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DIGESTIVE PROTEASES FROM THE STOMACHLESS CUNNER FlSH (TAUTOGOLABRUS ADSPERSUS): PREPARATION AND USE AS FOOD
PROCESSING AID
BY
MARY ABENA KYEl
A Thesis subrnitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Master of Science
Department of Food Science and Agricultural Chemistry Macdonald Campus of McGill University
Montreal, Canada
January, 1997
O Mary A.Kyei, 1997.
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ABSTRACT
Digestive proteases were isolated from the pancreas of the stomachless
cunner fish (Tautogolabrus adspersus) and characterized in terms of their
physicochemical properties, their ability to hydrolyze native pectin
methylesterase (PME) from orange and polyphenol oxidases (PPO) from
mushroom and the ability of the cunner enzyme(s) to maintain the stability of
orange juice cloud.
Optimization of the steps used for the preparation of the proteases
resulted in an increase in specific activity from 0.55 to 2.04 unitslrng (i.e. from the
crude to the trypsin fraction), a purity of 3.7 fold and a yield of 1.4 %. The
ammonium sulfate, acetone and trypsin fractions showed good stability when
stored for four weeks on ice and -20°C, with observed specific activity decreasing
in the order trypsin fraction, acetone fraction, and ammonium sulfate fractions.
The trypsin fraction had two protein bands with pl in the ranges of 6.24-6.83 and
9.66-10.25. The molecular weights of the 2 proteases were 24,000 and 14,400
@ kDA respectively. The 24,000 kDA protease was described as trypsin and the
14,000 kDA protease as trypsin-like, based on their action on the synthetic
substrate BAPNA. The cunner trypsin had 220 amino acid residues with a
relatively low basic amino acid content. Soybean trypsin inhibitor (SBTI)
competitively inhibited the cunner trypsin with V,,, ranges of 1.39 ic 0.001 to 1.42
k 0.002, and Km values of 0.99 f 0.002 to 4.94 i 0.040 mM. The K, values
decreased from 3.48 to 3.3 x IO ' mM. Aprotinin showed a mixed type of
inhibition, with V,, decreasing from 0.74 k 0.037 to 0.42 t 0.051 unitslminlmg
and Km also decreased from 1.81 f 0.23 to 0.3 f 0.10 mM. The K, values also
decreased from 13.7 k 0.001 to 1.9 k 0.003 x 10' mM. Furthermore, the cunner
trypsin exhibited a type 3 Km dependence on temperature, suggesting that the
affinity of the enzyme for its substrate was highest at 10°C which coincides with
the habitat temperature of cunner.
a The cunner trypsin fraction exhibited exceptional capacity to hydrolyze
native proteins versus the bovine trypsin. Incubation of native PME with cunner
or bovine trypsin resulted in a loss of 75% or 35% in PME activity respectively.
Similarly, a 75% or 55 % loss in PPO activity was observed after treatment with
cunner and bovine trypsin respectively. Bovine trypsin, however, hydrolyzed the
heat-denatured PME and PPO better than the cunner trypsin. Also, there was no
reactivation of both PME and PPO activity after treatment with either the cunner
or bovine enzyme during storage at 4 O C for 3 weeks. However, PPO retained up
to 20% or 50% of the initial activity after treatment with cunner or bovine trypsin,
respectively.
A 3 x 3 factorial design involving the factors of temperature, enzyme
concentration and incubation time carried out gave an r' of 0.92 and 0.95 for
cunner and bovine trypsin treated PME respectively. On the other hand, an r' of
0.91 and 0.94 was obtained for the combined effects using cunner and bovine
trypsin for PPO inactivation. Validation of the mode1 of PME inactivation
measured as the % cloud remaining revealed that the cunner trypsin fraction
upheld the cloud stability of cloud juice better than bovine trypsin, with cunner
trypsin retaining more than 90% of the cloud whereas the juice treated with
bovine trypsin only resulted in a 70% retention of the juice cloud.
RÉSUMÉ
Des protéases digestives ont été extraites du pancréas d'un poisson, la
tanche-tautogue (Tautogolabrus adspersus) et caractérisées en termes de leurs
propriétés physico-chimiques, de leur capacité à hydrolyser des pectine-
methylestérases natives d'oranges et des polyphénol oxydases de champignons
et de leur capacité a maintenir la turbidité d'un jus d'orange.
Les différentes étapes du protocole utilisé pour la préparation des
protéases ont permis une augmentation de I'activité spécifique de 0,55 à 2'04
unitéshg (de l'extrait brut a la fraction trypsine), un taux de purification de 3,7 et
un rendement de 1,4%. Les fractions sulfate d'ammonium, acétone et trypsine
ont montré une bonne stabilité pendant quatre semaines de conservation a - 20°C, l'activité spécifique diminuant le plus dans la fraction trypsine, puis dans la
fraction acétone et enfin dans les fractions sulfate permettait d'ammonium. La
fraction trypsine possède deux bandes de protéines avec des pl de 6,24-6,83 et
9,66-10'25. Les poids moléculaires des deux protéases sont respectivement de
0 24.000 et 14.400 kDa. La protéase de 24.000 kDa a été décrite comme une
trypsine alors que celle de 14.400 kDa comme étant de type trypsine, en se
basant sur leur action sur le substrat synthétique BAPNA. La trypsine de la
tanche-tautogue comprend 220 résidus d'acides aminés avec une teneur en
acides aminés basiques relativement faible. L'inhibiteur de la trypsine de soja
(SBTI) a inhibé de façon compétitive la trypsine de la tanche-tautogue avec des
V,,, compris entre 1,39 + 0,001 et 1,42 I 0,002. et des valeurs de Km comprises
entre 0,99 + 0,002 et 4'94 t 0,040 mM. Les valeurs de Ki ont diminué de 3,48 a
3,3 x 10-4 mM. L'aprotinine a démontré une inhibition de type mixte, avec un V,,,
diminuant de 0,74 k 0,037 à 0,42 k 0,051 unitéslminlmg et un Km diminuant de
1'81 + 0,23 à 0'3 +_ 0,10 mM. Les valeurs de Ki ont également chuté de 13,7 k
0,001 à 1,9 I 0,003 x 10-3 mM. De plus, la trypsine de la tanche-tautogue a
démontré une dépendance à la température de type 3 Km, ceci suggérant que
a l'affinité de l'enzyme pour son substrat est plus élevée à 10°C et coïncidant avec
la température d'habitat de la tanche-tautogue
0 La fraction trypsine de la tanche-tautogue a montré une capacité
exceptionnelle d'hydrolyse de protéines natives en comparaison de la trypsine
bovine. L'incubation de PME native avec la trypsine de la tanche-tautogue ou la
trypsine bovine s'est soldée, respectivement, par une perte de 75% ou 35 % de
I'activité de la PME. De même, une perte de 75% ou 55 % de l'activité de la PPO
a été observée apres traitement avec la trypsine de la tanche-tautogue et la
trypsine bovine, respectivement. Cependant, l'hydrolyse de la PME et la PPO
dénaturées par la chaleur a été meilleure par l'action de la trypsine bovine que
celle de la trypsine de la tanche-tautogue. De plus, il n'y a pas eu de réactivation
des activités de la PME et de la PPO après traitement avec la trypsine de la
tanche-tautogue ou la trypsine bovine et après conditionnement à 4°C pendant 3
semaines. Cependant, la PPO a retenu 20 % ou 50% de son activité initiale,
respectivement, après traitement avec la trypsine de la tanche-tautogue ou la
trypsine bovine.
Un modèle factoriel de type 3x3 incluant les facteurs de température, de
a concentration de l'enzyme et du temps d'incubation mis en oeuvre a donné,
respectivement, un de 0,92 et de 0'95 pour la PME traitée avec trypsine de la
tanche-tautogue et la trypsine bovine. D'autre part, un ? de 0.91 et de 0,94 a été
obtenu par les effets combinés de la trysine de la tanche-tautogue et de la
trypsine bovine pour l'inactivation de la PPO. La validation du modèle
d'inactivation de la PME mesurée comme le pourcentage de turbidité restante a
révélé que la fraction trypsine de la tanche-tautogue de maintenir la turbidité
d'un jus plus efficacement que la trypsine bovine, avec la trypsine de la tanche-
tautogue retenant plus de 90% de la turbidité alors que le jus traité avec la
trypsine bovine ne retenait que 70% de la turbidité.
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to my supervisor Dr. B. K.
Simpson for his guidance, patience, encouragement in various manner,
assistance and invaluable advice throughout the course of this study.
I wish to also acknowledge the assistance of Mr. Benny Gibbs in the
amino acid analysis, to Dr. 1. Alli (Chair of Food Science Department), Ms Lise
Stiebel, Ms Barbara Laplaine, and to al1 the staff of the Food Science
Department. I also express my sincere thanks to my colleagues, Mr. Amaral
Sequeira, Ms Luzette Teruel, Mr. Victor Awafo and Mr. Farag Eltaib, for their
help and advice any time I called on them. I also want to thank al1 the students of
Food Science Department for creating an atmosphere conducive for both
studying and socializing. For supporting me financially, I would like to thank ... I would like to thank the following for their friendship, and also making a home
away from home for me: Dr. and Mrs. 1. N. A. Ashie, Mr. and Dr. (Mrs.) F.
Yeboah, Dr. and Mrs. S. Aggrey, Dr. and Mrs. G. B. Awuah, Mrs. Eva Yapo, Ms
Wigdan Madani, Mr. and Mrs. S. Chimpango, Mr. and Mrs. R. Molenaar, Mr. and
Mrs. V. Awafo, Ms Ramon Sareevoravitkul, Mr. Michael Owusu-Manu and Mr.
Virgil Mensah Dartey.
I would like to deeply thank my husband, Mr. Nana Kwesi Agyepong, for his love,
enormous support, assistance and patience during rny absence from home. i
also appreciate deeply my parents, Mr. and Mrs. Kyei and my brothers and
sisters, my parents-in-law Mr. and Mrs. Agyepong and family for their support
love and unceasing prayers.
Above all, I am extremely grateful to the Lord God Almighty for strength and
comfort during my long stay away from my family.
vii
TABLE OF CONTENTS
. . ........................................................................................................... ABSTRACT II .............................................................................................................. RÉSUMÉ iv
................................................................................... ACKNOWLEDGEMENTS vi ........................................................................................ TABLE OF CONTENT vii
LIST OF FIGURES .............................................................................................. x ... .............................................................................................. LIST OF TABLES XIII
LIST OF ABBREVIATIONS .............................................................................. xiv
.......................................................................................... I INTRODUCTION 1
.................................. 2.1 Endogenous and exogenous enzymes in food processing 3 ................................................................................. 2.1.1 Endogenous enzymes 3
....................................................................................... 2.2 Exogenous enzymes 3 ...................................................................................... 2.2.1 Microbial sources 5
............................................................................................ 2.2.2 Plant sources 5 ......................................................................................... 2.2.3 Animal sources 7
2.3 Marine enzymes ................................................................................................... 7 ..................................................... 2.3.1 Trypsin(ogen)s from the marine sources 10
. . . .
2.3.2. Trypsin from the stomachless cunner ..................................... 2.3.2. Hydrolysis of native and denatured proteins ..........................
.......................................................... 2.3.3: Comparative biochemistry 2.3.4: Applications and potential applications of marine enzymes ir
.......................................................... ............................... industry .. 2.4 Pectin methylesterase and the citrus juice industry ......................
................... IO
..........S........ 12
................... 13 the food ................... 16 ................... 19
.......................................... 2.4.1 .Pectins. the substrate of pectin methylesterase 20 2.4.2. Citrus juices. pectin and cloud stability ...................................................... 21
...................................................................... 2.4.3. Methods of PME inactivation 22 O .................................................................. (a) Thermal inactivation (90-1 15 C) 22
(b) Low pH inactivation of PME ......................................................................... 23 .............................. (c) Supercritical carbon dioxide (Coq) inactivation of PME 23
............................................................ (d) High pressure inactivation of PME 2 4 (e) Degradation of PME substrates using pectinase ......................................... 24 (9 Use of PME inhibitors ................................................................................... 24
................................................ (g) Inactivation of PME by proteolytic enzymes 25 ...................................... 2.5 Polyphenol oxidases (PPO) and enzymatic browning 26
2.5 Inactivation of polyphenol oxidases (PPO) .................................................... 27 ..................................................................................... (a) Thermal inactivation 28
(b) Reducing agents .......................................................................................... 28 .......................................................................................... (c) Chelating agents 29
............................................................................................. (d) PPO in hibitors -29
viii
(e) Oxygen exclusion ........................................................................................ 30 ............................................................................................ 2.6. Rationale for study 30
....................................................................................................... 2.7. 0 bjectives 3 2
................................................................... I II MATERIALS AND METHODS 33 .......................................................................................... 3.1 Biological specimen: 33
............................................................................................................ 3.2 Reagents 33 ...................................................................................................... 3.3 Methodology 3 4
.......................................................... 3.3.1 Recovery and purification of trypsin : 3 4 ................................................................................................ 3.3.2 Enzyme assay 3 5
....................................................................................... 3.3.2.1 Amidase activity 3 5 3.4 Protein determination ......................................................................................... 36
............................................................ 3.5 SDS polyacrylamide gel electrophoresis 36 ............................................................................................. 3.5.1 Casting of gels 37
.............. 3.5.2 Sample preparation and SDS polyacrylamide gel electrophoresis 37 ................................................................ 3.5.6 Molecular weight determinations -40
............................................................................................. 3.6 lsoelectric focusing 41 ...................................................................................... 3.6.8 Fraction screening -43
........................................................................................... 3.6.7 Refractionation -43 ....................................................................... 3.7 Amino acid composition analysis 43
............................................................................................... 3.8 Inhibition studies -45 ..................................................... 3.8 Pectin methylesterase activity determination 46 ...................................................... 3.8.1 Effect of cunner trypsin on PME activity 47
......................................... 3.8.2 Effects of cunner trypsins on heat-treated PME 47 ............................................................................. 3.9 PPO activity determination 47
.................................................................. 3.9.1 Effect of cunner on PPO activity 48 3.9.2 Effect of cunner trypsin on heat-treated PPO activity .................................. 48
................................................... 3.10.1 Optimization studies 1 experimental design 48 .................................................................................... 3.10.2 Statistical analysis 49
............................................................................. 3.11 . 1 Treatment of orange juice 49 3.1 1.2 pH measurements ..................................................................................... 51
................................................................. 3.1 1.3 Storage of treated orange juice 51
................................................................ IV RESULTS AND DISCUSSIONS 52 ............................................................................. 4.1.1 Recovery of trypsin fraction 52
4.1.2 Elution profile of the trypsin fraction on SBTI-Sepharose Affinity media ...... 52 .......................................................... 4.1.3 Electrophoresis of the trypsin fraction 52
4.1.4 Stability of Various Fractions at Different Storage Temperatures ................ 58 .................................................... 4.1.5 lsoelectric focusing of the trypsin fraction 58
................................................................. 4.1.6 Amino acid composition analysis 62 .................................................... 4.2 The Effect of Inhibitors on the trypsin fraction 65
.................................................................. 4.3. Dependence of Y, on Temperature 68 4.4 Effects of trypsins on Native PME and PPO ...................................................... 73
........................................ 4.5 Effects of trypsins on heat-denatured PME and PPO 79 4.6. Reactivation studies of trypsin-treated PME and PPO ...................................... 83 4.7 Combined effect of [E]/[S] ratio. incubation time and temperature on PME and PPO ................................................................................................................... 86
................................................................................................. 4.8 Model Validation 95
V CONCLUSIONS ............................................................................................................................................. 98
LIST OF FIGURES
Flgure Title
Calibration curve for protein content determination using the enhanced lowry method and bovine serurn albumin (BSA) as standard.
Elution Profile of the acetone fraction on SBTI-Sepharose Affinity media.
Calibration curve for molecular weight determination for SDS-PAGE electrophoresis.
