that increasing the amount of beeswax resulted in
decreased potassium sorbate diffusivity in whey protein films.
As film-forming biopolymers have the ability to act as carriers of small
molecules, various additives have been applied in the films and coatings.
Incorporation of essential oils into the films and coatings may not only
improve the mechanical characteristics of the films, but also enhance their
antimicrobial properties. Combination of naturally occurring antimicrobial
components, chitosan and essential oils, may provide a unique system with
enhanced antimicrobial properties. Incorporation of essential oils into the
chitosan films could reduce loss of active components due to evaporation and
establish possibilities for prolonged antimicrobial action and improved safety
of foods. Furthermore, the hydrophobic compounds of the oils may enhance
barrier and mechanical properties of the films.
52
The usual approach to improve mechanical properties of edible films is
to add a plasticizer such as glycerol, which is a low molecular weight non
volatile substance, into the film to reduce biopolymer chain to chain interaction
resulting in the improvement of film flexibility and stretch ability. However
plasticizers also increase the film permeability (Yang and Paulson, 2000).
2.5.4.1 Cellulose
Cellulose is the most abundant natural polymer on earth and it is an
essentially linear natural polymer of anhydroglucose. As a consequence of its
chemical structure, it is highly crystalline, fibrous and insoluble. Several water-
soluble, composite coatings are made commercially from cellulose, carboxy
methylcellulose (CMC) with sucrose-fatty acid esters. Derivatives of cellulose,
such as methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC),
form strong and flexible water-soluble films (Baldwin et al., 1996).
2.5.4.2 Alginate
Alginates which are extracted from brown seaweeds of the Phaephyceae
class, are the salts of alginic acid, a linear co-polymer of D-mannuronic and L-
guluronic acid monomers. The ability of alginates to react with di and trivalent
cations is being utilized in alginate film formation. Calcium ions which are
more effective than magnesium, manganese, aluminum, ferrous and ferric ions,
have been applied as gelling agents.
Wu et al. (2001) described that starch-alginate-based edible films had
the ability to limit moisture loss and lipid oxidation of pre-cooked beef patties
but the abilities differed with the composition of films. Films made from high
amylose starch showed lower WVP and GP than regular corn starch films
while addition of oil decreased WVP of starch-based films (Garcia et al.,
2000).
53
2.5.4.3 Zein
Corn zein, the prolamin fraction of corn protein has been used
commercially in coating formulations for shelled nuts, candy and
pharmaceutical tablets. Corn zein coating provides a good barrier to oxygen
and its WVP is about 800 times higher than that of a typical shrink-wrap film.
The aqueous alcohol-soluble protein extracted from corn gluten has been
reported to form films with relatively good water barrier properties. Park et al.
(1999) reported that corn zein film had an effect on delaying ripening and
colour change in tomatoes during storage and confirmed that the degree of
colour change which is an indicative of ripening was mainly dependent on the
coating thickness.
2.5.4.4 Banana powder
Banana mainly consists of starch and pectin (Kotecha and Desai, 1995);
these compounds might possibly provide sufficient properties to form
renewable, biodegradable and inexpensive films and packages. Recently
starches isolated from banana, okenia and mango were used to form edible
films (Romero-Bastida et al., 2005). Banana flour obtained from the whole
banana consists mainly of both starch and pectin, which might form films with
good oxygen barrier and mechanical properties. These films would reduce the
need to isolate the starch, making material preparation easier. Therefore,
obtaining films with good oxygen permeability (OP) and desirable film
mechanical properties would be an indication of the possible use of banana
flour as an alternative secondary packaging. Banana films can be used for dried
products and may have a potential to be commercial.
2.6 Antimicrobial agents in film coating
Antimicrobial agents incorporated into edible films or coatings are
released onto the surface of food to control microbial growth. Such coatings
can also serve as a barrier to moisture and oxygen. Edible coatings have
54
become popular in the food industry because they produce less waste, are cost
effective and offer protection after the package has been opened. Hershko and
Nussinovitch (1998) reported on the behavior of hydrocolloid coatings on
vegetative materials. Park reviewed the development of systematic means of
selecting edible coatings to maximize quality and shelf life of fresh fruits and
vegetables.
Fresh foods may contain microorganisms both on their surfaces and
within. These microorganisms, if not destroyed, lead to food spoilage. The
prevention of food spoilage by inhibiting or destroying microorganisms is the
basis of food preservation. Antimicrobial agents incorporated into edible films
or coatings are released onto the surface of food to control microbial growth.
Such coatings can also serve as a barrier to moisture and oxygen.
2.6.1 Ethylene diamine tetra acetic acid (EDTA)
It has been recognized since 1960s that susceptibility of gram-negative
organisms to lysis by Lysozyme can be increased by the use of membrane
disrupting agents such as detergents and chelators. EDTA, a chelator, exhibits
antimicrobial effect by limiting the availability of cations and can act to
destabilize the cell membranes of bacteria by complexion of divalent cations
which act as salt bridges between membrane macromolecules, such as
lipopolysaccharrides (Boziaris and Adams, 1999). Cutter et al. (2001)
reported improved antimicrobial activity of nisin-incorporated PE or PE oxide
blend films by formulation change and addition of food grade chelator-EDTA.
EDTA and other antimicrobial agents such as nisin, lysozyme and GFSE were
mixed and incorporated into Na-alginate and κ-carrageenan for hurdle
effectiveness to Gram-positive and Gram-negative bacteria.
55
2.6.2 Bacteriocin
The bacteriocins produced by lactic acid bacteria (LAB) offer several
desirable properties that make them suitable for food preservations are: (i)
generally recognized as safe substances, (ii) are not active and nontoxic on
eukaryotic cells, (iii) become inactivated by digestive proteases, having little
influence on the gut micro biota, (iv) usually pH and heat-tolerant, (v) have a
relatively broad antimicrobial spectrum, against many food-borne pathogenic
and spoilage bacteria, (vi) they show a bactericidal mode of action, usually
acting on the bacterial cytoplasmic membrane: no cross resistance with
antibiotics and (vii) their genetic determinants are usually plasmid-encoded,
facilitating genetic manipulation. The accumulation of studies carried out in
recent years clearly indicate that the application of bacteriocins in food
preservation can offer several benefits (Thomas et al., 2000) are: (i), an
extended shelf-life of foods, (ii) provide extra protection during temperature
abuse conditions, (iii) decrease the risk for transmission of food borne
pathogens through the food chain, (iv) ameliorate the economic losses due to
food spoilage, (v) reduce the application of chemical preservatives, (vi) permit
the application of less severe heat treatments without compromising food
safety: better preservation of food nutrients and vitamins, as well as
organoleptic properties of foods, (vii), permit the marketing of “novel” foods
(less acidic, with a lower salt content, and with a higher water content), and
(viii) they may serve to satisfy industrial and consumers demands.
In this respect some of the trends of the food industry in Europe, such as
the need to eliminate the use of artificial ingredients and additives, the
demands for minimally-processed and fresher foods, as well as for ready-to-eat
food or the request for functional foods and nutraceuticals (Robertson et al.,
2004) could be satisfied, at least in part, by application of bacteriocins. The
present review will address different aspects related to food preservation by
bacteriocins including factors influencing bacteriocin activity in food systems,
56
hurdle technology, and the impact of recent advances in molecular biology and
the analysis of bacterial genomes on bacteriocin studies and application.
2.6.3 Lysozyme
Lysozyme is a single peptide protein which possesses enzymatic activity
against the β-1-4 glycosidic linkages between N-acetylmuramic acid and N-
acetylglucosamine found in peptidoglycan. Peptidoglycan is a major
component of the cell wall of both Gram-positive and Gram-negative bacteria.
Hydrolysis of the cell wall by lysozyme can damage the structural integrity of
cell wall and result in the lysis of bacterial cells. Lysozyme is of interest for
use in food systems as it is a naturally occurring enzyme that is produced by
humans and many animals, which have activity against cellular structure
specific to bacteria (Gill and Holley, 2000).
Appendini and Hotchkiss, (1997) investigated the feasibility of
incorporating lysozyme into polymers suitable for food contact. Among the
immobilized polymers (polyvinyl alcohol, nylon and cellulose triacetate)
tested, cellulose triacetate yielded the highest activity. The possible
inadequacies of ice preservation of fresh fish for distribution in the expanding
marketplace were noted and the need for its replacement and supplementation
was affirmed. The combination of the antibacterial effect of the enzyme and
acid production could prove to be an effective agent in the preservation of fresh
fish. Field et al. (1986) evaluated the preservative capabilities of GOx on fish
and attempted to design and implement a GOx system for the preservation of
fresh fish. Soares and Hotchkiss (1998) developed cellulose acetate films with
naringinase immobilized to reduce the naringin concentration in grapefruit
juice.
57
2.7 Microbial contaminates in vegetable products
The increasing popularity of salad bars containing freshly cut vegetables
in supermarkets and convenience stores has introduced new environments
which support the growth of food-borne pathogens including
L. monocytogenes. During handling and packaging of these foods, it is
sometimes difficult to maintain cold temperatures which may provide an
opportunity for pathogens to grow in food. In such cases it may be possible to
exploit bacteriocin producing cultures to provide an extra hurdle to these
pathogens. In this respect, Cai et al., (1997) demonstrated that a nisin
producing L. lactis strain HPB 1688, when co-inoculated with
L. monocytogenes on fresh-cut ready-to-eat salad, was able to reduce the
number of L. monocytogenes by approx. 10-fold after storage for 10 days at 7
and 10°C. This group also showed that a bacteriocin-producing E. faecium was
able to reduce the numbers of L. monocytogenes in Caesar salad.
Another problem associated with fresh vegetables is the possibility of
carry-over contamination with coliforms as a result of poor hygienic practices.
This possibility was addressed by Vescovo et al. (1995) who showed that
strains of Lb. casei had a remarkable inhibitory effect, strongly reducing or
eliminating coliforms or enterococci from the third day of refrigerated storage
of ready-to-eat vegetables.
Lactic acid fermentation of cabbage and other vegetables is a common
method for preserving fresh vegetables in the Western World, China and
Korea. In typical sauerkraut fermentation, Lc. mesenteroides initiates growth,
producing carbon dioxide which creates an anaerobic environment, and organic
acids which lower the pH thereby inhibiting the development of undesirable
microorganisms. However, microorganisms such as food spoilage lactobacilli
which can survive in these adverse conditions can lead to spoilage of the
fermented food.
58
2.8 Spices and edible oil in film making
The possibility of edible film or edible coating to carry some food
additives such as antioxidants, antimicrobials, colourants, flavours, fortified
nutrients and spices are being studied (Pena and Torres 1991). The method is
different from direct application, as the incorporation of antimicrobial agents
into edible film or edible coating localizes the functional effect at the food
surface. The antimicrobial agents are slowly released to the food surface, and
therefore, they remain at high concentrations for extended periods of time
(Ouattara et al., 2000a). Antimicrobial agents used in food application include
organic acids, bacteriocins, enzymes, alcohols and fatty acids (Han, 2000). In
addition, spice extracts have been introduced for their ability to control meat
spoilage (Ouattara et al., 2000b). The beneficial effects obtained by using
edible film and coating in terms of physical, mechanical, and biochemical
benefits have been reported in many publications (Krochta and Mulder-
Johnston, 1997). Gennadios and Weller (1990) reported the ability of edible
film in retarding moisture, oxygen, aromas and solute transport.
Spices such as garlic, onion, cinnamon, cloves, thyme and sage have
been investigated for their antimicrobial activity. The antimicrobial compounds
in plant materials are commonly present in the essential oil fraction and it has
more inhibitory effect than the corresponding ground form (Nychas, 1995).
2.9 Apple puree
Browning is due to condensation reactions between phenolic
compounds. The oxidation of o- and p-diphenols give condensation reactions
with the formation of brown polymers, which are more stable than the
monomer forms (Sims and Morris, 1984). Circumstances like normal storage
temperatures (Sommers and Pockock, 1990), light and oxygen dissolution are
favorable for apple browning. The first step of this process is the oxidation of
59
phenols to quinones and it can be a non-enzymatic chemical reaction catalyzed
by metals like copper and iron (Singleton, 1987) or an enzymatic reaction with
the intervention of polyphenol oxidase (Metche, 1986). The non-enzymatic
chemical reaction is predominant in apples is because the polyphenol oxidase
activity, present within itself (Rapp et al., 1977). Besides, the polyphenol
oxidase enzyme is able to catalyze the oxidation of phenols to quinines but
polymerization reactions are non-enzymatic.
The control of purees browning has always been a challenge for the
fruit-processing industry, the use of chemical antioxidant (e.g., ascorbic acid
and sulfites) and high temperature being the most common solutions. In the
recent years, however, there is an increasing interest in the market of foods wit
natural ingredients; in particular, the presence of sulfites in foods has been
related to adverse health effect, whereas ascorbic acid plays also a pro-oxidant
role. Thus, the research for natural antibrowning agents has been stimulated.
Enzymes represent a great potentiality for food processing.
These differences in the mechanism of inhibition may allow the use of
combinations of antibrowning agents that may result in enhancement of
inhibition. Most combinations of antibrowning agents or commercially
available are ascorbic acid-based compositions. Mixtures of ascorbic and
cyclodextrins were reported to be effective in the inhibition of apple juice
browning (Pizzocarno et al., 1993).
2.9.1 GOx-catalase system
The GOx-catalase system is able to scavenge the oxygen and thus
stabilizes foods and beverages against problems related to product oxidation
and browning (Mistry and Min, 1992a). The GOx has recently been used to
control the colour change in grape juice during high-pressure processing
60
(Castellari et al., 2000) and to produce wine with low alcohol content
(Pickering et al., 1998).
2.9.2 Lactoperoxidase
Lactoperoxidase (EC 1.11.1.7) is a member of the peroxidase family, a
group of natural enzymes, widely distributed in nature and found in plants and
animals, including man (Kussendrager and Van Hooijdonk, 2000).The
structure, function and antimicrobial properties of LPS have been reviewed
recently by De Wit and Hooydonk (1996). Next to xanthine oxidase, LPS is
the most abundant enzyme in milk and is found almost exclusively in the whey
after cheese making.
Lactoperoxidase has been identified as an antimicrobial agent in milk,
saliva and tears. LPS is a natural bacterial defence system through the
oxidation of thiocyanate ions by hydrogen peroxide. LPS has proven to be both
bactericidal and bacteriostatic to a wide variety of microorgnisms, while
having no effect on the proteins and enzymes of the organisms producing LPS
(Ekstrand, 1994).
The mechanism of action of the LPS has been explained in detail by
DeWit and Hooydonk (1996). LPS in more active at acidic pH levels (Wever et
al., 1982), but is less stable under acidic conditions showed that the LPS-
catalysed reactions yield short lived intermediary oxidation products of SCN-,
providing antibacterial activity. The major intermediary oxidation product is
hypothiocyante (OSCN-), which is produced in an amount of about 1 mol per
mol of hydrogen peroxide. At the pH optimum of 5.3, the OSCN- is in
equilibrium with HOSCN. The unchanged HOSCN is considered to be more
bactericidal of the two forms (Thomas et al., 1983).
The action of LPS against bacteria is reported to be caused by sulfydryl
(-SH) oxidation (Aune and Thomas, 1978). The oxidation of -SH groups in
61
the bacterial cytoplasmic membrane results in loss of the ability to transport
glucose and also in leaking of potassium ions, amino acids and peptides .
The microbial specificity of LPS has been reviewed by Kurhonen
(1980). Gram-negative, catalase positive organisms are more readily inhibited
by LPS than are Gram-positive, catalase negative bacteria. Gram-negative,
catalase positive organism, (coliforms, Salmonella, etc.,) are not only inhibited,
but are killed if sufficient hydrogen peroxide is provided chemically,
eznymatically or by hydrogen peroxide producing microorganism (Bjorck,
1992). On the other hand, the action of LPS against Gram-positive organisms
is generally bacteriostatic and not lethal.
Gould, (1995) found that there was a critical combination of LPS, GOx
glucose, iodide and thiocyanate to be effective in cosmetics. The treatment
was effective against a range of yeasts, fungi and viruses, as well as bacteria
for periods of up to 4 months. Although the system has been shown to have
potential as a bio-preservative, its potency has so far been extensively
investigated in only dairy and meat products. Its effectiveness against
pathogens in other foods is generally unexplored. Therefore, use of the system
against S. enteritidis in several foods suspected of harboring the pathogen was
investigated.
New preservation techniques are being applied and researched that will
increase the number of inactivating techniques that are available. These include
the addition of bactericidal enzymes such as lysozyme and the LPS, GOx, non-
enzymic proteins such as lactoferrin and lactoferricin, and bacteriocins (Gould,
1995). In this present investigation an attempt is made to control microbial
contaminations and browning during processing and storage of apple puree by
using GOx, LPS and catalase enzyme system.
62
2.10 Minimally processed fruits and vegetables
Minimally processed vegetables sold in ready-to-eat (salads) or ready-to
use forms have become a very important area of potential economic growth for
fresh-cut industry. Today MPV products have gained popularity mainly
because consumers perceive such products, besides their well known
nutritional qualities, as fresh, healthy, convenient, tasty and easy to use (Garret
et al., 2003). For reasons of expense, labour and hygiene, the catering industry
aims to purchase vegetables and fruit that are already peeled and possibly also
sliced, grated or shredded, that is, minimally processed. Consumers are
increasingly demanding convenient, ready-to-use and ready-to-eat fruit and
vegetables with a fresh-like quality, and containing only natural ingredients’.
Minimal processing of raw fruit and vegetables has two purposes. First, it is
important to keep the produce fresh, yet supply it in a convenient form without
losing its nutritional quality. Second, the product should have a shelf-life
sufficient to make its distribution feasible to its intended consumers4. In an
ideal case, minimal processing can be seen as ‘invisible’ processing
(Ahvenainen et al., 1994).
2.11 Reasons for quality changes in minimally processed produce
As a result of peeling, grating and shredding, produce will change from
a relatively stable product with a shelf-life of several weeks or months to a
perishable one that has only a very short shelf-life, even as short as l-3 d at
chill temperatures. Minimally processed produce deteriorates because of
physiological ageing, biochemical changes and microbial spoilage, which may
result in degradation of the colour, texture and flavour of the produce. During
peeling and grating operations, many cells are ruptured, and intracellular
products such as oxidizing enzymes are liberated (Ahvenainen et al., 1994).
63
2.12 Methods to improve the shelf-life and safety of minimally processed
produce
Minimally processed vegetables can be manufactured on the bases of
several different working principles (Table 2.1). If the principle is that products
are prepared today and consumed tomorrow, then very simple processing
methods can be used. Most fruit and vegetables are suitable for this type of
preparation. Such products are suitable for catering but not for retailing
purposes. The greatest advantage of this principle is the low requirement for
investment. If products are required to have a shelf-life of several days up to
one week, or even more in the case of products intended for retailing, then
more advanced processing methods and treatments using the hurdle concept
are needed, as well as the correct choice of raw materials that are suitable for
minimal processing. Preservation is based on combination of several
treatments. As the table shows, not all produce is suitable for this type of
preparation.
2.13 Microbial spoilage of vegetables and fruits
Contamination of vegetable products with food borne pathogens is very
common (Nguyen-The and Carlin, 1994). Effective and feasible means are
needed to remove pathogens and also to prevent food borne diseases associated
with consumption of fresh fruits and vegetables (Roever, 1998; Francis et al.,
1999).
Sanitizers studied for their effectiveness in removing pathogens from
fruits and vegetables include generally chlorine and various acids such as
acetic, ascorbic, citric, and lactic acids (Weissinger et al., 2000; Burnham et
al., 2001; Singh et al., 2002). Natural products may have applications in
controlling pathogens in foods (Bowles and Juneja, 1998), especially in ready-
to-eat foods. There are many studies which have investigated the antimicrobial
activity of different kinds of plant extracts in vitro system (Hsieh et al., 2001;
Cuspinera et al., 2003; Jayaprakasha et al., 2003). On the other hand,
64
Table 2.1. Requirements for the commercial manufacture of pre-peeled and/or sliced, grated or shredded fruit and vegetables
Working principle Demands for processing Customers Shelf-life at5°C (d)
Examples ofsuitable fruit and
vegetables
Preparation todayConsumptionTomorrow
Standard kitchen hygiene and tools.No heavy washing for peeled andshredded produce; potato is anexceptionPackages can be returnable,Containers.
Cateringindustry,restaurants,Schools,industry.
1–2 most fruit andvegetables
Preparation today,theCustomer uses theproduct within 3-4d
DisinfectionWashing of peeled and shreddedproduce with waterPermeable packages; potato is anexception
Cateringindustry,restaurants,schools, industry
3–5carrot, cabbages, Iceberg lettuce ,potato,beetroot, strawberry
Products are alsointended forretailing
Good disinfectionChlorine or acid washing for Peeledand shredded producePermeable packages; potato is anexceptionAdditives
Retail shops inaddition to thecustomers listedabove
5–7
Carrot, Chinesecabbage redcabbage, potato, beetroot, acid fruit,berries.
65
reports on the antimicrobial effects of table type acidulants that are used with
salad vegetables at household applications such as lemon juice (fresh and
commercial) and vinegar are very limited (Sengun and Karapinar, 2004, 2005a,
2005b; Vijayakumar and Wolf-Hall, 2002a, 2002b).
Illnesses caused due to the consumption of foods contaminated with
pathogens such as L. monocytogenes have a wide economic and public health
impact worldwide (Gandhi and Chikindas, 2007). L. monocytogenes can adapt to
survive and grow in a wide range of environmental conditions as well as in a large
variety of raw and processed foods, including milk and dairy products, or fresh
produce. Food spoilage includes physical damage, chemical changes, such as
oxidation, colour changes, or appearance of off-flavours and off-odors resulting
from microbial growth and metabolism in the product (Gram et al., 2002). The
spoilage of refrigerated meat is caused in part by Pseudomonas species which are
responsible for the off-odors, off-flavours, discolouration, gas production and
slime production (Oussalah et al., 2006). In some cases, a change in atmosphere
by vacuum-packing inhibits the aerobic pseudomonads causing a shift in the
microflora to lactic acid bacteria (LAB) and Enterobacteriaceae (Gram et al.,
2002). The pseudomonads are also found in pasteurized milk and are generally
from post-process contamination (Eneroth et al., 2000).
The spoilage microflora associated with fresh vegetables includes
Pseudomonas spp. as well as other Gram-negative bacteria, such as Enterobacteria
(Ragaert et al., 2007). Current technologies for preservation and shelf-life
extension of food include chemical preservatives, heat processing, modified
atmosphere packaging (MAP), vacuum packaging (VP) or refrigeration.
Unfortunately, these steps do not eliminate undesirable pathogens such as
L. monocytogenes from these products or delay microbial spoilage entirely.
Alternative preservation techniques such as novel non-thermal technologies and
66
naturally derived antimicrobial ingredients are under investigation for their
application to food products.
Staphylococcus aureus is highly vulnerable to destruction by heat treatment
and nearly all sanitizing agents. Thus, the presence of this bacterium or its
enterotoxins in processed foods or on food processing equipment is generally an
indication of poor sanitation. S. aureus can cause severe food poisoning. It has
been identified as the causative agent in many food poisoning outbreaks and is
probably responsible for even more cases in individuals and family groups than
the records show. Foods are examined for the presence of S. aureus and/or its
enterotoxins to confirm that S. aureus is the causative agent of food borne illness,
to determine whether a food is a potential source of "staph" food poisoning, and to
demonstrate post-processing contamination, which is generally due to human
contact or contaminated food-contact surfaces. Conclusions regarding the
significance of S. aureus in foods should be made with circumspection. The
presence of a large number of S. aureus organisms in a food may indicate poor
handling or sanitation; however, there is not sufficient evidence to incriminate a
food as the cause of food poisoning. The isolated S. aureus must be shown to
produce enterotoxins. Conversely, small staphylococcal populations at the time of
testing may be remnants of large populations that produced enterotoxins in
sufficient quantity to cause food poisoning. Therefore, the analyst should consider
all possibilities when analyzing a food for S. aureus.
Methods used to detect and enumerate S. aureus depend on the reasons for
testing the food and on the past history of the test material. Processed foods may
contain relatively small numbers of debilitated viable cells, whose presence must
be demonstrated by appropriate means. Analysis of food for S. aureus may lead to
legal action against the party or parties responsible for a contaminated food.
67
Escherichia coli are one of the predominant species of facultative
anaerobes in the human gut and usually harmless to the host; however, a group of
pathogenic E. coli has emerged that causes diarrheal disease in humans. Referred
to as Diarrheagenic E. coli (Nataro and Kaper, 1998) or commonly as pathogenic
E. coli, these groups are classified based on their unique virulence factors and can
only be identified by these traits. Hence, analysis for pathogenic E. coli often
requires that the isolates be first identified as E. coli before testing for virulence
markers. The pathogenic groups includes enterotoxigenic E. coli (ETEC),
enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC),
enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely
adherent E. coli (DAEC) and perhaps others that are not yet well characterized
(Levine, 1987; Nataro and Kaper, 1998). Of these, only the first 4 groups have
been implicated in food or water borne illness.
ETEC is recognized as the causative agent of travelers' diarrhea and illness
is characterized by watery diarrhea with little or no fever. ETEC infections occurs
commonly in under-developed countries but, in the U.S., it has been implicated in
sporadic waterborne outbreaks as well as due to the consumption of soft cheeses,
Mexican-style foods and raw vegetables. Pathogenesis of ETEC is due to the
production of any of several enterotoxins. ETEC may produce a heat-labile
enterotoxin (LT) that is very similar in size (86 kDa), sequence, antigenicity, and
function to the cholera toxin (CT).
EIEC closely resemble Shigella and causes an invasive, dysenteric form of
diarrhea in humans (Dupont et al., 1971). Like Shigella, there are no known
animal reservoirs; hence the primary source for EIEC appears to be infected
humans. Although the infective dose of Shigella is low and in the range of 10 to
few hundred cells, volunteer feeding studies showed that at least 106 EIEC
organisms are required to cause illness in healthy adults. Unlike typical E. coli,
EIEC are non-motile, do not decarboxylate lysine and do not ferment lactose, so
68
they are anaerogenic. Pathogenicity of EIEC is primarily due its ability to invade
and destroy colonic tissue. The invasion phenotype, encoded by a high molecular
weight plasmid, can be detected by invasion assays using HeLa or Hep-2 tissue
culture cells (Mehlman et al., 1982, Dupont et al., 1971) or by PCR and probes
specific for invasion genes.
EPEC causes a profuse watery diarrheal disease and it is a leading cause of
infantile diarrhea in developing countries. EPEC outbreaks have been linked to the
consumption of contaminated drinking water as well as some meat products.
Through volunteer feeding studies the infectious dose of EPEC in healthy adults
has been estimated to be 106 organisms. Pathogenesis of EPEC involves intimin
protein (encoded by eae gene) that causes attachment and effacing lesions (Hicks
et al., 1998); but it also involves a plasmid-encoded protein referred to as EPEC
adherence factor (EAF) that enables localized adherence of bacteria to intestinal
cells (Tobe et al.,1999).
Numerous epidemiologic studies exhibited, that a reduced risk of
degenerative diseases correlates with a high intake of fruits and vegetables
(Steinmetz and Potter, 1996). Ready-to-use salads can suit as useful sources of
minerals and physiologically active substances such as polyphenols, as the
increasing popularity of this convenience product indicates. Due to high bacterial
counts of raw vegetables after harvest up to 106–109 colony forming units per
gram fresh salad (CFU/g), ready-to-use sliced salads are usually contaminated by
microorganisms, too (Jaques and Morris, 1995). Even bacterial pathogens have
been detected in prepackaged salads (Lin et al., 1996) and lettuce (Park and
Sanders, 1992). High initial counts are not substantially reduced during
conventional cold washing.
69
2.14 Pesticides as a source of microbial contamination of salad vegetables
Pesticides are routinely used in the cultivation of vegetables to control
insects, weeds, spoilage bacteria and fungi, and other pests. Pesticides are
commonly grouped into three categories: insecticides, herbicides and fungicides
(Hislop, 1976). These categories have different target populations and active
ingredients, and usually come in a concentrated powder or liquid form.
