studies on extraction of stevioside from stevia (stevia ... · dr. (mrs.) gagandeep kaur sidhu er....
TRANSCRIPT
-
STUDIES ON EXTRACTION OF STEVIOSIDE FROM
STEVIA (Stevia rebaudiana Bertoni) LEAVES
Thesis
Submitted to the Punjab Agricultural University
in partial fulfillment of the requirements
for the degree of
MASTER OF TECHNOLOGY
in
PROCESSING AND FOOD ENGINEERING
(Minor Subject: Food Science and Technology)
By
Gursimran Singh Sangha
(L-2011-AE-150-M)
Department of Processing and Food Engineering
College of Agricultural Engineering and Technology
PUNJAB AGRICULTURAL UNIVERSITY
LUDHIANA-141004
2014
-
CERTIFICATE I
This is to certify that the thesis entitled, “Studies on extraction of stevioside from
stevia (Stevia rebaudiana Bertoni) leaves” submitted for the degree of M.Tech. in the
subject of Processing and Food Engineering (Minor subject: Food Science and
Technology) of the Punjab Agricultural University, Ludhiana, is a bonafide research work
carried out by Gursimran Singh Sangha (L-2011-AE-150-M) under my supervision and
that no part of thesis has been submitted for any other degree. The assistance and help received during the course of investigation have been fully
acknowledged.
______________________________
Dr. (Mrs.) Gagandeep Kaur Sidhu
Major Advisor
Research Engineer
Deptt. Of Processing and Food Engineering
Punjab Agricultural University
Ludhiana- 141004
-
CERTIFICATE II
This is to certify that the thesis entitled, “Studies on extraction of stevioside from
stevia (Stevia rebaudiana Bertoni) leaves” submitted by Gursimran Singh Sangha
(L-2011-AE-150-M) to the Punjab Agricultural University, Ludhiana, in partial fulfillment of
the requirements for the degree of M.Tech. in the subject of Processing and Food
Engineering (Minor subject: Food Science and Technology) has been approved by the
Student‟s Advisory Committee along with Head of the Department after an oral examination
on the same.
___________________________ ____________________________
Dr. (Mrs.) Gagandeep Kaur Sidhu Er. Navdeep Jindal
Major Advisor External Examiner
Assistant Professor
Deptt. of Food Engg. and Technology
SLIET, Longowal
_______________________
Dr. A.K. Singh
Head of the Department
_______________________
Dr. Gursharan Singh
Dean, Post-Graduate Studies
-
ACKNOWLEDGEMENT
I have been able to bring this study in the present shape only because of heartily cooperation
of number of heads and hands. There are some who have blessed, some who assisted and
some who have supplemented. Firstly, I wish to thank “Akaalpurakh” the almighty being for
all the blessings and providing me strength to complete this work.
I feel great pleasure to place on record my deep sense of appreciation and heartfelt thanks to
my major advisor Dr. (Mrs.) Gagandeep Kaur (Research Engineer), Department of
Processing and Food Engineering, for her constant supervision, valuable guidance, kindness,
encouragement and constructive criticisms from the initial stage. I am also greatly indebted
to my advisory committee members Dr. Mahesh Kumar (Professor), Department of
Processing and Food Engineering and Dr. (Mrs.) Amarjit Kaur (Sr. Milling Technologist
cum Head), Department of Food Science and Technology and Dr. Ashok Kumar (Professor),
Department of Processing and Food Engineering (Dean PG Nominee) for their valuable
comments, suggestions and support during the course of research work.
I am grateful to Dr. A.K. Singh (Sr. Research Engineer cum Head, Department of Processing
and Food Engineering), for providing necessary facilities for successful completion of this
work. His positive attitude and regular enquiry throughout the period is notable.
I would like to pay my sincere gratitude to Atinderpal Singh Samra (M.Sc., Agronomy) for
his encouraging words and continuous support during hard times, I cannot thank him with
words.
I wish to express my deepest gratitude to my mother, who nursed me with great affection and
my brother. Without their help, I would not have brought this study to fruition.
I acknowledge the wonderful support and work of my friends Gurpreet Gosal, Gurnaz,
Harman Sandhu, Narjeet, Pippal Singh, Kanwaljeet Singh and Gurleen Mann and thank
them for their flourishing inspiration during my student life at PAU, Ludhiana.