SDS-PAGE gel showing the migration of the proteins
Effect of storage temperature on the stability of the ammonium sulfate, acetone and trypsin fractions, (a) storage on ice and (b) storage at -20°C
(a) Fractionation and (b) Refractionation of the trypsin fraction after isoelectric focusing at 4 O C on Biorad Rotofor System
Chromatogram of amino acid standards at 570 nm using norleucine as interna1 standard
Effect of increasing BAPNA concentration on s bti-ln hibition. (a ) cunner trypsin and (b) bovine trypsin
Effects of increasing BAPNA concentration on aprotinin inhibition. (a) cunner trypsin and (b) bovine trypsin
Lineweaver-Burk plots for the trypsins using SBTl as inhibitor. (a) bovine trypsin and (b) cunner trypsin, with 1 mM BAPNA (pH 8.2) as substrate
Lineweaver-Burk plot for the trypsins using aprotinin as inhibitor. (a) bovine trypsin and (b) cunner trypsin with 1 mM BAPNA (pH 8.2) as substrate
Dependence of cunner trypsin Km on temperature.
Calibration curve for PME activity assay
Page
54
Effect of cunner and bovine trypsins on native PME activity using an [E]/[S] ratio 0.5 and (a) 0.35% and (b) 0.5% pectin solutions.
Effect of cunner and bovine trypsins on native PME activity using an [E]I[S] ratio of 1 and (a) 0.35% and (b) 0.5% pectin solutions
Effect of cunner and bovine trypsins on native PME activity using an [E]/[S] ratio of 2 and (a) 0.35% and (b) 0.5% pectin solutions
Effect of cunner and bovine trypsins on native PPO activity, using 5 mM DOPA as substrate at 25°C
(a) Effect of heat treatment on PME activity and the effect of cunner and bovine trypsins on heat-denatured PME at (b) 50°C and (c) 55°C.
(a) Effect of heat treatment on PPO activity and the effect of cunner and bovine trypsins on heat-denatured PPO ai (b) 40°C and (c) 50°C.
Reactivation studies of trypsin treated PME with (a)cunner and (b) bovine.The substrate was 0.5 % pectin at 25°C. The storage studies were carried out at 4°C.
Reactivation studies of trypsin treated PPO at 4"C, using 5 mM DOPA (pH 6.5) as substrate.
Three dimensional response surface plots showing the effects of [cunner trypsin]/[S], incubation time and temperature, on PME activity. (a) effect of temperature and [E]/[S] ratio at constant time (2.5h) (b) effect of [E]I[S] ratio and time at constant temperature (25°C) and (c) effect of time and temperature at a constant [E]/[S] ratio (1).
Three dimensional response surface plots showing the effects of [bovine trypsin]l[S] ratio, incubation time and temperature, on PME activity. (a) effect of temperature and [E]/[S] ratio at constant time (2.5h) (b) effect of [E]/[S] ratio and time at constant temperature (25°C) and (c) effect of tima and temperature at a constant [E]/[S] (1).
xii
@ 4.24 Three dimensional response surface plots showing the 93 effects of [cunner trypsin]/[S] ratio, incubation time and temperature, on PPO activity. (a) effect of temperature and [E]I[S] ratio at constant time (2.5h), (b) effect of [E]/[S] ratio and time at constant temperature (25°C) and (c) effect of time and temperature at a constant [E]/[S] ratio (1).
4.25 Three dimensional response surface plots showing the 94 effects of [bovine trypsin]l[S] ratio incubation time and temperature, on PPO activity. (a) Effect of temperature and [E]/[S] ratio at constant time (2.5h), (b) effect of [E]/[S] ratio and time at constant temperature (25°C) and (c) effect of tirne and temperature at a constant [E]/[S] ratio (1).
4.26 (a) An [E]I[S] ratio of 0.3 and 1.5 bovine and cunner trypsin 96 at 35" for 1.3 and 0.4 h respectively and (b) [E]/[S] ratio of 1.5 and 0.9 of bovine and cunner trypsin at 15°C for 2.5 h. The juice were stored at 4OC
. . . X l l l
LIST OF TABLES
Table Title
Examples of some endogenous enzymes and their effects
Examples of Microbial Enzymes used in Food Processing
Examples of plant enzymes used in food processing
Some examples of animal enzymes used in food
processing
Some examples of animal enzymes used in food
processing
Hydrolysis of protein substrates by cunner enzymes
Relative Activities of Trypsins from various sources
Formulations of SDS PAGE Resolving Gel
Formulations of SDS-PAGE stacking Gel
Solutions for running electrophoresis and staining Gets
Program for amino acid analysis on Beckman HPLC
System 6300
Values of coded Ievels in Experimental design
Recovery of trypsin from the pancreas of the cunner fish
One-way ANOVA of storage at O OC and -20 OC
Amino acid composition of trypsin fraction
Kinetic data on SBTl and aprotinin inhibition of cunner and
bovine trypsin
Parameter Estimates for percent PME and PPO
inactivation
Page
4
6
8
9
xiv
4 8 0
Ala
ANOVA
Arg
Asn
A ~ P BAPNA
BSA
CYS
dA/min
DOPA
Gln
Glu
G ~ Y
H is
IIE
KDa
Ki
Kr" Leu
LYS
Met
MPa
Phe
PME
PPO
Pro
R f
S BTl
LIST OF ABBREVIATIONS
Absorbance at 280 nm
Alanine
Analysis of variance
Arginine
Asparagine
Aspartic acid
N-a-Benzoyl-arginine-p-nitroanilide
Bovine serum alburnin
Cystein
Change in absorbance pet- minute
Dihydroxyphenylalanine
Glutamine
Glutamic acid
Glycine
Histidine
lsoleucine
Kilodaltons
Inhibitor dissociation constant
Substrate affinity constant
Leucine
Lysine
Methionine
Megapascal
Phenylanine
Pectin methylesterase
Polyphenol oxidase
Proline
Mobility
Soybean trypsin inhibitor
a PAGE
Ser
TAME
Thr
T ~ P
TY r Val
v m a x
Polyacrylamide gel electrophoresis
Serine
Tosylargine methyl ester
Threonine
Tryptophan
Tyrosine
Valine
Maximum velocity
CHAPTER 1
INTRODUCTION
Enzymes have been used to accomplish specific desirable changes in foods, for centuries. In brewing, malted barley have been used for starch conversions, the addition of saliva to starchy products in preparing fermented liquors, and the tenderization of meat by wrapping it in bruised leaves of papaya tree to tenderize it are a few examples of ancient use of enzymes (Whitaker, 1994). These techniques were handed down from generation to generation without complete understanding of the reactions and changes involved. Sorne of these traditional practices have been refined to the present-day uses of enzymes like chymosin, a-amylase, and papain in the food industry (Simpson and Haard,
their 1987a). The importance of enzymes in food processing has resulted in the more recent application of irnmobilized enzyme systems to the processing of foods and the production of food and chemical ingredients (Whitaker, 1994).
I ) Examples of some of the applications of immobilized enzyme technology are (a) immobilization of a-galactosidase used in the hydrolysis of raffinose in sugar
beet juice which hinders crystallization of sucrose in sucrose production (Reilly, 1980), and (b) the production of fructose from glucose using immobilized glucose isomerase (Burke, 1980).
Also with the advent of genetic engineering, a wider range of enzymes will become available on a larger scale and thus increase the scope of enzyme technoiogy (Price and Stevens, 1985). In genetically engineered enzymes, one or two aspects of the enzyme are improved and include (i) modification of the effect of pH andlor heat on the stability, (ii) altering the temperature andlor pH optimum, (iii) adjusting the substrate specificity and binding efficiency, (iv) increasing the resistance to solvents, or inhibitors, and (v) modifying the enzyme to catalyze an aitogether new reaction. Examples of applications of genetically engineered enzymes in food processing are (i) improving the quality of tomatoes by modifications of the enzymes polygalacturonase and PME. The modifications lowers the production of these enzymes in tomatoes. The modification also helps
O to improve the appearance of tomato paste made from the engineered tomato (Schuch, 1994), (ii) the production of genetically engineered acetolacetate decarboxylase for use in reducing the maturation time in brewing (Dornenburg
a and Lang-Hinricks, 1994) and (iii) the production of chymosin, which is used in the curdling of milk, by cloning the eukaryotic gene into microorganism so that the enzyme can be produced by normal fermentation (Dornenburg and Lang- Hinricks, 1994).
Enzymes have remarkable catalytic properties, especially when compared with other catalysts. The advantages include high catalytic activity under mild conditions of pH and temperature, high specificity, thus obviating undesirable side reactions. lndustrial catalysts lack this specificity of reaction, which precludes their use for modifying specific components of food systems. In addition, enzymes are natural and non-toxic substances, they are active at low concentrations. Furthermore, the rate of the reaction catalyzed by the enzymes can be controlled by adjusting the temperature, pH and the amount of enzyme employed, and they can be inactivated after use. These features of enzymes make them highly desirable for use as processing aids for various industrial products. In recent years they have been exploited on an increasing scale in food, pharmaceutical and chemical industries (Richardson and Hyslop, 1985).
Enzymes are widely distributed in biological systems and the main
a sources of enzymes used in food processing are from plants, animais and microorganisms. In plants, the enzymes are distributed in the latex, fruits, leaves, seed, flower, rhizome, sprouts and Sap of the plant (Macdonald et al., 1993; Robinson and Dry, 1992; Rillo et al., 1992; Seymour et al., 1991; Wicker and Temelli, 1988; Schwimmer, 1983). In animal sources, the enzymes are distributed mainly in the stomach, oral tissues, the pancreatic tissues, intestines, liver and the muscles. Other underutilized animal source is the protease-rich fish offal which often pose a disposa1 and pollution problem (Simpson et al. 1991; Schwimmer, 1981). In microbial sources the type of microorganism and the type of media is a prerequisite for the type of enzyme to be produced.
Homologous (anaiogous) enzymes from different sources and species exhibit different physical, chemical and biochemical properties. These properties include temperature optimum and stability, pH optimum and stability. These in turn affect the catalytic efficiency and thereby its applicability as food processing aid for a particular industrial bioconversion. In some conversions, a high temperature stability is of benefit, e.g., the production of corn-syrups, the step of
O the conversion of dextrins to glucose by glucoamylase, an enzyme with a high temperature stability is advantageous and makes the selection of rnicrobial glucoamylases beneficial over those from other sources (Spradlin, 1989).
CHAPTER II
2.1 ENDOGENOUS AND EXOGENOUS ENZYMES IN FOOD PROCESSING
2.1 .l ENDOGENOUS ENZYMES
Foods are complex biological materials, and as such subject to a wide variety of modifying agents. Among these are microorganisms that cause undesirable spoilage or beneficial fermentation, endogenous biochemicals that undergo such changes as autoxoidation, and endogenous enzymes that cause nurnerous desirable and undesirable changes (Richardson and Hysslop, 1 985). Majority of food-related enzymes can either improve or impair food quality depending on the extent of enzyme action on (i) the food material, (ii) the processing, or (iii) the final product, as well as such variables as time,
0 temperature, pH, and past history of the food (Schwimmer, 1981). Examples of some endogenous enzymes and their effects on foods are shown in Table 2.1
2.2 EXOGENOUS ENZYMES
There are three main sources of enzymes used in food processing, namely microorganisms, plants and animals. The greatest variety of industrial enzymes are presently derived from microbiat sources, with lesser diversity coming from plant and animal sources (Godfrey and Reichelt, 1983). The group of enzymes that find the most use in industry is proteolytic enzymes. Proteases from various sources differ in their catalytic and physical properties, and whether
a particular enzyme would be suitable for use in a particular industrial application depends on several factors such as the type of transformation desired and the nutritional constituents of the food material. For many uses, the specificity of the
Table 2.1 : Examples of sorne endogenous enzymes and their effects.
Enzyme Desirable Effect Undesirable Effect Pectic enzymes
Cathepsins
Phenolases
Lipoxygenases
Peroxidases
Firmness of green beans
Liquefaction of fish tissues in fish saucelpaste manufacture Developrnent of color in tea
Bleaching of hard wheat flour; lmprovement of the texture wheat flour doughs.
Degradation of H,O, and other peroxides in foods
Loss of juice cloud in orange juice Postharvest spoilage and textural changes in fresh fruits and vegeta bles
Postrnortem deterioration of muscle foods.
Enzymic browning of fruits vegetables such as apples, banana potatoes, and crustacean species such as shrimp
Destruction of vitamin A and provitamin A, carotenoids pigments, and essential polyunsaturated fatty acids Production of off-flavors in frozen vegetables and in stored cereals.
Oxidative destruction of vitamin C, bleaching of carotenoids and anthocyanins
Adapted from Richardson and Hysslop, 1985
9 Protease is of paramount importance (Simpson and Haard, 1987a), especially
when it is desired to modify a single component during food processing.
2.2.1 MlCROBlAL SOURCES
Microbial enzymes are obtained from non-pathogenic, non-toxigenic cultures and also those cultures that do not produce anti-biotics. Microbial enzymes form the greatest proportion of the food grade enzymes because the source microorganisms are very versatile, for example, they can be altered by
mutation or genetic engineering to produce a greater quantity or a different enzyme; the recovery of enzymes is often very easy since many microbial enzymes are extracellular; there are readily available, and rnicroorganisms have a very high rate of enzyme production.
lnspite of the potential diversity that can be achieved by use of microbial enzymes, only few species of microorganism have been extensively evaluated and approved as safe for use to produce industrial enzymes. The reason for this being the high cost of getting a microorganism approved by regulatory authorities
a (Simpson and Haard, 1987). Table 2.2 shows a list of some microbial enzymes used in food processing and their sources.
2.2.2 PLANT SOURCES
By far the two major sources of enzymes from higher plants used in food processing are the green papaya melon for the preparation of products rich in proteolytic enzymes, and germinated barley, malt, which contains starch- digesting enzymes used in brewing and to some extent as an adjunct in breadmaking. Minor sources include pineapples and figs.
The main plant sources of enzymes in the food industry are listed in Table 2.3.
Table 2.2: Examples of Microbial Enzymes used in Food Processing Enzyme Source Food Use
Carbohvdrases a-amylases Aspergillus niger; In baking and brewing
Bacillus subtillus indus t ry P-amylases Bacillus polymyxa In baking and brewing
industry Glucoamylases Aspergillus oryzae In preparation of thinned
Rhizopus oryzae starches
Cellulases
lnvertase
Trichoderma reesei In complex enzyme systems for oil extraction
Saccharomycetes Formation of invert sugar and in confectionery industries
Pectinases Aspergillus niger Fruit juice and wine Rhizopus oryzae industries
Proteases Fungal Proteases Aspergillus niger In the baking and the dairy
Aspergillus oryzae indu stries
9 Bacterial Proteases Bacillus subtilis In the baking industry Baccillus lich formis
Rennets Mucor mehei In cheese production Mucor pusillus
Esterase Lipases Aspergillus niger Lipid hydrolysis in fish oil
Aspergillus oryzae concentrate Oxidoreductases
Catalase Aspergillus niger Rernoval of H202 from milk Micrococcus and egg white lysodeikticus
Glucose oxidases Aspergillus niger desug aring of eggs
lsomerase Glucose isomerase Actinoplanes preparation of high fructose
missouriensis corn syrup Bacillus coagulans
Adapted from Richardson and Hysslop, (1 985)
2.2.3 ANIMAL SOURCES
Various animals have been used traditionally as sources of enzymes for food and the biornedical industry. The best known food enzyme obtained from animals is rennin (EC 3.4.4.3) found in the stomachs of calves (Taylor, 1991).
The hydrolytic activity of digestive enzymes derived from organs such as the stomach or pancreas has important consequence for the food industry (Yamamoto, 1975). Animal enzymes used in food processing are mainly proteolytic in nature. Proteolytic enzymes obtained from livestock offal currently do not adequately meet the demand on world basis, and the future availability of
traditional enzyme sources is dependent on the political and agricultural policies
that control the production of iivestock for slaughter (Godfrey and Reichelt,
1983). Moreover, traditional animal enzymes have been restricted to few species
namely bovine and porcine offal (Simpson and Haard, i987a). Table 2.4 shows some examples of animal enzymes used in food processing and their sources.