Reconstitution and dilution of the pesticide with water is required before
application to vegetables, and this is usually done on the farm by the farmer. In
recent years, there has been increased interest in the microbiological quality and
safety of fresh produce such as salad vegetables, as they have been linked to
outbreaks of food borne microbial disease (Beuchat, 1996, 2002; Heard, 1999,
2002; Nguyen-The and Carlin, 2000). While postharvest contamination is often
the source of the implicated microorganisms, there is increasing concern that
preharvest contamination presents significant public health risks. The main
sources of preharvest contamination are fertiliser, irrigation water and soil (Lund,
1992; Beuchat, 1996, 2002). As mentioned already, a diversity of pesticides are
regularly applied to vegetable produce, but they are rarely considered to be a
source of microbial contamination.
2.15 Cut vegetable and fruit preservation techniques in practice
Minimally processed vegetable sold in ready-to-eat or ready-to use form
have become a very important area of potential economic growth for fresh-cut
industry. Today MPV products have gained popularity mainly because consumers
perceive such products, besides their well known nutritional qualities, as fresh,
healthy, convenient, tasty and easy to use (Garret et al., 2003). Nevertheless
potential growth of food-borne pathogens is greater on MPV than on raw produce,
since their characteristics such as high humidity and large number of cut surfaces
that can create ideal conditions for several microorganisms growth (Nguyen-The
and Carlin, 1994; Francis et al., 1999; Alzamora et al., 2000). Although microbial
70
numbers in MPV products are kept low by the combined effect of washing,
modified atmosphere packaging (MAP) and low temperature, several pathogens,
including Campylobacter jejuni, Salmonella spp., E. coli O157:H7, Shigella spp.,
Aeromonas hydrophila, Yersinia enterocolitica and L. monocytogenes can grow
and cause diseases depending on the type of product, storage conditions (time,
temperature and atmosphere composition) and the presence of competitive
microorganisms (Gleeson and O’Beirne, 2005; Mukherjee et al.,2006).
With the objective to improve quality and safety of MPV products, and
reduce preservatives the industry is seeking novel and alternative technologies
with the objective of improving quality and safety of food products. Recently, the
use of lactic acid bacteria (LAB) and/or their natural products for the preservation
of foods (biopreservation) looks as a promising strategy, for reducing growth of
pathogens according to the hurdle technology strategy (Leistner and Gorris, 1995;
Leistner, 2000; Allende et al., 2006; Galvez et al., 2007; Settanni and Corsetti,
2008; Trias et al., 2008).
The preparation of fresh-cut products causes damage to plant tissues,
rendering a more perishable product with shortened shelf-life, compared to intact
fruits and vegetables (Guerzoni et al., 1996; Watada et al., 1996). This problem is
primarily due to a higher respiration rate and the significant damage resulting
from cutting (Pirovani et al., 1997). Fresh-cut processing affects quality factors
such as appearance, flavour, and colour, and product deterioration may proceed
rapidly. The MAP is effective in prolonging the shelf-life of horticultural
commodities by decreasing oxygen (O2) and increasing carbon dioxide (CO2)
concentrations in the package atmosphere achieved via the interaction between
respiratory O2 uptake and CO2 evolution of packaged produce, and Gas transfer
from the package films (Schlimme and Rooney, 1994; Jacxsens et al., 1999;
Makino, 2001).
71
In general, major factors affect the equilibrium gas concentrations of
packaged produce include packaged product weight and its respiration rate,
package film oxygen/carbon dioxide transmission rate and the respiring surface
area (Bell, 1996), and storage temperature. However, for packaged fresh-cut
vegetables in the retail market, package surface area and product fill weight are
often pre-determined to certain degree to achieve a market appeal, and the
respiration rate is also influenced by numerous factors, including storage
temperature, cut size and vegetables types etc. Therefore, selecting package films
with suitable OTRs plays an important role in developing MA packages for
improved quality and shelf-life of fresh-cut produce.
2.15.1 Combination preservation
Food preservation is carried out during food processing in an attempt to
maintain the raw material quality, physico chemical properties and functionality
whilst providing safe products that have a low spoilage potential. This is mainly
achieved through purposely designed processing that varies from one product to
the next. Hence, in preservation processes, which form about a third of the unit
operations used in food processing (Farkas, 1977), the general aim is to employ
combination processes where, for e.g., a mild heat stress and a low concentration
of preservatives are combine in order to fulfil all the above listed objectives.
Additionally, there is currently significant interest in using alternative physical
treatments such as ultra high pressure (UHP) or pulsed electric fields (PEF) to
replace the classical heat treatment. Progress in these areas has been discussed in
this issue by UHP presumably denatures microbial cell wall proteins such that
access to the rest of, e.g., the fungal wall and membrane is greatly facilitated
(Brul, 1999).
Indeed combination preservation treatments are often advocated and where
possible patented (Knorr, 1998). Combinations of preservation treatments allow
72
the required level of protection to be achieved while at the same time retaining the
organoleptic qualities of the product such as, colour, flavour, texture and
nutritional value. The potential use of some of the novel, “natural” preservatives,
discussed previously in this review, in combinations with physical treatments (i.e.,
mildheat, UHP and PEF), has not been extensively evaluated and may lead to the
development of novel mild preservation regimes tailored to the organolyptic
quality needs of individual products.
2.15.2 Chemical preservative agents
The most common classical preservative agents are the weak organic acids,
for example acetic, lactic, benzoic and sorbic acid. These molecules inhibit the
outgrowth of both bacterial and fungal cells and sorbic acid is also reported to
inhibit the germination and outgrowth of bacterial spores (Sofos and Busta, 1981;
Blocher and Busta, 1985).
In solution, weak acid preservatives exist in a pH dependent equilibrium
between the undissociated and dissociated state. Preservatives have optimal
inhibitory activity at low pH because this favors the uncharged, undissociated
state of the molecule which is freely permeable across the plasma membrane and
is thus able to enter the cell. Therefore, the inhibitory action is classically believed
to be due to the compound crossing of plasma membrane in the undissociated
state. Subsequently, upon encountering the higher pH inside the cell, the molecule
will dissociate resulting in the release of charge anions and protons which cannot
cross the plasma membrane.
Microbial resistance to weak organic acid can involve various mechanisms.
For bacteria, significant knowledge exists on their intrinsic, noninducible
resistance mechanisms against these compounds (Russel, 1991). Gram-positive
bacteria do not possess an outer membrane and the exclusion limit of the cell wall
73
of vegetative cells of bacillus megaterium has even been calculated to be as high
as 30,000D (Lambart, 1983). Hence preservatives can easily enter the cells and
their intrinsic resistance is relatively low. In Gram-negative bacteria, resistance
mechanisms are more complicated since these organisms’ posses an inner and
outer membrane.
2.16 Calcium ions on vegetables and fruits
Titchenal and Dobbs (2007) point out some dark green leafy cabbage
family vegetables and turnip greens as good calcium sources, and most leafy
vegetables as potential calcium sources. The major source of calcium in the
United States diet is dairy products, which supply 75% of the intake, and
vegetables, fruits and grains which supply the rest (Allen, 1982). The awareness of
consumers on the benefits of calcium is relatively high. The calcium content in the
diet is critical in most stages of life (Gras et al., 2003). Dietary calcium raises
concern for consumers and health specialists due to the number of processes it is
involved in, the high amount present in the body, and the continuous research
highlighting the benefits of an adequate intake.
Nowadays, an increasing part of the products in the food industry are
fortified, especially dairy products followed by beverages and snacks (Caceres et
al., 2006). A result of evidences linking osteoporosis, hypertension and cancer to
calcium deficiency. While the cause of these diseases is multifactor and poorly
understood, there is some evidence to support the hypothesis that increased
calcium intake might reduce the risk of suffering from these diseases (Appel et al.,
1997; Cumming et al., 1997).
Also, the use of phosphorous-free sources of calcium, such as gluconate,
citrate, lactate, acetate and carbonate calcium salts, can help to obtain a good
balance of calcium and phosphorous in the diet. To give consumers the
74
opportunity to increase their calcium intake without resorting to supplementation,
the industry has been encouraged to fortify food and beverages with calcium
(Cerklewski, 2005). This opens new ways of supplementing calcium intake by
increasing the calcium content in these commodities. For this reason, the use of
natural sources of calcium as preservative with a nutritional fortification effect
presents an advantage for the industry and for the consumer.
2.16.1 Calcium sources to maintain the shelf-life of fresh vegetables and fruits
Different calcium salts have been studied for decay prevention, sanitation
and nutritional enrichment of fresh fruits and vegetables. Calcium carbonate and
calcium citrate are the main calcium salts added to foods in order to enhance the
nutritional value (Brant, 2002). Other forms of calcium used in the food industry
are calcium lactate, calcium chloride, calcium phosphate, calcium propionate and
calcium gluconate, which are used more when the objective is the preservation
and/or the enhancement of the product firmness (Luna-Guzman and Barrett, 2000;
Alzamora et al., 2005; Manganaris et al., 2007).
The selection of the appropriate source depends on several factors:
bioavailability and solubility are the most significant, followed by flavour change
and the interaction with food ingredients. Calcium chloride has been widely used
as preservative and firming agent in the fruits and vegetables industry for whole
and fresh-cut commodities. Sams et al. (1993) and Chardonnet et al. (2003)
studied the effect of calcium chloride on fruit firmness and decay after the harvest
of whole apples.
Saftner et al. (2003) work was also focused on the firming effect of calcium
chloride treatment on fresh-cut honeydew. Luna-Guzman and Barrett (2000)
compared the effect of calcium chloride and calcium lactate dips in fresh-cut
cantaloupe firmness, microbial load, respiration and sensorial evaluation. Other
75
researchers (Garcia-Gimeno and Zurera-Cosano, 1997; Main et al., 1986; Morris
et al., 1985; Rosen and Kader, 1989; Suutarinen et al., 1998) used calcium
chloride as firming agent for processed strawberries.
Wills and Mahendra (1989) examined the effect of calcium chloride on
fresh-cut peaches from a quality point of view, meanwhile Conway and Sams
(1984) evaluated the safety of strawberries treated with calcium chloride. Other
fruits and vegetables, in which the effect of calcium chloride was studied, showing
significant improvement in the quality of the final product, are grape fruit, hot
peppers and diced tomatoes (Mohammed et al., 1991; Floros et al., 1992).
The use of calcium chloride is associated with bitterness and off-flavours
(Bolin and Huxsoll, 1989; Ohlsson, 1994), mainly due to the residual chlorine
remaining on the surface of the product. Calcium lactate, calcium propionate and
calcium gluconate have shown some of the benefits of the use of calcium chloride,
such as product firmness improvement, and avoid some of the disadvantages, such
as bitterness and residual flavour (Yang and Lawsless, 2003). Also, the use of
calcium salts other than calcium chloride could avoid the formation of
carcinogenic compounds (chloramines and trihalomethanes) linked to the use of
chlorine.
Manganaris et al. (2007) compared the effect of calcium lactate, calcium
chloride and calcium propionate dipping in peaches. Calcium increased in tissues
with no dependence on the source used. Calcium incorporation by impregnation
with two calcium sources, calcium lactate and calcium gluconate, was studied in
fresh-cut apple (Anino et al., 2006). Another source of calcium is the calcium-
amino acid chelate formulations which had been patented as nutritionally
functional chelates. Lester and Grusak (1999) showed that the use of calcium
chelate doubled the shelf-life of whole honeydew melon.
76
2.17 Koruk juice
Koruk juice (Unriped grape juice) is commonly used with salad
vegetables as an acidifying and flavouring agent in Turkey and neighboring
countries. It is also consumed as a drink after being sweetened. Currently grape
compounds have attracted increased attention especially in the Welds of nutrition,
health, and medicine (Waterhouse and Walzem, 1998). Phenolic compounds in
grape juice, grape seed and wine have been investigated by many researchers to
show their potent antioxidant, antimutagenic, antibacterial, antiviral, antifungal
and antiulcer activities (Takechi et al., 1985; Liviero et al., 1994; Caccioni et al.,
1998; Saito et al., 1998; Jayaprakasha et al., 2001; Baydar et al., 2004). However,
there is no information about the antimicrobial activity of koruk juice. The
antimicrobial effect of koruk juice varied depending on the culture strains and
products used (P<0.05). There was no significant difference in cell reduction in
samples exposed to koruk juices for 15, 30 and 60 minutes (P>0.05) whereas
reduction obtained at 0 time differed significantly (P<0.05). However, koruk juice
exerted a shock antimicrobial effect on S. typhimurium strains.
Although there is no previous microbiological study related to the
antimicrobial effect of koruk juice, other grape products such as grape seed
extracts, grape juice and wine have been studied by several researchers to
investigate the antimicrobial activity of these products. It has been reported that
grape seed extract reduced the number of S. typhimurium attached on chicken skin
between 1.6 and 1.8 log at 0.1% and 0.5% concentrations (Xiong et al., 1998). In
another study, Baydar et al. (2004) reported that the grape baggase extracts had no
inhibitory effects on the 15 bacteria tested, while the grape seed extracts inhibited
all the bacteria except B. amyloliquefaciens at 20% concentration. Jayaprakasha
et al. (2003) also studied the antimicrobial effect of grape seed extract on three
bacteria and found that, Gram-positive bacteria were inhibited at lower
concentrations of grape seed extracts than Gram-negative bacteria.
77
2.18 Effect of blanching
Generally, the antioxidant potential of vegetables is affected by thermal
processing. (Puupponen-Pimia et al., 2003) found that blanching reduced the
antioxidant capacity by 23% for cauliflower, but increased it by 9% for cabbage.
Wu et al. (2004) found a reduction of 14% in ORAC values for cooked broccoli
and an increase of 41% for cooked red cabbage. The blanching temperature
ranged between 96–98oC. Significant effects of various processing methods on the
reduction of vitamin C by leaching and thermal breakdown have been reported
(Davey et al., 2000). Household and industrial processing might thus affect the
flavanoid, GLS and vitamin contents and in turn affect the health related quality.
2.19 Cold storage
Heat treatments of vegetables are mostly intended to tenderize the
vegetable for consumption or, as a pretreatment for freezing or canning, to
inactivate enzymes and to remove air. The decontamination treatments and low
temperature storage can be combined with modified atmosphere packaging as a
multiapproach stratergy to prolong he shelf-life of MPV. After minimal
processing, a relatively stable agricultural product with a shelf-life of several
weeks or months will become one that has only a very short shelf-life. MPFV
should have storage lives of at least 4–7 days, but preferably longer, up to 21 days,
depending on the market (Ahvenainen, 1996; Barry-Ryan and O’Beirne, 1997).
Cut and damaged surfaces in MPV release nutrients and some intracellular
enzymes, such as polyphenol oxidase, and provide good conditions for some
enzymatic activities and possible microbial spoilage. Enzymatic and microbial
deterioration in MPV continues and shortens their shelf-life even when they under
the recommended chilling conditions (0–8ºC). O’Connor and Skarshshewski
(1992) reported that the shelf-life of some MPV products is less than 5 days at
4oC.
78
2.20 Economic loss
The economic potential is shown by the solid growth of the industry in the
recent past as illustrated by increasing consumption and increasing space devoted
to fresh-cut vegetable products in super markets and on restaurant menus in most
parts of the world (Kaufman et al., 2000).The cost of the chemical preservatives
used for storage of the fruits is higher than the cost of the fruits used for our
consumption or export. This leads to the greater economic loss in most of the food
industries.
79
CHAPTER-3
OBJECTIVE OF THE PRESENT WORK
With this background, in the present study was taken up to address the following
objectives:
· Isolation of glucose oxidase producing fungi from various sugar rich
products.
· Optimization of medium composision and suitable culture condition for the
selected fungus for GOx production.
· Production of extracellular glucose oxidase by laboratory fermentor,
purification and characterization.
· Application of glucose oxidase in food processing and preservation.
80
CHAPTER-4
MATERIALS AND METHODS
4.1 General
The glassware were soaked for overnight in chromic acid solution (10%
potassium dichromate solution in 25% concentrated sulphuric acid) and then
washed thoroughly in running tap water. Finally, they were rinsed in distilled
water and dried in a hot air oven.
4.1.2 Sterilization
The glasswares and medium were sterilized in an autoclave at 121°C at 15
psi for 20 minutes.
4.1.3 Chemicals
The Horse radish peroxidase, o-dianisidine were purchased from Sigma
Chemicals Co. Ltd, USA. Standard protein markers were purchased from Geni
laboratories, Bangalore, India. All other laboratory chemicals were purchased
from SRL and Qualigen, Mumbai, India.
4.2 Isolation and screening of glucose oxidase producing fungi and
optimization of medium
4.2.1 Isolation of GOx producing fungi
Different fungi were isolated from various sugar rich products such as
honey hive collected from Salem, dates, fruits and soil samples from
Maduravoyal, Chennai, Tamil nadu. Isolation was performed by serial dilution
and direct method. In direct method the segments were plated on Potato Dextrose
Agar (PDA) medium amended with streptomycine (30 mg/l). All the plates were
incubated at 28°C for 4–6 days. Single colonies were transferred to fresh plates
and screened their GOx secretion.
81
PDA medium composition
Dextrose - 20 g
Potato extract - Extracted from 200 g of potato
Agar - 20.0 g
The above constituents were made up to 1000 ml by adding distilled water and the
pH was adjusted to 5.8±0.2.
4.2.2 Screening of GOx producing fungi
The isolated fungi were screened for their GOx producing capability
according to the method described by Eun-Ha Park et al. (2000). The fungi were
grown on the media consists (g/l);
Glucose - 80
Peptone - 3.0
(NH4)2HPO4 - 0.388
KH2PO4 - 0.188
MgSO4 - 0.156
Agar - 20.00
The above composition was prepared in sodium acetate buffer (pH 5.5). A
disc of fungal culture was taken from the peripheral zone of the colony and
transferred to the middle of the Petri plate. It was incubated at 35°C for 3 days.
Then the plate was treated with the following mixture;
Glucose - 5 % (w/v)
Glycerol - 2% (v/v)
o-dianisidine - 0.1% (w/v)
Horse radish peroxidase - 60 IU/ml
Agar - 1% (w/v)
82
The above composition was prepared in sodium acetate buffer (pH 5.5) and
overlayed on the fungal culture medium and incubated for one hour. Then the
colour changes were observed. The appearance of brown colour indicates that the
fungi produced the GOx. The following enzymatic reaction will occur giving rise
a brown colour.
The positive colonies were selected and grown on PDA at 28°C.
4.2.3 Identification of fungi
The higher GOx producing fungus was identified as Aspergillus awamori
under the group of A. niger by Prof. B.P.R Vittal, Mycologist, Centre for
Advanced Studies in Botany, University of Madras, Chennai, India. The identified
fungi was submitted to Microbial Type Culture Collection (MTCC), Chandigarh,
India and designated as Aspergillus awamori MTCC 9645. The selective cultures
were maintained on PDA slants at 4°C and sub-cultured at an interval of 30 days.
The fungus A. awamori MTCC 9645 was used for further studies.
4.2.4 Preparation of spore suspension
The Malt Extract Agar (MEA) medium for sporulation of A. awamori
MTCC 9645, that consists of (g/l);
Malt extract - 20.00
Glucose - 20.00
Agar - 20.00
Distilled water - 1000 ml
pH - 5.5±0.2
83
The conidia suspension was prepared from the fungi that grown on the
MEA medium for 3 days at 28°C. After that the fungal colonies were washed with
sterile tween 80 solution (0.1% w/v) to yield a stock spore suspension of 2×107
conidiospores/ml using hemocytometer.
4.3 Analytical methods
4.3.1 Assay of GOx activity (Bergmeyer et al., 1988)
Principle
β-D-glucose + O2 + H2O GOx D-glucano-1,5-lactone+H2O2
H2O2+O-Dianisidine (reduced) POD O-Dianisidine
Where,
GOx- GOx; POD-Peroxidase
Conditions : Temperature =35ºC, pH = 5.1, A500nm, light path =1cm
Method: Continuous spectrophotometric rate determination
Reagents
A. Sodium acetate buffer (50 mM), pH 5.5.
B. o-Dianisidine solution (0.21mM), 50mg of o-Dianisidine
dihydrochloride was dissolved in 7.6 ml of Millipore water, this solution
was diluted to 1.0 ml to 100ml with reagent A.
C. β-D-glucose (10% w/v) substrate solution (this was prepared using β-
D-glucose in 10ml of Millipore water).
D. Reaction cocktail was prepared by o-Dianisidine (0.17 mM) and 1.72%
(w/v) of glucose solution. (The combination of 24 ml of reagent B and 5
ml of reagent C were mixed and adjusted the pH to 5.5. This solution
was prepared immediately before use as fresh solution).
E. Peroxidase enzyme solution (POD)
84
(Freshly prepared solution containing 60 purpurogallin units/ml of
peroxidase, type II was prepared in cold Millipore water).
Procedure
The following reagents were pipette out in to quartz cuvettes:
Reagents Test (ml) Blank (ml)
Reaction cocktail
(reagent D)2.90 2.90
Reagent E (POD)0.10 0.10
Then the mixture was mixed well by inversion and equilibrates to 35ºC.
The colour a change was read in a UV-Visible spectrophotometer at A500nm. After
that following reagents were added,
Reagents Test (ml) Blank (ml)
Reagent F
(enzyme solution)0.10 -
Reagent A (buffer) - 0.10
The above solutions were mixed thoroughly by inversion and recorded the
changes in A500nm for approximately 5 minutes. Maximum linear rate for both test
and blank were obtained at A500nm.
Calculations
(D A500nm / minutes test - D A500nm / minutes blank)(3.1) (df)Units/ml enzyme = (7.5) (0.1)
....... (4.1)
3.1 = volume (in millilitres) of assay
df = dilution factor
85
7.5 = millimolar extinction coefficient of oxidized o-dianisidine at 500 nm.
0.1 = volume (in millilitres) of enzyme used
Units/ml enzymeUnits/mg solid = .......(4.2) mg solid/ml enzyme
Unit definition
One unit of GOx will oxidize 1.0 µmole of β-D-glucose in to D-
gluconolactone and H2O2 per minute in the pH of 5.5 at 35ºC (equivalent to an O2
uptake of 22.4 µl per minute).
4.3.2 Estimation of protein
The protein content of the sample was estimated according to the method of
Bradford (1976).
Reagents
Coomassie Brilliant Blue G-250 (100 mg) was dissolved in 50 ml of
ethanol. Along this 100 ml of 85% (v/v) o-phosphoric acid was added and made
up to one litre with glass distilled water. The concentrations in the reagent were
0.01% (w/v) of CBB G-250, 4.7% (v/v) of ethanol and 8.5% (v/v) of ortho
phosphoric acid.
Procedure
Protein sample (100 µl) was added to 900 µl of sodium acetate buffer (pH
6.5), then, 3 ml of CBB-G250 solution was added and incubated at room
temperature for 5 minutes. Absorbance was read in UV-Visible spectrophotometer
at 595 nm against the blank containing one millilitre of sodium acetate buffer and
3 ml of CBB-G250 solution. The amount of protein was calculated using Bovine
Serum Albumin as a standard protein (Sigma Chemicals Co., USA).
86
4.3.3 Estimation of fungal biomass
The fungal biomass was estimated by by Sandip et al. (2008) method. The
fermented broth sample (50 ml) was acidified to pH 2.5 using 4 M HCl, because
the insoluble calcium carbonate was converted in to soluble calcium chloride and
carbon dioxide. Unless CaCO3 is used, the above procedure was omitted. The
samples were centrifuged at 4,000 g for 15 minutes. The mycelial pellets from the
samples were washed with distilled water and dried (90°C, 36 h) to a constant
mass. The biomass concentrations were expressed in dry weight of biomass per
litre of culture medium and the experiments were done in triplicates.
4.3.4 Analysis of glucose
The glucose content was estimated by dinitro-salicylic acid (DNSA)
method using glucose as the standard (Miller, 1959). The DNSA reagent consisted
of one gram DNSA dissolved in 20 ml of 2N sodium hydroxide and 50 ml
Millipore water. Thirty grams of potassium sodium tartarate tetrahydrate was
added and the volume was brought up to 100 ml with Millipore water. The
glucose was measured as follows: Fifty microlitre of sample was added to 1.95 ml
Millipore water and two millilitre DNSA reagent were boiled for 5 minutes
followed by cooling to room temperature and diluting to 24 ml. Glucose content
was determined by using linear glucose standard curve.
4.3.5 Analysis of gluconic acid
The quantity of gluconic acid was determined by titration of the culture
solution with 0.1 N sodium hydroxide at room temperature. Excess alkali was
added and the solution was maintained at the temperature of 50–55°C. At this
temperature it was found that any glucose present did not react with appreciable
quantities of the sodium hydroxide and that the gluconic acid lactone was
completely hydrolyzed to the acid in from 1 to 2 minutes. After 2 minutes at this
temperature, the solution was titrated with 0.1 N sulfuric acid and the gluconic
87
acid resulting from the hydrolysis of the lactone was added to the first titration. As
a rule the lactone equaled from 5 to 10% of the total quantity of gluconic acid.
The total acid was then calculated in gram of gluconic acid. To check these
results several determinations of gluconic acid, as calcium gluconate, were made
by neutralizing an aliquot portion of the culture liquor with calcium carbonate,
heating to boiling, filtering and precipitating the salt with 3 volumes of 95% (v/v)
ethanol. The mixture was allowed to stand for 2 days to insure complete
precipitation and was then filtered in a weighed crucible. The precipitate was
washed with 60% (v/v) ethanol, after which it was dried to constant weight at
90°C. The agreement between the quantity of acid calculated from the titration
and the quantity actually recovered was satisfactory.
4.4 Culture condition for GOx production
The five millilitre of conidiospore suspension (2×107 spores/ml) was
inoculated in to the Erlenmeyer flask (500 ml) containing 100 ml of the medium.
The cultures were incubated in an orbital shaker (200 rpm) at 32±2ºC for 120 h.
The cell free supernatant was subjected to GOx activity.
4.4.1 Selection of suitable medium for GOx production
Seven different media were chosen to find out a suitable medium for
maximum GOx production (Table 4.1). Among the seven different media, the
production medium GOxM 3 supported maximum GOx production from A.
awamori MTCC 9645 and therefore, it was chosen for further investigation.
88
Table 4.1: Different production medium for GOx
Media No. Media composition (g/l) Reference
GOxM 1
Glucose, 60.0; NH4NO3, 0.3; KH2PO4, 0.25;MgSO4. 7H2O, 0.25; urea, 2.0; CSL, 8 ml; pH,6.0
Li and Chen,1994
GOxM 2
Glucose, 80.0; peptone, 3.0; NaNO3, 5.0; KCl,0.5; KH2PO4, 1.0; FeSO4. 7H2O, 0.01; CaCO3,35.0; pH, 6.0
Petruccioli etal., 1995
GOxM 3
Glucose, 80.0; peptone, 3.0; (NH4)2HPO4,0.388; KH2PO4, 0.188; MgSO4. 7H2O, 0.156;CaCO3, 35.0; pH, 6.0
Fiedurek andSzczodrak,1995
GOxM 4Glucose, 40.0; NH4NO3, 1.0;KH2PO4, 1.0; MgSO4.7H2O, 0.25; pH 6.5
Träger et al.,1992
GOxM 5
Glucose, 40.0; NaNO3, 2.0; KCl, 0.5;KH2PO4,1.0; MgSO4 7H2O, 0.5; FeSO4.7H2O,0.01; yeast extract, 2.0; pH, 6.0
Nakamatsu etal., 1975
GOxM 6
Sucrose, 50.0; Ca(NO3)2, 2.0; citric acid, 7.5;KH2PO4, 0.25; KCl, 0.25; MgSO4. 7H2O, 0.25;FeCl3. 6H2O, 0.01; CSL, 20.0; pH, 6.0
Zetelaki, 1970
GOxM 7
Starch hydrolysate, dextrose basis, 200.0;(NH4)2HPO4, 0.2; CSL, 0.4; KH2PO4, 1, MgSO4.7H2O, 0.1; urea, 0.4; antifoam H-601, 0.5 ; pH,6.5
Shah andKothari, 1993
*Glucose and CaCO3 were sterilized separately and added to the medium beforeinoculation.
89
4.5 Media optimization
The production medium was optimized by classical Single Factor Analysis
(SFA) method and a statistical method of Response Surface Methodology (RSM).
The optimization procedures were carried out by above described shake flask
method.