I fully acknowledge the assistance and aid provided by the laboratory technicians during the
experiment.
Lastly, I offer my regards and blessings to all of those who supported me in any respect
during the completion of the thesis.
Kalgidhar Patshah, who continues to look after me despite my flaws.
_______________________
Gursimran Singh Sangha
-
Title of the thesis : Studies on extraction of stevioside from stevia
(Stevia rebaudiana Bertoni) leaves
Name of the Student : Gursimran Singh Sangha
and Admission No. L-2011-AE-150-M
Major subject : Processing and Food Engineering
Minor subject : Food Science and Technology
Name of Major Advisor : Dr. (Mrs.) Gagandeep Kaur Sidhu
& Designation Research Engineer
Degree to be Awarded : M.Tech.
Year of award of Degree : 2014
Total Pages in Thesis : 85 + Appendices + VITA
Name of University : Punjab Agricultural University, Ludhiana-141 004,
Punjab, India
ABSTRACT
A study was conducted on Stevia rebaudiana Bertoni leaves in order to study the
impact of varying process conditions on stevioside extraction. Four experiments were
conducted by varying three factors viz. time, temperature/power and leaf-solvent ratio to
three levels. In first experiment, levels of time were 20, 70 and 120 min, temperature were
30, 60 and 90°C and leaf-water ratio were 1:5, 1:15 and 1:25. In second experiment,
methanol was used as a solvent with levels of time as 10, 25 and 40 min, levels of
temperature as 30, 45 and 60°C and leaf-methanol ratio as 1:5, 1:7.5 and 1:10. In third and
fourth experiment, microwave was used as source of power. In both experiments, time and
power were varied as 0.5, 1.25 and 2.0 min and 180, 540 and 900 watt. In experiment where
water was used as solvent, leaf-solvent ratio was varied as 1:5, 1:15 and 1:25 and with
methanol it was varied as 1:5, 1:7.5 and 1:10. The extraction conditions were optimized
keeping in consideration five responses, namely Stevioside content (%), Color (A420), Sugar
content (%), TSS (°Brix) and pH using Response Surface Methodology. Stevioside content
increased with extraction time up to 70 min, temperature up to 77°C and leaf-water ratio up
to 1:16 with water and increased with time, temperature and leaf-methanol ratio up to 21 min,
53.5°C and 1:7.6 when methanol was used. When microwave assisted extraction was
performed using water as a solvent, stevioside increased with time, power and leaf-water ratio
up to 1.12 min, 635 watt and 1:18 respectively. With methanol it increased with time, power
and leaf-water ratio up to 1.09 min, 696 watt and 1:7.7 respectively, and decreased
afterwards. Microwave assisted extraction using water was found to be most suited method
for stevioside extraction from Stevia rebaudiana Bertoni leaves yielding highest stevioside
content.
Keywords: Stevioside, MAE, Stevia, Sweeteners, Steviol glycosides