According to Simpson et al. (1991), microbial enzymes that have replaced animallplant enzymes may exhibit certain subtle differences in some properties which render them less suitable for a process application, as in the case of the use of micrabial proteases in cheese making. It has been reported that sornetimes there is increase in curd hardness, acidity development and drainage of the whey when M. meihei is used (Schwimmer, 1985).
2.3 MARINE ENZYMES
Enzymes from marine organisms rnay also be utilized as food processing
aids. Since animals cary out essentially the same types of metabolism, it is to be expected that different organisms such as fish, shellfish, marnmals, birds or reptiles will contain the same functional classes of enzymes (Simpson et al.
1991). Some fish enzymes such as the digestive proteases, pepsin(ogen)s,
trypsin(ogen)s and chymotrypsin(ogen)s, have been extensively studied.
Table 2.3: Examples of plant enzymes used in food processing
Enzyme Source Food Uses Carbohvdrases
a-amylases Barley malt
Wheat Barley wheat Barley malt
Proteases
m Bromelain Pineapples, Amas cosmosus,
Ficin Figs, Ficus sp. Papain Papaya, Carica papaya
Oxidoreductases
In brewing and distilling industries In baking and brewing industries In brewing industries and in coffee making
In the meat industry, brewing; fats and oils industries and in protein modification
Lipoxygenases Soybean flour In baking industry
Adapted from Richardson and Hysslop, (1 985)
Table 2.4: Sorne examples of animal enzymes used in food processing
Enzvme Source Food Uses - - ... . -. - .
Proteases Pepsin Porcine or other animal In dairy as rennet
stomachs extenders; production of
Trypsin protein hydrolysates
Animal Pancreas In the dairy industry; production of protein hydrolysates
Rennets Fourth stomach of In cheese production ruminants
Esterases Lipases Edible stomach; animal, In flavor development in
pancreatic tissues cheese Oxidoreductases
Catalase Bovine liver Removal of H202, Cold sterilization of milk
Adapted from Richardson and Hysslop, (1 985)
2.3.1 TRYPSIN(0GEN)S FROM THE MARINE SOURCES
Trypsinogens have been recovered from digestive tract of several fish
species and some exarnples are shown in Table 2.5 . The fish trypsins thus far isolated and characterized include those from
fish species with a functionally distinct stomach. Exarnples include rainbow trout, Salmo gairdenerri, (Kiarnikado and Taichino, 1960); the chinook salrnon, Oncorhynchus tshawytcha, (Croston, 1965); Green chromide, Etropplus suratensis (Sundara and Sarma, 1960) Atlantic cod, Gadus morrhua, (Overnel, 1973); sardine, Sardinus rnelanostica (Murakami and Noda, 1981); Arctic capelin, Mallotus viliosus (Hjemeland and Raa, 1982); African lungfish,
Protopterus cuthiopicus, (de Haen et al., 1977); catfish, Parasilurus asotus,
(Yoshinda et al., 1984) and Greenland cod, Gadus agac (Simpson and Haard,
1984a).Trypsins have also been characterized from fish species without a
functionally distinct stomach, include crayfïsh, Astacus flavatillis, (Pfleiderer et
al., 1967); bonefish, Carasuis auratus gibello (Jany, 1976); cunner,
Tautogolabrus adpsersus (Simpson and Haard, 1985); crawfish, Cambrus virillis and mullet, Mugii cephalus, (Jeong et al. 1994).
2.3.2: TRYPSIN FROM THE STOMACHLESS CUNNER
Cunner, Tautogolabrus adsperus, is one of the group of fishes with no morphologically or physiologically distinct stornach (Chao, 1973). Other group of stomachless fish are the bonefish, Carassuis auratus gibello (Jany, 1976) and
crayfish, Astacus flaviatilis (Pfleiderer et al., 1967). The lack of stomach is a
phylogenetic characteristic of the family and is not related to the feeding habits of
the species.
A trypsin fraction was isolated from the pancreas of cunner and shown to contain two different enzymes, which were classified as trypsin (rnolecular weight of 24,000 kDa) and trypsin-like (molecular weight 14,000 kDa) (Simpson and Haard, 1985). The specific activities for the trypsin fraction, trypsin and trypsin- like enzymes were found to be 1.54, 1.21 and 0.58 respectively.
The trypsin fraction from cunner exhibited two pH optima of 7.0 and 8.5
and a maximum and minimum range of stability were 2-3 and 6-7 respectively
(Simpson and Haard, 1985)
Table 2.5: Trypin(ogen)s from selected marine species
Fish Source Tissue Reference Sardine, (Sardinus melanostica)
Goldfis h (Carassuis auratus)
Capelin (Mallotus villosus)
Caîfis h ( Parasilurus asotus)
Rain bow trout, (Salmo gairdenerri) Chinook salmon, (Oncorhynchus tshawytcha)
Green chromide (Etropplus suratensis)
African lungfish, (Protopterus cuthiopicus)
Atlantic cod (Gadus rnorhua),
Greenland cod (Gadus ogac)
Cunner, (Tautogolabrus adsperus)
Crayfis h (Astacus flaviatilis)
Crawfis h (Cambrus virillis)
Mullet (Mugii cephalus)
Pyloric ceca
Intestinelhepatopancreas
Digestive track
Pancreas
Digestive tract
Pyloric ceca
Digestive tract
Pancreas
Pyloric ceca/messentries
Pancreas
Cardiac fluid
Hepatopancreas
Pyloric ceca
Murakami and Noda, 1981
Jany, 1976
Hjerneland and Raa, 1982 Yoshinda et al., 1984
Kitamando and Taichino, 1960 Croston, 1 965
Sundara and Sarma, 1960
de Haen et al., 1977
Overnel, 1973
Simpson and Haard, l984a Simpson and Haard, 1985
Pfleiderer et al., 1967
Jeong et al., 1994
Jeong et al., 1994
The temperature optimum of the trypsin fraction was reported as 450C The enzyme was found to possess unusual thermal stability, retaining more than
75% of its activity after heating at 80% for 1 hr and more than 50% of its activity after 30 min. at 1000C (Simpson and Haard, 1987). In this aspect, trypsins from cunner were more heat stable than trypsins from other fish species thus far characterized, which tended to be readily inactivated by temperatures as Iow as 40-50oC (Simpson and Haard, 1984a; Hjelmeland and Raa, 1982; Murakami and
Nada, 1981). The activation energy for the hydrolysis of DL-BAPNA by the trypsin and
trypsin-like enzyme from cunner, were found to be 1791Jlmol and 2054JImol respectively (Simpson and Haard, 1985). These values are comparable to those
reported for Greenland cod (Simpson and Haard, 1984a) but much lower than those of mammalian trypsins, e.g., 3153JImol for bovine trypsin (Simpson and Haard, 1984b).
2.3.2. HYDROLYSIS OF NATIVE AND DENATURED PROTEINS
The stomach is known to aid protein digestion in several ways, notably by
its secretion of stomach acid and acid proteases that denature and initiate
hydrolysis of proteins. Thus stomachless fish like cunner, are deprived of the
acid denaturation and acid proteolysis which takes place in the stomach,
expected to make proteins in food more amenable to subsequent degradation in
the intestines by trypsin, chymotrypsin and other proteases (Simpson et al., 1991). However, trypsins from stomachless fish, such as crayfish (Pfleiderer et al., l967), cunner (Simpson and Haard, l987b) and the crawfish (Jeong et al., 1994) have been reported to be more capable of hydrolyzing native ribonuclease A, lactate dehydrogenase, and hemoglobin as compared with trypsins and chyrnotrypsin from higher vertebrates with a functional stomach, (Simpson and
Haard, 1987; Mihalyi, 1978; Jany, 1976). Simpson and Haard (1984b) reported that bovine trypsin hydrolyzed
native hemoglobin only 18% and ribonuclease A 20 % as fast as their denatured
a counterparts, while the rate at which cunner trypsin hydrolyzed the same
proteins was 58 or 40% compared to its hydrolysis of the denatured forms of the
a protein substrates (Table 2.6). Approximately 12% of RNase activity was lost with bovine trypsin, while the cunner trypsin and the trypsin-like enzyme
destroyed about 58% and 72% respectively of RNAse activity, after 4 hr
incubation at 23%. Similar findings were made with a trypsin-like enzyme from
the stornachless crayfish (Pleiderer et al., 1967) as summarized in Table 2.6.
A comparison of the rates of hydrolysis of the native versus denatured
proteins of trypsins from various sources in terms of their relative activities showed that cunner trypsin appears to be have a higher relative activity for al1 the substrates with the exception of casein (Simpson et al. 1989).
2.3.3: COMPAMTIVE BIOCHEMISTRY
Marine enzymes in general and cunner trypsin in particular are similar in
8 many ways to their mammalian counterparts. Generally their pH-activity profiles,
substrate specificities, molecular weight, and response to inhibitors or activators
resemble those from mamrnalian counterparts (Simpson and Haard, 1989; Tanji
et al., 1 988; Squires et al., 1986).
However the digestive proteases from marine sources differ from their
mammalian counterparts in certain respects namely
(i) they are less stable under acid conditions
(ii) they have a higher catalytic activity
(iii) they are generally more heat-labile
(iv) they are more stable at cold temperatures
(v) they hydrolyze (undenatured) proteins to a greater extent (Simpson et al., 1991 ; Tanji et al. 1988; Ramaskrishna et al., 9987;
Simpson and Haard, 1984)
Table 2.6 Hydrolysis of protein substrates by cunner enzymes
Enzyme Su bstrate initial rate of DH, %
hydro lysisa
Bovine trypsin hernoglobin 0.73 0.96
Cunner trypsin 1.24 0.95
Trypsin - like 0.43 2.53
Bovine trypsin UT- hernoglobinb 4.02 2.83
Cunner trypsin 2.14 1.68
Trypsin - like 1 .O5 5.50
Bovine trypsin ribonuclease . 0.25 0.60
Cunner trypsin 0.67 7.22
Trypsin - like 0.46 2.50
Bovine trypsin HT-ribonucleasec 1.27 7 -41
Cunner trypsin 1.69 1.29
Trypsin - like 0.85 3.72
aln milliequivalents/min per micromole enzyme; b Urea-denatured hemoglobin
CHeat-denatured ribonuclease. Adapted from Simpson and Haard, (1 987b)
Table 2.7: Relative Activities of Trypsins from various sources
% Relative Activity
Source BAPNA TAME Casein RNasea Hemoglobin
Greenland cod 64.5 96.1 77.4 ndb ndb
Atlantic cod 68.5 1 O0 85.5 40.3 52.4
Cunner 1 O0 91.0 82.2 I O 0 1 O0
Bovine 48.8 81.5 1 O0 37.3 58.9
a Undenatured substrates ; Not determined; Data from Simpson et al., (1989)
2.3.4: APPLICATIONS AND POTENTIAL APPLICATIONS OF MARINE ENZYMES IN THE FOOD INDUSTRY
The application of an enzyme as a food processing aid in an industrial
conversion depends on its catalytic efkiency and physical properties (Godfrey
and Reichelt, 1983, Richardson and Hysslop, 1985). Furtherrnore, in order to use
a particular enzyme as a food processing aid it should confer sorne commercial
benefiîs (Taylor, 1991). The effectiveness of an enzyme for a given industrial
conversion is species dependent as analogous enzymes from different
sources/species display a varied catalytic activity and physical properties.
Digestive proteolytic enzymes utilized in the food industry have been
obtained froin the livestock offal of bovine and porcine (Simpson and Haard,
e 1987) and the stomachs of suckling calves (Schwimmer, 1981). In general,
animals are a poor source of enzymes and animal production lacks the flexibility
if enzyme production needs to be suddenly increased or decreased (Taylor,
1991). This has led to the development of microbially derived alternatives,
making microorganisrns source for greater variety of industrial enzymes. lnspite
of the potential that can be achieved by the use of microbial enzymes, very few
of the species are used to produce industrial enzymes due to the stringent
evaluation required for a microorganism to be regarded as safe and the
substantial cost involved in getting microorganism approved (Godfrey and
Reichelt, 1983).
An alternative animal source for enzyme production is the fish offal.
However, few attempts have been made to recover enzymes from these
sources. Apart from being a very good source for nutrients, the fish offal is also a
very good source for enzymes (Simpson et al. 1991). The underutilization of this
a rich enzyme source is of concern because according to Simpson and Haard,
(1991), only about half of the fish offal produced are converted into products of
economical importance such as fish meal. The rest serve as disposal and
pollution problem. As with a typical animal source for enzyme production, the use
of fish offal as source for industrial enzymes is limited by fluctuations in
availability due to seasonal harvest. The enzymes from the fish offal can be used
to augment the production of enzymes from animal sources, as well as make use
of their unique property of having a high rnolecular activity at low ternperatures.
Also, the fish offal can be turned into a proper economic use thereby reducing
disposal /pollution problems associated with the fish offal.
To date, there are few marine enzymes that are used on a commercial
scale. The following are a few examples:
(i) lcelandic Fisheries Laboratories use trypsin-like enzymes from cod viscera for
a the removal of skins, membranes and scales from fish and production of fish
gelatin from fish skins and scales (Raa, 1989).
(ii) In Nonnray pepsin from cod viscera is used for the hydrolysis of fish frames to
produce marine peptones for subsequent use in microbiological media
preparations (Almas, 1989) and incorporation in fish feeds for the production of
immune stimulants for fish reared in captivity (Raa, 1989).
The laboratory scale applications are as follows:
Fish enzymes have been used to prepare various cheeses. Rennin or
chymosin (EC 3.4.4.3) is the enzyme traditionally used in cheese rnaking. This
enzyme converts the colloidal milk casein into a curd giving high yields of cheese
and desirable proteolysis in the aged cheese. The source of this enzyme has
been the abomassum of suckling calves (Schwimmer, 1983). There is increasing
O concern about the decreasing supply of rennin from the calves. Moreover, the
slaughter of young calves is economically wasteful (Taylor, 1991). Most
e proteases are inferior to chymosin for cheese production. This is due to their
broader specificities for protein substrates, which leads to excessive proteolysis
during curdling and subsequent steps in the cheese making operations. Such
activities lead to inadequate yield of curd, unacceptable rheological properties of
the cheese, and off-flavor (de Koning, 1978). According to Berridge (1951),
however, nearly al1 proteolytic enzymes will clot milk under appropriate
conditions.
Studies by Brewer et al., (1984) have shown that it is possible to prepare
a satisfactory cheddar cheese with Atlantic cod pepsin as the rennet agent.
However, when the conventional cheddar cheese process was employed, there
was loss of some fat and proteins to the whey fraction indicating that additional
protein degradation occurs during curd formation and the early stage of
cheddaring. The high molecular activity of fish pepsins at low temperatures also
I ) occurs when milk is treated to initiate the clotting process. The enzymic phase of
milk clotting, involving hydrolysis of the phenylalanine-methionine bond of K-
casein, can be separated from the non enzymic phase, ii-ivolving transformation
of sol to gel, by carrying out the process at a temperature below 10°C. Cold
renneting of milk with a catalyst having a low temperature coefficient for the
enzyme, can be accomplished with lower enzyme concentration, thereby
conserving rennet and minimizing the presence of residual curd pepsin. Utilizing
the temperature stability of the cod pepsin, the temperature of milk treated with
the enzyme can be increased to 39°C after the enzymic phase at 0°C to cause
the inactivation of the enzyme (Simpson and Haard, 1987a).
Bovine trypsin is capable of preventing the copper induced-oxidized flavor
in milk (Lim and Shipe, 1972). Greenland cod trypsin has also been shown to be
effective in preventing oxidized flavor in milk (Simpson and Haard, 1984~).
While Greenland cod trypsin is thermally unstable, the high thermal stability of
a bovine trypsin allows it to survive the pasteurization treatment used and this
helps to prevent the subsequent hydrolysis of milk proteins.
Greenland cod trypsin was used to accelerate the fermentation of
herrings, and was able to achieve greater solubilization of fish protein and
sensory score of the fish sauce during low temperature fermentation than bovine
trypsin (Simpson and Haard, 1984~).