4.5.1 Single factor analysis for GOx production
The media components were optimized by SFA. The following factors
were analyzed for the optimization of basal production medium components
which includes the effect of different carbon, nitrogen sources, environmental
factors (pH and temperature) and fermentation time.
4.5.1.1 Effect of carbon source on GOx production
The various carbon sources like glucose, sucrose, fructose, maltose,
xylose and rhamnose were analyzed. The carbon sources were checked in different
concentrations such as 20, 40, 60, 80, 100, 120, 140 and 160 g/l. The carbon
sources were separately sterilized and added in to the medium.
4.5.1.2 Effect of nitrogen sources on GOx production
The effect of different nitrogen sources on the production of GOx was
studied. The nitrogen sources such as mycological peptone, proteose peptone,
bacteriological peptone, yeast extract and beef extract were studied in different
concentrations like 1, 2, 3, 4, 5 and 6 g/l.
4.5.1.3 Effect of Di-ammonium hydrogen phosphate, potassium di-hydrogen
phosphate and magnesium sulphate on GOx production
The effect of MgSO4 and (NH4)2HPO4 were evaluated at the concentration
of 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 g/l. The KH2PO4 was supplemented in
the production medium at the concentration of 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 g/l.
90
4.5.1.4 Effect of calcium carbonate supplementation on GOx production
The effect of the addition of CaCO3 in to the media was analyzed for
biosynthesis of GOx at the concentration of 10, 20, 30, 40, 50 and 60 g/l.
4.5.1.5 Effect of pH and temperature on GOx production
The different pH (3, 4, 5, 6, 7 and 8) was employed for the production of
GOx. To determine the optimum temperature for GOx production the
fermentation medium was incubated at 20, 25, 30, 35, 40 and 45ºC.
4.5.1.6 Effect of fermentation time on GOx production
The fermentation time was determined for the economic production of
GOx. The production profile of GOx and biomass were calculated at every 12
hour interval of 0 to 144 h.
4.5.2 Statistical optimization by Response Surface Methodology (RSM)
To find out the optimum concentration of medium constituents were
analyzed by RSM using central composite design. The experiments were carried
out by using Design-Expert 7.1.6 software package.
RSM is a collection of statistical and mathematical techniques useful for
developing, improving and optimizing the process. RSM defines the effects of the
independent variables, alone or in combination, on the process. In addition to
analyzing the effects of the independent variables, this experimental methodology
generates a mathematical model that accurately describes the overall process. It
has been successfully applied to optimizing conditions in food, chemical and
biological processes.
91
4.5.2.1 Experimental design of RSM for optimization of media components
The optimal levels of eight variables such as glucose, mycological peptone,
CaCO3, MgSO4, KH2PO4, (NH4)2HPO4 and environmental factors (temperature
and pH) were optimized for GOx production. It was carried out by three sets of
experiments. The first set was carried out by the first three variables like glucose,
mycological peptone and CaCO3. After that the second set of variables [MgSO4,
KH2PO4 and (NH4)2HPO4] were done with the optimized concentrations of first
set variables. The third set was optimized by the two variables like temperature
and pH. For that purpose, the response surface approach by using a set of
experimental design (central composite design with five coded levels) was
performed. For the three factors, this design was made up of a full 23 factorial
design with its eight points augmented with three replications of the center points
(all factors at level 0) and the six star points, that is, points having for one factor
an axial distance to the center of ±α, whereas the other two factors are at the level
of 0. The axial distance α was chosen to be 1.68 to make this design orthogonal. A
set of 20 experiments were carried out for the three variables and 13 experiments
for two variables. The central values (0 level) chosen for experimental design
were (g/l):
Set-1: glucose (90.0), mycological peptone (4.0) and CaCO3 (35.0) –Table 4.2.1–4.2.2
Set-2: (NH4)2HPO4 (0.48), KH2PO4 (0.32) and MgSO4 (0.23) –Table 4.3.1–4.3.2
Set-3: pH (6.0) and temperature (35°C) –Table 4.4.1–4.4.2
In developing the regression equation, the test factors were coded according to the
following equation:
....... (4.3)
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Table 4.2: Set 1 Optimization of glucose, proteose peptone and calcium carbonate for GOx production
Table 4.2.1: Design summary
Variables UnitsLevels
-1 -α 0 +1 +-α
Glucose (A) g/l 40 5.910 90 140 174.089Proteosepeptone (B) g/l 1 -1.045 4 7 9.045
CaCO3 (C) g/l 10 -7.044 35 60 77.045
Table 4.2.2: Experimendal design and of 23 factorial design
RunA:
Glucose(g/l)
B:Proteose peptone
(g/l)
C:CaCO3
(g/l)1 90 -1.045 352 40 1 103 90 4 354 40 7 105 5.91 4 356 140 1 607 90 4 358 40 1 609 174 4 3510 90 4 77.0411 90 4 3512 90 4 3513 90 9.045 3514 140 7 6015 140 1 1016 90 4 3517 90 4 3518 140 7 1019 40 7 6020 90 4 -7.044
93
Table 4.3: Set 2 Optimization of (NH4)2HPO4, KH2PO4 and MgSO4 for GOx
production
Table 4.3.1: Design summary
Variables UnitsLevels
-1 -α 0 +1 +-α
(NH4)2HPO4 (A) g/l 0.1 -0.13863 0.45 0.8 1.038627
KH2PO4 (B) g/l 0.05 -0.13749 0.325 0.6 0.787493
MgSO4 (C) g/l 0.05 -0.1034 0.275 0.5 0.653403
Table 4.3.2: Experimendal design and of 23 factorial design
Run A:(NH4)2HPO4
(g/l)
B:KH2PO4
(g/l)
C:MgSO4
(g/l)1 0.45 0.787 0.2752 0.45 0.325 0.2753 0.45 0.325 0.2754 0.1 0.05 0.55 0.45 0.325 -0.1036 0.1 0.05 0.057 0.1 0.6 0.58 0.45 0.325 0.2759 1.038 0.325 0.27510 0.45 0.325 0.27511 0.8 0.05 0.512 0.1 0.6 0.0513 0.8 0.6 0.514 0.8 0.6 0.0515 0.45 0.325 0.27516 0.45 -0.137 0.27517 -0.138 0.325 0.27518 0.8 0.05 0.0519 0.45 0.325 0.65320 0.45 0.325 0.275
94
Table 4.4: Set 3 Optimization of pH and temperature for GOx production
Table 4.4.1: Design summary
Variables UnitsLevels
-1 -α 0 +1 +-α
pH (A) 3.0 1.757 6.0 9 10.242
Temperature (B) °C 20.0 13.786 35.0 50.0 56.213
Table 4.4.2: Experimendal design and of 22 factorial design
Run A:pH
B:Temperature (°C)
1 3 502 9 503 1.79 354 6 355 6 13.76 6 56.27 6 358 6 359 6 3510 10.24 3511 3 2012 6 3513 9 20
95
Where as xi is the coded value of the ith independent variable, Xi the natural value
of the ith independent variable, X0 the natural value of the ith independent variable
at the center point, and ∆Xi the step change value of variables. For a three-factor
system, the model equation is:
...... (4.4)
Where: Y-predicted response; b0-intercept; b1, b2, and b3 - linear coefficients; b11,
b22, and b33-squared coefficients; and b12, b13, and b23-interaction coefficients
(Myers and Montgomery, 2002).
4.5.3 Modified composition of production medium GOxM 3
Based on the above observation the following composition was formulated
for the maximum production of GOx medium. The environmental factors are also
proposed for the high production of GOx from A. awamori MTCC 9546. The
modified (GOxM 3) media consists of (g/l);
Glucose - 92.7
Proteose peptone - 3.24
(NH4)2HPO4 - 0.48
KH2PO4 - 0.32
MgSO4 - 0.23
CaCO3 - 36.8
Distilled water - 1000 ml
pH - 5.83
Temperature - 30.7°C
96
4.6 Production of GOx by laboratory batch fermentor
In this study two litre laboratory batch fermentor was used (Lark
Innovation, Chennai, India). After the optimization of medium, the GOx
production was carried out in the fermentor equipped with the instrumentation for
the measurement and control of temperature and pH with working volume of one
litre culture medium. The fermentor vessel containing the optimized production
medium (one litre) was moist heat-sterilized at 121ºC for 20 minutes. The medium
was inoculated with 5% of conidiospore suspensions (2x107 spores/ml) of a 24 h
culture from A. awamori MTCC 9645.
Agitation was performed with a double four bladed impeller at 200-500
rpm. It was carried out at 200 rpm for the initial five hours of the fermentation and
increased up to 500 rpm for the rest of the fermentation period. Aeration was
performed by membrane filtered sterile air passed through the sparger. In the
initial hours the agitation and aeration were decreased to prevent the spore
flotation and adhesion to the walls of the fermentation vessels. The dissolved
oxygen content was maintained at 11 to 12 mg/l during the fermentation. The pH
level was maintained at 5.83±0.2 and it was maintained by the addition of 2N
NaOH or 2N HCl. Fermentation was carried out up to 90 h at 30.7ºC.
4.7 Morphological studies
Fungal morphological characteristics like spore aggregation and pellet
formation were checked during the early stage of fermentation in the bioreactor,
that was checked by slightly modified method of Hesham El-Enshasy (2006).
4.7.1 Quantification and qualification of different types of bioparticles
Any discrete biomass in a culture was considered as `bioparticle`;
bioparticles may consist of a single spore, an aggregation of spores or fungal
pellet.
97
4.7.2 Large pellets (>3 mm diameter)
A sample of the suspension was transferred to a petriplate and the diameter
of pellets was measured using a varnier caliper. Calibration was achieved by
means of a standard millimeter scale. Pellet diameters were measured and the
mean value of the pellet diameter was calculated.
4.7.3 Small bioparticles (< 3 mm diameter)
Small bioparticles (spores, aggregates and small pellets) were obtained
during the cultivation and measured by an ocular micrometry. The maximal
magnification of this unit was 1500X. The average diameter of both spores and
small aggregates were measured manually. The microscopic magnification was set
at 1000X and the bioparticles were observed randomly. In all cases, the data
represented in this study are an average of randomly selected (80–100)
bioparticles.
4.7.4 Measurement and calculations
The average diameter of each bioparticle and length in case of germ tube
was measured. The microscope magnification was set at 50X for pellet
measurement and 450X for spore, germ tube and hyphae measurement. For each
sample, the process was repeated at least 25 times using new positions on the
same and on different bioparticles. The microscopic morphology, that is the
average total hyphal length and the average diameter of the hyphal element, has
been quantified during batch cultivation.
98
4.7.5 Time course study on cell growth, GOx production, substrate utilization
and acid formation during fermentation period
The GOx production, substrate glucose utilization, gluconic acid formation,
protein secretion and cell growth were studied during the fermentation period at
12 h of intervals up to 120 h.
4.8 Purification and characterization of GOx
4.8.1 Preparation of enzyme for purification
After the fermentation, the fermented broth was filtered through Whatman
No.1 filter paper and centrifuge at 10,000 g for 10 minutes. The cell free extract
(600 ml) was mixed with 150 ml of GOx buffer (Sodium acetate buffer 50 mM,
pH 5.5) because ammonium sulphate is known to slightly acidify the extract. The
gradient ammonium sulphate precipitation was carried out [40–85% (w/v)] at 4ºC
with constant stirring. The aggregated proteins were separated by centrifugation at
10,000 g for 15 minutes. All the gradient precipitations were analyzed for protein
and GOx activity. The maximum GOx activity producing precipitates were
dialysised and concentrated by lyophilization.
4.8.2 Dialysis
Dialysis membrane of 110 kDa cut-off range was selected (Hi-Media,
Mumbai, India) and required length was taken. The dialysis membrane consists of
glycerate a plasticizer and some sulphorous compound as stabilizer. It was
activated by immersing the membrane in hot water for 15 minutes at 80°C. The
dialysis tube was then washed with Millipore water. One end was tighten with the
help of dialysis tube closure and the sample was loaded leaving a sufficient head
space the other end was also tightened and then immersed in sodium acetate buffer
(10 mM, pH 5.5). It was kept in a magnetic stirrer and the buffer was changed in
an interval of 4, 12 and 24 h.
99
4.8.3 Lyophilization
The dialyzed protein sample was concentrated by lyophilization. Freeze
drying was performed at the machine working (pump) temperature of -70±5 °C
and 0.01 mbar vacuum. The lyophilized enzyme was stored at -20 °C and it was
used for further purifications.
4.8.4 Ion exchange chromatography
The partially purified enzyme was loaded in to DEAE cellulose anion
exchange column (16 h x 2.4 dia. cm gel bed) pre-equilibrated with 100 ml of 50
mM sodium acetate buffer (pH 5.5). After that the protein sample was loaded and
the bound protein was eluted with linear gradient from 50–400 mM NaCl in
sodium acetate buffer (50 mM, pH 5.5). Elution was performed at 2.0 ml/minute
flow rate and the 5.0 ml fractions were collected separately. The GOx activity and
protein content were analyzed in all the fractions. The high GOx activity
contained fractions were pooled together and dialyzed. The dialyzed suspension
was lyophilized and stored at -20 °C.
4.8.5 Size exclusion chromatography
Sephacryl S-200 (Pharmacia Biotech, Sweden) was allowed to swell
overnight in sterilized distilled water. The floating gel beads were removed and
the gel slurry was packed into a glass column (10 h X 1.6 dia. cm), which
contained sintered filter at bottom. While packing, care was taken to avoid air
bubbles. The packed gel column was equilibrated with 10 mM sodium acetate
buffer pH 5.5.
Concentrated enzyme was loaded on to Sephacryl S-200 column and eluted
with 10 mM sodium acetate buffer (pH 5.5) at the flow rate of 3 ml per 10
minutes. The GOx activity and protein content were analyzed by previously
100
described method and the active fractions were pooled together and concentrated
by lyophilization.
4.9 Polyacrylamide Gel Electrophoresis (PAGE)
The purity of the GOx sample was checked by SDS-PAGE. SDS-PAGE
was performed in 10% polyacrylamide and the proteins were detected with
coomassie brilliant blue R-250 (Laemmli, 1970).
4.9.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE)
The SDS-PAGE is a standard technique used for the qualitative analysis of
protein mixtures. Since the method involves the separation of proteins according
to size, it is useful in determining the relative molecular weight of proteins and
monitoring enzyme purification. SDS is an anionic detergent and under defined
experimental conditions the relative electrophoretic mobilities of proteins in the
presence of SDS are related to their relative molecular masses. Samples to be run
on SDS-PAGE are boiled for 5 minutes in the presence of β-mercaptoethanol and
SDS. The β-mercaptoethanol results in the reduction of any tertiary structure
disulphide bonds and the SDS strongly binds the proteins and denatures them.
Each protein in the mixture is fully denatured into a rod-shaped structure
containing a series of negatively charged SDS molecules along the polypeptide
chain.
The SDS-poly acrylamide gel electrophoresis was performed on slab gel
with separating gels (10 and 5% w/v) by the following method of Laemmli
(1970).
101
Reagents
Stock solutions
Solution A 1.5 M Tris-HCl buffer (pH 8.8) with 0.4% (w/v) SDS
Solution B 0.5 M Tris-HCl buffer (pH 6.8) with 0.4% (w/v) SDS
Solution C 30% (w/v) acrylamide with 0.8% bisacrylamide
Solution D 1.4% ammonium persulphate
Solution E 1% SDS
Solution F N,N,N,N’ tetramethyl ethylene diamine (TEMED)
Preparation of gel
Separating gel [10% (w/v)] Stacking gel [5% (w/v)]
Solution A 0.75 ml Solution B 0.38 ml
Solution C 2.0 ml Solution C 0.50 ml
Solution D 0.3 ml Solution D 0.15 ml
Solution E 0.6 ml Solution E 0.3 ml
Millipore water 2.6 ml Millipore water 1.98 ml
Solution F 0.005 ml Solution F 0.005 ml
102
Sample buffer
Glycerol 2.0 ml
β-mercaptoethanol 1.0 ml
10% SDS (w/v) 4.0 ml
Solution B 1.7 ml
Bromophenol blue (aqueous) 0.2 ml
Millipore water 0.6 ml
Tank buffer (pH 8.3)
Tris 3.0 ml
Glycine 14.4 ml
SDS 1.0 ml
Millipore water 1.0 ml
Procedure
The enzyme solution was mixed with an equal volume of sample buffer,
boiled in a water bath for 5 minutes, cooled and added to the wells. Then the
power supply was connected with cathode in the upper tank and anode in the
lower tank. Electrophoresis was carried out at room temperature with at 20mA
current supply until the tracer dye reached 0.5 cm above the lower end.
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Staining of separated proteins
At the end of electrophoresis, gel was removed and stained with CBB stain
[40% methanol, 0.7% acetic acid, 0.075% Coomassie dye (CBB-R250)] and
destained using 40% methanol, 0.7% acetic acid. The subunit molecular mass of
GOx from A. awamori MTCC 9645 was determined by calibration against a set of
protein standards.
4.9.2 Native polyacrylamide gel electrophoresis
Native-PAGE is also known as non-denaturing gel in which the entire process
does not contain SDS. It helps in studying the character of the protein such as
without denaturing the protein. It is used to study the enzymes. As the protein will
be intact, the protein bonds will be less in native-PAGE than SDS-PAGE. The
sample buffer does not contain SDS and β-mercapto ethanol. The sample is not
heated here. In native the bands are resolved based on molecular weight and net
charge. Native-PAGE was performed as per the method of Davis, (1964).
Reagents
Solution A Solution B
1 N HCl 48.0 ml Acrylamide 30.0 g
Tris 36.6 g N,V’-methyl bisacrylamide 0.8 g
Millipore water 100 ml
Millipore water 100 mlSeparating gel (pH 8.8)
Stacking gel (pH 6.8)
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Solution C Solution D
Ammonium persulphate 0.14 g N,N,N,N’-tetramethyl ethylene diamine
(TEMED)Millipore water 100 ml
Separating gel [8% (w/v)] Stacking gel [5% (w/v)]
Solution A 0.74 ml Solution A 0.35 ml
Solution B 2.0 ml Solution B 0.50 ml
Solution C 0.3 ml Solution C 0.15 ml
Millipore water 2.89 ml Millipore water 2.0 ml
Solution D 0.005 ml Solution D 0.005 ml
Tank buffer (pH 8.3)
Tris 60.0 g
Glycine 28.8 ml
Millipore water 100 ml
Tank buffer stock solution, was made up to 100 ml with sterile Millipore
water, adjusted to pH of 8.3 and used as tank buffer. Slab gel electrophoresis was
carried out on glass plates of 10.5 x 10.5 cm.
105
Electrophoresis procedure
Polymerization of separating gel was carried out on the glass plates.
Stacking gel was polymerized over the separating gel after inserting a comb. The
known amount of enzyme sample mixed with sample buffer with bromophenol
blue was loaded into the wells and then the power supply was connected with
cathode in the upper tank and anode in the lower tank. Electrophoresis was carried
out at 4°C with constant voltage and 20mA current supply for 2 h until the tracer
dye reached 0.5 cm above the lower end.
4.9.2.1 Zymogram analysis
The native page was developed by overlaying of 1.5% soft agar containing
glucose, o-dianisidine and horse radish peroxidase and kept in dark for 10 minutes
for development of brown colour.
Preparation of soft agar
Agar 1.5% (w/v)
o-dianisidine 1.0 ml [0.21mM(w/v)]
Horse radish peroxidase 0.3 ml (100U)
Glucose 2% (w/v)
4.9.3 Confirmation of enzyme activity using plate assay
Two percentage (w/v) of agar with substrate [5% (w/v) of glucose] was
prepared and poured in a Petri plate. Three wells of 6mm size were made with the
help of cork borer. One kept as control, one well with 50 μl of purified enzyme
and the other well with 50 μl of authentic commercial GOx (Sigma, GOx from A.
niger) enzyme and observed for brown colour formation.
4.10 Characterization of glucose oxidase
4.10.1 Kinetic charecterization
106
100samplecontrolinactivityEnzymesamplein treatedactivityEnzymeactivityrelativeof% ´=
The apparent Km for glucose of the purified GOx was determined by
measuring initial velocities over a range of glucose concentration (100mM to
1000mM) and constant enzyme concentration (20 µg protein). The kinetic
constants for the purified GOx was determined from Lineweaver’s Burk plot,
Hanes-Woolf linear plot and Eadie-Hofstee plot. The Michaelis constant (Km), the
maximal limiting rate velocity (Vmax) were all calculated.
4.10.2 Effect of temperature and pH on GOx activity
The effect of temperature and pH on GOx activity was determined by
Simpson et al. (2006) method. The GOx assay reagents were equilibrated for 10
minutes at the temperatures ranging from 20 to 80°C, before initiating the
reactions with the addition of the GOx at 0.2 U/ml. The pH profile for GOx was
performed in universal buffer containing 50mM potassium dihydrogen
orthophosphate, 33mM citric acid and 50.7 mM boric acid, adjusted the pH values
ranging from 3 to 9 with potassium hydroxide. The assay reagent buffer was
replaced with universal buffer and a GOx concentration of 0.4 U/ml was used to
initiate the reaction. The activity of GOx was expressed as relative activity and it
was calculated by the formula as,
……. (4.5)
4.10.3 Stability testing
The stability of purified GOx was determined based on the method
described by Simpson et al. (2006). It was performed at 25 and 37°C, since these
temperatures correlated to potential applications of GOx, namely implantable
glucose biosensors for human application and food processing (operating at 37°C
and physiological pH of 7.2) and glucose determination test strips (room
temperature operation 25°C). Purified GOx at a concentration of 0.15U/ml was
prepared by dissolution in Millipore water. Volumes of 20 ml were placed in
107
water baths pre-equilibrated to ~25 and 37°C. Samples (0.5 ml) were removed
periodically (10-90 minutes) and analyzed for GOx activity. The GOx activities of
the samples were compared to an initial sample taken at the onset of the
experiment. A lyophilized GOx preparation was stored at -20°C and assayed
monthly for 6 months to provide an indication of shelf-life.
……. (4.6)
4.10.4 Stability and inhibitory studies of GOx
4.10.4.1 Effect of metal ions on GOx activity
The effects of metal ions on enzyme activity were studied at 1 mM
concentration of different metal ions such as lead acetate, cobaltous chloride,
copper acetate, silver sulphate, copper sulphate and mercuric chloride. The 100µl
of enzyme with enzyme activity of 1U/ml was incubated along with 0.9 ml of 1
mM metal ions for 20 minutes. After the incubation period the enzyme activity
was analyzed.
4.10.4.2 Effect of calcium ions on GOx activity
Enzyme (0.1 ml) with the activity of 1 U/ml was incubated with 0.9 ml of 1
mM calcium ions such as calcium carbonate, calcium lactate and calcium
propionate. The enzyme activity was measured after 20 minutes.
4.10.5 Preparation of carrier based enzyme
The enzyme active fractions were mixed with the aqueous stabilizing solution
(Poly ethylene glycol, 1% w/v). The above mixture was freeze dried and stored at
-20°C. The carrier based enzyme was used for further application processes.
4.11. APPLICATION OF GLUCOSE OXIDASE IN FOOD PROCESSING
AND PRESERVATION
100incubationafteractivityEnzymeincubationbeforeactivityEnzymeactivityresidualof% ´=
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4.11.1 Enhancing the storage stability of vegetable by coating of edible film
incorporated with glucose oxidase, lactoperoxidse and lysozyme
This present study was to evaluate the applicability of antimicrobial effect
by purified GOx, partially purified LPS and lysozyme with EDTA for enhancing
the shelf-life of carrot.
4.11.1.1 Microorganisms and culture conditions
The pure culture of E. coli (MTCC 443) and S. aureus (MTCC 96) were
obtained from Microbial Type Culture Collection, Chandigargh, India. The
cultures were frequently subcultured and maintained in Luria Bertani (LB) and
nutrient agar slants at 4ºC respectively. The composition of the LB medium are:
tryptone 10.0 g; yeast extract 5.0 g; NaCl 10.0 g distilled water 1.0 litre and the
nutrient agar medium are: beef extract 1.0g; yeast extract 2.0 g; peptone 5.0 g
NaCl 5.0 g; agar 15.0 g and distilled water one litre.
4.11.1.2 Preparation and analysis of antimicrobial enzymes
Glucose oxidase
The GOx was produced from Aspergillus awamori MTCC 9645. GOx
activity was measured according to Bergmeyer, (1988).
Lactoperoxidase
The partially purified LPS was obtained from whey permeate by Martin
Morrison (1957) method. The initial step in the preparation of whey permeate was
prepared from one litre of milk was taken and heated to 50ºC. Then 10 ml of
acetic acid solution was added to the milk. After that the milk was stirred at room
temperature until coagulation occurred. All subsequent steps were carried out at
4ºC. The coagulated milk was centrifuged for 15 minutes at 3000 g. The pellet and
the solid casein were removed from milk and the supernatant was collected for the
109
preparation of partially purified LPS. The collected sample was subsequently
subjected to ammonium sulphate for protein precipitation.
Forty millilitre of wet Amberlite IR-120 (NH4+, form) was added to one
litre of whey permeate and then the suspension was stirred for 3 h and allowed to
stand for 30 minutes to let the resin settle. The resin was then transferred to a
Buchner funnel, and was washed with water and 50 mM sodium acetate (1 litre
and two litres each per litre of whey, respectively). Then resin was transferred to a
column (1.5 X 50 cm) and elution was carried out in two steps using 0.5 M
sodium acetate and 1.0 M sodium acetate at a flow rate of 2-5 ml/minutes. The
ammonium sulphate precipitated protein sample containing LPS was eluted using
0.5M sodium acetate solution and this elution was lyophilized. The partially
purified LPS was stored at -8 ºC.
The enzyme activity of partially purified LPS was measured according to
the method described by Shindler et al., (1976). The oxidation of 3’3, 5,5’-
tetramethylbenzidine (TMB) (Sigma) by LPS was measured
spectrophotometrically at 413 nm using UV-Visible spectrophotometer. The
reaction was conducted in 0.1 M acetate buffer, pH 5.2 at 20°C containing
appropriate concentrations of TMB, HZ02 (Fisher) and LPS in a volume of 1 ml.
Result was expressed in units/ml, where one unit (U) is defined as the amount of
enzyme that oxidizes 1µmol TMB/min.
Lysozyme
The partial purification of lysozyme was carried out by Çifdem Mecitoflu
et al. (2006) method. Lysozyme was prepared from the hen egg. The egg whites
and yolks were separated and the egg white portion was used for the partial
purification. The separated egg white was diluted with two volumes of 0.05M
NaCl solution. The lysozyme was precipitated from the mixture of proteins in the
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egg white by lowering the pH. It was performed by bringing down the pH up to
4.0 by using 1N acetic acid and it was diluted with equal volume of 60% (v/v)
ethanol. It was stored at room temperature for 6 h and the mixture was centrifuged
at 15,000 g for 15minutes at 4°C. Precipitate was removed and the supernatant
containing lysozyme was first dialyzed with cellulose acetate membrane (5 kDa,
Hi-Media, India) at 4°C for 21 h by three changes of 2000ml distilled water. Then
it was concentrated by using lyophilizer at the machine working (pump)
temperature of -70±5°C and 0.01 mbar vacuum. The lyophilized enzyme was
stored at -20 °C and it was used for the film preparation.
The lysozyme enzyme activity was analyzed by spectrophotometrically at
A660nm adopted by Çifdem Mecitoflu et al. (2006). The reaction mixture was
formed by mixing 0.1 ml of enzyme extract or enzyme containing solution can be
prepared by dissolving films in distilled water. Then 2.9 ml of Micrococcus
lysodeikticus cell suspension was prepared (0.26 mg/ml, prepared in 0.05 M Na-
phosphate buffer at pH 7) and incubated at 30°C. The mixture was rapidly
vortexed and immersed into a water bath at 30°C. The absorbance of the reaction
mixture was determined at the end of first minute and the difference between
absorbance value and the initial absorbance value was used for the calculation of
enzyme activity (the absorbance-time curves were linear for 1.5–2 minutes). The
enzyme activity was expressed as units (0.001 absorbance change in one minute).
The average of three measurements was used in all tests.
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4.11.1.3 Evaluation of antimicrobial activity of GOx, LPS and lysozyme with
EDTA
The antimicrobial activity of GOx, LPS and lysozyme with EDTA was
determined against E. coli and S. aureus. The E. coli and S. aureus were taken
from the stored culture and grown in 10 ml of fresh nutrient broth at 37ºC for 24 h.