________________________ ________________________
Signature of Major Advisor Signature of Student
-
Koj pRbMD dw isrlyK : stIvIAw (stIvIAw irbwaufIAwnw brtonI) dy p`iqAW qoN stIvIEsweIf inSkrSq krn dw AiDAYn
ividAwrQI dw nwm Aqy dwKlw nMbr
: gurismrn isMG sMGw (AYl-2011-eyeI-150-AYm)
mu`K ivSw : pRosYisMg Aqy PUf ieMjIinAirMg
inmn ivSw : Puf swieMs Aqy qknwlojI
pRmu`K slwhkwr dw nwm Aqy Ahudw
: fw. (sRImqI) ggndIp kOr is`DU Koj ieMjInIAr
ifgrI : AYm.tYk
ifgrI imlx dw swl : 2014
Koj pRbMD iv`c ku`l pMny : 85 + AMiqkw + vItw
XUnIvristI dw nwm : pMjwb KyqIbwVI XUnIvristI, luiDAwxw – 141 004 pMjwb, Bwrq
incoV
stIvIEsweIf inSkrSq krn aupr v`Ko-v`KorIAW pRosYs hlwqW dy pRBwv dw mulWkx krn leI mOjUdw AiDAYn stIvIAw irbwaufIAwnw brtonI dy p`iqAW aupr kIqw igAw[ AiDAYn dOrwn iqMn v`Ko-v`Kry mwpdMfW ijvyNik iqMn p`DrW q`k smW, qwpmwn/pwvr Aqy p`qw Golk Anupwq nwl cwr qzrby kIqy gey[ pihly qzrby ADIn, smW, qwpmwn Aqy p`qw-pwxI Anupwq dw p`Dr kRmvwr 20, 70 Aqy 120 imMt; 30, 60 Aqy 90°C Aqy 1:5, 1:15 Aqy 1:25 q`k inrDwrq kIqw igAw[ dUjy qzrby dOrwn, smW, qwpmwn Aqy p`qw-pwxI Anupwq dw p`Dr kRmvwr 10, 25 Aqy 40 imMt; 30, 45 Aqy 60°C Aqy 1:5, 1:7.5 Aqy 1:10 q`k inrDwrq krky mIQynol nUM Golk vjoN vriqAw igAw[ qIjy Aqy cOQy qzrby iv`c mweIkRovyv dI vrqoN pwrv dy sroq vjoN kIqI geI[ donW qzribAW iv`c, smW Aqy pwvr dw p`Dr 0.5, 1.25 Aqy 2.0 imMt Aqy 180, 540 Aqy 900 vwt r`iKAw igAw[ auh qzrbw ijs ivc̀ pwxI nUM Golk vjoN vriqAw igAw sI aus ivc̀ p`qw-Golk Anupwq 1:5, 1:15 Aqy 1:25 q`k r`iKAw igAw Aqy mIQynol nUM Golk vjoN vrqx vwy qzrby iv`c nwl p`qw-Golk Anupwq 1:5, 1:7.5 Aqy 1:10 q`k r̀iKAw igAw[ irspoNs srPys mYQyfolojI dI vrqoN krky pRqIikirAwvW – stIvIEsweIf dI imkdwr (%), rMg (ey420), SUgr dI imkdwr (%), tI.AYs.AYs. (°ibRks) Aqy pI.AYc. nUM md̀ynzr r`Kdy hoey inSkrSn hlwqW dI AnukUlqw inrDwrq kIqI geI[ pwxI nUM Golk vjoN vrqx nwl inSkrSn smyN nUM 70 imMt, qwpmwn nUM 77°C Aqy p`qw-pwxI Anupwq nUM 1:16 q`k vDwaux nwl stIvIEsweIf dI imkdwr iv`c vwDw hoieAw jdoNik mIQynol nUM Golk vjoN vrqx nwl inSkrSn smyN nUM 21 imMt, qwpmwn nUM 53.5°C Aqy p`qw-mIQynol Anupwq nUM 1:7.6 qk̀ vDwaux nwl stIvIEsweIf dI imkdwr ivc̀ vwDw hoieAw[ pwxI nUM Golk vjoN vrq ky mweIkRovyv dI vrqoN nwl inSkrSn krn dOrwn inSkrSn smyN, pwvr Aqy p`qw-pwxI Anupwq nUM kRmvwr 1.12, 635 vwt Aqy 1:18 qk̀ vDwaux nwl stIvIEsweIf dI imkdwr ivc̀ vwDw hoieAw[ jdoNik mIQynol vwly qzrby iv`c smW, pwvr Aqy p`qw-pwxI Anupwq nUM kRmvwr 1.09, 696 vwt Aqy 1:7.7 q`k vDwaux nwl stIvIEsweIf dI imkdwr iv`c vwDw hoieAw Aqy ies mgroN ies iv`c kmI AwauxI SurU ho geI[ AiDAYn dy nqIijAW qoN ieh q`Q swhmxy Awey ik pwxI nUM Golk vjoN vrq ky mweIkRovyv dI vrqoN nwl stIvIAw irbwaufIAwnw brtonI dy p`iqAW qoN stIvIEsweIf dI vDyry imkdwr pRwpq huMdI hY[
mu`K Sbd: stIvIEsweIf, AYm.ey.eI, stIvIAw, svItnr, stIvEl glweIkosweIfs
_____________________ _____________________ pRmu`K slwhkwr dy hsqwKr ividAwrQI dy hsqwKr
-
CONTENTS
Chapter
No.
Topic Page
No.