Trypsin from Atlantic cod was used to facilitate the extraction
carotenoproteins from crustacean shells (Cano-Lopez et a/., 1987). The fish
enzyme recovered more pigment (64 %) and protein (81%) in the complex than
bovine trypsin (49 % pigment and 68 % protein).
The applications and the potential applications of the proteases from the marine
sources are thus many and diverse. As reported in Table 1, there are several
@ endogenous enzymes like the pectic enzymes which causes destruction of cloud
stability in fruit juices (e.g. citrus and tomato) as well as postharvest texture
softening in fresh fruits and vegetables; and polyphenol oxidases which causes
enzymic browning of fruits and vegetables such, as apples, banana, potatoes
and crustacean species such as the shrimp. The financial losses due the effect
of these endogenous enzymes are enormous and several methods to control
their effect have been investigated.
2.4 PECTIN METHYLESTERASE AND THE CITRUS JUlCE INDUSTRY
Pectin methylesterase (PME), (E.C. 3.1.1.1 1) causes loss in cloud stability
a of citrus juices by releasing pectyl methyl esters from pectin (Chaplin and Bucke,
a 1990). PME has been reported to operate in a sequential manner with
polygalacturonase (E.C.3.2.1.5) (Dahodwala et al., 1974).
PME from mandarin orange has been described as a single glycosylated
polypeptide with molecular weight of 37000. Although this enzyme exhibited ad a
pH optimum of 9.0, it has been shown to be active at pH's below pH 5.50. Such
studies observed a Km of 0.84rnglml and a V,, 0.38pmol of galacturonic acid
produced /min for hydrolysis of pectin.
Mandarin PME retained about 50% of its activity after heating at 62OC for
1 min with the activity decreasing to 20% at 70'~. At 9U°C the activity is
undetectable (Rillo et al., 1992).
2.4.1: PECTINS, THE SUBSTRATE OF PECTIN METHYLESTERASE
Chemically, pectins are polymers of galacturonic acid linked by a,?-4
glycosidic linkages into long chains (Schwimrner, 1981).
Pectins have been classified as
(i) pectic substances, Le., material comprising al1 polygalacturonic acid
containing substances; (ii) protopectin, Le., water insoluble materials in bound
form that yield pectins upon hydrolysis; (iii) pectins, i.e., partly esterified
polygalacturonic acids (generally rnethyl esters) further divided into low rnethoxy
and high methoxy pectins depending on whether they contain less or more than
7% methoxy-esterified polygalacturonic acids; and (iv) pectinic acids which have
al1 carboxyl groups in the free form and are water insoluble (Fellers, 1991).
The solubility of pectins increase with increasing esterification and
decreases with size. The usefulness of pectins stems mainly from their capacity a
to form stable gels or films, and to increase the viscosity of acidified sugar
solutions (Pomeranz and Meloan, 1987).
2.4.2: CITRUS JUICES, PECTIN AND CLOUD STABILITY
Fresh citrus juice, like orange juice, contains finely divided particulates in
a suspension, that give it a 'cloudy' appearance. Analysis has shown that this
particulate material is composed almost exclusively of pectin, protein and lipid
(Baker and Breummer, 1972). When this stable colloidal system collapses, the
juice separates into a clear liquid and solid sediment. Once converted to an
unattractive two-phase system of a flocculant sedirnent in a clear serum, the
I) juice looses its consumer appeal (Castaldo et al., 1991) since cloud stability is a
most important quality attribute of orange juice (Balaban et al., 1991). In addition,
the cloud contains most of the characteristic orange flavor and color. During
citrus processing, large juice vesicles (sacs) are ruptured to release the juice
(Bradock and Marcy, 1987) and large juice vesicles are removed during the
finishing operation as a secondary product of juice processing. Juice vesicles,
referred to as pulp have unique physical and chemical properties and are used
in non juice drink bases and juice containing beverages (Braddock and Marcy,
1 987).
Multiple forms of PME are reported to be associated with cell fractions of
peel, rag and juice sacs from citrus fruits (Versteeg et al., 1980). PME therefore
enters the juice from the pulp during extraction and deesterifies pectin to
a produce low methoxy pectins (Castoldo et al., 1991). The product, pectinic acid
chelates with monovalent and divalent ions (e.g., ~ a + and Ca++) present in the
juice to form insoluble calcium pectates, which precipitates along with cloud
particles resulting in clarification of juice cloud (Versteeg et al., 1980).
2.4.3: METHODS OF PME INACTIVATION
The cloud of citrus juice has been stabilized by protecting pectin from
PME (Owusu-Yaw et al., 1988). Several technological approaches have been
used to solve the problem of juice clarification. These approaches include:
(A) THERMAL INACTIVATION (90-1 1 5 ' ~ )
This is the most common method in PME inactivation (Wicker and Temelli,
1988; Veersteg et al., 1980; Eagerman and Rouse, 1976). Heat treatments
however, affects aroma and flavor (Balaban et al., 1991) and causes
development of a brown color due to non-enzymatic browning reactions to affect
the quality of the single strength orange juice (Nurniki and Hayashi, 1983). The
extent or severity of the heat treatment was found to be highly dependent on the
total soluble solids and increasing total soluble solids resulted in less
inactivation. Severe heat treatment of foods is known to induce racemization of
amino acids and proteins (Gandolfi et al., 1994) and instantaneous heating and
cooling is difficult to achieve (Cohen et al., 1994). Thus there is the potential for
losses of heat-labile components of the juice by heat treatrnents. Wicker and
Temelli (1988) indicated the difficulty in obtaining a logarithmic fold reduction in
a PME activity, and for industrial purpose there is the necessity of introducing
tubular heat exchangers of relatively large diameter, and the addition of water to
enhance pumpability and reduce disruptions in continuous process.
(B) LOW pH INACTIVATION OF PME
This method is based on the fact that PME from orange has less activity
at pH 5.5 than at their optima pH of 9.0 or close to that (Rillo et a/., 1992;
Versteeg, 1980; 1978). In this method, pH 5 2.0 is used in conjunction with low
temperature storage. Very little cloud loss was observed at 40C in orange juice at
pH 2.0, however, with this method there was a major destruction of (> 95%) of
vitamin C (Owusu-Yaw et al., 1988). Moreover, the addition of HCI or the use of
cation exchange resins to lower the pH of the juice resulted in unacceptable juice
quality (Balaban et a/., 1991).
(C) SUPERCRITICAL CARBON DlOXlDE (COp) INACTIVATION OF PME
PME may be inactivated with supercritical CO2 below temperatures
necessary for thermal inactivation. This treatment is reported to be based on the
hypotheses that at high pressures, COn dissolves in water, producing carbonic
acid, thereby lowering the pH temporarily and inactivating PME. On returning the
pressure to atmospheric, COn would be separated from the juice and the pH
restored to the original value. This treatrnent combines the effect of low pH and
high pressure in the inactivation process. There is a problem of enzyme
reactivation with this treatment (Balaban et a/., 1991) and this rnay cause loss of
cloud stability during storage. Supercritical treatment conditions employed in this
a treatment range between 7-34 MPa with corresponding temperatures of 35-60°C
(Arreola et al., 1991 ).
(D) HlGH PRESSURE INACTIVATION OF PME
High pressures of about 500 MPa for a treatment period of 10 min partially
inactivate PME. This treatment is highly dependent on the soluble solids content.
At pressures up to 600 MPa, pulp particle size distribution was slightly changed
after treatment at room temperature for 30 min (Takahashi et al., 1993) Higher
soluble solid content is known to protect PME from inactivation (Ogawa et al.,
1990). In addition, complete inactivation was not possible at any pressure Ievel
used, and the
a (Ogawa et al.,
sta bility.
problem of enzyme reactivation during storage was observed
1990), which may subsequently contribute to the Ioss in cloud
(E) DEGRADATION OF PME SUBSTRATES USlNG PECTINASE
In this method, soluble pectin is degraded to a low degree of rnethylation
with enzymes such as pectin lyase or polygalacturonase that prevent pectin
precipitation by calcium through reduction of pectin size. This method has found
limited application (Castaldo et al., 1991) as there are potential problems of
enzymic flavor deterioration (Baker and Breummer, 1972)
(F) USE OF PME INHIBITORS.
A glycoprotein isolated from the kiwi fruits is known to be a powerful
enzyme inhibitor (Balesteri et al., 1990) and is capable of inactivating PME from
orange juice and other fruit juices (Castaldo et a/., 1990). The activity of the
inhibitor was found to be dependent on the amount of soluble solids. Above a
certain minimum soluble solids content, a decrease in cloud stability was
observed (Castaldo et al. 1991).
(G) INACTIVATION OF PME BY PROTEOLYTIC ENZYMES.
It has been reported that the cloud in citrus beverages could be stabilized
by subjecting pasteurized concentrate to one or more enzymes with protease
activity and that the cloud stability depended on the degree of protein hydrolysis
in the juice (Castaldo et al. 1991).
In al1 the methods reported so far, the amount of total soluble solids
tended to be the determining factor in the success of the inactivation procedure
employed. In addition, there was the possibility of enzyme reactivation when the
inhibiting effect was removed. In other cases involving the use of low pH,
although no cloud loss was reported, there was a loss of almost al1 the vitamin C
contained in the juice.
The possibility of using a combination of proteolytic and pectinases
(Castaldo et al., 1990; Baker and Breummer, 1972) precludes the effect of the
amount of total soluble solids, which tends to be the major determining factor in
a most of the methods reported. The cloud stability depends on the degree of
enzyme hydrolysis in the juice.
Alternatively, the unique ability of digestive proteases from the
stomachless cunner to inactivate native protein molecules may be exploited to
inactivate PME and thereby achieve cloud stability in citrus and other fruit juices.
The trypsin from cunner may be utilized in combination with other
treatments like low temperature and1 or low pressure to aid in the inactivation of
PME, and thereby stabilize the juice cloud of selected fruit juices, as well as to
preserve heat labile components in the juice.
2.5 POLYPHENOL OXIDASES (PPO) AND ENZYMATIC BROWNING
PPO (EC.1.10.3.1) has several names depending on the type of substrate
used in the enzymatic conversion viz. tyrosinase, cathecol oxidase, cresolase,
polyphenolase, cathecolase and phenolase, (Whitaker, 1994). PPO is widely
distributed in biological systems and have been isolated from several fruits, e.g.
apples (Sapers et al., 1989) banana, kiwi (Park and Luh, 1985), grapes (Valero
et al., 1988), pears (Zhou and Feng, 1991) and avocado (Kahn, 1983);
vegetables, such as spinach leaves (Katoh et al., 1989); mushrooms (Golan-
Goldhirsh and Whitaker, 1984); crustacean species (Chen et al., 1993) and
potato (Friedman and Bautista, 1995).
PPO causes undesirable browning reactions in fruits, vegetables and
crustacean species (Chen et al., 1993; Valero et al., 1988). The browning
reactions observed during postharvest storage or processing fruits, vegetables
and crustacean species is a widespread phenornenon (Zhou and Feng, 1991)
a and its net only cornmercially undesirable, but it has been considered to
generally affect food quality from both sensory and nutritional point of view
(Rouet-Mayer et al., 1993).
PPO, a multifunctional copper-containing protein, is known to catalyze the
orthohydroxylation of monophenols to o-diphenols, and the subsequent oxidation
of O-diphenols to O-quinones. The quinones so formed react with themselves
protein or amino acids to give characteristic brown pigments (Valero et al. 1992;
Garcia-Gomona et al., 1988)
The characteristics of PPO Vary as the type of substrates available. The
temperature optimum of PPO range from 35°C in airen grapes (Valero et al.,
1988) to 45°C for phenolase-DOPA in shrimp (Simpson et al., 1987). The
temperature stability range from 35°C to 75°C in kiwifruit (Park and Luh, 1985).
The pH optimum for the different PPO range from as low as 3.5 in airen grapes
@ (Valero et al., 1988) to 7.5 in shrimp (Chen et al., 1993). Most of the PPOs are
reported to be stable in the alkaline pH regions (- 8.0) and less stable in acid pH
range.
There are several PPO substrates and the specific substrate requirernent
differs markedly depending on the source. The main PPO substrate is tyrosine
(Richardson and I-lyslop, 1985). The other substrates are di- or tri- phenolic
compounds like cathechol, d-cathecin, chlorogenic acid and Dopa and
rnonophenolic compounds are pcresol and pcoumaric acid (Whitaker, 1994).
2.5 INACTIVATION OF POLYPHENOL OXIDASES (PPO)
Several methods have been employed for PPO inactivation. They include:
(A) THERMAL INACTIVATION
Thermal inactivation of PPO consists of a treatment behveen 80-90°C for
6 S. Although the process is swift, there is a considerable loss in flavor, loss of
solids due to leaching, weakened texture of products like prepeeled potato and
even the possibility of non-enzymic browning (Schwimmer, 1981).
(B) REDUCJNG AGENTS
The reducing agents normally used in PPO inactivation include ascorbate,
bisulfites and thiols. Sulfites are the rnost commonly used reducing agents.
Thiols, sulfites and ascorbate act in a complex way by reacting with the quinones
4) formed by enzymatic reactions and reducing them back to their O-diphenolic
compounds and other colorless complex (Walker, 1975; Golan-Goldhirsh and
Whitaker, 1984). In addition, the enzyme is known to loose activity in the
presence of sulfites by direct suicide inactivation of the enzyme (Golan-Goldhirsh
and Whitaker, 1984). Thiols are reported to directly affect PPO by displacing the
histidine ligated to the copper due to the high affinity of the thiols for copper. This
results in modifications of the active sites and therefore inactivation (Friedman
and Bautista, 1995). However, because of the greater awareness of its possible
damaging effect to the health especially in asthmatics (Friedman and Bautista,
1995) reduction or its complete elimination is one of the highest priority (Valero
et al., 1992). Equally good alternatives to sulfites are being investigated.
(C) CHELATING AGENTS
Several chelating agents have been used to inhibit enzymatic browning.
These agents are known to cause inhibition by interacting with copper on the
prosthetic group (Sapers et al., 1989). Examples of such agents are cyanide,
diethyldithiocarbamate, azide, EDTA, an acidic polyphosphate (sporix), and
copper-metallothionein (Goetghebeur and Kermasha, 1996; Sapers et al., 1989;
Simpson et al., 1987). De pletion of the agents, however, causes browning.
(D) PPO INHIBITORS
Among several chernicals proven to inhibit PPO activity, sorbic acid, kojic
O acid and some aromatic carboxylic acids of the benzoic, cinnarnic and
phenylalkanoic series have been widely studied and proven to be effective
inhibitors of PPO of various origins (Janovitz-Klapp et al., 1990; Gunata at al.,
1987; Simpson et al., 1987). These inhibitors act by the binding of both the
neutral and dissociated forms of the acids to the free and cornplexed enzymes
causing the inhibition (Billaud et al., 1996). However, in the application of these
inhibitors, the degree of inhibition is highly dependent on the pH of the medium
since the form of inhibitor (free or dissociated) which acts best is pH dependent.
In addition, Sapers et al. (1989) have reported the possibility of large proportions
of benzoic acid inducing browning by stimuiating the hydroxylation of cinnamic
acid to p-coumaric acid, a PPO inhibitor which can be further hydroxylated to
give caffeic acid, a substrate of PPO.
(E) OXYGEN EXCLUSION
An atmosphere modified with carbon dioxide (CO,) has been used to
inactivate several enzymes including PPO (Arreola et al., 1991; Chen et al.,
1993). This treatment is effective in inactivating lobster PPO and the combination
on heat and carbon dioxide treatment resulted in further loss of PPO activity.
Carbon dioxide affects PPO by the acidification of the environment, leading to
the inactivation of PPO (Chen et al., 1993)
Many chemicals have been extensively studied for the inactivation of
PPO. Although large proportions of these chemicals show effectiveness in
inhibiting PPO activity in fruits vegetables and crustaceans, several problems
plague their successful application. These include their toxicity, off-flavor the
e possibility of the chemicals re-inducing browning and above al1 the economic
feasibility of such an inactivation procedure. An alternative method to inactivate
PPO may be the use of digestive proteases from stomachless marine organism
as the cunner. The digestive proteases are naturally occurring compounds and
their use in food application would meet with less resistance from consumers.