One millilitre of the culture was centrifuged at 8000 g for 20 minutes. After
centrifugation, the supernatant was discarded and the bacterial pellet was
resuspended with phosphate buffer solution (PBS - pH 7.0). The effect of
antimicrobial enzyme activity was evaluated by the formulations like, (I) GOx,
(II) LPS (III) Lysozyme with EDTA (IV) GOx and LPS, (V) GOx, lysozyme with
EDTA, (VI) LPS and lysozyme with EDTA, (VII) GOx, LPS and lysozyme with
EDTA, (VIII) Control. For every formulation one millilitre of PBS bacterial
suspension was added. The enzyme concentration in the test solution contains
GOx (5 mg/ml), LPS (10 mg/ml) and lysozyme (0.5 mg/ml) with EDTA (0.3
mg/ml). The control treatment contains no antimicrobial enzymes. Triplicates
were incubated at 37ºC for 48 h on rotary platform shaker at 250 rpm. The treated
formulations were analyzed for every 6 h of incubation up to 48 h. The inhibitory
activity of the E. coli and S. aureus was analyzed by serial dilution in PBS by
plating in duplicate on nutrient agar and incubating plates at 37ºC for 48 h. The
results were expressed in colony forming units per millilitre (log CFU/ml).
4.11.1.4 Film making
Film making was carried out by the modified method of Çifdem Mecitoflu
et al. (2006). One gram of sodium alginate was dissolved in 10 ml of distilled
water and glycerol (0.50 ml) was then added to the alginate solution. This mixture
was dissolved thoroughly by using boiling water bath with constant stirring and
boiled for 5 minutes.
112
The hydrocolloid was brought down to room temperature. Then the film
solution was prepared by mixing with GOx, LPS and lysozyme and di-sodium
EDTA.2H2O to get final concentrations of GOx (5 mg/ml), LPS (10 mg/ml) and
lysozyme (0.5 mg/ml) with EDTA (0.3 mg/ml) di-sodium EDTA 2H2O. This bio-
film hydrocolloids was made to stand by for 30 minutes to avoid air bubbles. Then
the film solution was poured on a clean glass plate to form a uniform layer. The
films were dried at room temperature and 6 mm diameter film discs were cut by
using standard stainless steel well cutter. This film discs were used for
antimicrobial test. The above mentioned different formulations of enzymes were
added to the film (alginate) forming solution.
4.11.1.5 Antimicrobial film activity
Antimicrobial activity of the film was performed by using E. coli and
S. aureus as test microorganisms. The over night cultures of E. coli and S. aureus
were prepared in nutrient broth and incubated at 37ºC for 48 h incubation. For
antimicrobial test, discs (six mm in diameter) were prepared from each film by
sterile well cutter under aseptic condition. These discs were placed carefully onto
Petri dishes containing nutrient agar on which 0.1ml culture was spreaded. The
Petri dishes were incubated at 37°C for 24 h and the zone of inhibition (diameter)
was measured with the help of centimeter scale.
4.11.1.6 Surface sterilization of carrots
Fresh carrots (Daucus carota sp.) were purchased in a local supermarket
and stored at 4°C before processing. In this study chlorine dioxide has been
chosen for surface sterilization of carrots. The concentration and soaking time
were optimized for efficient and minimal use of chlorine dioxide. The samples
were sanitized with chlorine dioxide at different concentrations (2, 4, 6, 8 and
10ppm) prepared from the stock solution. Carrots were surface washed with these
different concentrations of chlorine dioxide solution at different time periods (10,
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20 and 30 minutes). After surface sterilization, the sterility was determined by
soaking in sterile distilled water. The microbial population in soaked water were
measured by viable plate method. Number of microbes ware measured as colony
forming units.
4.11.1.7 Coating procedure
Carrots were surface sterilized with chlorine dioxide (6 ppm) for 20
minutes. Surface sterilized whole carrots were taken for alginate coating with
different enzyme formulations. Fresh carrots were dipped completely into the
coating solution for 5 seconds at room temperature and then taken out. This
dipping procedure was repeated thrice and excess coating material was allowed to
drain completely. Then it was immersed in 2% (w/v) of calcium chloride solution
for 5 minutes and the coating was dried with an air blower. Coated and uncoated
carrots were kept in both room temperature (~26°C) and (6°C) before the analysis
of water loss.
4.11.1.8 Determination of weight loss
The shelf-life can be defined as the length of time which the vegetable can
maintain their appearance, safety and nutritional value that appeals to the
consumer. So water loss (weight loss) and soluble protein content were monitored
in carrots stored at both ~26°C and at 6 °C. The coated and uncoated carrots were
weighed and packed in perforated polypropylene covers and kept at both ~26°C
and 6 °C. They were reweighed every 48 h in order to follow the moisture loss as
a function of time over a period of 10 d. A digital electronic balance (Shimadzu,
Japan) was used to determine the product weight. Weight loss percentage
calculated was calculated using the formula,
……. (4.7)
114
where: %WL(t) is the percentage weight loss at time t, W0 is the initial sample
weight and W(t) is sample weight at time t.
4.11.1.9 Determination of soluble protein content
Soluble protein content was measured by taking 0.1 g of freeze dried carrot
sample mixed with of two millilitre distilled water and vortexed for 5 minutes at
room temperature. The tubes were centrifuged for 20 minutes at 5000 rpm. The
supernatant was collected in eppendorf tubes and stored at 4°C for 2 h before
analyzing. The soluble protein content of the extracted samples was estimated
according to the method of Bradford (1976). Protein extract was diluted 1:50 in
distilled water and one millilitre of sample was mixed with 5 ml of Coomassie
Brilliant Blue G-250 solution and incubated at room temperature for 20 minutes.
Absorbance value was measured at 595nm in UV-Vissible spectrophotometer.
4.11.1.10 Microbial analysis by viable plate count method
Ten grams of carrot sample was taken and mixed with 90 ml of sterile
distilled water and vortexes for 10 minutes. Then it was serially diluted up to 10-8
dilutions. One millilitre of 10-6 dilution was delivered to Petri dishes containing
nutrient agar medium and spread uniformly. The plates were incubated at 37ºC for
24 h and the results were expressed in colony forming units (CFU/g) of sample.
4.11.1.11 Sensory analysis
The sensory qualities of treated and untreated carrots were performed
during the storage period using five member trained panel with an age of 25–35.
The panel consists of three females and two males who were trained to be familiar
with sensory properties of carrots. The sensory testing method was an acceptance
test in which the sensory parameters were scored on a descriptive scale of 1–5.
The investigated sensory parameters include: (i) taste, (ii) colour, (iii) texture, (iv)
appearance and (v) overall acceptance. Descriptions for each score were as
115
follows: 5-likely very much, 4-likely slightly, 3-neither like nor dislike 2-dislike
slightly and 1-dislike very much. Sensory trials were replicated thrice.
4.11.2 Control of browning and enhancing shelf-life of apple puree by
applying glucose oxidase-catalase system with lactoperoxidase
4.11.2.1 Preparation and analysis of antimicrobial enzymes
GOx: The GOx was produced from Aspergillus awamori MTCC 9645 and the
activity was measured according to Bergmeyer et al. (1988).
Lactoperoxidase: The partially purified LPS was obtained from whey permeate
by Martin Morrison (1957). The enzyme activity of partially purified LPS was
measured according to the method described by Shindler et al., (1976).
Catalase: Enzyme Catalase was brought from M/s Speciality enzymes and
Biochemicals Co, USA. Catalase activity was measured spectrophotometrically by
observing the decrease in light absorption at 525 nm during decomposition of
H2O2 by enzyme. The reaction mixture contained one millilitre of 0.1 M McIl-
Valine buffer pH 6.8, 0.05 ml of suitability diluted enzyme, and 0.03 mg of 100
mM H2O2 solution and was incubated for 30 minutes at 30º C. The reaction was
developed with 0.2 mg o-di-anisidine, 0.06 mg peroxidase (500/mg), and 0.8 ml
glycerol in 2ml of 0.2 M. Tris-phosphate buffer pH 6.8. After 30 minutes
incubation at 30ºC, the enzymatic reaction was stopped by adding 4 ml of 5 N
HCl. The absorbance at 525 nm was measured against a blank, i.e., a control
mixture composed and incubated as described above but deprived of hydrogen
peroxide and peroxidase. One unit (U) of catalase activity was defined as the
amount of enzyme catalyzing the decomposition of 1µmol hydrogen peroxide per
minutes at 30ºC.
116
4.11.2.2 Determination antimicrobial activity of GOx, LPS and catalase
The antimicrobial activity of GOx, LPS and Lysozyme with EDTA was
determined against E. coli (MTCC 443) and S. aureus (MTCC 96. The E. coli and
S. aureus were taken from the stored culture and grown in 10 ml of fresh nutrient
broth at 37°C for 24 h. One millilitre of the culture was centrifuged at 8000 g for
20 minutes using eppendorf centrifuge model. After centrifugation, the
supernatant was discarded and the bacterial pellet was resuspended with
phosphate buffer solution (PBS-pH 7.0). The effect of antimicrobial enzyme
activity was evaluated by the formulations (mg or ml /10ml) like, of (I) GOx (100
mg), (II) LPS (40mg), (III) Catalase (0.1ml), (IV) GOx (100mg) +LPS (40mg) (V)
GOx (100mg)+Catalase (0.1ml), (VI) LPS (40mg)+Catalase (0.1ml) and (VII)
GOx (100mg)+Catalase (0.1ml)+LPS (40mg), (VIII) Control.
For every 10 ml of enzyme formulation one millilitre of PBS bacterial
suspension was added. The control treatment contains no antimicrobial enzymes.
Triplicate were incubated at 37°C for 48 h on rotary platform shaker at 250 rpm.
The treated formulations were analyzed for every 6 h of incubation up to 48 h.
The inhibitory activity of the E. coli and S. aureus was enumerated by serial
dilution in PBS by plating in duplicate on nutrient agar and incubating plates at
37°C for 48 h. The results were expressed in colony forming units per millilitre
(CFU/ml) of sample.
4.11.2.3 Preparation of apple puree
Fresh fruits of golden delicious apples were purchased in local super
market, Chennai, India. Apples were culled and apples with fine shiny skins which
were free of physical damage. The apple puree sample was prepared based on the
method described by Parpinello et al. (2002). The apple puree was extracted by
the following process. The selected apples were washed thoroughly, peeled with
stainless steel clean knife and the peeled apples were cut in to small pieces. It was
117
heated at 95ºC for 5 minutes in hot water bath and ground with domestic juice
extractor (Preethi Chepro Plus, India) until puree was obtained. Then it was heated
at 95ºC for 10 minutes, cooled up to 25ºC, homogenized and stored at 4ºC. The
fruit puree was characterized by the following parameters: colour (browning),
dissolved oxygen content, microbial analysis and sensory analysis.
4.11.2.4 Effect of GOx and ascorbic acid on dissolved oxygen consumption
Measurement of dissolved oxygen (DO) was performed in digital DO
Meter Model 801(EI), India. The DO probe was placed in 2% (w/v) of sodium
sulphite solution. The display is allowed to attain equilibrium. The zero knob was
brought to display to read 0.0 by keeping the temperature knob to actual
temperature of the solution. Calibrate knob was then brought to extreme right
position. As given in the instrument manual chart water at 25ºC was taken as the
standard which has an oxygen solubility of 8.2. The above mentioned steps were
repeated with the apple puree samples which were treated with GOx 50, 100 and
150 mg; ascorbic acid 50, 100 and 150 mg/l concentration per litre of apple puree.
4.11.2.5 Experimental design
The effect of browning (colour changes) and antimicrobial activity on apple
purees for combination of GOx-catalase system and LPS. The effect of
antimicrobial enzyme activity was evaluated by the formulations (mg or ml /l of
puree) like, of (I) GOx (100 mg), (II) LPS (40mg), (III) Catalase (0.1ml), (IV)
GOx (100mg) +LPS (40mg) (V) GOx (100mg)+Catalase (0.1ml), (VI) LPS
(40mg)+Catalase (0.1ml) and (VII) GOx (100mg)+Catalase (0.1ml)+LPS (40mg)
and (VIII) Control.
4.11.2.6 Evaluation of browning on apple puree
Right after the extraction of puree and the addition of respective
combination of enzymes and antibrowning agents, the puree was bottled and
118
stored at room temperature in 750 ml glass bottles with cork caps. Browning was
observed during storage by analyzing all the samples at an interval of 5 days up to
30 days. The sample of 20 ml apple puree was taken from each sample and
centrifuged at 10000 g for 15 minutes. The browning was evaluated in the
supernatant by measuring the absorbance at 420 nm. Browning is characterized by
spectro photometric measurements at 420 nm to detect browning pigments
(Toribio and Lozano, 1986; Nagy et al., 1990; Wong and Stanton, 1992).
4.11.2.7 Examination of microbial populations
The prepared samples with various combinations of enzymes and
antibrowning agents were bottled and corked. These corked bottles were stored at
room temperature. These samples were checked for the antimicrobial activity at an
interval of 5 days up to 30 days. The microbial population was enumerated from
the apple puree by viable plate count method as described earlier. One millilitre of
the apple puree sample was uniformly distributed to the nutrient agar medium and
incubated for 24 h at 37°C. The results were expressed in CFU/ml of apple puree.
4.11.2.8 Sensory analysis
The sensory qualities of apple puree were performed during the storage
period using 5 member trained panel with an age of 25 to 35 years. The panel
consists of two females and three males who were trained to be familiar with
sensory properties of apple puree. The sensory testing method was an acceptance
test in which the sensory parameters were scored on a descriptive scale of 1-6. The
sensory parameters investigated included the following: (i) taste, (ii) colour, (iii)
flavour, (iv) texture, (v) appearance and (vi) overall acceptance. Descriptions for
each score were as follows: 5–likely very much, 4–likely slightly, 3–neither like
nor dislike 2–dislike slightly and 1–dislike very much. Sensory trials were
replicated thrice.
119
4.11.3 Studies on the effect of glucose oxidase-catalase with calcium ions
in stabilizing and improving the fruit salad quality
4.11.3.1 Preparation of enzyme
The GOx was produced from A. awamori (MTCC 9645) and the activity
was measured according to Bergmeyer et al. (1988). Catalase was brought from
M/s Speciality enzymes and Biochemicals Co, USA and analyzed as described
earlier.
4.11.3.2 Collection of fruits
Fruits like apple, guava, orange, grapes, mango, banana, cherry and lemon
were purchased from local super market, Chennai. Fruits were selected based on
the characters like without physical damage, without microbial spoilage, matured
and equal in size.
4.11.3.3 Preparation of koruk juice
Unripe grapes were purchased from the agriculture farm located at
Dindigul in Tamilnadu. The stalks of the grapes were removed and the grapes
were washed. The grapes were weighed to one kilogram and crushed thoroughly
in grinder. The extracted juice was used for fruit salad preparation.
4.11.3.4 Analysis of antimicrobial activity of GOx, calcium ions and koruk
juice
The antimicrobial activities of calcium ions, GOx and koruk juice were
tested against E. coli (MTCC 443) and S. aureus (MTCC 96). Phosphate buffer
solution was prepared with E. coli and S. aureus (approximately 9 logs CFU/ml).
One ml of PBS solution was treated with 50 µl of following test formulations. The
effect of antimicrobial activity was evaluated by the different treatments like,
120
I) GOx (5mg/ml), II) calcium lactate (2% w/v), III) calcium chloride
(2% w/v), IV) calcium propionate (2% w/v), V) optimized calcium ions
combination (2% w/v), VI) koruk juice (2% v/v), VII) GOx + koruk juice
+calcium ions combination (2% w/v) and VIII) Control.
A control sample consisted of bacterial pellets dissolved in one millilitre of
PBS, which contained no antimicrobial formulation. Six sets of PBS cell
suspension were prepared for each formulation respectively. Triplicate samples
were then incubated at 37°C for 48 h on a rotary platform shaker at 250 rpm. The
E. coli and S. aureus were enumerated by serial dilutions in PBS, plating in
duplicate on nutrient agar and incubated at 37°C for 48 h. The treated samples
were analyzed by every six hours of incubation up to 48 h.
4.11.3.5 Preparation of treatment solution
1. Three different concentrations of GOx like 0.25, 0.5 and 1.0% (w/v) were
dissolved in sterilized distilled water.
2. Calcium lactate, calcium propionate and calcium chloride were prepared at
the concentrations of 0.5, 1.0 and 2% (w/v) in sterile distilled.
3. The extracted koruk juice at the concentration of 0.5, 1.0 and 1.5% (v/v)
mixed in sterilized distilled water.
4. Combined calcium ions (0.5, 1.0, 1.5, 2.0 and 2.5% w/v) with 0.25% (w/v)
of GOx-catalase and Koruk juice (1% v/v) were mixed sterile water.
4.11.3.6 General fruit salad preparation procedure
Fresh fruits like apple, guava, orange, grapes, mango, banana, cherry and
lemon were washed with water and cut it small pieces using sterilized stainless
steel knife. Then it was soaked for 15 minutes in the above treatment solutions.
After that the fruits were taken out from the solutions and used for fruit salad
preparation. Lemon juice (4 ml) was poured to the 1.0 Kg sliced fruits and 150 g
121
of half melted sugar solution added. Then it was transferred to a clean plastic
container and stored at room temperature.
4.11.3.7 Sensory analysis
The sensory quality was analyzed after a day of storage such as taste,
colour, texture, appearance and overall acceptance. These qualities were
performed using 5 member trained panel with an age of 25 to 35 years. The panel
consists of three females and two males who were trained to be familiar with
sensory properties of fruit salad. The sensory testing method was an acceptance
test in which the sensory parameters were scored on a descriptive scale of 1-6. The
sensory parameters investigated included the following: (i) taste, (ii) colour, (iii)
texture, (iv) odour and (vi) overall acceptance. Descriptions for each score were as
follows: 5–likely very much, 4–likely slightly, 3–neither like nor dislike 2–dislike
slightly and 1–dislike very much. Sensory trials were replicated thrice.
4.11.3.8 Experimental design of RSM for optimization of calcium ions
The optimization of calcium ions was performed using Design-Expert 7.1.6
software package by Response Surface Methodology (RSM). The optimal levels
of three variables like calcium chloride, calcium lactate, calcium propionate was
done. For that purpose, the response surface approach by using a set of
experimental design (central composite design with five coded levels) was
performed. For the three factors, this design was made up of a full 23 factorial
design with its eight points augmented with three replications of the center points
(all factors at level 0) and the six star points, that is, points having for one factor
an axial distance to the center of ±α, whereas the other two factors are at level 0.
The axial distance α was chosen to be 1.68 to make this design orthogonal. A set
of 20 experiments were carried out for the three variables. The central values (0
level) chosen for experimental design were (% w/v) Calcium lactate-1.50,
Calcium propionate-0.88, Calcium chloride-0.60 (Table 4.5.1–4.5.2).
122
In developing the regression equation, the test factors were coded
according to the following equation:
……… (4.8)
Were xi is the coded value of the ith independent variable, Xi the natural
value of the ith independent variable, X0 the natural value of the ith independent
variable at the center point, and ∆Xi the step change value (∆Xi is 1.50 for calcium
lactate, 0.88 for calcium propionate, 0.60 for calcium chloride). For a three-factor
system, the model equation is:
……… (4.9)
Where: Y, predicted response; b0, intercept; b1, b2, and b3, linear coefficients; b11,
b22, and b33, squared coefficients; and b12, b13, and b23, interaction coefficients
Results were analyzed by the experimental design module of the design expert
7.0. The model permitted evaluation of the effects of linear, quadratic and
interactive terms of the independent variables on the chosen dependent variables.
Three-dimensional surface plots were drawn to illustrate the main and interactive
effects of the independent variables on calcium ions optimization. The optimum
values of the selected variables were obtained by solving the regression equation
and also by analyzing the response surface contour plots (Myers and Montgomery,
2002).
123
Table 4.5: Optimization of calcium ions for salad preparation
Table 4.5.1: Design summary
Variables UnitsLevels
-1 -α 0 +1 +αCalcium lactate (A) % (w/v) 0.5 -0.181 1.5 2.5 3.181
Calcium propionate (B) % (w/v) 0.25 -0.176 0.88 1.5 1.926
Calcium chloride (C) % (w/v) 0.2 -0.072 0.60 1 1.272
Tanle 4.5.2: Experimendal design and results of 23 factorial design
RunA
Calcium lactate(%,w/v)
BCalcium
Propionate (%, w/v)
CCalcium chloride
(% ,w/v)
1 1.50 1.93 0.602 1.50 0.88 0.603 1.50 0.88 0.604 0.50 1.50 1.005 0.50 0.25 1.006 2.50 0.25 1.007 1.50 0.88 -0.078 2.50 0.25 0.209 -0.18 0.88 0.6010 1.50 0.88 0.6011 2.50 1.50 0.2012 2.50 1.50 1.0013 1.50 -0.18 0.6014 0.50 0.25 0.2015 1.50 0.88 0.6016 3.18 0.88 0.6017 1.50 0.88 1.2718 1.50 0.88 0.6019 0.50 1.50 0.2020 1.50 0.88 0.60
124
4.11.3.9 Effect of optimized calcium ions on fruit salad preparation
The RSM optimized calcium ions were; calcium lactate- 1.45% (w/v),
calcium propionate- 0.68% (w/v), calcium chloride- 0.55% (w/v). The optimized
combination of calcium ions were analyzed in fruit salad preparation. This
experiment was also done with GOx-catalase and koruk juice.
4.11.3.10 Measurement of weight loss
Weight loss percentage of fruit salad was calculated after 48 h of storage at
room temperature. A digital electronic balance (Shimadzu, Japan) was used to
determine the product weight. Weight loss percentage calculated was calculated
by the formula,
……… (4.10)
where: %WL(t) is the percentage weight loss at time t, W0 is the initial sample
weight and W(t) is sample weight at time t.
4.11.3.11 Microbial analysis of fruit salad by viable plate count method
Thirty gram of sample was taken and mixed with 270 ml of sterile distilled
water. With a sterile pipette, one millilitre of suspension was transferred to the
second tube containing 9.0 ml of sterile water. This was continued for up to 10-8
dilutions. One millilitre of 10-6 to 10-8 dilutions was delivered to Petri dishes
containing nutrient agar medium and spread uniformly. The results were
expressed in colony forming units (CFU/g) of sample.
125
4.12 Statistical analysis
All values are expressed as means ± standard deviation. The results were
analyzed using one-way analysis of variance (ANOVA) and the differences
among the treatments means were analyzed using the Tukey-Kramer multiple
comparison test. P value<0.05 was considered as least significant. The software
GraphPad InStat was employed for the statistical analysis.
126
CHAPTER-5
RESULTS AND DISCUSSION
The present study focused on the screening of potential GOx producing
fungi and their application in the food processing and preservation. The initial part
of the investigation was concentrated on isolation of fungi from various sources
and enhancing the GOx production through the optimization medium
compositions was carried out. Production of GOx was carried out in laboratory
batch fermentor. The cell free extract was purified by various purification
methods. The purified enzyme was characterized by enzyme kinetic constant,
environmental factors, inhibitory activity and stability. The final part of the study
was to applying the GOx in the novel areas such as enhancing shelf-life of
vegetables, preparation in apple puree and fruit salad prepared with calcium ions.
5.1 Isolation, media optimization and production of GOx
5.1.1 Isolation of GOx producing fungi
Glucose oxidase producing fungi were isolated from various sugar rich
products (fruits, dates and honey hive) and also from soil samples. The fungi were
screened based on their GOx producing capability. Screening was carried out from
the 35 isolates of Aspergillus niger group and 17 isolates of Penicillum sp. in
order to identify the highest GOx (extra cellular) activity in the screening medium.
The eleven strains were secreted considerable amount of GOx from the 52 isolates
(Table 5.1). The strains such as GOP3 and GOP7 (Figure 5.3 and 5.4)
(Aspergillus awamori belongs to the group of Aspergillus niger) isolated from
dates and honey hive produced the highest extracellular GOx activity in the basal
production medium as 4.2±0.14 and 5.1±0.22 U/ml respectively. The isolated
higher GOx producing fungus (GOP7) was submitted in Microbial Type Culture
Collection (MTCC), Chandigarh, India and it was designated as Aspergillus
awamori MTCC 9645. The fungus A. awamori MTCC 9645 was produced high
127
amount of extra cellular GOx from the different isolates and this fungus was used
for further optimization and production of GOx.
Table 5.1: Glucose oxidase production by different fungi
Organism
codeName of the fungi Source
GOx activity
(U/ml)
GOP1 Aspergillus awamori Dates 1.25±0.58
GOP2 Aspergillus niger Orange 1.15±0.067
GOP3 Aspergillus awamori Dates 4.2±0.14
GOP4 Aspergillus niger Soil 0.51±0.021
GOP5 Aspergillus niger Soil 2.5±0.092
GOP6 Aspergillus niger Honey hive 3.35±0.11
GOP7 Aspergillus awamori Honey hive 5.1±0.22
GOP8 Penicillium sp Soil 0.75±0.038
GOP9 Penicillium sp Honey hive 1.70±0.67
GOP10 Penicillium sp Sugarcane baggase 2.35±0.14
GOP11 Penicillium variale Dates 4.3±0.18
Values are mean of three replicates (±S.D)
128
Figure 5.3: Isolation, screening and identification of A. awamori (GOP-3)
(D) Spores of A. awamori (GOP-3)
(B) A. awamori (GOP-3) on PDA (C) Browning on screening media
(A) Dates
1000 X450 X
129
(A) Honey hive
(C) Browning on screening media(B) A. awamori (GOP-7) on PDA
(D) Spores of A. awamori MTCC 9645
450 X 1000 X
Figure 5.4: Isolation, screening and identification of A. awamori (GOP-7) MTCC 9645
130
5.2 Optimization of medium for glucose oxidase production
Seven different medium were used for the selection of suitable medium for
GOx production in which GOxM3 supported maximum GOx activity (Figure 5.5).
The optimization of medium was carried out by change the constituents and
concentrations of the cultivation medium through the utilization of various carbon,
nitrogen sources, di-ammonium hydrogen phosphate, potassium di-hydrogen
phosphate, magnesium sulphate, CaCO3 and environmental factors (pH and
temperature). For this investigation two type of optimization were performed one
is classical single factor analysis and another one is statistically by response
surface methodology using central composite design.
5.2.1 Single Factor Analysis (SFA) for GOx production
5.2.1.1 Effect of carbon source on GOx production
A number of carbon sources were analyzed in order to determine their
effect on GOx production in the single factor analysis. The different carbon
sources were supplemented to the basal medium constituents. Carbon source
exerts a great influence on the extracellular GOx production. It was supported
GOx production with different extent and results were expressed in the Figure 5.6.
The highest GOx production was obtained in glucose followed by sucrose. A.
awamori MTCC 9645 was able to grow in all the carbon sources evoluated, while
significant (P<0.05) GOx production was obtained in the glucose and sucrose.
Hatziuikolaou and Macris (1995) described that glucose is a principal inducer for
the transcription of GOx gene.
The GOx has a high specificity to glucose, when compared to other
carbohydrates (Rogalski, 1988; Schomburg and Stephan, 1995). During the
microbial fermentations, the carbon source not only acts as a major constituent for
building of cellular material, while it also used in synthesis of polysaccharide and
131
0 1 2 3 4 5 6
GOxM 1
GOxM 2
GOxM 3
GOxM 4
GOxM 5
GOxM 6
GOxM 7D
iffer
ent p
rodu
ctio
n m
ediu
m
GOx activity (U/ml)
b
b
a
a
c
b
c
0
1
2
3
4
5
6
7
20 40 60 80 100 120 140 160Concentrations (g/l)
GO
x ac
tivity
(U/m
l)
Glucose Fructose Sucrose Maltose Xylose Rhamnose
a
dde
d
a
a
e
ab
e
a
a
a
a
b
c
b
b
a
d
bccb
cc
cc
cd
d
cc
d
d
a
d
a
c
a
d
b
b
a
c
a
a
b
ab
Figure 5.5: Effect of different media for GOx production Legends followed by the same letter are not significantly different (P<0.05).
Values represent the mean of triplicates with standard deviation.