This alternative may also serve to preserve the flavor lost during thermal
inactivation of PPO and maybe lower the cost of the operation, in that the source
of these enzymes are from the fish waste (offal).
2.6: RATIONALE FOR STUDY
Traditionally, digestive proteolytic enzymes have been derived from
bovine and porcine offal, whereas a greater diversity of the industrial enzymes
are obtained from microbial sources (Godfrey and Reichelt, 1983).
Fish offal is known to be a good source of nutrients (e.g., proteins, fat,
and vitamins) and useful biochemicals (e.g., enzymes, pigments, flavorants),
which may be recovered and used as food ingredients or processing aids.
Proteases from fish so far isolated have been reported to have several unique
advantages over the proteases of the mammals from land. As a result of their
low temperature stability, some of them have the ability to hydrolyze native
proteins better, and give high turnover rate at low temperatures
In addition to curtailing the problem of pollution posed by the disposal of
the fish offaf, the offal of the fish can be put into a better economic use by
extracting the digestive enzymes and utilizing the unique properties of these
enzymes in the inactivation of some problematic enzymes such as PME and
PPO. Several inactivation procedures have been utilized to control the effect of
PME in fruit juices, and PPO in vegetables, fruits and crustacean species. These
a include thermal inactivation, low pH, high pressure, use of chemicals and several
protein inhibitors. In addition to the operating costs involved in the running of
thermal heat exchangers and high pressure systems, these methods of
inactivation influence the final product by affecting the final flavor since they
affect the heat-labile essential components responsible for the flavor. In addition
to the effects on heat-labile components, there is also the possibiiity of non-
enzymic browning. With some of the methods, the appearance of the products,
is comprornised as the particles are affected by the extent of the treatment, as in
the pressure treatments.
Certain chernicals such as sulfites are not permitted in foods beyond their
effective limits due to safety concerns. Lowering the pH below certain levels can
also create problems in the food systems, for example in orange juice al1 the
@ vitamin C present in the juice is destroyed.
An inactivation procedure where the PME and PPO are directly
hydrolyzed by digestive enzymes from marine sources, will not only lower the
operating costs involved in inactivating these enzymes but will also preserve the
heat-labile flavor components. The use of trypsins from the stomachless cunner
could be one such approach for inactivation of PME and PPO. In addition,
trypsins are naturally occurring components of food systems and will appeal to
consumers over the chernical additives.
Although there have been relatively few attempts to use fish enzymes
such as cunner trypsin as industrial processing aids, their effectiveness for
hydrolysis at low temperatures warrants more attention in exploiting such
properties.
2.7: OBJECTIVES
The overall objectives of this research are
(i) to extract and purify, ttypsin from the pancreas of the cunner fish,
(ii) to evaluate the effectiveness of cunner trypsin(s) to inactivate PME andlor
PPO and
(iii) to determine the effect of storage conditions on certain quality attributes of
single strength orange juice treated with the trypsin from cunner.
CHAPTER III
MATERIALS AND METHODS
Live cunner fish, Tautogolabrus adspersus, were held in tanks in the
laboratory between 5-10 OC and fed with capelin fish until needed. The fish were
killed by severing the cervical vertebra and the pancreatic tissue was removed
and rapidly frozen in liquid nitrogen and kept at -80 O C prior to extraction.
Orange juice: Orange juice (Tropicana) was bought from the local grocery
shop.
3.2 REAGENTS
Trichloroacetic acid (TCA), Folin-Ciocalteu's phenol reagent, acrylamide,
Coomaasie brilliant blue (R250), citric acid, pectinesterase (citrus), bovine
trypsin, soybean trypsin inhibitor, aprotinin, bovine serum albumin, cyanogen
bromide activated sepharose 4B, ammonium sulfate, N,N,N1,N'-tetra methyl
ethylenediamine (TEMED), phosphoric acid, dihydroxyphenylalanine (DOPA)
and bromophenol blue were purchased from Sigma Chemical Company (St.
Louis).
N, N' methylene-bis-acrylamide, glycine, tris(hydroxymethyl)
aminomethane, sucrose, urea, sodium dodecyl sulfate, 2-mercaptoethanol were
purchased from Biorad (Montreal)
aB Galacturonic acid and pectin (citrus), were purchased from ICN
Biomedicals (Toronto)
Bromothymol blue and acetone were obtained from ACP ( Montreal)
Sodium acetate, isopropanol, sodium hydroxide and sodium chloride
were obtained from BDH (Quebec)
Acetic acid, ether, dimethyl sulfoxide (DMSO), sulfuric acid (H2S04),
potassium phosphate monobasic, and potassium phosphate dibasic were
purchased from Anachemia (Quebec)
Hydrochloric acid and Brij 35 (enzyme grade) were purchased from Fisher
Scientific (Toronto)
Electrophoresis calibration kit (low molecular weight) was purchased from
Pharmacia (Montreal).
3.3 METHODOLOGY
3.3.1 RECOVERY AND PURIFICATION OF TRYPSIN :
Trypsin fraction from cunner pancreas was prepared according to the
method described by Simpson and Haard (1985).
The pancreatic tissue was rapidly frozen in liquid nitrogen and comminuted into
powder using a waring blender. About l o g of the powder was stirred in 0.05M
Tris-HCI buffer (5 mllg) pH 7.8 containing 0.5M NaCl and 0.02M CaC12 at 4 OC
for 3 hours, after which the resultant mixture was centrifuged at 3000g for 30
min. at 4 OC. The supernatant was made up to 0.2% with Brij 35 and stirred
overnight at 4 OC. It was then centrifuged at 10,000g for 30 min. at 4°C. This
second supernatant was fractionated with solid ammonium sulfate and the
fraction sedirnenting between 40% and 60% saturation was collected by
centrifugation at 60009 for 30 min at 4 OC. The precipitate from the ammonium
sulfate step was dissolved in 20 ml of Tris-HCI buffer (pH 7.8) and dialyzed
overnight against three changes of 6L of the same buffer. The dialysate was
mixed with three times its volume of cold acetone (-20'~) and kept at -20°C for
3h. The precipitate formed was collected by centrifugation at 60009 at 4OC for 30
min. The material from the acetone step was redissolved in 10 ml Tris-HCI buffer
(pH 7.8) and pumped into a SBTI-Sepharose 4B affinity chromatography column
according to the method of Katoh et al., (1978). The column was thoroughly
washed with elution buffer to remove the unbound material after which the bound
material was eluted with 5 mM HCI at a rate of 15 mlfhr and fractions of 4.8
mlltube collected.
3.3.2 ENZYME ASSAY
The amidase activity of the trypsin fraction, was estimated using the
method of Erlanger et al., (1 961 ).
A 200 pl aliquot of an appropriately diluted isolate was added to 2.8 ml of
1 mM N a-benzoyl-DL-arginine p-nitroanilide (BAPNA) in Tris-HCI (pH 8.2)
containing 0.02M CaCI2 and the release of p-nitroanliline was measured at 410
nm at 25 OC, using Hitachi U 2000 spectrophotometer.
One BAPNA unit was defined as AA,,,,m,mi,,, x 3 x 1000 1 8800 where 8800
is the extinction coefficient of p-nitroaniline. The stock enzyme used in the study
was prepared in 5 mM HCI (pH 2.5).
1 mM BAPA was prepared as follows: 0.04359 of BAPNA was dissolved
in 1 ml dimethyl sulfoxide (DMSO). The resulting solution was made up to 100 ml
with Tris-HCI buffer, pH 8.2, containing 0.02M CaCI2.
The protein content was determined by the modified method of Lowry et
al., (1 951) as reported by Stoscheck (1 990).
To 400 pl of sample, 400 pl of Lowry concentrate was added and incubated at
room temperature for about 12 min. 200~1 of 0.2 N Folin reagent was then added
and then vortexed immediately. The resulting mixture was incubated ai
approximately 24°C for another 30 min. The absorbance of the solution was read
at 750 nm. A calibration curve was perforrned using appropriately diluted solution
of bovine serum albumin stock solution of 2 mglml.
Lowry concentrate: This was prepared as follows:
20g of Na2C03 were dissolved in 260 ml water; 0.4 g of CuS04.5H20
was dissolved in 20 ml water; and 0.2g sodium potassium tatrate was also
dissolved in 20 ml water and mixed to form the copper reagent.
l og of sodium dodecyl sulfate (SDS) were dissolved in 100 ml of water to
give a 1 % solution
49 of NaOH were dissolved in 100 ml of water to give a 1 M solution
3 parts of copper reagent, 1 part NaOH and 1 part SDS were mixed just
before use
3.5 SDS POLYACRYLAMIDE GEL ELECTROPHORESIS
SDS polyacrylamide gel electrophoresis was carried out according to the
method of Laemmli, (1970).
3.5.1 CASTING OF GELS
The casting apparatus were assembled. A 12% solution of the monomer
was prepared by combining al1 the reagents in Table 3.1 except the ammonium
persulfate (APS) and TEMED. The monomer solution was deaerated under
vacuum for 15 min. The deaerated monomer solution was gently rnixed with APS
and TEMED. The rnonomer solution was poured between the plates and
immediately overlayed with water. The gel was allowed to polymerize for 45 min.
The stacking rnonomer solution was prepared by combining al1 the reagents in
Table 3.2, except the APS and TEMED and deaerated under vacuum for 15 min.
The top of the resolving gel was thoroughly rinsed with water and dried with filter
paper. A well-forming comb was placed in between the plates and tilted at a
slight angle to provide for bubbles to escape. APS and TEMED were added to
the degassed stacking monomer solution and poured on top of the resolving gel.
9 The comb was aligned in its proper position, and polymerization allowed to
proceed for 45 min.
3.5.2 SAMPLE PREPARATION AND SDS POLYACRYLAMIDE GEL ELECTROPHORESIS
The samples were diluted with at least 4 vol. of complete SDS-reducing
buffer and heated at 950C for 4 min. in boiling water bath and used for the
electrophoresis. The electrophoresis cell was assembled, and the lower and the
upper reservoirs filled with electrode buffer as per Table 3.3 and the comb was
removed from the stacking gel. The prepared samples were loaded into the wells
in the stacking gel by layering them under electrode buffer using a microliter
syringe. The leads were then attached to the power supply (BioRad 3000 Xi) and
Table 3.1 Formulations of SDS PAGE Resolving Gel
Component Resolving gel (1 2%)
Water 3.35 ml
1.5M Tris-HCI, pH 8.8 2.5 ml
10% SDS 0.1 ml
Acrylamidelbis 4.0 ml
10% ammonium persulfated 50 pI
TEMED 5 PI
d Prepared freshly each day
Table 3.2 Formulations of SDS-PAGE stacking Gel
Component Volume
Water 6.1 ml
0.5 M Tris HC1, pH 6.8 2.5 ml
Acrylamidelbis (30% T) 1.3 ml
10% SDS 0.1 ml
10% APS 50 pl
TEMED I O pl
Table 3.3 : Solutions for running electrophoresis and staining Gels
Solution Composition
- --
%ample buffer distilled water (4ml, Tris-HCI, pH 6.8 (1 ml), 10% SDS
(1.6 ml) 2-rnercaptoethanol (0,4 ml). 0.05% (wlv)
bromophenol blue (0.2 ml)
7 Electrode buffer
1 Fixating Solution
2Staining Solution
tris(l5g), glycine (72 g) SDS (5 g) in 1 L aqueous
solution pH 8.3
trichloroacetic acid (12.5g) in 100 ml deionized water
Coornassie brilliant blue R 250, (0.1 % wlv) in 25%
propanol, 10% acetic acid and deionized water
*Destainhg Solution acetic acid: propano1:water (ratio 1 :2:7)
l Laemmli, (1970)
2 Simpson et al., (1 987)
the electrophoresis allowed to proceed under constant voltage. The run was a stopped when the blue tracking dye was about 1 cm frorn the bottom of the glass
plates. The gels were removed from the glass plates and fixed in 12% TCA, table
3.3, solution overnight in a glass casserole container. After fixation, the gels were
soaked in an excess of staining solution, (Table 3.3), overnight, and then
destained with several changes of the destaining solution, (Table 3.3), until the
background stain was satisfactorily removed.
3.5.3 MOLECULAR WEIGHT DETERMINATIONS
The molecular weights of the trypsin fraction were determined by
cornparison of their mobilities with those of standard marker proteins of known
molecular weight which were run simultaneously on the same gel as the trypsin
O fraction. The molecular weight markers (Seeblue prestained markers from
Novex) used were myosin (250,000), bovine serum alburnin (98,000), glutamic
dehydrogenase (64,000), alcohol dehydrogenase (50,000), carbonic anhydrase
(36,000), myoglogin (30,000), lysozyme (16,000), aprotinin (6,000) and insulin P-
chah (4,000). The distance of migration of each protein was divided by the
distance traveled by the tracking dye. The norrnalized migration distances so
obtained are called the relative mobilities of the proteins (relative to the dye front)
and conventionally denoted as Rf.
The logarithms of the molecular weights were plotted as functions of the Rf
values. The unknown molecular weights were determined by linear regression
analysis.
3.6 ISOELECTRIC FOCUSING
lsoelectric focusing to determine the isoelectric point (pl) of the trypsin
fraction was carried out using the Rotofor ce11 (Biorad). The Rotofor cell is a
modification of an apparatus originally designed by Egen et al., (1984)
Prior to the run, the anion and cation exchange membrane of the rotofor
were equilibrated overnight in 0.1 M NaOH and 0.1 M H3PO4 respectively.
A gasket was placed over the aligning pins and seated on a flat surface of the
inner assembly. The anion exchange membrane was then placed on the gasket
by the aligning notches in the membrane and the "sandwich" with a second
gasket on top of the membrane. The pins and sockets in the two halves of the
assembly were aligned and fastened together with the captive threaded sleeve
to form the cathode assembly. The process was repeated using the cation
a exchange membrane to form the anode electrode assernbly. The electrode
chambers were filled with the electrolytes immediately after assembly. The
anode was filled with about 25 ml of 0.1 M H3PO4 and the cathode was filled
with 25 ml of 0.1 M NaOH.
The assembled anode electrode chamber was slided over the ceramic
cooling finger so that the two protruding screw heads fitted into the holes in the
black plastic base of the cooling finger support. A membrane core was then
slided ont0 the cerarnic cooling finger, in such a way that the core was abutting
the acrylic ridge on the anode chamber. The focusing chamber was then slided
over the membrane core and the metal pins were inserted into the small hole in
the anode. The black nylon retaining screws were then tightened. The
assembled cathode electrode was then slided over the cooling finger, aligning
e the rnetal pins and the hole in the cathode chamber, and the nylon retaining
a screws tightened. The assembled focusing chamber was then mounted in the
stand.
The sample was prepared by diluting 5 ml of the trypsin extract with 3 M
urea to 55 ml and 2% Bio-lyte ampholyte pH 3-10.
With the cell mounted, the cell was rotated so that the 20 collection ports
faced up. The ports were covered with some sealing tape and reinforced with an
acrylic cell-cover block and the screws lightly tightened.
The cell was then rotated so that the filling ports faced up. The cell was
filled with sample through the filling ports using a syringe. The filling ports were
then sealed with a second cell cover block.
The cooling finger was then connected to a recirculating coolant source
maintained at 40C. The high voltage leads were attached to the power supply
(Biorad 3000Xi) and the conditions for the run set as follows: voltage limit of
a 2000 V, current limit if 35 mA and power limit of 12 W.
The harvest box was loaded with twenty 12 X 75 mm culture tubes and
the lid of the box was placed in position such that each stainless steel was inside
a collecting test-tube and a vacuum source was then connected to the port on
the box. When focusing was completed, the black toggle switch was moved to
the harvest position, the power supply disconnected, the lid removed and the
Rotofor cell rnoved close to the harvest box. The cell cover blocks of the upper
and lower focusing cell chamber were then removed. Vacuum was applied to the
collection box. The needle array was mounted on the two alignment pins on the
bottom of the chamber. The needle array was grasped with fingers of both hands
while placing the thumbs on the top of the focusing chamber. The needles were
then pushed uniformly al1 the way through sealing tape into the chamber. The 20
fractions were then simultaneously aspirated from the cell and delivered to the
collection tubes. The run was completed within 4-6 h
a 3.6.2 FRACTION SCREENING
(1) The pH range of the collected fractions were read using a standard pH
meter (Corning, 220).
(2) The amidase activity of the fractions were assayed for using the
method of Erlanger et al., (1 961) on a Hitachi U 2000 spectrophotometer.
The fractions exhibiting amidase activity were pooled together, diluted to
55 ml using deionized water and refractionated.
3.7 AMINO AClD COMPOSITION ANALYSE
The trypsin fraction for amino acid analysis was freeze-dried using a
Labanco freeze drier. The dried samples were then transfered into culture tubes
(6 x 50 mm) which were previously muffled at 450°C overnight. The tubes were
then placed in a Waters reaction via1 and dried in a Waters Pico-Tag Work
Station (Waters, CT, USA). 200pl of 6N HCI containing 1 % phenol was added to
the vial. The via1 was alternately purged (3 times) with dried nitrogen then heated
at 150°C for I h under vacuum. The samples were cooled and again evacuated
to remove traces of hydrochloric acid. The hydrolysates were applied to a
Beckman System 6300 High Performance Analyzer using the method of
Vereeragan and Gibbs (1989), Table 3.4. A strong cation exchange was used
with a temperature gradient from 50 to 65°C at I0Clmin. The amino acids were
Table 3.4 Program for amino acid analysis on Beckman HPLC System 6300
Sodium hydroxide
Buffer Molarity Flow rate pH Time Temperature
(mllh) (min) (Oc)
Sodium citrate 0.2 20 3.0 52.0 50
0.4 20 5.1 17.2 65
0.7 20 6.2 27.4 77
51 .O 50
36.0 65
2. O 77
Ninhydrin reagent 53.0
End of data collection 50.0
Sample loop filled 61 .O
Sample to be injected 63.0
a separated using sodium citrate buffer, in a period of 50 min.
3.8 INHIBITION STUDIES
The sensitivity of the cunner trypsin to various inhibitors was investigated
according to the method of Simpson and Haard, (1984),
Soybean trypsin inhibitor (SBTI) was dissolved in deionized water to the
following concentrations: 0.006, 0.001, and 0.1 mglml. The trypsin solution was
added separately to equal volumes of the SBTl solution and incubated on ice for
30 min and the residual trypsin activity assayed using BAPNA (pH 7.8) as
substrate
Aprotinin was diluted with deionized water to the following concentration,
e in trypsin inhibitor units (TIU): 0.02, 0.03, 0.04 and 0.06 TIUIrnl. Trypsin solution
was mixed with an equal volume of aprotinin, incubated on ice for 30 min and the
residual activity determined by assay with BAPNA (pH 7.8) as substrate.
Different substrate concentration in the range of 1-5 mM were used for
aprotinin while 1-7 mM range were used for SBTl in the determination of the
kinetic parameters of Km, (substrate affinity), V,,,, (maximum velocity) and Ki
(inhibitor dissociation constant). Km and V,,, were determined using the
Lineweaver-Burk equation :
Ki was deterrnined using the 1, method, where
where 15, is the concentration of inhibitor required for 50 % inhibition of the
enzyme activity.
3.8 PECTIN METHYLESTERASE ACTlVlTY DETERMINATION
PME activity was monitored by the spectrophotometric rnethod of
Hagerman and Austin (1986)
The method is based on the color change of a pH indicator dye during the
PME-catalyzed reaction. As ester bonds are hydrolyzed, acid groups are
produced to lower the pH, which causes a color change in the indicator dye. The
a color change is continuously monitored spectrophotometricaIly to obtain the
initial rate of the reaction.
For the assay itself, PME was dissolved in deionized water whose pH has been
pre-adjusted to 7.5 with 2N NaOH. In a cuvette, 2 ml of 0.5 % solution were
mixed with 0.15 ml of 0.01 % bromothymol blue in 0.003 M potassium phoshpate
buffer pH 7.5 and 0.83 ml water, and the initial absorbance measured at 620 nm
using a Hitachi U 2000 spectrophotometer. Twenty (20) pl of 20pg solution of
PME was added to the mixture and the rate of decrease in absorbance A620 (A
A ) was recorded. 6201rnin.
The method was calibrated with appropriate dilutions of a 0.87 mM galacturonic
acid. To achieve constant starting pH for the reaction, al1 solutions (water, pectin
and dye) were adjusted to pH 7.5 with 2N NaOH just before each trial was
started.
3.8.1 EFFECT OF CUNNER TRYPSIN ON PME ACTIVITY
Trypsin concentrations of 10, 20 and 40 uglml were incubated with equal
amounts of PME in solution (20 uglml). The mixture was incubated on ice for 4 h.
Portions of the mixture were taken at 1 h interval and the residual PME activity
assayed using the method of Hagerman and Austin (1986). The trypsin-treated
PME were stored on ice for 21 days to test for reactivation of the PME activity.
3.8.2 EFFECTS OF CUNNER TRYPSINS ON HEAT-TREATED PME
Twenty (20) uglml portions PME were incubated at 40 and 45°C for 30
min., and immediately cooled on ice. Equal volumes of cunner and bovine trypsin
were added to the heat-treated PME, incubated on ice for 4h. Sample aliquots
were taken every l h and the residual enzyme activity assayed using the method
of Hagerman and Austin (1986).
3.9 PPO ACTlVlTY DETERMINATION
PPO activity was determined according to the method of Savagon and
Sreenivasan (1978). A 0.2 ml portion of mushroom PPO solution in sodium
phosphate buffer, pH 6.5, was added to 2.8 ml of 5 mM DOPA in 0.05 M sodium
phosphate buffer pH 6.5, and the formation of dopachrome was measured at
475 nm at 25°C in a Hitachi U 2000 spectrophotometer
One unit of activity was defined as AA4,,,,m,, 10.6
3.9.1 EFFECT OF CUNNER TRYPSIN ON PPO ACTlVlN
Equal volumes of appropriately diluted trypsin fraction were incubated with
equal volumes of PPO in solution. The mixture was incubated on ice for 4 h.
Portions of 0.2 ml were taken at 1 h interval and the residual PPO activity was
assayed by measuring the formation of dopachrome at 475 nrn at 25"C, on a
Hitachi U 2000 spectrophotometer. The trypsin treated PPO was stored on ice
for 21 days to test for reactivation of the enzyme activity.
3.9.2 EFFECT OF CUNNER TRYPSIN ON HEAT-TREATED PPO ACTlVlTY
O Portions of PPO were incubated at 50°C and 55°C for 30 min., and
immediately cooled on ice. Equal volumes of appropriately diluted cunner and
bovine trypsins were incubated with the heat-treated PPO for a period of 4h.
Sample aliquots (75 pl) were taken every 1 hr and the residual PPO activity
determined by measuring the formation of dopachrome at 475 nm at 25" on a
Hitachi U 2000 spectrophotometer.
3.10.1 OPTlMlZATlON STUDIES / EXPERIMENTAL DESIGN
The different factors and their levels used in this study were [E]/[S] ratio
(0.5 - 1.5), incubation time (1 - 4h) and temperature (15 - 35"C), where the
substrate, S, is considered to be PME or PPO. Table 3.5 shows the parameters
with their levels and the coded and uncoded variables. To study the
a simultaneous effect of these factors on the residual activities of PME and PPO
activities, a 3 x 3 factorial design (Schmidt and Launsby, 1992) was used.
The model is indicated below:
X,, X, and X, are the variables or the factors namely enzyme:substrate ratio,
incubation time and temperature respectively and p values represent the
corresponding regression coefficients
To predetermined volumes of PME, 20 pg/ml, (20 pl) and PPO, 25 p g h l
(75 pl) the calculated [E]/[S] ratio (0.5 - 1.5) added and the resulting mixture
incubated at the predetermined temperature (15 - 35 OC) for a specified time (1 -
4 h). There were in al1 20 different combinations.
Statistical analysis and three dimensional plots were performed using the
statistical tool pack of Microsoft Excel4.0 (1994).
3.1 1.1 TREATMENT OF ORANGE JUlCE
The trypsin-treated PME and the untreated PME were added to different
portions of commercial orange juice (Tropicana). The cloud stability and the pH
of the juice were measured over a storage time of 21 days as per the procedure
m described by Owusu Yaw et al. (1988). For this, 10 ml portions of the sample
were centrifuged at 3209 for 10 min. The supernatant was filtered through
Table 3.5 Values of coded levels in Experimental design
Process Variable Code Levels
-il O 1 -
[E]/[S] ratio XI 0.5 'l 1.5
Incubation time (h) x2 1 2.5 4
Temperature (OC) X, 15 25 35
Whatman #41 filter paper (Whatman Limited, England). Cloud loss was
determined by measuring the absorbance at 660 nm on a Hitachi U 2000
spectrophotometer, using 3 ml cuvettes with distilled water as blank. The
experiments were run in triplicates.
3.1 1.2 pH MEASUREMENTS
This was measured using standard pH meter (Corning 220) by placing the
pH electrode in 10 ml of sample in a beaker and stirring with a magnetic stirrer.
The pH was recorded when the readings were stable.
3.1 1.3 STORAGE OF TREATED ORANGE JUICE
The treated and untreated orange juice were then stored in glass
containers at 4°C for a period of 21 days and readings of cloud stability and pH
taken fotthnightly.
CHAPTER IV
RESULTS AND DISCUSSIONS
4.1.1 Recovery of trypsin fraction
The recovery of the trypsin fraction is summarized in Table 4.1, which
shows that the specific activity increased from 0.55 for the crude fraction to 2.02
unitlrng for the affinity fraction (trypsin fraction). The purity of the extraction was
3.7 fold with a yield of 1.4%. The protein content as determined by the enhanced
Lowry assay was about 2.3 mg/lOg, using the standard curve for the protein
determination in Figure 4.1.
4.1.2 Elution profile of the trypsin fraction on SBTI-Sepharose Affinity media
9 The elution profile of the trypsin fraction from the SBTI-affÏnity media after
the application of the acetone fraction is shown in Figure 4.2 which shows the
absorbance at 280 nm, (A2,,), and the corresponding change in absorbance per
minute (dA/min) at 410 nm. There was a gradual increase in the absorbance at
280nm, (4,) up to a maximum and then a decrease after fraction 9. A second
gradual increase was observed from fractions 13-15. The dA/min followed the
same trend as observed for the A,,. The fractions showing trypsin activity, tubes
4-8 and 13-15, were pooled together, concentrated and used in subsequent
analysis.
4.1.3 Electrophoresis of the trypsin fraction
The graph of R, versus log. molecular weights of standards used to
@ determine the molecular weights of the protein bands are shown in Figure 4.3.
Table 4.1 : Recovery of trypsin from the pancreas of the cunner fish.
Step Total Total Total Specific Yield % Purification volume Protein activity activity fold
(ml) (mg) (units) (unitslrng)
Crude extract (300Os>
Crude extract (1 000s)
Acetone fraction
Affinity fraction
10.09 of pancreatic tissue was used.
t 1 1 I
O 20 40 60 80 1 O0 BSA concentration (uglml)
Figure 4.1 : Calibration curve for protein content determination using the enhanced
Lowry method and bovine çerum albumin (BSA) as standard. Aliquot of stock BSA
concentration of 2 mglml were used to prepare the standard curve.
1 3 5 7 9 11 13 15 17 19 21 2 4 6 8 10 12 14 16 18 20 22
Tube number
abs 280 nm dAlmh 1-91
Figure 4.2: Elution Profile of the acetone fraction on SBTI-Sepharose
Aninity media. Three (3) ml of acetone fraction cuntaining 1.5 mglml protein
were injded onto the wlumn.
O 0.2 0.4 0.6 O. 8 1 Mobility (F+ )
Figure 4.3: Calibration curve for molecular weight detemination for SDS-PAGE
electrophoresis
Figure 4.4: SDS-PAGE gel showing the migration of the proteins. Lane 1
shows the trypsin fraction from the SBTI-Sepharose affinity media and
lane 2 shows the standards, (Seeblue prestained markers from Novex)
from top to bottom: myosin, 250.000; BSA, 98,000; glutamic
dehydrogenase, 64,000, alcohol dehydrogenase, 50,000; carbonic
anhydrase, 36,000; myoglogin, 30.000; lysozyme, 16,000; aprotinin,
6,000; and insulin p-chah, 4,000.
m The electrophoresis of the trypsin fraction on SDS-PAGE gel is shown in Figure
4.4. The electrophoregram showed a distinct band at 24,000 kDa. This was
consistent with the findings of Simpson and Haard (1 984).
4.1.4 Stability of Various Fractions at Different Storage Temperatures
The stability of an enzyme fraction is of importance to ensure that no loss
of activity occurs during the extraction process. The integrity of the three
dimensional structure of the active site is essential for maintenance of activity
(Whitaker, 1994). One of the several factors affecting the integrity of the three
dimensional structure include temperature. Generally, enzymes are stable at
lower temperatures (Schwimmer, 1981). The stability of the various fractions on
ice and -20°C are shown in Figures 4.5 a & b respectively. The trypsin fraction
had the highest specific activity followed by the acetone and then the ammonium
0 sulfate fraction for both storage temperatures. There was a significant difference
in the specific activities of the fractions kept at 0°C and -20°C (p 2 0.05) for the
ammonium sulfate fraction. On the other hand, there were no significant
differences for either the acetone or the trypsin fraction stored at both storage
temperatures. The results of the analysis of variance (ANOVA) performed on the
results are shown on Table 4.2 a - 4 . 2 ~
4.1.5 lsoelectric focusing of the trypsin fraction
The isoelectric pH (pl) of a protein is the pH at which the net charge on a
protein is zero and has no mobility in an electric field. Proteins are minimally
soluble at their pl (Cheftel et al. 1985; Whitaker, 1994). Figures 4.6 a & b show
the pH-activity profile of the trypsin fraction after isoelectric focusing. After
refractionation, the trypsin showad two peaks, the lowest peak corresponding to
the ranges 6.48-6.83 and the highest peak corresponding the ranges 9.86-1 0.25. * This is consistent with the observation that the trypsin fraction had two proteins.
Storage time (weeks)
ammonium sulfate
Storage time (weeks)
acetone fraction
trypsin fraction
Figure 4.5: Effect of storage temperature on the stability of the
ammonium sulfate, acetone and trypsin fractions. (a) storage
on ice (b) storage at - 20 C
Table 4.2a One-way ANOVA of the storage on ice and at -20°C for the
ammonium sulfate fraction at p 5 0.05
Parameter Mean Variance P
O O C 0.774 0.008
-20 OC O. 893 0.002 0.028
Table 4.2b One-way ANOVA of the storage on ice and at -20°C for the
acetone fraction at p I 0.05
Parameter Mean Variance P
Table 4 . 2 ~ One-way ANOVA of the storage on ice and at -20°C for the
affinity fraction at p 0.05
Parameter Mean Variance P
O O C 4.61 3 O. 074
-20 O C 4.672 0.078 0.743
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Tube number
1 2 3 4 5 6 7 8 9 10 I l 12 13 14 15 16 17 18 19 20 Tube number
Figure 4.6: (a) Fractionation and (b) refractionation of the trypsin fraction
after isoelectric focussing at 4 C on Biorad rotofor system
a A pl value reported for bovine trypsin is around 9.3 (Kiel, 1971). A trypsin
fraction recovered from the fungus, Metarhizium anisopliae, however, has been
reported to have two proteins fractions with rather low pl values of 5.4 and 4.6,
both of which are lower than the bovine or the cunner trypsins (Cole et al. 1993),
of 9.3 or 6.48-6.83 and 9.86-1 0.25.