Figure 5.6: Effect of carbon sources on GOx production Legends followed by the same letter are not significantly different (P<0.05) for the same concentration. Values represent the mean of triplicates with standard deviation.
132
as energy source (Stanbury et al., 1997). The present study was also correlated
with Traeger et al. (1991) and Sandip et al. (2008) report such as the glucose and
sucrose were more effective in the production of GOx in the A. niger.
5.2.1.2 Effect of nitrogen sources on GOx production
The various types of nitrogen sources were analyzed for the effective GOx
production. Proteose peptone was found to be effective nitrogen source for GOx
production when compared to other nitrogen sources. The detailed results were
expresed in figure 5.7. The optimum concentration of nitrogen sources for the
production of GOx was found to be 3–4 g/l. Increasing concentration of nitrogen
source produced higher biomass, but reduces the GOx production. A similar result
was obtained by Sandip et al. (2008). Hatziuikolaou and Macris (1995) reported
that peptone was an effective nitrogen source for the production of GOx.
Obviously, the low concentration of peptone has a positive effect on process
economy.
5.2.1.3 Effect of di-ammonium hydrogen phosphate, potassium di-hydrogen
phosphate and magnesium sulphate on GOx production
The enhancement of GOx production was obtained from the addition of
di-ammonium hydrogen phosphate and potassium di-hydrogen phosphate. In the
levels of 0.4 g/l of di-ammonium hydrogen phosphate and 0.2 g/l of potassium di-
hydrogen phosphate were found to be maximum production. Addition of
magnesium sulphate increased the amount of GOx production in the fermentation
medium, while above 0.2 g/l of magnesium sulphate affected the GOx production
(Figure 5.8 and 5.9). Nakamatsu et al. (1975) reported that ammonium di-
hydrogen phosphate, potassium di-hydrogen phosphate and Mg++ were found to be
increase the GOx production.
133
0
1
2
3
4
5
6
7
1 2 3 4 5 6Concentrations (g/l)
GO
x ac
tivity
(U/m
l)
Bacteriological peptoneMycological peptoneProteose peptoneYeast extractBeaf extract
a
aa
a
a
a
a
a
bb
bb
bb
cc
cc
cc
dd
b
b
c
d
dc
bb
Figure 5.7: Effect of different nitrogen sources for the GOx production
Legends followed by the same letter are not significantly different (P<0.05) for the same concentration. Values represent the mean of triplicates with standard deviation.
134
0
1
2
3
4
5
6
7
0 0.1 0.2 0.3 0.4 0.5 0.6Concentration (g/l)
GO
x ac
tivity
(U/m
l)a
c
bc
ab
a
b
a
0
1
2
3
4
5
6
7
0 0.05 0.15 0.2 0.25 0.3 0.35 0.4
Concentration (g/l)
GO
x ac
tivity
(U/m
l)
KH2PO4 MgSO4
a
b
B
bAabab AA
c
bc
A
D
bc
C
B
Figure 5.8: Effect of di-ammonium hydrogen phosphate on GOx production
Figure 5.9: Effect of potassium di-hydrogen phosphate and magnesium sulphate of GOx production
Legends followed by the same letter are not significantly different (P<0.05) for the same concentration. Values represent the mean of triplicates with standard deviation.
135
5.2.1.4 Effect of calcium carbonate on GOx production
The effect of calcium carbonate on the production of GOx is representing
in figure 5.10. Remarkably, GOx production was increased by the
supplementation of 30–40 g/l calcium carbonate. The level of 3.5% CaCO3 has
been reported to be essential for the induction of GOx synthesis in the production
medium by Sandip et al. (2008). Hatzinikolaou and Macris (1995) was also
reported that CaCO3 as a strong inducer for the production of GOx that
accompanied by a metabolic shift from glycolysis to the pentose phosphate
pathway. Liu et al. (1999) and Rogalski et al. (1988) were suggested that the GOx
synthesis was increased by CaCO3, due to a high calcium ion concentration or an
insoluble salt.
Fructose-6-phosphate, which is derived from glucose-6-phosphate
(catalyzed by glucose-6-phosphate isomerase) may enter the Embden–Meyerhof–
Parnas (EMP) or HMP (hexose mono phosphate) pathway. The addition of CaCO3
in to the growth medium made an alteration in the production of GOx by 6-
phosphofructokinase and glucose-6-phosphate dehydrogenase. The 6-
phosphofructokinase is a key regulatory enzyme in the EMP pathway in most
living cells. Cells grown in media without CaCO3 produced high levels of 6-
phosphofructokinase and low amounts of glucose-6-phosphate dehydrogenase and
GOx. The Addition of CaCO3 in to the growth medium was enhanced the
production of GOx and decreased the synthesis of 6-phosphofructokinase. The
induction of CaCO3 was accompanied by a metabolic shift from the glycolytic
pathway (EMP) that induced direct oxidation of glucose by GOx (Liu et al.,
2001).
5.2.1.5 Effect of pH on GOx production
Maximum GOx production was observed at pH between 5 and 6 (Figure
5.11). GOx production was minimal below the pH of 4.0, which could be due to
acidophilic characteristics of the fungi. The pH of the culture medium plays a vital
136
0 1 2 3 4 5 6
10
20
30
40
50
60C
onc.
of c
alci
um c
arbo
nate
(g/l)
GOx activity (g/l)
b
a
c
b
a
d
0
1
2
3
4
5
6
7
3 4 5 6 7 8pH
GO
x ac
tivity
(U/m
l)
0
2
4
6
8
10
12
14
16
Tota
l dry
mas
s (g
/l)
GOx activity TDMa
c
b
c
b
a
C
B
AA
B
C
Figure 5.10: Effect calcium carbonate on glucose oxidase production
Figure 5.11: Effect of pH on GOx production
Legends followed by the same letter are not significantly different (P<0.05). Values represent the mean of triplicates with standard deviation.
137
role for fungal growth and GOx production. The optimum pH range is necessary
for the production of GOx. Rogalski et al. (1988) concluded that calcium
carbonate appeared to be important in preventing the acidification of the broth
culture during cultivation. Petruccioli et al. (1995) stated that the addition of
calcium carbonate to the growth medium in shake flasks and fermenters prevented
pH drop during cultivation.
5.2.1.6 Effect of temperature on GOx production
The optimum temperature was found to be from 30 to 35ºC for the
production of GOx. The increase in temperature affects the GOx production as
well as biomass in the basal medium (Figure 5.12). The optimum temperature for
GOx production was observed at 27.5ºC (Hatzinikolaou and Macris, 1995). Other
researchers reported that the slight increase in temperatures (up to 35°C) enhances
the GOx production (Rogalski et al., 1988; Markwell et al., 1989; Traege et al.,
1991; Caridis et al., 1991).
5.2.1.7 Effect of fermentation time on GOx production
The optimum fermentation time for the GOx production was found to be 84
h and the GOx activity exhibited different patterns in the course of fermentation
(Figure 5.13). Hatzinikolaou and Macris (1995) reported that the optimum
cultivation time for GOx production was 70 h, while Sandip et al. (2008) found at
96 h. In this present investigation after 84 h, the enzyme activity was decreased
drastically and the total dry mass remained stationary.
138
0
1
2
3
4
5
6
7
20 25 30 35 40 45
Temperature (C)
GO
x ac
tivity
(U/m
l)
0
2
4
6
8
10
12
14
Tota
l dry
mas
s (g
/l)
GOx activity Total dry mass
A
C
AB
C
B
A
d
b
a
a
ab
c
0
1
2
3
4
5
6
7
0 12 24 36 48 60 72 84 96 108 120 132 144
Incubation period (h)
GO
x ac
tivity
(U/m
l)
0
2
4
6
8
10
12
14
16
18
20
Tota
l dry
mas
s (g
/l)
GOx activity Total dry mass
d
AAA
BBC
C
D
E
F
H
d
bc
ba
aaa
a
b
c
e
AA
Figure 5.12: Effect of temperature on GOx production
Figure 5.13: Effect of fermentation time on GOx production and cell growth
Legends followed by the same letter are not significantly different (P<0.05). Values represent the mean of triplicates with standard deviation.
139
5.2.2 Response surface methodology for GOx production
5.2.2.1 SET 1: Optimization of glucose, proteose peptone and calcium
carbonate for GOx production
The central composite design (CCD) was employed for the optimization of
glucose, proteose peptone and calcium carbonate on the GOx production and the
results are presented in Table 5.2.
The F-value of 14.30 represent as significant model. There is a chance of
0.01% variation in "model-F value" due to noice (Table 5.2.1). The values of
"Prob > F" and less than 0.05 indicate that model terms are significant. In this
case A, A2, B2 and C2 are significant model terms. Values greater than 0.1
indicate that the model terms are not significant.
The "Lack of Fit F-value" of 14.11 exhibit as significant and there is only
slight chance of 0.57% variation in "Lack of Fit F-value" due to noise. The lack
of fit value is not significant, so the model has to be change to fit as significant.
The "Pred R-Squared" of 0.4706 is not as close to the "Adj R-Squared" of
0.8630 (Table 5.2.2). "Adeq Precision" measures the signal to noise ratio and a
ratio greater than 4 is desirable. The ratio of 11.322 indicates an adequate signal.
This model can be used to navigate the design space. The model coefficient was
estimated by linear regression (Table 5.2.3).
Final equation in terms of coded factors
GOx = 9.346642634+1.238727515 × A +0.065968246 ×B +0.281339953 ×C
+0.0875×A ×B +0.1475 ×A ×C+0.0125 ×B ×C-1.668147103 ×A^2-1.275702839
×B^2-1.189082258 ×C^2
140
Table 5.2: Set 1 Optimization of glucose, proteose peptone and calcium carbonate for the GOx production by CCD of response surface methodology (23 factorial design)
RunA:
Glucose(g/l)
B:Proteosepeptone
(g/l)
C:CaCO3
(g/l)
ResponseGOx activity
(U/ml)
1 90 -1.045 35 5.52
2 40 1 10 4.363 90 4 35 9.56
4 40 7 10 5.16
5 5.91 4 35 1.23
6 140 1 60 5.947 90 4 35 9.75
8 40 1 60 4.72
9 174 4 35 7.21
10 90 4 77.04 6.6411 90 4 35 9.3
12 90 4 35 8.93
13 90 9.045 35 5.14
14 140 7 60 7.1415 140 1 10 6.22
16 90 4 35 9.56
17 90 4 35 9.12
18 140 7 10 6.1419 40 7 60 4.34
20 90 4 -7.044 4.51
141
Table 5.2.1: F-test analysis ( ANOVA for Response Surface Quadratic Model)
SourceSum ofSquares df
MeanSquare
FValue
p-valueProb > F
Model 92.876 9 10.319 14.298 0.0001 A-Glucose 20.955 1 20.955 29.034 0.0003 B-Peptone 0.059 1 0.059 0.082 0.7800 C- CaCO3 1.080 1 1.080 1.497 0.2491 AB 0.061 1 0.061 0.084 0.7768 AC 0.174 1 0.174 0.241 0.6340 BC 0.001 1 0.001 0.002 0.9676 A^2 40.102 1 40.102 55.563 < 0.0001 B^2 23.453 1 23.453 32.495 0.0002 C^2 20.376 1 20.376 28.232 0.0003Residual 7.217 10 0.721Lack of Fit 6.739 5 1.347 14.111 0.0057Pure Error 0.477 5 0.095Cor Total 100.091 19
Table 5.2.2: Comparition of R2 predicted and estimated
Std. Dev. 0.849 R-Squared 0.927
Mean 6.524 Adj R-Squared 0.862
C.V. % 13.021 Pred R-Squared 0.470
PRESS 52.994 Adeq Precision 11.322
Table 5.2.3: Model coefficient estimated by linear regression
FactorCoefficientEstimate df
StandardError
95% CILow
95% CIHigh VIF
Intercept 9.346 1 0.346 8.574 10.118A-Glucose 1.238 1 0.229 0.726 1.750 1B-Peptone 0.065 1 0.229 -0.446 0.578 1C- CaCO3 0.281 1 0.229 -0.230 0.793 1AB 0.087 1 0.301 -0.581 0.756 1AC 0.147 1 0.301 -0.521 0.816 1BC 0.012 1 0.300 -0.656 0.681 1A^2 -1.668 1 0.223 -2.16 -1.169 1.018B^2 -1.275 1 0.223 -1.774 -0.777 1.018C^2 -1.189 1 0.223 -1.687 -0.690 1.018
142
Final equation in terms of actual factors
GOx = -2.763181152+0.138417808×Glucose +1.097614161 ×Peptone
+0.133144144 ×CaCO3+0.000583333 ×Glucose ×Peptone 0.000118 ×Glucose
×CaCO3+0.000166667 ×Peptone ×CaCO3-0.000667259 ×Glucose^2-0.14174476
×Peptone^2-0.001902532 ×CaCO3^2
The contour and three-dimensional response surface curves were plotted
and expressed in Figure 5.14-5.16. Maximum GOx production (9.32 U/ml) was
achieved at 92.7 g/l of glucose, 3.24 g/l of proteose peptone and 36.82 g/l of
calcium carbonate. The higher amount of proteose peptone was enhanced the
biomass, but reduced the GOx production. This result suggested that it may reduce
the fermentation cost.
5.2.2.2 SET 2: Optimization of di-ammonium hydrogen phosphate, potassium
di-hydrogen phosphate and magnesium sulphate for GOx
production
The results of central composite design experiments for studying the effects
of second set of three independent variables such as (NH4)2HPO4, KH2PO4 and
MgSO4 on GOx production are presented in Table 5.3.
The F-value of 2.66 represent as significant model. There is a chance of
7.19% variation in "model-F value" due to noice (Table 5.3.1). The values of
"Prob > F" and less than 0.05 indicate that model terms are significant. In this
case A2, B2 and C2 are significant model terms. Values greater than 0.1 indicate
that the model terms are not significant.
The "Lack of Fit F-value" of 396.04 exhibit as significant and there is only
slight chance of 0.01% variation in "Lack of Fit F-value" due to noise.
143
Design-Expert® Software
Glucose oxidaseDesign Points9.75
1.23
X1 = A: GlucoseX2 = C: CaCo3
Actual FactorB: Peptone = 4.00
40.00 65.00 90.00 115.00 140.00
10.00
22.50
35.00
47.50
60.00Glucose oxidase
A: Glucose
C: C
aCo3
5.86409
6.61134
7.35859
8.10583
8.85308
6
Design-Expert® Software
Glucose oxidaseDesign points above predicted valueDesign points below predicted value9.75
1.23
X1 = A: GlucoseX2 = C: CaCo3
Actual FactorB: Peptone = 4.00
40.00
65.00
90.00
115.00
140.00 10.00
22.50
35.00
47.50
60.00
5.1
6.275
7.45
8.625
9.8
Glu
cose
oxi
dase
A: Glucose C: CaCo3
Figure 5.14: The contour and 3D response surface plot showing the effect of glucose and calcium carbonate on GOx production
144
Design-Expert® Software
Glucose oxidaseDesign points above predicted valueDesign points below predicted value9.75
1.23
X1 = A: GlucoseX2 = B: Peptone
Actual FactorC: CaCo3 = 35.00
40.00
65.00
90.00
115.00
140.00 1.00
2.50
4.00
5.50
7.00
5.1
6.275
7.45
8.625
9.8
Glu
cose
oxi
dase
A: Glucose B: Peptone
Design-Expert® Software
Glucose oxidaseDesign Points9.75
1.23
X1 = A: GlucoseX2 = B: Peptone
Actual FactorC: CaCo3 = 35.00
40.00 65.00 90.00 115.00 140.00
1.00
2.50
4.00
5.50
7.00Glucose oxidase
A: Glucose
B: P
epto
ne
5.88183
5.88183
6.62113
7.360438.09973
8.09973
8.839036
Figure 5.15: The contour and 3D response surface plot showing the effect of glucose and peptone on GOx production
145
Design-Expert® Software
Glucose oxidaseDesign Points9.75
1.23
X1 = B: PeptoneX2 = C: CaCo3
Actual FactorA: Glucose = 90.00
1.00 2.50 4.00 5.50 7.00
10.00
22.50
35.00
47.50
60.00Glucose oxidase
B: Peptone
C: C
aCo3
7.01656 7.01656
7.48607 7.48607
7.95559
7.955597.95559
8.4251
8.89461
6
Design-Expert® Software
Glucose oxidaseDesign points above predicted valueDesign points below predicted value9.75
1.23
X1 = B: PeptoneX2 = C: CaCo3
Actual FactorA: Glucose = 90.00
1.00
2.50
4.00
5.50
7.00
10.00
22.50
35.00
47.50
60.00
6.5
7.325
8.15
8.975
9.8
Glu
cose
oxi
dase
B: Peptone C: CaCo3
Figure 5.16: The contour and 3D response surface plot showing the effect of peptone and calcium carbonate on GOx production
146
"Adeq Precision" measures the signal to noise ratio and a ratio greater than
4 is desirable. The ratio of 5.013 indicates an adequate signal (Table 5.3.2). This
model can be used to navigate the design space. The model coefficient was
estimated by linear regression (Table 5.3.3).
Final equation in terms of coded factors
GOx = 9.68+0.32 ×A+0.025 ×B-0.24 ×C-0.025×A ×B 0.25 ×A ×C -0.60 ×B ×C-
0.62 ×A2-0.69 ×B
2-0.62 ×C2
Final equation in terms of actual factors
GOx =+6.09373+4.68728 ×(NH4)2HPO4+8.83236 ×H2PO4+7.42811
×MgSO4-0.25974 ×(NH4)2HPO4 ×KH2PO4+3.17460 ×(NH4)2HPO4 ×MgSO4-
9.69697 ×KH2PO4×MgSO4-5.08266 ×((NH4)2HPO4)2-9.16808 ×KH2PO4-
12.29877 ×(MgSO4)2
The contour and three-dimensional response surface curves were plotted
and represented in Figure 5.17-5.19. Maximum GOx production (9.71 U/ml) was
achieved at (NH4)2HPO4−0.48 g/l, KH2PO4–0.32 g/l and MgSO4–0.23 g/l.
Higher amount of MgSO4 significantly affected the GOx production followed by
KH2PO4.
147
Table 5.3: Set 2 Optimization of (NH4)2HPO4, KH2PO4 and MgSO4 for the GOx production by CCD of response surface methodology (23
factorial design)
RunA:
(NH4)2HPO4(g/l)
B:KH2PO4
(g/l)
C:MgSO4
(g/l)
ResponseGOx activity
(U/ml)
1 0.45 0.787 0.275 8.4
2 0.45 0.325 0.275 9.65
3 0.45 0.325 0.275 9.54
4 0.1 0.05 0.5 6.9
5 0.45 0.325 -0.103 8.1
6 0.1 0.05 0.05 7.8
7 0.1 0.6 0.5 6.2
8 0.45 0.325 0.275 9.72
9 1.038 0.325 0.275 9.9
10 0.45 0.325 0.275 9.6
11 0.8 0.05 0.5 7.8
12 0.1 0.6 0.05 8.6
13 0.8 0.6 0.5 6.1
14 0.8 0.6 0.05 8.4
15 0.45 0.325 0.275 9.7
16 0.45 -0.137 0.275 8.2
17 -0.138 0.325 0.275 7.118 0.8 0.05 0.05 6.8
19 0.45 0.325 0.653 8.9
20 0.45 0.325 0.275 9.65
148
Table 5.3.1: F-test analysis ( ANOVA for Response Surface Quadratic Model)
Source Sum ofSquares df Mean
SquareF
Valuep-value
Prob > FModel 20.64 9 2.293 2.658 0.0719A-(NH4)2HPO4 0.008 1 0.008 0.009 0.9239B- KH2PO4 0.775 1 0.775 0.898 0.3654C- MgSO4 0.005 1 0.005 0.005 0.9408AB 0.5 1 0.5 0.579 0.4641AC 2.88 1 2.88 3.337 0.0977BC 5.586 1 5.586 6.474 0.0291 A^2 6.927 1 6.927 8.023 0.0177 B^2 5.586 1 5.586 6.474 0.0291 C^2 8.629 10 0.862Residual 8.607 5 1.724 396.04 < 0.0001Lack of Fit 0.021 5 0.004Pure Error 29.272 19
Table 5.3.2: Comparition of R2 predicted and estimated
Std. Dev. 0.928926 R-Squared 0.70522Mean 8.353 Adj R-Squared 0.439918C.V. % 11.12087 Pred R-Squared -1.25073PRESS 65.88533 Adeq Precision 5.012998
Table 5.3.3: Model coefficient estimated by linear regression
Factor CoefficientEstimate df Standard
Error95% CI
Low95% CI
High VIFIntercept 9.67675 1 0.378861 8.832595 10.52091A-(NH4)2HPO4
0.315 1 0.251 -0.244 0.875 1
B- KH2PO4 0.024 1 0.251 -0.535 0.584 1C- MgSO4 -0.238 1 0.251 -0.798 0.321 1AB -0.025 1 0.328 -0.756 0.706 1AC 0.25 1 0.328 -0.481 0.981 1BC -0.6 1 0.328 -1.331 0.131 1A^2 -0.622 1 0.244 -1.167 -0.077 1.018B^2 -0.693 1 0.244 -1.238 -0.148 1.018C^2 -0.622 1 0.244 -1.167 -0.077 1.018
149
Design-Expert® Software
Glucose oxidaseDesign points above predicted valueDesign points below predicted value9.9
6.1
X1 = A: (NH4)HPO4X2 = B: KH2PO4
Actual FactorC: MgSO4 = 0.28
0.10
0.28
0.45
0.63
0.80
0.05
0.19
0.33
0.46
0.60
7.9
8.375
8.85
9.325
9.8
Glu
cose
oxi
dase
A: (NH4)HPO4 B: KH2PO4
Figure 5.17: The contour and 3D response surface plot showing the effect of (NH4)2HPO4 and KH2PO4 on GOx production
Design-Expert® Software
Glucose oxidaseDesign Points9.9
6.1
X1 = A: (NH4)HPO4X2 = B: KH2PO4
Actual FactorC: MgSO4 = 0.28
0.10 0.28 0.45 0.63 0.80
0.05
0.19
0.33
0.46
0.60Glucose oxidase
A: (NH4)HPO4
B: K
H2P
O4
8.28249
8.56934
8.56934
8.85619
8.85619
9.14304
9.42989
6
150
Design-Expert® Software
Glucose oxidaseDesign Points9.9
6.1
X1 = A: (NH4)HPO4X2 = C: MgSO4
Actual FactorB: KH2PO4 = 0.33
0.10 0.28 0.45 0.63 0.80
0.05
0.16
0.28
0.39
0.50Glucose oxidase
A: (NH4)HPO4
C: M
gSO
4
7.97798
8.32829
8.6786
9.0289
9.02899.37921
6
Design-Expert® Software
Glucose oxidaseDesign points above predicted valueDesign points below predicted value9.9
6.1
X1 = A: (NH4)HPO4X2 = C: MgSO4
Actual FactorB: KH2PO4 = 0.33
0.10
0.28
0.45
0.63
0.80
0.05
0.16
0.28
0.39
0.50
7.6
8.15
8.7
9.25
9.8
Glu
cose
oxi
dase
A: (NH4)HPO4 C: MgSO4
Figure 5.18: The contour and 3D response surface plot showing the effect of (NH4)2HPO4, and MgSO4 on GOx production
151
Design-Expert® Software
Glucose oxidaseDesign Points9.9
6.1
X1 = B: KH2PO4X2 = C: MgSO4
Actual FactorA: (NH4)HPO4 = 0.45
0.05 0.19 0.33 0.46 0.60
0.05
0.16
0.28
0.39
0.50Glucose oxidase
B: KH2PO4
C: M
gSO
4
7.90732
8.26754
8.26754
8.62776
8.62776
8.98798
8.98798
9.34819
6
Design-Expert® Software
Glucose oxidaseDesign points above predicted valueDesign points below predicted value9.9
6.1
X1 = B: KH2PO4X2 = C: MgSO4
Actual FactorA: (NH4)HPO4 = 0.45
0.05
0.19
0.33
0.46
0.60
0.05
0.16
0.28
0.39
0.50
7.5
8.075
8.65
9.225
9.8
Glu
cose
oxi
dase
B: KH2PO4 C: MgSO4
Figure 5.19: The contour and 3D response surface plot showing the effect of KH2PO4 and MgSO4 on GOx production
152
5.2.2.3 SET 3: Optimization of pH and temperature
The results of central composite design experiments for studying the effects
of pH and temperature on GOx production are presented in Table 5.4. The F-value
of 347.29 showed the model is significant. There is a onlychance of 0.01%
variation in "model-F value" due to noice (Table 5.4.1). The values of "Prob > F"
and less than 0.05 indicate that model terms are significant. In this case A2 and
B2 are significant model terms. Values greater than 0.1 indicate that the model
terms are not significant.
The "Lack of Fit F-value" of 22.76 exhibit as significant and there is only
slight chance of 0.57% variation in "Lack of Fit F-value" due to noise. The lack
of fit value is not significant, so the model has to be change to fit as significant.
The "Pred R-Squared" of 0.972 is in reasonable agreement with the "Adj R-
Squared" of 0.993. "Adeq Precision" measures the signal to noise ratio and a ratio
greater than 4 is desirable. The ratio of 37.456 indicates an adequate signal (Table
5.4.2). This model can be used to navigate the design space. The model
coefficient was estimated by linear regression (Table 5.4.3).
Final equation in terms of coded factors
GOx = 10.10-0.13 A-0.24 ×B-0.075 ×A×B-4.92×A2-4.40 ×B2
Final equation in terms of actual factors
GOx = -33.07707+6.57270 ×pH+1.36371 ×Temperature-1.66667E-003 ×pH ×
Temperature-0.54639 ×pH2 -0.019567 ×Temperature2
The contour and three-dimensional response surface curves were plotted
and presented in the Figure 5.20. Maximum GOx production (10.08 U/ml) was
achieved at the pH 5.83 and the temperature at 30.7°C.
153
Table 5.4: Set 3 Optimization of pH and temperature for the GOx production by CCD of response surface methodology (23 factorial design)
Run A:pH
B:Temperature (°C)
ResponseGOx activity
(U/ml)
1 3 50 0.72 9 50 03 1.79 35 0.544 6 35 10.15 6 13.7 2.16 6 56.2 1.17 6 35 9.98 6 35 10.29 6 35 10.110 10.24 35 0.611 3 20 0.812 6 35 10.213 9 20 0.4
154
Table 5.4.1: F-test analysis ( ANOVA for Response Surface Quadratic
Model)
Source Sum ofSquares df Mean
SquareF
Valuep-value
Prob > FModel 268.939 5 53.787 347.287 < 0.0001 A-pH 0.128 1 0.128 0.831 0.392 B-Temperature 0.458 1 0.458 2.957 0.129
AB 0.022 1 0.022 0.145 0.714 A^2 168.221 1 168.221 1086.142 < 0.0001 B^2 134.833 1 134.831 870.555 < 0.0001Residual 1.0841 7 0.154Lack of Fit 1.0241 3 0.341 22.759 0.005Pure Error 0.06 4 0.015Cor Total 270.023 12
Table Table 5.4.2: Comparition of R2 predicted and estimated
Std. Dev. 0.393 R-Squared 0.995
Mean 4.364 Adj R-Squared 0.993
C.V. % 9.016 Pred R-Squared 0.972
PRESS 7.376 Adeq Precision 37.456
Table Table 5.4.3: Model coefficient estimated by linear regression
Factor CoefficientEstimate df Standard
Error95% CI
Low95% CI
High VIF
Intercept 10.1 1 0.176 9.683 10.516
A-pH -0.126 1 0.139 -0.455 0.202 1B-Temperature -0.239 1 0.139 -0.568 0.089 1
AB -0.075 1 0.196 -0.540 0.390 1
A^2 -4.917 1 0.149 -5.270 -4.564 1.017
B^2 -4.402 1 0.149 -4.755 -4.049 1.017
155
Design-Expert® Software
Glucose oxidaseDesign points above predicted valueDesign points below predicted value10.2
0
X1 = A: pHX2 = B: Temperature
3.00
4.50
6.00
7.50
9.00
20.00
27.50
35.00
42.50
50.00
0
2.75
5.5
8.25
11
Glu
cose
oxi
dase
A: pH B: Temperature
Design-Expert® Software
Glucose oxidaseDesign Points10.2
0
X1 = A: pHX2 = B: Temperature
3.00 4.50 6.00 7.50 9.00
20.00
27.50
35.00
42.50
50.00Glucose oxidase
A: pH
B: T
empe
ratu
re
3.59365 3.59365
3.593653.59365
5.22106
5.22106
5.22106
6.84846
8.47587
5
Figure 5.20: The contour and 3D response surface plot showing the effect of pH and temperature on GOx production
156
Response surface methodology was reduced the fermentation time (65-70
h) and significantly improved the GOx production, when compared to single
factor analysis. According to Sandip et al. (2008), the changes in the cultivation
time and increase in GOx production was probably due to change in the
concentrations of media constituents obtained by the statistical analysis central
composite design.