4.1.6 Amino acid composition analysis
The elution of amino acid standards is shown on Figure 4.7. The acidic
and straight chain hydroxy amino acids were the first to be eluted. On increasing
the temperature, other small and neutral amino acids were eluted. lncreasing the
pH and temperature further, eluted the basic amino acids. The amino acid
composition of the trypsin fraction showing the mole fraction and number of
O residues is shown in Table 4.3. The trypsin fraction was found to have 220 amino
acid residues, whilst bovine trypsin has been reported to possess 223 residues
(Kiel, 1971). The residues were very rich in several of the common amino acid
residues including Asp, Glu, Ile, Leu and Ser, except Arg, Lys, Phe, Tyr and Val.
The amino acid composition was similar to those of trypsins from other sources
such as the Greenland cod and Atlantic cod, bovine, porcine and human
(Simpson et al. 1989; Simpson and Haard, 1984; and Kiel, 1971). Fish trypsins
including the cunner have been reported to be stable at alkaline pH values
(Simpson et al. 1989). The low levels of basic amino acid residues (Arg and Lys)
makes them resistant to autodigestion, since trypsins, are active in the alkaline
pH and hydrolyze peptide bonds at the carboxyl side of arnino acid residues
contributed by Arg or Lys. Furthermore if the amino acid next to the arginine or
lysyl residue in the primary structure of trypsin polypeptide chain is a proline,
then the trypsin molecule will be resistant to autodigestion. Moreover, if the
arginine or lysine residues form the carboxyl end of the amino acid chain, the
Figure 4.7: Chromatogram of amino acid standards at 570 nm using
norleucine as interna1 standard
Table 4.3 Amino acid composition of the cunner trypsin
Amino acid Mole % Number of
Residuesa
Asp + Asn 9.70 21
Thr 4.82 II
Ser 10.09 18
Glu + Gln 8.40 27
G ~ Y 12.23 15
Ala 6.68 14
Val 6.32 6
Met 2.94 II
IIE 4.91 20
Leu 8.95 20
Tyr 4.2 9
Phe 1.86 4
His 4.52 10
LYS 3.66 8
T ~ P - -
Arg 2.02 4
Pro 4.99 II
CYS 3.60 14
Total # residues 220
'Based on a molecular weight of 24,400 KDa
protein becomes less susceptible to autodigestion at alkaline pH (Segel, 1976).
4.2 The Effect of lnhibitors on the trypsin fraction
Both the cunner and the bovine trypsin were susceptible to inhibition by
SBTI. The extent of inhibition of cunner and bovine trypsin activity with
increasing substrate concentration is shown in Figures 4.8 a & b. lncreasing the
inhibitor concentration resulted in corresponding increase in the percent
inhibition of both trypsins. However, for the cunner trypsin, the effect of inhibition
was lowered as the substrate concentration, [SI, increased from 3 mM to 7 mM,
Fig 4.8a. The same trend was observed for the bovine trypsin, Fig. 4.8b, but a
higher substrate concentration of 5 mM was required before the percent
inhibition was lowered.
Both the cunner and the bovine trypsin were susceptible to inhibition by
aprotinin. The extent of inhibition of both cunner and bovine trypsin activity with
increasing [SI are shown in Figs. 4.9 a & b. lncreasing the inhibitor concentration
resulted in the corresponding increase in the percent inhibition of both trypsins.
The reverse of the observation with SBTI, however, resulted. A higher [SI was
required by the cunner trypsin, Fig. 4.9a, to relieve the inhibition. On the other
hand, increasing the [SI from 3 to 5mM resulted in a decrease in the inhibition of
the bovine trypsin by aprotinin. The extent of the inhibition relief was relatively
lower in aprotinin than in SBTI, suggesting that different types of inhibition may
be exhibited by both inhibitors.
The Lineweaver-Burk plots of SBTl inhibition of cunner and bovine trypsin
are shown in Figs 4.10 a & b. SBTI exhibited a predominantly cornpetitive type of
inhibition for cunner and bovine trypsin. The plots using aprotinin as inhibitor is
shown in Figs 4.1 1 a & b. The kinetic parameters for cunner and bovine trypsin
using SBTl and aprotinin are shown in Tables 4.4a - 4.4d. 4
Figure 4.8: Effect of increasing BAPNA concentration on SBTl inhibition.
(a) cunner trypsin (b) bovine trypsin
-- -
0.02 TIU 0.03 TI U 0.04 TIU 0.06 TIU -m- -8. + *
Figure 4.9: Effect of increasing BAPNA concentration on aprotinin inhibition.
(a) cunner trypsin (b) bovine trypsin
From table 4.4a, the substrate affinity, Km, increased 5-fold after
increasing [Il from 0.025 to 0.1 0 mg/ml SBTI, and a 1 .l fold decrease in the
maximum velocity, Vma,, value. From Table 4.4b, the Km values for bovine trypsin
exhibited a 12-fold increase whilst there was only a 7.4 fold increase in VmaX
values after an increase of [Il from 0.025 to 0.10 mglml of SBTI. The results
show a predominantly cornpetitive inhibition of both cunner and bovine trypsins
by SBTI. It was also observed from the effect of SBTI on the substrate affinity
that the inhibitor binds to the enzyme in such a way as to block its active sites
(Blow et al, 1974). A low K, value denotes a high susceptibility of an enzyme to
inhibition (Segel, 1978; Whitaker, 1994). From Tables 4.4a & b, the cunner
trypsin fraction had lower K, values than the bovine trypsin and thus appears to
be more susceptible to inhibition by SBTI than bovine trypsin.
The effects of aprotinin on both cunner and bovine trypsin showed a
mixed type of inhibition, as both V,,, and Km decreased with increase in [Il. There
m was a 1.76 and 2.8-fold decrease for Vmax and a 6 and 1.3-fold decrease in the Km
for the cunner trypsin fraction and bovine trypsin respectively. In contrast to
observations of the enzyme's susceptibility to SBTI, the bovine trypsin exhibited
lower K,, values than the cunner trypsin. Thus the bovine trypsin was more
susceptible to inhibition by aprotinin than the cunner trypsin.
4.3. Dependence of Y, on Temperature
The enzyme-substrate affinity, (Km), is reported to be affected by the
habitat temperature of the organism (Haard et al., 1982), and that the Km of
trypsins from various fishes correlated with the temperature preferendum of each
species. The dependence of Km, the substrate binding affinity, of cunner trypsin
on temperature is shown in Figure 4.12. The Km increased with increase in assay
control 0.025 mg 0.075 mg 0.1 mg - -$à- A ++ -
Figure 4.10: Lineweaver-Burk plot for the trypsins using SBTl as inhibitor.
(a) bovine trypsin and (b) cunner trypsin, with 1 mM BAPNA (pH 8.2) as
su bstrate
control0.02 TIU 0.03 TIU 0.04 TIU 0.06 TIU I * e + * +K 91
Figure 4.11: Lineweaver-Burk plot for the trypsins using aprotinin as inhibitor.
6 (a) bovine trypsin and (b) cunner trypsin, with 1 mM BAPNA (pH 8.2) as
substrate
Table 4.4a Kinetic data on SBTl inhibition of cunner trypsin
[il (mg) V,, (un itslmin .) Km (mM) K, (mM x10 ")
Table 4.4b Kinetic data on SBTl inhibition of bovine trypsin
Ill mg) V,,,, (unitslmin.) Km (mM) K, (mM x10
0.00 1.42 + 0.01 1 0.99 ir 0.085 0.00
0.025 1.91 1 0.060 3.41 k0.214 28.8 + 0.006
0.075 1.52 k0.002 5.20 + 0.089 4.06 t0.005
0.1 O0 2.02 & 0.001 12.47 + 0.041 4.67 t0.001
Table 4.4~ Kinetic data on aprotinin inhibition of cunner trypsin
[Il ( T u V,,, (units/min.) Km (mM) Ki (mM x10 ")
Table 4.4d Kinetic data on aprotinin inhibition of bovine trypsin
[Il (w Vm,, (un itslmin .) Km (mM) Ki (mM x i 0 -3)
0.00 1.42 I 0.01 1 0.99 a 0.085 0.00
0.02 2.08 k 0.123 0.97 ,+ 0.20 7.5 k0.0003
0.03 1.39 + 0.053 0.88 t 0.02 3.9 +0.0005
I O 20 30 40 50
Assay temperature (C)
Figure 4.12: Dependence of cunner trypsin Km on temperature. Cunner
trypsin fraction depicts a "type 3" Km -temperature dependence
e temperature being insensitive at lower temperatures. Greenland cod trypsin, and
trypsins from Salmo atra and Tnturus alpestris, have also been reported to
exhibit this type of response (Simpson and Haard, 1987; Hofer et ab, 1975). This
type of dependence is called the "type-3" response (Hultin, 1978). In this
response, the Km increases with temperature as the assay temperature is
increased, but at lower temperatures, insensitivity to temperature is observed.
The minimum Km observed in thiç Km-temperature profile coincides with
the habitat temperature of the cunner fish, which is around lQ°C, implying that
the cunner fish has adapted to have the highest substrate affinity at the habitat
temperature to be able to utilize the food ingested. Bovine trypsin on the other
hand has been reported to show a "type 2" Km temperature response, in which
the Km remains constant, neither increasing or decreasing with temperature
(Hultin, 1978; Simpson and Haard, 1987).
4.4 Effects of trypsins on Native PME and PPO
PME is known to cause the breakdown of juice cloud in citrus juices
(Chaplin and Bucke, 1990) and PPO is known to cause the enzymatic browning
observed in fruits, vegetables and crustacean species (Chen et al., 1993; Zhou
and Feng, 1991; Valero et al., 1988). Trypsins from the stomachless cunner
have been reported to hydrolyze native proteins better than trypsins from the
species with a physiologically and morphologically distinct stomach as in the
bovine (Simpson and Haard, 1987b). However, there are wide differences
among proteins in their susceptibility to proteolysis (Whitaker, 1994).
The effects of differing concentrations of cunner and bovine trypsin on
PME at differing pectin concentrations on PME activities are shown in Figures
4.14, 4.15 and 4.16. The hydrolysis of PME was measured as a loss of
enzymatic catalysis. The cunner trypsin treatment resulted in a loss of 75 % of
the initial activity of PME after 4h incubation at 4°C using 0.5 % pectin solution.
Treatment of PME with bovine trypsin on the other hand resulted in only a loss of
35 % of the initial activity. Lowering the pectin concentration to 0.35 %
decreased the difference observed to 60 and 50 % loss initial activity for the
cunner and bovine trypsins, respectively. Increasing the [enzyme]/substrate ratio
to 1 and 2, at a pectin concentration of 0.35 %, the bovine trypsin tended to
decrease the percent initial activity better than the cunner trypsin. This
observation is consistent with the report of Castaldo et al. (1991), of the effects
of proteolytic enzymes on PME activity. PME from orange has also been
reported to have 22 Arg and Lys residues (Castaldo et al., 1991) for the trypsins
to hydrolyze.
The effect of the trypsins on native PPO activity is shown in Figure 4.77.
Trypsin frorn cunner hydrolyzed native PPO better than bovine trypsin, resulting
in loss of 75 % and 55 % of initial activity upon incubation for 4h with cunrier and
bovine trypsins respectively. Generally proteolytic enzymes are known to
8 hydrolyze peptide bond when that portion of the protein molecule is in the
denatured state (Whitaker, 1994), and even if they do hydrolyze native bonds,
the rate of hydrolysis is very low. However, the observations with the cunner
trypsins show that the cunner trypsin hydrolyzes native proteins (PME and PPO)
better than the bovine trypsin. This observation is consistent with the results
observed with a trypsin-like enzyme from the stornachless crayfish (Pfleiderer et
al, 1967), trypsins from the stomachless cunner on ribonucease and hemoglobin
(Simpson and Haard, 1987b) and of trypsins from the stomachless crawfish on
PME activity (Jeong et al. 1994). This is an adaptation of the species without a
distinct stomach, to counterbalance the absence of the acid denaturation of
proteins that occur in the stomach which makes the proteins amenable for
digestion by the alkaline proteases in the small intestines.
-0.14 ' I I l I 1 I l
O. 1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Galacturonic acid concentration (umol)
Figure 4.13: Calibration curve for PME assay. Aliquots of stock galacturonic acid
solution (0.87 mM) were used to generate the standard curve, with 0.5 % pectin
solution at 25 C
O 1 2 3 4
Tirne (hr)
i 8 i i 1
4
Time (hr)
-*-. cunner trypsin -O- bovine trypsin
Figure 4.14: Effect of cunner and bovine trypsins on PME activity using
an enzyme concentration of 0.5 and (a) 0.35% and (b) 0.5% pectin solutions
O 1 2 3 4 Time (hr)
8 I 1 1 I
1 2 3 4
Time (hr)
-.m-- cunner trypsin -a- bovine trypsin
Figure 4.1 5: Effect of cunner and bovine trypsins on PME activity uskg
an enzyme concentration of i and (a) 0.35% and (b) 0.5% pectin solutions
Time (hr)
1 2 3 4
Tme (hr)
-- cunner trypsin -+ bovine trypsin
Figure 4.16: Effect of cunner and bovine trypsins on PME activity rising
an enzyme concentration of 2 and (a) 0.35% and (b) 0.5% pectin solutions
4.5 Effects of trypsins on heat-denatured PME and PPO
The effects of cunner and bovine trypsins on heat-denatured PME at 50
and 55°C are represented in Figures 4.18 a-c. Heat treating PME at 50 and 55°C
for 3' min., resulted in 20% and 70 % loss of the initial activity respectively.
Treatrnent of the heat-denatured PME at 50°C with cunner and bovine trypsins
resulted in a loss of 90% and 60% respectively of the remaining PME activity.
Conversely, treatment at 55°C of PME resulted in a loss of 30% and 45% PME
activity with cunner and bovine trypsin respectively. Thus heat denaturation at a
higher temperature (55°C) resulted in a higher degree of hydrolysis of PME with
bovine trypsin than the cunner trypsin.
Effects of trypsins on heat-treated PPO at 40 and 50°C are shown in
Figures 4.19 a-c. Heat treatment of PPO at 40 and 50°C resulted in a loss of
about 10% and 80% of the initial PPO activity respectively. Treatment of PPO
e denatured at 40°C, resulted in a loss of 25% and 40% of the PPO activity with
bovine and cunner trypsin respectively. On the other hand, 50°C treated PPO
resulted in a loss of 95% and 98.5% of the initial PPO activity with cunner and
bovine trypsins respectively. This observation was similar to the effects of the
trypsins on PME activity.
In solution, proteins (e.g., PPO and PME) are known to exists in at least
three forms narnely the native, reversibly denatured and irreversibly denatured
forms. Of the three forms, the reversibly denatured forrn is subject to extensive
proteolysis. The other two forms are somewhat susceptible to proteolysis, but the
contribution of these two forms to the entire proteolysis is very low or negligible
(Whitaker, 1994). Increaising the temperature of incubation of PME or PPO,
therefore, increases the denatured forms rather than the native form, thus
increasing the level of hydrolysis of the heat-denatured proteins over the native
proteins. Trypsins are known to hydrolyze peptide bonds at the carboxyl ends
a contributed by arginine or lysine residues (Yamamoto, 1975). The extent of
Incubation l ime (hr)
B--
bovine trypsin
O-
cunner tvpsin
Figure 4.17: Efiect of cunner and bovine trypsins on native PPO activity
using 5 mM DOPA as substrate at 25 C
Temperature (C)
O 1 2 3 4
incribation time (h)
incubalion tirne( h)
- D. -e- bovine trypsin cunner trypsin
Figure 4.18: (a) Effect of heat treatment on PME activity and the effect of cunner
and bovine trypsins on heat-denatured PME at (b) 50 C and (b) 55 C
O L
1 O 20 30 40 50 Tcmperature (C)
Incubation Time (h)
m- -@- bovine trypsin cunner trypsin
Figure 4.19: (a) Effect of heat treatinent on PPO activity and the effect of cunner
and bovine trypsins on heat-denatured PME at (b) 40 C and (b) 50 C
hydrolysis of a protein such as PME and PPO will also depend primarily on the
amount of lysine and arginine residues present. The type of amino acid residue
next to the arginine or lysine residue in the polypeptide chain such as proline will
also make a protein more resistant to trypsin attack (Kiel, 1971). Moreover, the
degree of hydrolysis can be affected if the lysine or arginine residues form the
carboxyl end of the proteins (Segel, 1976).