5.3 Production of glucose oxidase by laboratory batch fermentor
5.3.1 Spore aggregation and pellet formation during the early cultivation time
Aspergillus awamori MTCC 9645 was cultivated in the two litre of
laboratory batch fermentor (Figure 5.21) at constant pH 5.83. The dissolved
oxygen content and temperature were maintained during the fermentation as 11 to
12 mg/l and 30.7ºC respectively.
The number of spores at the beginning of cultivation time was 1.0×106/ml.
After 5 h, the numbers of free spores were decreased to 2.4X104/ml, when the
spore aggregates began to develop and reached 1.6X103/ml with an average
diameter of about 0.051±0.04 mm. Spore aggregates were varied in their shape
and the number of spores per aggregate could not be easily determined. After 10
h, the number of free spores and aggregates were continually decreased. After
spore germination, the aggregates number was reduce and subsequently the
average diameters of aggregates were increased to 0.18±0.06 mm. After 15 h of
cultivation, no free spores were observed in the cultivation medium followed by
the numbers of aggregates were decreased. The complete pellet structure was
formed after 20 h with a number of 46±0.3 pellets/ml and the average diameter of
1.4±0.3 mm (Table 5.5).
157
Culture conditions
Medium : Modified GOxM3pH : 5.83Temperature : 30.7°CDO : 11-12 mg/lAgitation : 200-500Duration : 84 hVolume : 1000 ml
Figure 5.21: Production of GOx in A. awamori MTCC 9645 in laboratory bioreactor
158
Table 5.5: Spore aggregation and pellet formation during the early
cultivation time in the laboratory batch fermentor
Cultivationtime (h)
No. of freespores/ml
No. ofaggregates/ml
AverageDiameters ofaggregates
(mm)
Total No.ofBioparticles/ml
0 1.0X106 - - 1.0X106
5 2.4X104 1.6X104 0.051±0.04 4.0X104
10 3.5X102 1.2X102 0.18±0.06 4.7X102
15 0.56X102 0.9X102 0.8±0.6 1.46X102
20 - 46.0 1.4±0.3 46±4.0
25 - 46.0 1.4±0.3 46±4.0
30 - 48.0 1.5±0.5 48±6.0
35 - 51.0 1.5±0.2 51±5.0
Values are mean of three replicates (±S.D)
159
5.3.2 Morphological studies
After the complete formation of compact pellet structure, both the pellet
number and diameter were constant at 46-51 pellets per ml and 1.5±0.2mm
respectively during the rest of cultivation time (Table 5.5). Further cell growth
was attributed in the inside of the pellets and also spores germinated in the pellet
core.
The mycelial stage of fungus may grow as dispersed hyphal fragments or
pellets. The growth of the pellet was exponential until the density of the pellet
causes diffusion limitation. In this limitation, the biomass was not increased
because of the insufficient nutrient supply. Further growth was started from the
outer shell of the pellet. New pellet was formed from the fragments of old pellets
(Sandip et al., 2008). The enzyme production rate was decreased after 86 h,
probably because of the depletion of nutrient in the medium (substrate limitation)
(Stanbury et al., 1997).
5.3.3 Time course study on cell growth, substrate utilization, GOx and
gluconic acid production during fermentation period
Based on the previous results, after a lag time of five hour, spores were
germinated giving rise to a short germ tube. The fungus apparently grew
exponentially up to 72 h and reached maximum concentration of 12.5±0.63 g/l cell
dry mass and maintained more or less constant for the rest of cultivation period. A
similar observation was observed by Hesham El-Enshasy, (2006). After this time,
the rate of increase in cell mass was less due to the depletion of glucose, since
glucose is converted to gluconic acid (a less suitable carbon source for cell
growth, Lakshminarayana et al., 1969) by the GOx. The utilization of glucose
started higher after 12 h and complete consumption of glucose was observed at
50–60 h of fermentation. Production of gluconic acid was started at 12 h and
maximum amount was observed at 48 h as 62.3±4.1 g/l. It was slowly decreased
160
in the rest of fermentation period due to the utilization of the fungus as carbon
source (Figure 5.22).
The extracellular GOx secretion was started about 10–12 h and then
reached maximum GOx activity at 72 h as 12±0.63 U/ml. The increase of the GOx
production was mainly through the increase in cell mass. Therefore, increasing
extracellular GOx titer with cultivation time is mainly due to excretion of the
enzyme rather than new production. The initial protein content in the fermentation
medium was high due to the presence of proteose peptone. It was decreased
drastically from 35.5±1.61 to 8.1±0.54 µg/ml at 36 h of fermentation. The level of
the protein content was increased after the secretion of protein by the fungus
(Figure 5.23).
The lower enzyme production and excretion in this culture were due to
morphological and physiological problems. It is known that the growth of fungal
cells in pellet form larger than 46 mm in diameter causes severe mass transfer
limitations between medium and pellet regarding substrate transport into the pellet
as well as product transport from the pellet into the medium (Schügerl et al.,
1983). Moreover, not all cells were biologically active and the biologically active
layer was restricted only in the outer layer of pellet. Also the conversion of
glucose (a good carbon source for both cell growth and enzyme induction) to
gluconic acid (a less suitable carbon source for cell growth and non-GOx inducer)
due to enzyme production resulted in termination of enzyme synthesis. This
problem was also observed in fed batch cultures feeded either with glucose or
yeast extract. The growth morphology was identical in form of dense pellet in all
cultures and the complete transformation of glucose to gluconic acid was observed
after a few hours of enzyme production.
161
0
20
40
60
80
100
120
0 12 24 36 48 60 72 84 96 108 120
Fermentation period (h)
Glu
cose
/Glu
coni
c ac
id (g
/l)
0
2
4
6
8
10
12
14
16
Cel
l dry
mas
s (g
/l)
Gucose Gluconic acid Cell dry mass
0
2
4
6
8
10
12
14
0 12 24 36 48 60 72 84 96 108 120Fermentation period (h)
GO
x ac
tivity
(U/m
l)
0
5
10
15
20
25
30
35
40
Prot
ein
cont
ent (
µg/m
l)
GOx Protein
Figure 5.22: Time course study on substrate utilization, production of gluconic acid and biomass during the fermentation
Figure 5.23: Time course study on production of GOx and protein during the fermentation
Values represent the mean of triplicates with standard deviation.
162
5.4 Purification and characterization of glucose oxidase produced from
A. awamori MTCC 9645
The total protein was taken from the cell free culture filtrate (600 ml) of 72
h old A. awamori MTCC 9645 culture. The protein was precipitated using
ammonium sulphate fractions from 40–85% (w/v). The 60–70% (w/v) of
ammonium sulphate fractions showed higher GOx activity than the rest of the
fractions. The activity was not observed from 40% (w/v) saturation.
The precipitated proteins were used for the purification of GOx. The
precipitate was dialyzed, concentrated by lyophilization and fractionated by
chromatographic techniques. The flow chart of GOx purification is presented in
Figure 5.24.
5.4.1 DEAE-Cellulose column chromatography
The concentrated protein was loaded in the DEAE-Cellulose column.
Eighty fractions of each 4 ml were collected. GOx was eluted from 26–38
fractions (Figure 5.25 a). The enzyme active fractions were pooled, concentrated
by lyophilization, dialyzed against sodium acetate buffer (10 mM, pH 5.5) and
used for the further purification.
5.4.2 Sephacryl S-200 column chromatography
The concentrated protein was loaded on Sephacryl S-200 column and
eluted with sodium acetate buffer (100 mM, pH 5.5). Eighty fractions of 3.0 ml
were collected and the active fractions were observed from 31 to 43 fractions. The
GOx active fractions were pooled dialyzed and freeze dried (Figure 5.25b).
The details of purifications of GOx are summarized in the Table 5.6. The
enzyme activity was purified up to 9.19 folds with a final recovery of 12.98%. The
specific activity of purified GOx was 282.27 U/mg proteins. There were slight
163
Cell free culture filtrate
Salting out with ammonium sulphate up to 70% (w/v) saturation
Centrifuged at 10,000 g for 15 minutes at 4°C
Dialyzed against sodium acetate buffer (10 mM, pH 5.5)
Lyophilized and dialyzed proteins
Loaded the proteins on DEAE-cellulose column chromatography
A. awamori MTCC 9645 grown in optimized medium for 72 h
Harvested the culture by filtration and centrifugation at 10,000 g for 10 minutes
Collected GOx active fractions, dialyzed (10 mM, pH 5.5) and lyophilized
Loaded the proteins on Sephacryl S-200 column chromatography
Collected GOx active fractions, dialyzed and lyophilized
SDS-PAGE,
Native-PAGE
Zymogram
Characterization
Enzyme kinetics
pH optimum and stability
Temperature optimum and stability
Effect of metal ions
Figure 5.24: Flow chart for the purification and characterization of extracellular GOxof A. awamori MTCC 9645
164
0
5
10
15
20
25
30
35
40
45
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76Fraction
GO
x ac
tivity
(U/m
L)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Abs
orba
nce
at 2
80 n
m
Enzyme Protein- 400
- 50
- 200
-100
- 300
- 150
- 250
- 350
Nac
l (m
M)
Nacl
0
5
10
15
20
25
30
35
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76
Fraction
GO
x ac
tivity
(U/m
L)
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
Abs
orba
nce
at 2
80 n
m
Enzyme Protein
Figure 5.25 (a): Elution profile on DEAE column chromatography
Figure 5.25 (b): Elution profile on Sephacryl S-200 column chromatography Figure 5.25: Purification of GOx from A. awamori MTCC 9645
165
Table 5.6: Summary of purification of GOx from A. awamori MTCC 9645
SampleDescription
TotalProtein
(mg)
Totalactivity(Units)
Specificactivity
(Units/ mgprotein)
Yield(%)
Foldpurification
Initial sample 314.5 9660.0 30.7 100 1
Calcium removal 292.2 9120.0 31.2 94.4 1.01Ammoniumsulphateprecipitation
42.2 5841.0 138.41 60.4 4.50
Dialysis 26.4 3847.0 145.71 39.8 4.74Anion exchangechromatograpy 9.51 2794.0 293.79 28.9 9.56
Dialysis 6.75 2264.0 335.4 23.43 10.92
Lyophilization 5.84 1816.0 310.9 18.8 10.1Size-exclusionchromatograpy 4.91 1669.0 339.9 17.2 11.07
Lyophilization 4.41 1254.0 282.27 12.98 9.19
166
decreases in specific activity during the freeze drying steps since no stabilizers
were added which could potentially interfere with subsequent purification steps.
5.4.3 Molecular mass determination of purified GOx by SDS-PAGE
The purity of GOx was analyzed by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) with 10% of gel. On SDS-PAGE, the purified
GOx showed a single band indicating electrophoretically homogenous (Figure
5.26). The molecular weight of 71.1 kDa by comparing with relative mobility of
the molecular weight of standard protein marker. Simpson et al., (2006) reports
that GOx had a molecular weight of 70 kDa, which was similar to the present
investigation.
5.4.4 Purified GOx activity on native-PAGE
The activity of purified GOx was determined on native-PAGE. It was
developed by overlaying 1.5% of soft agar containing glucose, o-dianisidine and
Horse radish peroxidase. Brown color band was formed in the native gel. Single
zymogram analysis is shown in Figure 5.27a.
5.4.5 Confirmation of enzyme activity using plate assay
Dark brown colour was formed immediately in purified GOx and the
authentic GOx. It was confirmed the activity of the GOx in the final product
(Figure 5.27b). The purified GOx was further subjected to kinetic characterization.
167
kDa
205
97.4
66
43
29
KDa
71.5
L1 L2 L3
L1-Marker
L2-Ion exchange chromatography
L3-Purified GOx (Size exclusion)
Figure 5.26: Molecular mass determination on SDS-PAGE (10%) of
GOx from A. awamori MTCC 9645
168
2
1
3
L1 L2
L1-Purified GOx
L2-Authentic GOx
Figure 5.27 (a): Zymogram of GOx (Iso enzyme patterning) on native-PAGE
(8%) developed with horse radishproxidase, o-dianisidine
and glucose
1. Control (Buffer)
2. Purified GOx
3. Authentic GOx
Figure 5.27(b): Confirmation of GOx from A. awamori MTCC 9645 by
plate assay
169
5.4.6 Kinetic characterization of GOx
The Lineweaver’s Burk plot, Eadie-Hofstee plot and Hans-Woolf linear
plots were used to determine the Vmax and Km values of the GOx from A. awamori
MTCC 1945. The initial velocity was determined by the o-dianisidine-horseradish
peroxidase assay described in materials and method. The enzyme was kinetically
characterized and displayed characteristics with a Vmax of 5.77 (U/20µg) and Km
of 119.44 (mM ) in Lineweaver’s Burk plot, Vmax of 5.73 (U/20µg) and Km of
115.65 (mM ) in Eadie-Hofstee plot, Vmax of 5.71 (U/20µg) and Km of 114.05
(mM ) in Hans-Woolf linear and presented in figure 5.28–5.30.
5.4.7 Effect of temperature and pH on GOx activity
The optimum temperature of GOx was found at 30±2ºC and about 62.3%
of relative activity was observed at the temperature between 20 and 50ºC. More
than 50ºC decreased the GOx activity rapidly. Gouda et al. (2003) reported that
the dissociation of FDA from free holoenzyme in aqueous medium was at 59ºC
and concluded that dissociation of FDA from the holoenzyme was responsible for
the thermal inactivation of GOx. Gul Ozyilmaz et al. (2005) also observed that,
temperature affected the activity of the GOx sharply and maximum activity at
35ºC. At 60ºC the relative activity of GOx were found as 33%. The optimum pH
was observed at 5.5 and showed more than 70% of the maximum relative activity
between pH 4 and 7 (Figure 5.31). A similar observation was reported by Gul
Ozyilmaz et al. (2005) as the optimum pH of 5.5.
5.4.8 Stability testing
The stability of the purified GOx was tested at 25 and 37ºC and the relative
activity not affected over 12 h at 25ºC, while showed a half life of 40 minutes at
37ºC (Figure 5.32). It indicates that the GOx would not be effective at 37ºC
without prior stabilization. Combes and Monsan (1988) reported that GOx
170
Figure 5.28: Kinetic parameters (apparent Km and Vmax) for purified GOx ofA. awamori MTCC 9645 by Lineweaver's Burk plot
The initial velocity was determined by the o-dianisidine-horseradish peroxidase assay described in materials and method
y = 20.7x + 0.1733R2 = 0.983
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
-0.015 -0.01 -0.005 0 0.005 0.01 0.0151/[S0]
1/[V
0]
Slope=km/Vmax=20.7Intercept=1/Vmax=0.1733km=20.7/0.1733=119.44Vmax=1/0.1733=5.77
171
y = -115.64x + 5.7329R2 = 0.9435
-5
-4
-3
-2
-1
0
1
2
3
4
5
6
7
8
9
10
-0.1 -0.075 -0.05 -0.025 0 0.025 0.05 0.075 0.1 0.125 0.15
[V0]/[S0]
[V0]
Slope=-km=-115.65km=115.65Intercept=Vmax=5.7317
Figure 5.29: Kinetic parameters (apparent Km and Vmax) for purified GOx ofA. awamori MTCC 9645 by Eadie-Hofstee plot
The initial velocity was determined by the o-dianisidine-horseradish peroxidase assay described in materials and method
172
Figure 5.30: Kinetic parameters (apparent Km and Vmax) for purified GOx ofA. awamori MTCC 9645 by Hanes plot
The initial velocity was determined by the o-dianisidine-horseradish peroxidase assay described in materials and method
y = 0.175x + 19.96R2 = 0.995
-100
-50
0
50
100
150
200
250
-300 -200 -100 0 100 200 300 400 500 600 700 800 900 1000 1100
[S0]
[S0]
/[V0]
Slope=1/Vmax=0.175Vmax=5.714Intercept=km/Vmax=19.96km=19.96X5.714=114.05
173
0
20
40
60
80
100
120
20 30 40 50 60 70 80
Temperature (C)
Rel
ativ
e G
Ox
activ
ity (%
)3 4 5 6 7 8 9
pH
pH profile Temperature profile
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70 80 90Incubation at 37 ºC (minutes)
Res
idua
l GO
x ac
tivity
(%)
6 12 18 24 30 36 42 48 54 60Incubation at 25 ºC (h)
37 ºC 25 ºC
Figure 5.31: Effect of temperature and pH on GOx activity
Figure 5.32: Effect of temperature stability of purified GOx
174
required effective prior stabilization like immobilization, polyhydric alcohols
(polyethylene glycol). The lyophilized freeze dried GOx powder stored at -20ºC
remained same without loss of activity for 6 months. A similar observation also
reported by Simpson et al. (2006).
5.4.9 Stability and inhibitory activity of GOx
5.4.9.1 Effect of metal ions on GOx activity
The mercuric chloride, copper sulphate, cobaltous chloride and silver
nitrate remarkably inhibited the GOx activity even at 1mM concentration. The
detailed results expressed in figure 5.33. Satoshi Nakamura and Yasuyuki Ogura,
(1986) reported that the reaction catalyzed by the GOx from A niger was markedly
inhibited by Ag+, Hg++ and Cu++. All the metal ions shows various degrees of
inhibition; the inhibition sequence was in the order: copper acetate > silver
sulphate > cobaltous chloride > mercuric chloride > copper sulphate at same
concentration.
The reason for the activation and inhibitions is that lower concentrations of
metal ions can stabilize the conformation of GOx and cause change in
conformation to a more active form, so GOx is activated by metal ions. GOx
having interactions of the FAD molecule at the active site of GOx are 23 potential
hydrogen bonds mostly involving the ribose and pyrophosphate groups. At higher
metal ion concentration, the great number of ions will compete with FAD for the
binding sites on the pyrophosphate or ribose groups, which causes the interactions
of the hydrogen bonds between the FAD molecule and pyrophosphate or ribose
groups to become weakened. They will also compete with the substrate for the
binding sites on the enzyme, so GOx activity was partially inhibited. Divalent
metal ions bind to pyrophosphate and ribose more strongly than monovalent ions,
so the inhibition of divalent ions is stronger than that of monovalent ions.
175
5.4.9.2 Effect of calcium ions on GOx activity (1mM)
Effect of calcium ions on GOx activity was analyzed and it was found that
relative activity of calcium lactate was more i.e., 119.5% when compared to
control. Calcium lactate, calcium carbonate, calcium propionate shows a broad
range of activation. The activation sequence is calcium lactate > calcium
carbonate > calcium propionate (Figure 5.34).
5.4.9.3 Effect of calcium ions on pH stability of GOx
Figure 5.35 shows the effect of calcium ions on pH stability of GOx.
Maximum GOx activity was achieved using calcium lactate and calcium
propionate at pH level 5–6. All these values were compared with control in which
maximum activity was obtained at pH 5–6 while, it was shifted to 7.0 for the
calcium lactate followed by calcium propionate treated GOx. Activity of the
calcium lactate treated GOx was less sensitive to pH changes at acidic and
alkaline pH as compared with that of untreated control enzyme.
5.4.9.4 Effect of calcium ions on temperature stability of GOx (1mM)
The effect of calcium ions on temperature stability of GOx was found to
be effective and it was observed a maximum stability at 50°C in calcium lactate
treated GOx as 86.67% but in control showed only 43.3% of relative activity
(Figure 5.36). It was observed that calcium ions provide temperature stability of
GOx and less sensitive at lower and higher temperature when compared with
control.
176
0
20
40
60
80
100
120
Leadacetate
Cobaltouschloride
Copperacetate
Coppersulphate
Silversulphate
Silvernitrate
Mercuricchloride
Zincchloride
Nickelchloride
Control
Rel
ativ
e ac
tivity
(%)
0
20
40
60
80
100
120
140
Calcium lactate Calcium chloride Calcium propionate Calcium carbonate Control
Rel
ativ
e ac
tivity
(%)
Figure 5.33: Effect of metal ions (1mM) on GOx activity
Figure 5.34: Effect of calcium ions (1mM) on GOx activity
177
0
20
40
60
80
100
120
140
3 4 5 6 7 8 9pH
Rel
ativ
e ac
tivity
(%)
ControlCalcium chlorideCalcium lactateCalcium Propionate
0
20
40
60
80
100
120
140
20 30 40 50 60 70
Temperature (C)
Rel
ativ
e ac
tivity
(%)
Control
Calcium chloride
Calcium lactate
Calcium Propionate
Figure 5.35: Effect of calcium ions (1mM) on pH stability of GOx activity
Figure 5.36: Effect of calcium ions (1mM) on temperature stability of GOx
178
5.5 Application of GOx in food processing and preservation5.5.1 Edible films incorporated with glucose oxidase, lactoperoxidase
and lysozyme for carrot preservation
5.5.1.1 Assay of antimicrobial enzymes
The formulated GOx activity was found to be 5.20 U/mg. This enzyme
works in a pH range 4–6. The total activity of lyophilized LPS (0.5 M sodium
acetate elutes) showed 35 U/mg and the optimum pH between 5.5 and 6.5. The
total activity of lyophilized enzyme was 2400 U/mg.
5.5.1.2 Evaluation of antimicrobial activity of GOx, LPS and lysozyme with
EDTA
After 48 h of incubation, formulation VII treatment showed a significant
activity (P<0.05) against both E. coli and S. aureus by decreasing the cell
population to 0.052 and 0.21 CFU/ml respectively. However, the inhibitory effect
of formulation-VII was found to be more potent when compared with other
formulation. It was followed by a significant activity (P<0.05) obtained from the
formulation-V showing 3.1 CFU/ml with E. coli and 3.65 CFU/ml with S. aureus.
Other formulations showed moderate activity against both the test organisms.
There was no reduction obtained from control (Figure 5.37 and 5.38).
A number of naturally occurring antimicrobials have been investigated,
including GOx, LPS, lactoferrin, lysozyme, avidin, various plant extracts such as
spices and their essential oils, sulfur and phenolic compounds (Davidson et al.,
2001). GOx is one of the antimicrobial enzymes immobilized onto various
substrates. GOx is a typical example of oxidoreductase systems that do not
themselves possess antimicrobial activity. However, the reaction products from
reaction catalyzed by a given antimicrobial oxidoreductase system exhibit
antimicrobial activity (Appending et al., 2002; Suye et al., 1998). In spite of these
known activities, there is still a controversy as to whether the effects of the system
179
0
2
4
6
8
10
12
0 6 12 18 24 30 36 42 48Incubation period (h)
E. c
oli
popu
latio
n (L
og C
FU/m
l)
GOx LPSLysozyme with EDTA GOx and LPSGOx, Lysozyme with EDTA LPS and Lysozyme with EDTAGOx, LPS and Lysozyme with EDTA Control
a
aa
aa
aaa
c
bb
b
bb
bb
b
d
c
d
ecc
c
c
c
e
dd
d
cd
d
e
ee
e
e
f
d
0
2
4
6
8
10
12
0 6 12 18 24 30 36 42 48
Incubation period (h)
S. a
ureu
s p
opul
atio
n (L
og C
FU/m
l)
GOx LPSLysozyme with EDTA GOx and LPSGOx, Lysozyme with EDTA LPS and Lysozyme with EDTAGOx, LPS and Lysozyme with EDTA Control
a a a a a a
a ab bb b
bb
bb
c
cc
c
cc
c c
d
d
d
dd
d
de
e
d
ee
e
Figure 5.37 : Effect of antimicrobial activity of GOx, LPS and lysozyme with EDTA against E. coli
Figure 5.38: Effect of antimicrobial activity of GOx, LPS and lysozyme with EDTA against S. aureus
Legends followed by the same letter are not significantly different (P<0.05) for thesame incubation period.Values represent the mean of triplicates with standarddeviation.
180
on gram-negative and gram-positive bacteria are the same. In addition, its
effectiveness against such pathogens like E. coli O157 and L. monocytogenes in
foods has recently been questioned by a number of investigators. Difference
between reported findings may have resulted from differences in experimental
conditions.
The LPS, when used in conjunction with GOx, is a very useful
antimicrobial agent. LPS is part of the immune system’s innate defense
mechanism against foreign microorganisms and can be found in mammalian
secretions such as milk, tears and saliva. This system consists of three components
like LPS, thiocyanate (SCN-) and hydrogen peroxide. LPS activation occurs only
in the presence of thiocyanate and hydrogen peroxide. Catalysis by LPS generates
active intermediates, which has antimicrobial properties and is completely safe to
humans. The presence of GOx allows hydrogen peroxide required by LPS to be
continuously generated and replenished (Seifu et al., 2005). The hydrogen
peroxide produced by GOx is utilized by LPS for cold, i.e. room temperature
sterilization, while the gluconic acid produced is used for direct acidification (Fox
and Stepaniak, 1993). It should be noted that this LPS-GOx antimicrobial system
is not limited to food and has been used in toothpaste (Biotene 2006; National
Library of Medicine 2007a), lotions (National Library of Medicine 2007b),
shampoos, cosmetics, meat processing (Food Standards Australia New Zealand
2002) and fish farming (Seifu et al., 2005).
5.5.1.3 Antimicrobial activity of alginate film
The discs bored from these films were used for the antimicrobial testing
(Figure 5.39). The film disc with formulation-VII showed significant (P<0.05)
zone of inhibition inferring good antimicrobial activity. There was no clear zone
of clearance found with S. aureus but when compared with E. coli showed lager
partial lysis in all enzyme formulations.
181
0
5
10
15
20
25
I II III IV V VI VII VIIIEnzyme formulations
Zone
of i
nhib
ition
(mm
)
E. coli S. aureus
b
a a
cc
d
e
A
A
A
B
A
C
D
Figure 5.39: Effect of antimicrobial alginate films incorporated with partially purified enzymes of different formulations
I-GOx, II-LPS, III-Lyz with EDTA, IV-GOx and LPS, V-GOx, Lyz with EDTA, VI-LPS and Lyz with EDTA, VII-GOx, LPS and Lyz with EDTA, VIII-Control.
Legends followed by the same letter are not significantly different (P<0.05) for the same microbial treatment. Values represent the mean of triplicates with standard deviation.
182
5.5.1.4 Surface sterilization of carrotA significant inhibitory activity (P<0.05) obtained from above 6 ppm of
chlorine dioxide showed higher inhibitory activity. Six ppm of chlorine dioxide
with soaking of 20 minutes was found to be optimum for surface sterilization of
vegetables. The plate count readings were given in Table 5.7 in terms of CFU/ml.
Chlorine dioxide had received a lot of attention in the past few years because its
effectiveness is less affected by pH and organic matter content than that of
chlorine. Another advantage is its high oxidative action, which has been observed
to be 2.5 times greater than chlorine (Benarde et al., 1967).
5.5.1.5 Measurement of weight loss
The alginate coated films extended the shelf-life of carrots by retarding the
evaporation rate and hence there was no weight loss from the carrot. Carrots
coated with the formulations-VII examined to have lower weight loss than
uncoated ones and retain freshness of the carrots (Figure 5.40). The effect of
lowering the water loss was found to be significant (P<0.05) with the coat
containing formulation-VII. The water loss of the sample increased significantly
during storage. This was expected since fresh vegetables usually lose water after
processing and throughout storage. There was a drastic increase in water loss
reported for the first 2 days then the water loss was stabilized and increased
relatively slow until the tenth day. The difference in weight loss between carrots
stored at 6ºC and ~26°C was minimal, though there was relatively less weight loss
in the carrots stored at 6ºC. The loss of water is a natural process of the catabolism
in fresh-cut vegetables and is attributed to the respiration and other senescence-
related metabolic processes during storage (Watads and Qui 1999). The
percentage weight loss due to water loss until the end of the 10th day for coated
and uncoated carrots is represented in Figure 5.41a and 5.41b.
183
Table 5.7: Bacterial counts (CFU/ml± SD) on surface washing carrot treated
with chlorine dioxide at different concentration and time of exposure
Values are mean of three replicates (± S.D)
Incubationtime inminutes
Bacterial count (CFU/ml ±S.D)Concentration of chlorine dioxide (ClO2)
2ppm 4ppm 6ppm 8ppm 10ppm
10 36±6 14.33±2.52 7.66±1.53 3±1 Absent
20 21.33±4.16 7.33±1.53 Absent Absent Absent
30 11±2 3.33±1.58 Absent Absent Absent
184
I II III IV
V VI VII
(a) Stored at ~26°C
(b) Stored at 6°C
Figure 5.40 (a&b): Effect of GOx, LPS and lysozyme incorporated with
alginate film coated carrots stored at (A) Stored at ~26°C and 6°C (I) Control (II) Sodium alginate (III) GOx (IV) LPS (V) Lysozyme with EDTA
(VI) GOx+LPS (VII) GOx, LPS and Lysozyme with EDTA
I II III IV
V VI VII
185
0
5
10
15
20
25
30
0 2 4 2 8 10Storage period (d)
Wei
ght l
oss
(%)
Alginate film+F-VII Alginate film Control
b
a
a
aa
a
b
b
bb
b
b
c
bcb
0
5
10
15
20
25
30
35
40
45
50
0 2 4 2 8 10Storage period (d)
Wei
ght l
oss
(%)
Alginate film+F-VII Alginate film Control
b
a
a
aa
a
bb
bb
b
b b cbc
c
Figure 5.41 (a&b): Effect of alginate coated (Formulation VII) and uncoated carrots stored on weight loss (%) during the storage period at (a) 6°C and (b) ~26ºC
Legends followed by the same letter are not significantly different (P<0.05) for the same storage period. Values represent the mean of triplicates with standard deviation.
(a): Stored at 6°C
(b): Stored at ~26°C
186
5.5.1.6 Measurement of soluble protein content
There was no significant variation obtained in soluble protein content
during the storage period. The initial soluble protein content of treated carrot
(Formulation-VII) was 0.51±0.0271 mg/g dry weight which dropped significantly
and reached its lowest value of 0.423±0.088 mg/g dry weight. The increment in
soluble protein during the last few days of storage may be attributed to the
formation of some stress proteins, or to the senescence, degradation and enzymatic
activities that soften the texture and lead to the formation of more soluble proteins.
The decline in SPC activities between 2–6 days could be related to the utilization
of soluble proteins in some metabolic activities such as a substrate for respiration,
when the carbohydrate sources become very limited. Similar conclusion was
reported by King et al. (1990), who indicated that more CO2 was released from
asparagus spears over 3–5 days than could be accounted by carbohydrates loss
(Table 5.8).
5.5.1.7 Enumeration of bacterial population from treated and control carrots
The plate count observations were expressed in the Figure 5.42 in terms of
CFU/g. The results showed that treated carrots formulation-VII was less in
microbial population when compared to the control.
5.5.1.8 Sensory analysis
The results of sensory analysis of treated and untreated carrots are
expressed in Figure 5.43(a) and 5.43(b). The carrot treated with formulation-VII
[GOx (5 mg/ml), LPS (10 mg/ml) and Lysozyme (0.5 mg/ml) with EDTA (0.3
mg/ml)] were accepted throughout the storage period both 6ºC and ~26°C. There
was no significant difference found in taste of alginate coated and alginate with
formulation-VII but good score was obtained on colour and texture. However,
higher overall acceptances of carrots were received from the treatment of alginate
with formulation-VII with respect to appearance, taste, texture and colour.
187
Table 5.8: Soluble protein content (mg/g of dry wt±SD) in alginate coated (Formulation VII) and uncoated
control carrots
Values are mean of three replicates (S.D). Numbers followed by the same letter are not significantly different (P < 0.05) for the same incubation period.
TreatmentSoluble protein content (mg/g of dry wt ± SD
Storage (days)0 2 4 6 8 10
Formulation VII 0.51±0.0271a 0.532±0.051a 0.513±0.038a 0.47±0.036a 0.453±0.022a 0.423±0.088a
Alginate film 0.524±0.024 a 0.512±0.068 a 0.492±0.048 a 0.462±0.0584a 0.442±0.054 a 0.418±0.087 a
Control 0.520±0.062a 0.512±0.038a 0.470±0.082a 0.43±0.03a 0.411±0.034a 0.378±0.068a
188
0
20
40
60
80
100
120
Alginate film+F-VII Alginate film Control
Mic
robi
al p
opul
atio
n (C
FU/g
)
~26°C 6°C
A
B
C
a
b
c
Figure 5.42: Showed the microbial population (10-6 dilution) the treated and control carrots after the storage period (10 d) stored at 6ºC and ~26ºC
Legends followed by the same letter are not significantly different (P<0.05) for thesame storage temperature. Values represent the mean of triplicates with standarddeviation.
189
0
1
2
3
4
5Appearance
Colour
TextureTaste
Overall acceptance
Formulation VII Alginate film Control
0
1
2
3
4
5Appearance
Colour
TextureTaste
Overallacceptance
Formulation VII Alginate film Control
Figure 5.43 (a&b): Evaluation of the sensory profile of treated and control carrots after the storage period (10 d) stored at (a) 6ºC and (b) ~26ºC. Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much.
Values represent the mean of triplicates.5.5.2 Control of browning and enhancing the shelf-life of apple puree by
glucose oxidase, catalase and lactoperoxidase
(b) Stored at ~26°C
(a) Stored at 6°C
190
5
6
7
8
9
10
log
CFU
/ml
Control
GOx (100 mg)
LPS (40mg)
Catalase (0.1ml)
GOx (100mg) +LPS (40mg)
a
cc
c cc
ab
b bb
bb
b
aa a a a
aa
dd
bc
c
c
cdc
b
cc
bc
cd
dc
cd
5.5.2.1 Assay of antimicrobial enzymes
The formulated GOx activity was found to be 5.2 U/mg and the optimum
pH range between 5and 6. The total activity of lyophilized LPS (0.5 M
sodium acetate elutes) showed 40 U/mg and the optimum pH between 5.5 and 6.5.
The Catalase activity was found to be 2000U/ml and the optimum pH for its
activity was 5–6.
5.5.2.2 Evaluation of antimicrobial activity of GOx, catalase and LPS
The effect of antimicrobial activity of enzyme was evaluated by the
formulations containing; (mg or ml /10ml) like, of (I) GOx (100 mg), (II) LPS
(40mg), (III) Catalase (0.1ml), (IV) GOx (100mg) +LPS (40mg) (V) GOx
(100mg)+Catalase (0.1ml), (VI) LPS (40mg)+Catalase (0.1ml) (VII) GOx
(100mg)+Catalase (0.1ml)+LPS (40mg) and (VIII) Control. The inhibitory
activity of the above enzymes and their combinations were evaluated against
E. coli and S. aureus. After 48 hours of incubation, the formlation-VII
combinationshowed effective control against both E. coli and S. aureus, that
decreasing the cell population to 2.01 and 2.1 log CFU/ml respectively (Figure
5.44a and 5.44b).
Davidson et al. (2001) studied a number of naturally occurring
antimicrobials including GOx, LPS, lactoferrin, lysozyme, avidin, plant extracts
(spices, essential oils, sulfur and phenolic compounds). Seifu et al. (2005) state
that the presence of GOx allows hydrogen peroxide required by LPS to be
continuously generated and replenished. The hydrogen peroxide produced by
GOx is utilized by the LPS for room temperature sterilization, while the gluconic
acid produced is used for direct acidification (Fox and Stepaniak, 1993).
191
0
1
2
3
4
5
6
7
8
9
10
0 6 12 18 24 30 36 42 48Incubation period (h)
log
CFU
/ml
Control
GOx (100 mg)
LPS (40mg)
Catalase (0.1ml)
GOx (100mg) +LPS (40mg)
GOx (100mg)+Catalase (0.1ml)
LPS (40mg)+Catalase (0.1ml)
GOx (100mg)+Catalase(0.1ml)+LPS (40mg)
a
eef
f
d
ddc
b
c
d
ed
dd d
b
cc
c c
cc
aa
a bb
bb b
a aa
a aa
a
a
ff
b
cdc
c c
b
cc
d
Figure 5.44 (a&b): Effect of antimicrobial activity of GOx, catalase and LPS against (a) E. coli and (b) S. aureus
Legends followed by the same letter are not significantly different (P<0.05) for the same incubation period. Values represent the mean of triplicates with standard deviation.
The LPS, when used in conjunction with GOx, is a very useful
antimicrobial agent. LPS is part of the immune system’s innate defense
mechanism against foreign microorganisms and can be found in mammalian
secretions such as milk, tears and saliva. This system consists of three components
like LPS, thiocyanate and hydrogen peroxide. The LPS activation occurs only in
(b) S .aureus
(a) E. coli
192
the presence of thiocyanate and hydrogen peroxide. Catalysis by LPS generates
active intermediates, which has antimicrobial properties and is completely safe to
humans. The LPS and GOx combination was not only used as a antimicrobial
agent and interestingly they also used in preparation of toothpaste (Biotene 2006;
National Library of Medicine 2007a), lotions (National Library of Medicine
2007b), shampoos, cosmetics and also applied in meat processing (Food Standards
Australia New Zealand 2002) and fish farming (Seifu et al., 2005).
5.5.2.3 Effect of GOx and ascorbic acid on removal of dissolved oxygen from
apple puree
Three concentrations of ascorbic acid (50, 100 and 150 mg/l) were tested on
the apple puree for the curb of oxygen. The complete oxygen disappeared
happened on 100 and 150 mg/l of ascorbic acid at 10 and 8 minutes respectively.
The GOx also applied in three concentrations such as 50, 100 and 150 mg/l and
the complete removal of dissolved oxygen appeared at 8 (50 mg/l), 6 (100 mg/l)
and 5 (mg/l) minutes. Based on the observation, GOx exhibit better response than
the ascorbic acid (Figure 5.45).
Parpinello et al. (2002) reported that the treatment of fruit purees with GOx
removed the 99% of dissolved oxygen within 120 seconds. On the other hand,
fruit purees treated with ascorbic acid required up to 200 minutes to achieve
similar results. These findings are supported to this study for the controlling of
oxidative browning reactions of fruit purees controlled by the treatment of GOx.
193
5.5.2.4 Effect of GOx, catalase, LPS and ascorbic acid on controlling of
browning in apple puree
The formulations containing GOx-catalase and ascorbic acid were showed
effective control of browning when compared with other treatments (Figure 5.46).
The LPS and catalase did not showing significant antibrowning activity. However,
GOx was very effective in the presence of catalase combination (Figure 5.47a and
5.47b). It is clearly exhibiting the oxygen is a key factor in fruit purees browning.
The preparation of apple puree with the combination of GOx-catalase and LPS
was exhibited good antibrowning and antibacterial activity. A similar observation
was reported by Mistry and Min (1992a) that the GOx-catalase system is able to
scavenge the oxygen and thus stabilizes foods and beverages against problems
related to product oxidation and browning.
Sathiya moorthi et al. (2007) state that browning reaction occurs during
storage of fruits as well as purees and juices. In other words, this reaction has a
negative effect on the quality and shelf-life of the product. GOx-catalase system
helps to scavenge the oxygen and it thus helps in preventing oxidation and
browning. Heating has been the unique treatment able to control the enzymatic
browning of fruit purees, whereas GOx showed efficient in reducing to low level
the dissolved oxygen content in apple purees and showed an interesting capability
to control the non-enzymatic browning during apple purees storage.
Enzymic browning starts with the initial enzymic oxidation of phenols to
quinones by the enzyme polyphenol oxidase in the presence of oxygen. Then these
quinones are subjected to further reactions, enzymically catalyzed or not, leading
to the formation of pigments. Cloudy apple juice has increasing market value due
to its sensory and nutritional qualities. Although a typical amber-like hue is
commercially desirable in clarified apple juice, both apple puree and cloudy juice
194
0
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ControlGOx (100mg)LPS (40mg)Catalase (0.1ml)GOx (100mg) +LPS (40mg)GOx (100mg) +Catalase (0.1ml)LPS (40mg)+Catalase (0.1ml)GOx (100mg)+Catalase (0.1ml)+LPS (40mg)Ascorbic acid (100mg)
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Figure 5.45: Effect GOx and ascorbic acid on removal of dissolved oxygen from apple puree Legends followed by the same letter are not significantly different (P<0.05) for the same incubation period. Values represent the mean of triplicates with standard deviation.
Figure 5.46: Effect GOx, catalase, LPS and ascorbic acid on controlling of browning in apple puree Legends followed by the same letter are not significantly different (P<0.05) for the same storage period (d). Values represent the mean of triplicates with standard deviation
195
Control Ascorbic acid GOx
(b) Apple puree
Control Ascorbic acid Formulation-VII
(a) Cut apple
Figure 5.47 (a&b): Effect GOx and ascorbic acid on controlling of browning in (a) Cut apple and (b) Apple puree
Formulation-VII: GOx (100mg) + catalase (0.1ml)+LPS (40mg)
196
are expected to have the yellowish colour which characterizes the fresh product
(Lozano et al., 1994). The food industry has given increasing attention to
minimally processed products. This might be achieved in raw juices by membrane
filtration, ultra high pressure treatment and preservation by freezing.
The control of enzymatic browning has great importance just at the start of
the processes. An approach for the prevention of enzymatic browning of fruit
juices has been the use of antibrowning agents. The most widely used
antibrowning agents are sulfiting agents. Due to adverse health effects, several
studies have been devoted to the non sulfite anti browning agents such as reducing
agents (ascorbic acid and analogs, glutathione, L-cysteine), enzyme inhibitors
(aromatic carboxylic acids, substituted resorcinols, anions, peptides), chelating
agents (phosphates, organic acids), acidulants (citric acid, phosphoric acid),
complexing agents (cyclodextrins) (Labuza et al., 1992; Lambrech, 1995;
Martinez and Whitaker, 1995).
However, ascorbic acid decreased the non-enzymatic browning of fruit
purees to a larger extent. Browning reactions occur during post harvest of fruits
processing, storage of purees and juices that may have a negative effect on the
quality and shelf-life of the products. These differences in the mechanism of
inhibition may allow the use of combinations of antibrowning agents that may
result in enhancement of inhibition. Most combinations of antibrowning agents or
commercially available are ascorbic acid-based compositions (Pizzocarno et al.,
1993).
197
5.5.2.5 Examination of microbial populations
The formulation-VII [(GOx (100mg) + Catalase (0.1ml) + LPS (40mg)]
was found to be effective control of microbial growth, when compared with other
treatments (Figure 5.48). Seifu et al. (2005) state that GOx is one of the most
important enzyme in food processing industry for food preservation. The
combinations of LPS and GOx were very useful antimicrobial agent. Further,
reported that the presence of GOx allows hydrogen peroxide required by LPS to
be continuously generated and replenished. The hydrogen peroxide produced by
GOx is utilized by the LPS for cold, i.e. room temperature sterilization, while the
gluconic acid produced is used for direct acidification (Fox and Stepaniak, 1993).
5.5.2.6 Sensory analysis
The sensory analyses of treated and untreated apple puree are expressed in
figure 5.49. The apple puree treated with Formulation-VII contains GOx
(100mg)+Catalase(0.1ml)+LPS(40mg) was accepted throughout the storage
period. The ascorbic acid treated apple puree had a good sensory score on colour
and appearance, while GOx-catalase and LPS treatment produces a similar result
along with good sensory scores with respect to taste, flavour and overall
acceptance.
198
0
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140
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Storage period (d)
Bac
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ulat
ion
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/ml)
ControlGOx (100 mg)LPS (40mg)Catalase (0.1ml)GOx (100mg) +LPS (40mg)GOx (100mg)+Catalase (0.1ml)LPS (40mg)+Catalase (0.1ml)GOx (100mg)+Catalase (0.1ml)+LPS (40mg)Ascorbic acid (100mg)
a
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0
1
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4
5Taste
Colour
Texture
Appearance
Flavour
Overalacceptance
Control GOx+Catalase+LPS Ascarbic acid
Figure 5.48: Showed the microbial population of treated and control apple puree Legends followed by the same letter are not significantly different (P<0.05) for the same storage period. Values represent the mean of triplicates with standard deviation.
Figure 5.49: Evaluation of the sensory profile of treated and control apple puree Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.5.5.3 Studies on the effects of glucose oxidase-catalase with calcium ions
199
for the improvement of fruit salad quality
5.5.3.1 Evaluation of antimicrobial activity of GOx, catalase, calcium ions and
koruk juice
The GOx, calcium propionate, koruk juice and also combined treatment
showed Significant (P<0.05) inhibitory activity against E. coli and S. aureus.
There were no significant inhibitory activities exhibited with calcium chloride and
calcium lactate treatments when compared with other treatments against both the
tested microbes (Figure 5.50a and 5.50b). The hydrogen peroxide produced by the
GOx acts as a good bactericide and can be later removed using catalase which
converts hydrogen peroxide to oxygen and water. The GOx was also found to be
antagonistic potential against different food borne pathogens such as Salmonella
infantis, S. aureus, Clostridium perfringens, Bacillus cereus, Campylobacter jejuni
and L. monocytogenes (Tiina and Sandhlm, 1989). Phenolic compounds in grape
juice, grape seed and wine have been investigated by many researchers to show
their potent antioxidant, antimutagenic, antibacterial, antiviral, antifungal and
antiulcer activities (Takechi et al., 1985; Liviero et al., 1994; Caccioni et al.,
1998; Saito et al., 1998; Baydar et al., 2004; Jayaprakasha et al., 2003). Koruk
juice showed good antimicrobial effect against S. aureus
5.5.3.2 Sensory analysis
After 24 h of storage period the sensory analysis scores were expressed in
the radar plots. The effect of antibrowning activity of calcium ions, GOx and
koruk juice on cut apple, pomegranate and guava were shown in figure 5.51
(A-G).
200
a
b
a a
bb
0
1
2
3
4
5
6
7
8
9
10
0 6 12 18 24 30 36 42 48Incubation period (h)
Log
CFU
/ml
GOx Koruk juiceC. lactate C.chlorideC.propionate C. combinationGOx+koruk juice+c.combination Control
a
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0 6 12 18 24 30 36 42 48Incubation period (h)
Log
CFU
/ml
GOx Koruk juiceC. lactate C.chlorideC.propionate C. combinationGOx+koruk juice+c.combination Control
a
f
g
g
ff
f
b
cb
ee e
dcd d
dd
b b cc c
c
ab a a a b bb b
a aa a a a a a
c
Figure 5.50 (a&b): Effect of antimicrobial activity of GOx, calcium ions and koruk juice against (a) E. coli and (b) S. aureus
Legends followed by the same letter are not significantly different (P<0.05) for the same incubation period. Values represent the mean of triplicates with standard deviation
(a) E. coli
(b) S. aureus
201
5.5.3.3 Sensory analysis of GOx-catalase treated fruit salad
The fruit salad prepared with GOx-catalase was found to be no changes in
their appearance, odour and browning in the sensory analysis. The concentration
of 0.25% (w/v) of GOx-catalase produced a maximum sensory score of 4.6
(Figure 5.52a and 5.55a). Mistry and Min (1992b) reported that the GOx-catalase
system scavenge the oxygen and resulted to stabilizes foods and beverages against
problems related to product oxidation and browning. According to Bao et al.
(2001 and 2003) the food grade GOx preparation used typically contains a mixture
of GOx and catalase because the two enzymes are found naturally together in the
mycelium of the cell wall (Witteveen et al., 1992). Catalase assists in the
breakdown of hydrogen peroxide produced by GOx, thereby reducing inhibition
and deactivation by hydrogen peroxide.
5.5.3.4 Sensory analysis of calcium chloride treated fruit salad
Calcium chloride was produced better texture, but browning was appeared
at 0.5% (w/v) concentration. Higher concentration of calcium chloride revealed
bitter taste and off odour. It was found to be optimum with a sensory score of 4.2
at 1% (w/v) (Figure 5.52b). Ohlsson (1994) reported that the use of calcium
chloride is associated with bitterness and off-flavour.
5.5.3.5 Sensory analysis of calcium propionate treated fruit salad
The fruit salad prepared with calcium propionate was found to have a
sensory evaluation of good taste, slightly better appearance and odour but slightly
browning was observed in the 0.5% (w/v) concentration. Higher concentration
exhibited sour taste and lowering the colour of the fruit salad. Satisfactory sensory
qualities were obtained at the concentration of 1.0% (w/v) with the sensory score
of 4.4 (Figure 5.53a).
202
0
1
2
3
4
5Texture
Colour
TasteOdour
Overalacceptance
0.50% 1.0% 2.0% Control
0
1
2
3
4
5Texture
Colour
TasteOdour
Overal acceptance
0.25% 0.5% 1.0% Control
Figure 5.52 (a&b): Evaluation of the sensory profile of different concentration of (a) GOx and (b) calcium chloride treated and control fruit salad Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.
(b) Calcium chloride
(a) Glucose oxidase
203
5.5.3.6 Sensory analysis of calcium lactate treated fruit salad
The fruit salad prepared with calcium lactate was found to have a sensory
report of good taste, odour, colour but the texture of the fruit salad remained
slightly bad than the other parameters at the concentration of 0.5% (w/v).
However, the other two concentrations 1.0 and 2.0% (w/v) exhibited good score
and 1.0% (w/v) was found to be optimum with a sensory score of 4.6 (Figure
5.53b) Calcium lactate, calcium propionate and calcium gluconate have shown
some of the benefits of the use of calcium chloride, such as product firmness
improvement, and avoid some of the disadvantages, such as bitterness and residual
flavour (Yang and Lawsless, 2003).
5.5.3.7 Sensory analysis of koruk juice treated fruit salad
The fruit salad prepared with koruk juice exhibits no change in taste, very
less odour, little change in appearance and with less browning, when compared the
control fruit salad. It was found to be the optimum concentration at 1.0% (v/v)
with a sensory score of 4.4 (Figure 5.54 and 5.55b). Koruk juice is commonly used
in the preparation of fruit and vegetable salads as an acidifying and flavouring
agent in Turkey and neighboring countries. It is also consumed as a drink after
being sweetened. Currently grape compounds have attracted increased attention
especially in the Welds of nutrition, health and medicine (Waterhouse and
Walzem, 1998).
204
0
1
2
3
4
5Texture
Colour
TasteOdour
Overalacceptance
0.50% 1.0% 2.0% Control
0
1
2
3
4
5Texture
Colour
TasteOdour
Overalacceptance
0.50% 1.0% 2.0% Control
Figure 5.53 (a&b): Evaluation of the sensory profile of different concentration of (a)calcium propionate and (b) calcium lactate treated and control fruit salad Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.
(b) Calcium lactate
(a) Calcium propionate
205
0
1
2
3
4
5Texture
Colour
TasteOdour
Overal acceptance
0.5% 1.0% 1.5% Control
Figure 5.54: Evaluation of the sensory profile of different concentration of koruk juice treated and control fruit salad Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.
206
(a) GOx
(b) Extract of koruk juice
Figure 5.55 (a&b): Effect of GOx and koruk juice on fruit salad
207
5.5.3.8 Optimization of calcium ions concentrations by response surface
methodology
The results of central composite design experiments for studying the effects
of set of three independent variables such as calcium lactate, calcium propionate
and calcium chloride on combination are presented in the table 5.9.
The Model F-value of 6.92 implies the model is significant. There is only
0.34% chance that a "Model F-Value" that large could occur due to noise. Values
of Prob > F" less than 0.05 indicate model terms are significant. In that case A, C
were significant model terms. Values greater than 0.1 indicate the model terms are
not significant. If there are many insignificant model terms (not counting those
required to support hierarchy), model reduction may improve the model (Table
5.9.1).The "Lack of Fit F-value" of 9.87 exhibit as significant and there is only
slight chance of 1.02% variation in "Lack of Fit F-value" due to noise.
The "Pred R-Squared" of 0.2886 is in reasonable agreement with the "Adj
R-Squared" of 0.4831. "Adeq Precision" measures the signal to noise ratio and a
ratio greater than 4 is desirable. The ratio of 9.246 indicates an adequate signal.
This model can be used to navigate the design space. The model coefficient was
estimated by linear regression (Table 5.9.2).
Final equation in terms of coded factors
Weight loss = 27.14-2.56×A-1.44×B-1.65×C
Final equation in terms of actual factors
Weight loss = 35.46090-2.55814×Calcium lactate-2.30105×Calcium
propionate-4.11712×Calcium chloride
208
Table 5.9: Optimization of calcium ions for salad preparation by CCD of response surface methodology (23 factorial design)
Run
ACalciumlactate
(%,w/v)
BCalcium
Propionate(%,w/v)
CCalciumchloride(%,w/v)
Response1weight loss
(%,w/v)
Response2overall
acceptence(sensoryscore)
1 1.50 1.93 0.60 21.2 4
2 1.50 0.88 0.60 24.5 4.6
3 1.50 0.88 0.60 25.7 4.8
4 0.50 1.50 1.00 30.5 3.2
5 0.50 0.25 1.00 32.3 2.8
6 2.50 0.25 1.00 24.7 3.6
7 1.50 0.88 -0.07 28.5 3.4
8 2.50 0.25 0.20 29.4 4
9 -0.18 0.88 0.60 31.5 3
10 1.50 0.88 0.60 23.7 4.8
11 2.50 1.50 0.20 29.7 4.2
12 2.50 1.50 1.00 24.7 3.8
13 1.50 -0.18 0.60 30.5 4
14 0.50 0.25 0.20 34 3.8
15 1.50 0.88 0.60 25.5 4.6
16 3.18 0.88 0.60 22.5 4.2
17 1.50 0.88 1.27 22.5 4
18 1.50 0.88 0.60 26.1 4.8
19 0.50 1.50 0.20 31.5 3.8
20 1.50 0.88 0.60 23.8 4.6
209
Table 5.9.1: F-test analysis ( ANOVA for Response Surface Quadratic Model)
SourceSum ofSquares
df MeanSquare
FValue
p-valueProb >
Model 154.6567 3 51.55223 6.920266 0.0034
A- Calcium lactate 89.3715 1 89.3715 11.99705 0.0032
B-Calcium propionate 28.24633 1 28.24633 3.79173 0.0693C- Calcium chloride 37.03885 1 37.03885 4.97202 0.0404
Residual 119.1913 16 7.449457
Lack of Fit 113.943 11 10.35845 9.868326 0.0102
Pure Error 5.248333 5 1.049667
Cor Total 273.848 19
Table 5.9.2: Comparition of R2 predicted and estimated
STD DEV 2.729369 R-SQUARED 0.564754
Mean 27.14 Adj R-Squared 0.483145
C.V. % 10.05663 Pred R-Squared 0.288577PRESS 194.8217 Adeq Precision 9.246421
210
The results of central composite design experiments for studying the effects of
set of three independent variables, calcium lactate, calcium propionate, calcium
chloride on overall acceptance are presented in Table 5.9.
The Model F-value of 7.09 implies the model is significant. There is only a
0.26% chance that a "Model F-Value" that large could occur due to noise.Values
of "Prob > F" less than 0.0500 indicate model terms are significant.
In that case A, A2, B2, C2 were significant model terms. Values greater than
0.100 indicate the model terms are not significant. The "Lack of Fit F-value" of
14.35 implies the Lack of Fit is significant (Table 5.9.3). There is only 0.55%
chance that a "Lack of Fit F-value" that large could occur due to noise.
The "Pred R-Squared" of 0.0211 is not as close to the "Adj R-Squared" of
0.7427 as one might normally expect."Adeq Precision" measures the signal to
noise ratio and a ratio greater than 4 is desirable. The ratio of 7.609 indicates an
adequate signal (Table 5.9.4). This model can be used to navigate the design
space. The model coefficient was estimated by linear regression.