4.6: Reactivation studies of trypsin-treated PME and PPO.
Enzyme reactivation is of great importance in the food industry since this
can result in spoilage of the particular food during storage (Schwimmer, 1987).
The effects of storage time on the reactivation of trypsin-treated PME and PPO
are shown in Figures 4.20 and 4.21 respectively. The control PME solution
@ retained al1 its activity throughout the 21 day storage, indicating that the enzyme
is very stable at 4°C. There was no reactivation of the trypsin treated PME or
PPO during storage. Increasing the cunner trypsin concentration (Figure 4.20a)
from 0.5 to 2, it was observed that PME activity was reduced to zero at day
21,12 and 3. On the other hand, for bovine trypsin, (Figure 4.20b) the days were
21, 18 and 9 suggesting that the inactivation of PME by the trypsins was
irreversible. PME treated with supercritical carbon dioxide has been reported to
show sorne reactivation after 15 days of storage at 4.4"C (Balaban et al., 1991 ),
indicating that the treatment was reversible. The observation with the cunner
trypsin therefore, makes it more desirable as an alternate way for PME
inactivation.
Figure 4.21 shows the reactivation studies of PPO during storage at 4°C.
PPO did not show any reactivation, but PPO retained a substantial amount of its
initial activity, up to 20 % and 45 % for cunner and bovine trypsin treatrnent
O 3 6 9 P 2 15 18 21 Days
control 0.5 A.0 2.0 -wt -*+
Figure 4.20: Reactivation studies of trypsin treated PME with (a) cunner
and (b) bovine. The substrate was 0.5 % pectin solution. The storage
studies were carried out at 4 C
control cunner bovine r-i- +
Figure 4.21: Reactivation studies of trypsin treated PPO at 4 C using 5 mM
DOPA (pH 6.5) as substrate.
respectively for the 21 day storage. The residual activity so observed turned the @ substrate DOPA brown, indicating that the residual activity was enough to cause
some undesirable discolorations in foods. In this respect, PPO showed some
resistance to proteolysis during storage, and this may be due to some of the
factors discussed in section 4.3 above. Also, Robinson and Dry (1992) reported
that a PPO fraction from the broad bean leaf retained its activity even after
proteolysis to remove a 15-18 kDa peptide. This suggests that the active sites of
these PPO molecules had not been fully destroyed by the proteolysis.
Furthermore, in some proteins, after initial hydrolysis of some of the bonds, the
remainder of the molecule becomes less susceptible to hydrolysis (Whitaker,
1994; Schwimmer, 1981). This changes in susceptibility may be due to changes
in the conformation of the protein after the initial hydrolysis resulting in a less
susceptible protein to enzyme hydrolysis.
a 4.7 Cornbined effect of [E]/[S] ratio, incubation time and temperature on
PME and PPO.
Analysis of variance for the response of % PME inactivation, using the
model in section 3.10.1 was significant (pc0.05) and had f values of 0.92 and
0.95 for cunner and bovine trypsin respectively. For % PPO inactivation iL values
of 0.91 and 0.94 were obtained for cunner and bovine trypsin treatrnents
respectively. The parameter estimates for % inactivation of PME and PPO for
both cunner and bovine trypsins are shown in Table 4.5. The parameters that
were insignificant were dropped from the model, and the data re-analyzed. The
mathematical model for PME inactivation and the response surface plots were
then generated from the reanalyzed parameter estimates.
The equations used in the generation of the three dimensional response
surface plots are as follows:
Table 4.5 Parameter Estimates for percent PME and PPO inactivation
% PME inactivation % PPO inactivation
Parameter Cunner trypsin Bovine trypsin Cunner trypsin Bovine trypsin
l ntercept 99.24" 99.52" 57.57" 78.40"
X1 14.1 8" 10.67" -1 0.48" -5.99"
X2 3.1 6"' 2-78"' -0.48"' -1.65"'
X3 32-63" 18.69" -3.74"= -4.5gnS
XI2 -7 0.58b -1 1 .6Ia 1.72"' -4.8gb
x: -22.22" -1 9.02" -9 -25" -9.64"
x t -1 1.71 -7.00~ -6. 34" -1 5.97a
X1X2 -6.36"" -5.18"" -3.3Sns -3.1 5""
XI% -5. 72"$ 3.98"" -4.98"" -2.34"'
'2'3 4.33ns 3.98"' -8.33" -4.77""
" Significant at 5 % level; Significant at 10 % level; "" Not significant; X, =[E]I[S]
ratio; X, = incubation time (h); X, = Temperature (OC)
Cunner trypsin
% inactivation PME, Y, = 90.58 + 14.18X, + 32.63X3 - 21.16Xt-10.56~: (1)
% PPO inactivation, Y, = 58.98 - 10.48 XI - 9.42 X l - 6.51 X t - 8.33X2X, (2)
Bovine trypsin
% inactivation PME, Yb= 99.52 + 10.67X, + 78.69X3 - 11 .60XlZ- 19.02X; -
2.03 X,' (3)
% PPO inactivation, Yb, = 78.46 - 5.99 X, + 4.89 X,' - 9.64 X l - 15.97 XJ? (4)
From the results of sections 4.3 and 4.5 a response of importance is that
between 80-100% inactivation of these enzymes, since these ranges resulted in
e no reactivation during storage.
Using cunner trypsin for PME hydrolysis, the response surface plots,
(Figure 4.22a), showed an increase in % PME inactivation on increasing both the
[E]/[S] ratio and temperature at a constant time, giving a linear response. Figure
4.22b depicted an increase in the % PME inactivation with both an increase in
[E]/[S] ratio and incubation time at a constant temperature, and that gave a
pronounced quadratic response. Similarly, at a constant [E]/[S] ratio, there was
an increase in % PME inactivation with increasing the temperature and
incubation time, and that also gave a quadratic response (Figure 4.22~). Using
bovine trypsin for PME hydroiysis, at a constant incubation time, increasing the
[E]I[S] ratio and temperature increased the % PME inactivation (Figure 4.23a). At
a constant temperature while varying [E]/[S] ratio and incubation tirne (Figures
4.23 b), gave a pronounced quadratic response. A similar response was
observed at a constant [E]I[S] ratio and varying the incubation time and
0 temperature for bovine trypsin treatment (Figure 4.23~). According to Ashie
(1996), such an absence of either a unique maximum or a minimum for one of
the factors with a variation in the other factors, indicates that there is a critical or
narrow range of values for one of the factors. Thus to generate a maximum level
of PME inactivation, there exist a broad range of conditions given by such a
response. This type of response is advantageous in the food industry because
there exist, a broad range of selection by the food processor to achieve the
same results, and as such minimum levels of expensive ingredients or additives
can be selected. Example of such a response is seen in figure 4.22~. To obtain
an 80-100 % inactivation of PME at a constant ternperature of 25"C, an [E]I[S]
ratio of 0.8 for an incubation of 2.5 h or [E]I[S] ratio of 1.1 for the same
incubation time can be utilized.
This type of quadratic response may be due to a number of factors
causing either the activation or the inactivation of the trypsins. Cunner and
bovine trypsins are known to have temperature optima around 45°C (Simpson et
9 al. 1989), and PME is also known to have a temperature optimum at 45°C
(Seymour et al. 1991). The temperatures used were below the optima, thereby
decreasing the possibility of inactivation after the optima had been reached. Also
these combined treatments resulted in about 100% inactivation of PME, which
was hitherto impossible to achieve by other methods (pressure and heat
inactivation) used previously (Takahashi et al., 1993; Ogawa et al., 1990; Wicker
and Temelli, 1988), due to thermostable form of PME which was resistant to the
heat treatments. The heat treatments also affected the aroma and quality since it
led to the loss of heat labile aroma compounds of the finished product.
Furthermore, some treatments with supercritical carbon dioxide has been
reported to reactivate the PME during storage (Balaban et al., 1991). The
varying combination of treatments of temperature, time and [E]/[S] ratio provide a
wide selection of treatments as alternate treatments for cloud stabilization in
products like the citrus juices. The low ternperature used is also desirable since it
will have no effect on the arorna and quality of the juice.
a Contrary to our expectations, using both bovine and cunner trypsins for
PPO hydrolysis, at constant temperature, increasing the [E]I[S] ratio with variable
incubation time, resulted in an initial decrease in % inactivation of PPO and then
an increase in the PPO activity (Figure 4.24 a & 4.25 a). For cunner trypsin
fraction, at a constant tirne increasing the [E]/[S] ratio and temperature resulted
in an increase in % PPO inactivation (Figure 4.24 b). Bovine trypsin treatment
resulted in an initial decrease in % PPO inactivation to a minimum and then an
increase (Figure 4.25b) just as was o b s e ~ e d for the cunner trypsin. At a
constant [E]/[S] ratio with increasing time and temperatures both cunner and
bovine trypsins, exhibited a pronounced quadratic response (Figure 4.24 c 81
4.25 c). These observations presupposes that both trypsins had an activation
effect on the PPO at certain combination levels. The temperature optimum of
PPO range from 35°C - 45°C (Valero et a/., 1988; Simpson et ai., 1987). The
temperature of operation coincided with the temperature optima of PPO, and
thus instead of inhibitory effects, there was an activation of the PPO. It was
e impossible to obtain 90-100% inactivation with any treatment. These resufts
showed that this mode1 would not be good in inhibiting the enzymic browning
observed in several food products and as such an alternate and more effective
model should be sought.
t O0
4 0
h 6 O
4 0 yr
2 0
O
Figure 4.22 Three dimensional response surface plots showing the effects of
[cunner trypsin]l[S] ratio, incubation time and temperature, on PME
activity. (a) Effect of temperature and [E]I[S] ratio et constant time (2.5h)
(b) effect of [E]l[S] ratio and time at constant temperature (25 OC) and
(c) effect of tirne and temperature at a constant [E]I[S] ratio (1).
T h e ( h )
1 O0
8 0
6 0 4 0
ua 2 0
O T a m p ( C )
T lm a ( h )
Figure 4.23 Three dimensional responsa surface plots showing the effects of
[bovine trypsin]/[S] ratio, incubation time and temperature, on PME
activity. (a) Effect of temperature and [E]l[S] ratio at constant time (2.5h)
(b) effect of [E]/[S] ratio and time at constant temperature (25'C) and (c)
effect of time and temperature at a constant [E]/[S] ratio (1).
10 T lm e ( h )
O O d -
Figure 4.24 Three dimensional response surface plots showing the effects of
[cunner trypsin]/[S] ratio, incubation time and temperature, on PPO
activity. (a) Effect of [E]I[S] ratio concentration and time at constant
temperature (25 O C ) and (c) effect of time and temperature at a constant
[E]/[S] ratio (1).
Figure 4.25 Three dimensional response surface plots showing the effects of
[bovine trypsin]/[S] ratio. incubation time and temperature, on PPO activity.
(a) effect of temperature and [E]/[S] ratio at constant tirne (2.5h) (b) effect of
[E]/[S] ratio and tirne at constant temperature (25 'C) and (c) effect of time
and temperature at a constant [E]I[S] ratio (1).
4.8 Model Validation
The effect of some of the optimum factors were used to treat orange juice.
The effectiveness of PME hydrolysis was measured as the change in pH and the
loss in cloud in the orange juice during storage at 4°C. Cloudiness is an
important attribute in orange juice, and the cloud suspended contains most of the
characteristic orange flavor and color (Balaban et al., 1991; Castaldo et al.,
1991). The changes in the pH values so measured ranged from 3.96 to 3.85 for
the control, and 3.96 and 4.0 for al1 the other treatments. The changes in cloud
stability of the juice are shown in Figures 4.26 a & b. The control was treated
with 0.f4 units/ml PME, and there was an initial decrease of about 15% of the
cloud after day 1. This value then stayed the same until day 9, after which the
cloud deteriorated for the remainder of the study, with a loss of up to 30% of the
cloud. For the treatment of the juice with bovine trypsin at 35°C (Figure 4.25 a),
the cloud was stable up to day 7, after which there was a cloud loss of up to 30
a % of the initial cloud. Treatment of the juice with cunner trypsin at 35"C, however,
resulted in an initial loss of about I O % in the cloud. The cloud was stable over
the storage period up to day 13, after which there was an increase in the cloud
up to the end of the storage period.
At lower temperature treatments (15"C), there was cloud loss for both
cunner and bovine trypsin throughout the storage period, with a loss of 20% and
35% of the cloud using cunner and bovine respectively. The juice treated with
the cunner trypsin appeared to hold the integrity of the cloud better than the
bovine trypsin treated juice. Owusu-Yaw et al. (1988), reported a similar
observation when they used a combination of low pH and low temperature
treated orange juice over a storage period of 12 weeks. The control resulted in
more cloud loss than the juice whose pH has been lowered to 2.2. However, the
juice treated this way has been reported to have unacceptable sensory quality
(Arreola et al., 1991), and also caused a loss of up to 99% of vitamin Cl ascorbic
O 1 2 4 7 9 11 ?3 15 18 21 Days of storage
I I I I l i 1 1 I l l
O 1 2 4 7 9 1 13 15 18 21 Days of storage
Figure 4.26: (a) An [E]I[S] ratio of 0.3 and 1.5 bovine and cunner at 35 C
for 1.3 and 0.4 h respectlvely ard (b) [E]l[S] ratio of 1.5 and 0.9 bovine and
cunner trypsin at 15 C for 2.5 h. The juice was stored at 4 C
a acid content of the juice (Owusu-Yaw et al., 1988). Although treatment with
supercritical carbon dioxide has been reported to give a somewhat stable cloud
in orange juice (Balaban et al, 1991), the presence of residual PME activity
reported earlier at 4 OC may cause cloud loss under certain storage conditions.
Some pectinases have also been reported to stabilize the cloud of the orange
juice to varying extents (Baker and Breummer, 1972), however a strict control of
the depolymerizing action of the pectinase is important, since a high level of
depolymerization results in the loss of juice cloud.
Treatment of PME with the cunner trypsin fraction in combination with a
temperature of 35°C was able to maintain the stability of the orange juice cloud
better than treatments with bovine trypsin at the same temperature. This
treatment can therefore serve as an alternate method in PME inactivation.
However, further studies on the effects of the treatments on other quality
attributes need to be studied.
CHAPTER V
CONCLUSIONS
Based on the findings, the following conclusions may be made:
Digestive proteases recovered from the pancreas of the cunner fish had two
pl ranges of 6.24-6.83 and 9.66-1 0.25, suggesting the cunner trypsin fraction
possess two proteins.
The amino acid composition data revealed a polypeptide of 220 amino acid
residues, low in basic amino acids explaining the trypsin's stability at alkaline
PH.
The trypsin fraction displayed a "type 3" Km dependence on temperature,
with the highest affinity coinciding with the habitat temperature of the cunner
fish, (1 O°C).
The ammonium sulfate, acetone and affinity fractions al1 showed a great
amount of stability after storage on ice and -20°C for a period of 4 weeks
Soybean trypsin inhibitor competitively inhi bited both the cunner and bovine
trypsin. However, a mixed type of inhibition was observed for aprotinin.
The cunner trypsin fraction was more capable of hydrolyzing the native PME
and PPO better than the bovine trypsin. Conversely, bovine hydrolyzed the
heat-denatured PME and PPO better than the cunner trypsin. On storage at
4OC for 3 weeks, there was no reactivation observed for either PME or PPO
that had previously been treated with either the bovine or the cunner trypsin.
The effects of combination treatments of temperature, time and Kamel
enzyme concentration (bovine or cunner trypsin), using a 3 x 3 factorial
design, on PME gave an f of 0.92 and 095 using cunner and bovine trypsin
respectively; and on PPO an f of 0.91 and 0.94 using cunner and bovine
trypsin respectively. A complete (1 00%) inactivation of PME was possible for
some combinations; however no complete inactivation of PPO was obtained
for al1 the levels of combination treatments. Certain combinations rather had
activatory effects on PPO.
8. The trypsin from the cunner fish was capable of upholding the cloud integrity
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obsewed for some combination treatments on the cunner and thus, cunner
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