That may indicate a large block effect or a possible problem with the
model and/or data. Things to consider are model reduction, response
tranformation, outliers, etc. "Adeq Precision" measures the signal to noise ratio. A
ratio greater than 4 is desirable (Table 5.9.5). The ratio of 7.609 indicates an
adequate signal. That model can be used to navigate the design space and the
model coefficient estimated by linear regression
211
Table 5.9.3: F-test analysis ( ANOVA for Response Surface Quadratic Model)
Source Sum ofSquares df Mean
SquareF
Valuep-value
Prob > FModel 5.879 9 0.653 7.094 0.003 A-Calcium lactate 1.182 1 1.182 12.839 0.005 B-Calcium propionate 0.046 1 0.046 0.508 0.491 C-Calcium chloride 0.141 1 0.1416 1.538 0.243 AB 0 1 0 0 1.000 AC 0.08 1 0.08 0.868 0.373 BC 0.02 1 0.02 0.217 0.651 A^2 2.324 1 2.324 25.240 0.0005 B^2 0.975 1 0.975 10.593 0.008 C^2 1.932 1 1.932 20.991 0.001Residual 0.920 10 0.092Lack of Fit 0.860 5 0.172 14.346 0.005Pure Error 0.06 5 0.012
Table 5.9.4: Comparison of R2 predicted and estimated
STD DEV 0.30344 R-SQUARED 0.864Mean 4 Adj R-Squared 0.742C.V. % 7.586 Pred R-Squared 0.021PRESS 6.656 Adeq Precision 7.608
212
Table 5.9.5: Model coefficient estimated by linear regression
Factor CoefficientEstimate
Df StandardError
95% CILow
95% CIHigh
VIF
Intercept 4.701 1 0.123 4.426 4.977A-Calcium lactate 0.294 1 0.082 0.112 0.477 1B-Calciumpropionate 0.058 1 0.082 -0.124 0.241 1
C-Calcium chloride -0.101 1 0.082 -0.284 0.081 1AB 0 1 0.107 -0.239 0.239 1AC 0.1 1 0.107 -0.139 0.339 1BC 0.05 1 0.107 -0.189 0.289 1A^2 -0.401 1 0.079 -0.579 -0.223 1.018B^2 -0.260 1 0.079 -0.438 -0.082 1.018C^2 -0.366 1 0.079 -0.544 -0.188 1.018
Final equation in terms of coded factors
Overal acceptancy =+4.70+0.29×A+0.059×B-0.10×+0.000 ×A ×B+0.10 ×A×C
+0.050 × B × C-0.40×A2-0.26×B
2-0.37×C
2
Final equation in terms of actual factors
Overal acceptancy = 2.42389+1.34896×Calcium lactate+1.13924×Calcium
propionate +1.94207 ×Calcium chloride +2.32374E-015×Calcium lactate
×Calcium propionate + 0.25000 ×Calcium lactate ×Calcium
chloride+0.20000×Calcium propionate ×Calcium chloride - 0.40158×Calcium
lactate2-0.66601×Calcium propionate2-2.28891×Calcium chloride2
The contour and three dimensional response surface curves were plotted.
Maximum overall acceptance was achieved at: Calcium lactate-1.45% (w/v),
calcium propionate-0.68% (w/v), calcium chloride-0.55% (w/v). For the optimized
values the weight loss and overall acceptance was observed maximum of 27.2%
and 4.70 respectively. If the values of the calcium ions concentration was changed
the weight loss and overall acceptance were changing in the response (Figure
5.56-5.61).
213Design-Expert® Software
Weight lossDesign Points34
21.2
X1 = A: Calcium lactateX2 = B: Calcium propionate
Actual FactorC: Calcium chloride = 0.60
0.50 1.00 1.50 2.00 2.50
0.25
0.56
0.88
1.19
1.50Weight loss
A: Calcium lactate
B: C
alci
um p
ropi
onat
e
24.4758
25.807927.14
28.4721
29.8042
6
Design-Expert® Software
Weight lossDesign points below predicted value34
21.2
X1 = A: Calcium lactateX2 = B: Calcium propionate
Actual FactorC: Calcium chloride = 0.60
0.5
1
1.5
2
2.5
0.25 0.56 0.88 1.19 1.50
23.1
25.125
27.15
29.175
31.2
Wei
ght l
oss
A: Calcium lactate
B: Calcium propionate
Figure 5.56: The contour and 3D response surface plot showing the weight loss % of calcium lactate and calcium propionate treatment on fruit salad
214Design-Expert® Software
Weight lossDesign Points34
21.2
X1 = B: Calcium propionateX2 = C: Calcium chloride
Actual FactorA: Calcium lactate = 1.50
0.25 0.56 0.88 1.19 1.50
0.20
0.40
0.60
0.80
1.00Weight loss
B: Calcium propionate
C: C
alci
um c
hlor
ide
25.0833
26.1117
27.14
28.1683
29.1967
6
Design-Expert® Software
Weight lossDesign points below predicted value34
21.2
X1 = B: Calcium propionateX2 = C: Calcium chloride
Actual FactorA: Calcium lactate = 1.50
0.25
0.56
0.88
1.19
1.50
0.20 0.40 0.60 0.80 1.00
23.6
25.275
26.95
28.625
30.3
Wei
ght l
oss
B: Calcium propionate
C: Calcium chloride
Figure 5.57: The contour and 3D response surface plot showing the weight loss % of calcium propionate and calcium chloride treatment on fruit salad
215Design-Expert® Software
Weight lossDesign Points34
21.2
X1 = C: Calcium chlorideX2 = A: Calcium lactate
Actual FactorB: Calcium propionate = 0.88
0.20 0.40 0.60 0.80 1.00
0.50
1.00
1.50
2.00
2.50Weight loss
C: Calcium chloride
A: C
alci
um la
ctat
e
24.3367
25.7383
27.14
28.5417
29.9433
6
Design-Expert® Software
Weight lossDesign points below predicted value34
21.2
X1 = C: Calcium chlorideX2 = A: Calcium lactate
Actual FactorB: Calcium propionate = 0.88
0.20
0.40
0.60
0.80
1.00
0.50 1.00 1.50 2.00 2.50
22.9
25.025
27.15
29.275
31.4
Wei
ght l
oss
C: Calcium chloride
A: Calcium lactate
Figure 5.58: The contour and 3D response surface plot showing the weight loss % of calcium chloride and calcium lactate treatment on fruit salad
216Design-Expert® Software
Overal acceptancyDesign Points4.8
2.8
X1 = A: Calcium lactateX2 = B: Calcium propionate
Actual FactorC: Calcium chloride = 0.60
0.50 1.00 1.50 2.00 2.50
0.25
0.56
0.88
1.19
1.50Overal acceptancy
A: Calcium lactate
B: C
alci
um p
ropi
onat
e
3.86601
4.04462
4.223234.40184
4.40184
4.58045
6
Design-Expert® Software
Overal acceptancyDesign points above predicted valueDesign points below predicted value4.8
2.8
X1 = A: Calcium lactateX2 = B: Calcium propionate
Actual FactorC: Calcium chloride = 0.60
0.50
1.00
1.50
2.00
2.50
0.25 0.56
0.88 1.19
1.50
3.6
3.925
4.25
4.575
4.9
Ove
ral a
ccep
tanc
y
A: Calcium lactate
B: Calcium propionate
Figure 5.59: The contour and 3D response surface plot showing the overall sensory acceptability profile of calcium propionate and calcium lactate treatmenton fruit salad
217
Design-Expert® Software
Overal acceptancyDesign Points4.8
2.8
X1 = A: Calcium lactate
0.80
1.00Overal acceptancy
C: C
alci
um c
hlor
ide
3.65818
3.87829
4.31852
Design-Expert® Software
Overal acceptancyDesign Points4.8
2.8
X1 = B: Calcium propionateX2 = C: Calcium chloride
Actual FactorA: Calcium lactate = 1.50
0.25 0.56 0.88 1.19 1.50
0.20
0.40
0.60
0.80
1.00Overal acceptancy
B: Calcium propionate
C: C
alci
um c
hlor
ide
4.00624.14728
4.28835 4.28835
4.28835
4.42943
4.429434.5705
6
Design-Expert® Software
Overal acceptancyDesign points above predicted valueDesign points below predicted value4.8
2.8
X1 = B: Calcium propionateX2 = C: Calcium chloride
Actual FactorA: Calcium lactate = 1.50
0.25
0.56
0.88
1.19
1.50
0.20 0.40
0.60 0.80
1.00
3.86
4.095
4.33
4.565
4.8
Ove
ral a
ccep
tanc
y
B: Calcium propionate
C: Calcium chloride
Figure 5.60: The contour and 3D response surface plot showing the overall sensory acceptability profile of calcium propionate and calcium chloride treatment on fruit salad
218
Design-Expert® Software
Overal acceptancyDesign points above predicted valueDesign points below predicted value4.8
2.8
X1 = A: Calcium lactateX2 = C: Calcium chloride
Actual FactorB: Calcium propionate = 0.88
0.50
1.00
1.50
2.00
2.50
0.20 0.40
0.60 0.80
1.00
3.4
3.775
4.15
4.525
4.9
Ove
ral a
ccep
tanc
y
A: Calcium lactate
C: Calcium chloride
Figure 5.61: The contour and 3D response surface plot showing the overall sensory acceptability profile of calcium lactate and calcium chloride treatment on fruit salad
219
5.5.3.9 Effect of RSM optimized combined of calcium ions, GOx-catalase and
koruk juice on fruit salad
The calcium ions have its own merits and demerits with respect to sensory
qualities. To overcome this problem fruit salad was prepared with the combination
of calcium ions. The fruit salad prepared with the combination of calcium ions was
found to have a good sensory score of better appearance, good taste, odour and
texture with less browning and a sensory score of 4.6 (Figure 5.62 and Figure
5.63). Manganaris et al. (2007) state that the calcium lactate, calcium chloride,
calcium phosphate, calcium propionate and calcium gluconate, which are used
more when the objective is the preservation and/or the enhancement of the product
firmness.
Fruit salad prepared with the combination of optimized calcium ions, GOx-
catalase and koruk juice exhibited good sensory score with respect to colour,
texture, taste, odour and overall acceptance. In this treatment was found to be very
effective in all the parameters analyzed (Figure 5.64a , 5.64b and 5.6c).
Indeed combination of preservation treatments are often advocated (Knorr,
1998). Combinations of preservation treatments allow the required level of
protection to be achieved while at the same time retaining the organoleptic
qualities of the product such as, colour, flavour, texture, taste and nutritional
value. The potential use GOx, calcium ions and koruk juice in combinations in the
preparation of cut fruit and vegetable may lead to preserve the organolyptic quality
needs to individual products.
220
0
1
2
3
4
5Texture
Colour
TasteOdour
Overalacceptance
RSM optimized calcium ions+GOx-catalase+koruk juiceRSM optimized calcium ionsControl
Figure 5.62: Effect of RSM optimized calcium ions on fruit salad
Figure 5.63: Evaluation of the sensory profile of combined calcium ions (obtained from RSM) and GOx-catalase and koruk juice treated and control fruit salad Descriptions for each score: 5=likely very much, 4=likely slightly, 3=neither like nor dislike 2=dislike slightly and 1=dislike very much. Values represent the mean of triplicates.
221
(c) Control
(b) GOx-catalase+combined calcium+koruk juice
ions
(a) GOx-catalase+combined calcium ions
Figure 5.64 (a,b&c): Effect of GOx-catalase, calcium ions and koruk juice on fruit salad
222
5.5.3.10 Measurement of weight loss
The fruit salad treated with all the formulations examined to have lower
weight loss than treated fruit salads. The effect of lowering the water loss was
found with the treatment of (IV) optimized calcium ions combination and GOx-
catalase + calcium ions combination + koruk juice. The water loss of the sample
increased significantly during storage period (after 48 h). This was expected since
fresh-cut fruits and vegetables usually lose water after processing and throughout
storage. The loss of water is a natural process of the catabolism in fresh-cut
vegetables and is attributed to the respiration and other senescence-related
metabolic processes during storage (Watads and Qui 1999). The percentage
weight losses due to water loss of the fruit salad in the different formulations are
represented in Figure 5.65.
5.5.3.11 Enumeration of bacterial population from treated and control fruit salads
The plate count observations were expressed in terms of CFU/g (Figure
5.66). The GOx-catalase, koruk juice, calcium propionate and combined treated
fruit salad with were exhibited less in microbial population when compared with
untreated. Numerous epidemiologic studies exhibited, that a reduced risk of
degenerative diseases correlates with a high intake of fruits and vegetables
(Steinmetz and Potter, 1996). Ready-to-use salads can suit as useful sources of
minerals and physiologically active substances such as polyphenols, as the
increasing popularity of this convenience product indicates. Due to high bacterial
counts of raw vegetables after harvest up to 106–109 colony forming units per
gram fresh salad (CFU/g), ready-to-use sliced salads are usually contaminated by
microorganisms (Jaques and Morris, 1995). Even bacterial pathogens have been
detected in prepackaged salads (Lin et al., 1996) and lettuce (Park and Sanders,
1992). High initial counts are not substantially reduced during conventional cold
washing.
223
0 5 10 15 20 25 30 35 40 45
Calcium chloride (1%)
Calcium lactate (1%)
Calcium Propionate (1%)
Combined calcium ion (1%)
Koruk juice (1%)
Glucose oxidase (0.25%)
GOx+combined calcium ion
GOx+combined calcium ion+koruke juice
Control
Weight loss (%)
a
e
d
c
c
b
b
d
d
0
50
100
150
200
250
Calciumchloride (1%)
Calcium lactate(1%)
Calciumpropionate (1%)
Combinedcalcium ion
(1%)
Koruk juice(1%)
Glucoseoxidase(0.25%)
GOx+combinedcalcium ion
GOx+combinedcalcium ion+koruke juice
Control
Mic
robi
al p
opul
atio
n (C
FU/g
)
bc
bb
c
b
c c
b
a
Figure 5.65: Effect of GOx, calcium ions and koruk juice for controlling weight loss on fruit salad Legends followed by the same letter are not significantly different (P<0.05). Values represent the mean of triplicates with standard deviation.
Figure 5.66: Enumeration of microbial population of different treated and control fruit salad Legends followed by the same letter are not significantly different (P<0.05). Values represent the mean of triplicates with standard deviation.
224
CHAPTER-6
SUMMARY
The current study focused on the screening of GOx producing fungi and
their application in the food processing and preservation. GOx producing fungi
were isolated from various sugar rich products and the strains GOP3 and GOP7
(Aspergillus awamori belongs to the group of niger) isolated from dates and honey
hive produced the highest extra cellular GOx activity in the basal production
medium of 4.2±0.14 and 5.1±0.22 U/ml, respectively. The isolated higher GOx
producing fungus (GOP7) was submitted in Microbial Type Culture Collection
(MTCC), Chandigarh, India and it was designated as Aspergillus awamori MTCC
9645. This fungus was used for further studies.
Seven different medium were used for the selection of suitable medium for
GOx production in which GOxM3 supported maximum GOx activity. Production
medium was optimized by classical single factor analysis and also statistically by
response surface methodology using central composite design.
In single factor analysis, the highest GOx production was obtained in
glucose followed by sucrose at 80–100 g/l. Proteose peptone (3–4 g/l) was a
suitable nitrogen source for GOx production when compared to other nitrogen
sources. In the levels of 0.4 g/l of di-ammonium hydrogen phosphate and 0.2 g/l of
potassium di-hydrogen phosphate were found to be maximum production.
Addition of magnesium sulphate increase of the amount of GOx production in the
fermentation medium, while above 0.2 g/l of magnesium sulphate affects the GOx
production. Remarkably, GOx production was increased by the supplementation
of 30–40 g/l of calcium carbonate. Maximum GOx production was observed at pH
between 5 and 6. The optimum temperature was found to be from 30 to 35ºC for
the production of GOx and the optimum fermentation time for the GOx production
was at 84 h.
225
In Response Surface Methodology, the maximum GOx production (10.08
U/ml) was achieved at 92.7 g/l of glucose, 3.24 g/l of proteose peptone, 36.82 g/l
of calcium carbonate, 0.48 g/l of (NH4)2HPO4, 0.32 g/l of KH2PO4 and 0.23 g/l of
MgSO4. Higher amount of MgSO4 significantly affects the GOx production
followed by KH2PO4. Maximum GOx production was achieved at the pH 5.83 and
the temperature at 30.7°C. Response Surface Methodology was reduced the
fermentation time (65–70h) and significantly improved the GOx production, when
compared to single factor analysis.
Aspergillus awamori MTCC 9645 was cultivated in the two litre laboratory
batch fermentor. The fungal morphological structure was studied. The fungus
apparently grew exponentially up to 72 h and reached maximum concentration of
12.5±0.63 g/l cell dry mass and maintained more or less constant for the rest of
cultivation period. The utilization of glucose started higher after about 12 h and
complete consumption of glucose was observed 50–60 h of fermentation.
Production of gluconic acid was started at 12 h and maximum amount was
observed at 48 h as 62.3±4.1 g/l. The extracellular GOx secretion was started from
10 to 12 h and then reached maximum GOx activity at 72 h as 12±0.63 U/ml.
The concentrated protein obtained from ammonium sulphate precipitation
was purified in the DEAE-Cellulose column followed by Sephacryl S-200 column.
The enzyme activity was purified up to 9.19 folds with a final recovery of 12.98%.
The specific activity of purified GOx was 282.27 U/mg protein. On SDS-PAGE,
the purified GOx showed a single band indicating electrophoretically homogenous
with the molecular weight of 71.1 kDa. A single brown color band was formed in
the native gel. The Lineweaver’s Burk plot, Eadie-Hofstee plot and Hans-Woolf
linear plots were used to determine the Vmax and Km values of the GOx from A.
awamori MTCC 1945.
226
The optimum temperature and pH of GOx activity were found to be 30±2ºC
and pH of 5.5, respectively. The stability of the purified GOx was tested at 25 and
37ºC and the relative activity not affected over 12 h at 25 ºC, while showed a half
life of 40 minutes at 37ºC. All the metal ions showed various degrees of inhibition;
the inhibition sequence was in the order: copper acetate > silver sulphate >
cobaltous chloride > mercuric chloride > copper sulphate at same concentration.
The activity of calcium lactate treated GOx was less sensitive to pH
changes at acidic and alkaline pH as compared with that of untreated control
enzyme. The effect of calcium ions on temperature stability of GOx was found to
be effective and it was observed a maximum stability at 50°C in calcium lactate
treated GOx as 86.67% but in control showed only 43.3% of relative activity.
The combined effect of GOx, LPS and Lysozyme with EDTA were showed
good inhibitory activity than the other formulations against Escherichia coli and
Staphylococcus aureus. The enzymes incorporated into the alginate film (GOx,
LPS, Lysozymes with EDTA) showed good antimicrobial and minimizing the
microbial contamination during storage period. Good sensory score was obtained
in the carrot treated with formulation VII [GOx (5 mg/ml), LPS (10 mg/ml) and
lysozyme (0.5 mg/ml) with EDTA (0.3 mg/ml)] and accepted throughout the
storage period both 6 ºC and ~26°C.
The control of browning and enhancing the shelf-life of apple puree by
GOx, catalase and LPS were studied. The LPS and catalase did not show
significant antibrowning activity. However, GOx was very effective in the
presence of catalase combination. The formulation-VII [(GOx (100mg) + Catalase
(0.1ml) + LPS (40mg)] was found to be effective control of browning and
microbial growth in apple puree. GOx-catalase and LPS treatment produced good
sensory scores.
227
The effects of GOx-catalase with calcium ions for the improvement of fruit
salad quality were evaluated. Fruit salad prepared with the combination of GOx-
catalase, optimized calcium ions exhibited effective antimicrobial, antibrowning
activities. Further, it provided good sensory score with respect to colour, texture,
taste, odour and overall acceptance.
228
CHAPTER 7
CONCLUSIONS AND SCOPE FOR FUTURE WORK
In the present investigation, glucose oxidase producing
Aspergillus awamori MTCC 9645 was isolated and optimum media constituents
were analyzed by single factor analysis and central composite design. The single
factor analysis method indicated glucose, proteose peptone, calcium carbonate and
di-ammonium hydrogen phosphate contributing higher GOx production. Statistical
method for media optimization resulted significant increase in GOx production.
Alginate film coated carrot with the formulation of GOx, LPS and lysozyme with
EDTA exhibited good shelf-life. Apple puree was processed with the combination
of GOx-catalase with LPS exhibited good antibrowning and antibacterial
properties. The combination of GOx-catalase with calcium ions showed
antibacterial and antibrowning activity in salad that increased the storage stability
and high sensory score. The observations from the present findings were clearly
showed that potential of glucose oxidase would use in food processing and
preservation with combined preservation treatments.
Currently, the industrial and food applications of the commercialization of
GOx have an important role. Although, it is remains very less in comparison to
that of hydrolytic enzymes. This enzyme can be used in different formulation for
innovative food processing, preservations. This leads to substantial increase of
utilization in food industry.
Nowadays, consumers are more concerned over the safety of food and so,
the demand for natural foods has spurred the search for biopreservatives. The
antimicrobial packaging is a rapidly developing technology that can be employed
for controlling food borne microbial outbreaks caused mainly by the easily
prepared and minimally processed fresh vegetables and fruits. A greater emphasis
229
on safety features associated with the addition of natural antimicrobial agents
maybe the next area for food processing and preservation. Many of the
antimicrobial compounds studied are not permitted for food application, as they
need to migrate to the food to be effective. Technical challenges exist in
implementing appropriate antimicrobial agents into the process. Researchers have
focused primarily on developing new approaches and testing new methods on
model systems and not quite as much on applications in real food products.
Antimicrobial enzymes and natural compounds must focus on the technical
feasibility, consumer acceptance and food safety aspects of antimicrobial agents in
addition to their chemical, microbiological and physiological effects.
230
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258
LIST OF PUBLICATIONS, PRESENTATIONS AND
CONFERENCES
List of publications in National and International Journals
· Sathiya moorthi P, Rajkumar R and Kalaichelvan PT (2007). Applications of glucoseoxidase enzyme in food Industries. Modern Food Processing 3(1): 74–76.
· Sathiya moorthi P, Periyar selvam S, Deecaraman M and Kalaichelvan PT (2008).
Efficiencies of alternate carrier electives for Rhizobium bio-fertilizer. ICFAI Journal of
Life Sciences 2(2): 41–51.
· Sathiya moorthi P, Deecaraman M and Kalaichelvan PT (2009). Co-immobilization ofBacillus megaterium and Rhizobium leguminoserum for efficient phosphorus andnitrogen fixation on Cicer arietinum. Journal of Plant Disease Sciences 4(1): 41–51.
· Sathiya moorthi P, Jijendrakumar P, Jayakumar and Kalaichelvan PT (2008).Lactoperoxidase enzyme system: The future of preservation. Modern Food Processing3(11): 60–64.
· Sathiya moorthi P, Periyar selvam S, Sasikalaveni A, Murugesan K and KalaichelvanPT (2006). Biological decolorization of textile dyes and their effluents using white rotfungi. African Journal of Biotechnology, 6(4): 425–429.
· Sathiya moorthi P, Periyar selvam S, Deecaraman M, Murugesan K and KalaichelvanPT (2008) Biosorption of textile dyes and effluents by Pleurotus florida, Tameteshirsuta and evaluation of their laccase enzyme activity. Iranian Journal ofBiotechnology, 5(2): 1–5.
· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT (2008). Bioremediation ofauto mobile oil effluent by Pseudomonas sp. Journal of Advanced Biotechnology,6(12): 34–37.
· Sathiya moorthi P, Deecaraman M, Praveen kumar K, Kishan vaidyanat N andKalaichelvan PT (2009). Screening of α- Amylase inhibitors from Natural sources;with particular reference to Stevia rebaudianl. Journal of Advanced Biotechnology,8(10): 33–36.
· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. Enhancing shelf-life ofcarrot by using sodium alginate film incorporated with glucose oxidase,lactoperoxidase and lysozyme. Journal of Food Science (Under Revision).
259
List of paper presentation
· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. “Screening of glucoseoxidase producing fungi and factors regulating production”. National Conferenceon “Resent Trends in Mycological Research” on December 28th and 29th, 2006,Mycological Society of India at J.J. College of Science, Pudukkottai, Tamil Nadu.
· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. “Preparation of ediblecarboxy methyl cellulose film incorporated with glucose oxidase, lactoperoxidaseand lysozyme for prawn preservation” at Young Scientist conference at LoyolaCollege, Chennai 3rd and 4th December 2007.
· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. “Incorporation ofpartially purified antimicrobial enzymes in to sodium alginate films for carrotpreservation”. Safety assessment and consumer production with reference toDairy and Food industry. Dept. of Dairy Science, Madras Veterinary University,Chennai. 28th and 29th November 2007.
· Sathiya mooorthi P, Deecaraman M and Kalaichelvan PT. “Industrial enzymes forfood processing and preservation”. Safety assessment and consumer productionwith reference to Dairy and Food industry. Dept. of Dairy Science, MadrasVeterinary University, Chennai. 28th and 29th November 2007.
· Sathiya moorthi P, Rekha G, Sasikalaveni A, and Kalaichelvan PT.“Decolorization of Textile dyes by white rot fungi” in the National Conference onCurrent Perspectives in Aquatic Biology, held at University of Madras, Chennai.During 17th and 18th March 2006.
· Praveeven kumar K, Kishan vaidyanat N and Sathiya moorthi P. "Production ofedible films incorporated with anti microbial enzymes for the preservation ofcarrots" in the International conference, Bangalore Bio-2007, during 7th to 9th
June 2007.
260
List of conference and work shop participated
· Training course in “Fruit and Vegetable preservation and nutrition” coducted byGovernment of India, Ministry of Food and Nutrition Board, during the period of9th –13th July 2007.
· Participated in the Annual Conference on “Strategies for food safety and qualityin India. Jointly Organized by TNAU Coimbatore and University of California,Dvis, USA at TNAU, Coimbatore, during 26-28th October 2006.
· Participated in the National workshop on “Recent trends in Biotechnology andanalytical instrumentation” on 5–7th March 2008 conducted by Department ofPlant Biology and Biotechnology, Presidency College and SophisticatedAnalytical Instrument Facility, IIT Chennai.
· Participated in the conference on “Agro Food Processing Technologies” (Qualityand Safety in Fruits and Vegetables Processing), Organized by Confederation ofIndian Industry and USAID during 24th July 2009.
· Participated in the Biotechnology: UK and Indian Perspectives Organized byBritish council and Anna University, durind 17th and 18th Febraury 2008.
· Participated in the seminor on Herbal analysis and applications, Centre for HerbalSciences, University of Madras, Chennai, 29th October 2008.
· Participated in the “Theme workshop on Emerging Trends in EnvironmentalBiotechnology” held during 12th–14th January 2009 at National Institute ofTechnology Karnataka, Surathkal.
· Workshop on “Medical Biotechnology” at Biomedical Research Unit and LabAnimal Centre (BRULAC), Saveetha University, Chennai, during 4th–8th June2007.
· Attended the advanced technical workshop on “Water treatment solutions andwaste water management at Living Waterfine Technologies Pvt Ltd, Chennai,during 11th August 2006.
· Participated in the training programme at CIMAP Hyderabad on“Entrepreneurship development through medicinal and aromatic plantstechnologies” 9th–11th January 2008.
· Participated in the National Conference at University of Madras on “Trends inAlgal Biotechnology” 17th–19th February 2008.
261
Curriculum vitae of P. Sathiya moorthi
He was born on 2nd May 1981 at Salem, Tamilnadu. He have completed
under graduation in B.Sc., Microbiology (2001) at A.V.S. College of Science,
Periyar University and successfully finished his post graduation in M.Sc.,
Industrial Microbiology (2003) at Centre for Advanced Studies in Botany,
University of Madras. After completion of his Master Degree, he joined at Chinnu
Exports Bio-Products Division as Microbiologist and plant in-charge from 2003 to
2005. During this period he was gained the experience in various optimizations of
industrial processes, post harvest technology, value addition of agricultural
product, natural food processing and preservation, drinking water and effluent
treatment.
After that he was joined as a full time Research Scholar in the department
of Industrial Biotechnology, Dr. M.G.R. Educational and Research Institute
University, Maduravoyal, Chennai under the supervision of Prof. P.T.
Kalaichelvan and co-supervision of Prof. M. Deecaraman. He started his research
work on January 2006 in the production of glucose oxidase from fungi and their
applications in food processing and preservation. During the research period he
published several research papers and articles at National, International Journals
and scientific magazines. Further, he was also participated and presented many
research papers in various conferences and seminars. Moreover, he acquired the
following experience during his studies like Microbiological Techniques,
Biochemical analysis, Bioprocess techniques and assesses the research activities to
project students.
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