I INTRODUCTION 1-3
II REVIEW OF LITERATURE 4-13
III MATERIAL AND METHODS 14-23
3.1 Experimental design and equipment used 14
3.2 Preparation and procurement of material 15
3.3 Experimental design for optimization of process
parameters
15
3.3.1 Analysis of design 17
3.3.2 Optimization of stevioside extraction process. 19
3.4 Extraction of stevia extract 20
3.4.1 Extraction with water 20
3.4.2 Extraction with methanol 20
3.4.3 Microwave assisted extraction 21
3.5 Determination of different physico-chemical
characteristics of stevia extract
21
3.5.1 Estimation of stevioside content 21
3.5.2 Determination of color (A420) 22
3.5.3 Estimation of total sugars 22
3.5.4 Determination of total soluble solids 23
3.5.5 Determination of pH 23
IV RESULTS AND DISCUSSION 24-77
4.1 Extraction of stevioside with water 24
4.1.1 Effect of different process parameters on stevioside
content
26
4.1.2 Effect of different process parameters on color
(A420) of extract
28
-
Chapter
No.
Topic Page
No.
4.1.3 Effect of process parameters on sugar content 31
4.1.4 Effect of process parameters on total soluble solids
(TSS)
33
4.1.5 Effect of different process parameters on pH values 35
4.1.6 Optimization of process parameters for extraction
of stevioside using hot water
37
4.2 Extraction of stevioside by methanol 38
4.2.1 Effect of different process parameters on stevioside
content
39
4.2.2 Effect of different process parameters on color
(A420)
42
4.2.3 Effect of different process parameters on sugar
content
44
4.2.4 Effect of different process parameters on total
soluble solids (TSS)
46
4.2.5 Effect of process parameters on pH 48
4.2.6 Optimization of process parameters for extraction
of stevioside using methanol
50
4.3 Microwave assisted extraction of stevioside with water 51
4.3.1 Effect of different process parameters on stevioside
content
52
4.3.2 Effect of different process parameters on color
(A420) of stevia extract
55
4.3.3 Effect of process parameters on sugar content (%) 57
4.3.4 Effect of process parameters on total soluble solids
(TSS)
59
4.3.5 Effect of process parameters on pH 61
4.3.6 Optimization of process parameters for extraction
of stevioside using MAE with water
63
4.4 Microwave assisted extraction of stevioside with methanol 64
-
Chapter
No.
Topic Page
No.
4.4.1 Effect of process parameters on stevioside content 65
4.4.2 Effect of process parameters on color (A420) 67
4.4.3 Effect of process parameters on sugar content (%) 69
4.4.4 Effect of process parameters on total soluble solids
(TSS)
71
4.4.5 Effect of process parameters on pH 73
4.4.6 Optimization of process parameters for extraction
of stevioside using MAE with methanol
75
4.5 Comparison of different methods of extraction of
stevioside content
76
V SUMMARY 78-80
REFERENCES 81-85
APPENDIX I-II
-
LIST OF TABLES
Table
No.
Title Page
No.
3.1 Design of experiment 14
3.2 Equipments used in study 14
3.3 The level of variables chosen for the Box-Behnken design for
extraction with water
16
3.4 The level of variables chosen for the Box-Behnken design for
extraction with methanol
16
3.5 The level of variables chosen for the Box-Behnken design for
microwave assisted extraction with water
16
3.6 The level of variables chosen for the Box-Behnken design for
microwave assisted extraction with methanol
17
3.7 Three factor Box-Behnken experimental design for extraction with
water
17
3.8 Three factor Box-Behnken experimental design for extraction with
methanol
18
3.9 Three factor Box-Behnken experimental design for microwave
assisted extraction with water
18
3.10 Three factor Box-Behnken experimental design for microwave
assisted extraction with water
19
4.1 Effect of extraction conditions on product responses 25
4.2 Statistics of various parameters 25
4.3 ANOVA for stevioside content 26
4.4 ANOVA for color (A420) of stevia extract 29
4.5 ANOVA for sugar content 31
4.6 ANOVA for TSS 33
4.7 ANOVA for pH 35
4.8 Optimization of the extraction conditions for stevioside using water 37
4.9 Effect of extraction conditions on product responses 38
4.10 Statistics of various parameters 39
4.11 ANOVA for stevioside content 39
-
Table
No.
Title Page
No.
4.12 ANOVA for color (A420) of stevia extract 42
4.13 ANOVA for sugar content 44
4.14 ANOVA for TSS 46
4.15 ANOVA for pH 48
4.16 Optimization of the extraction conditions for stevioside using
methanol
50
4.17 Effect of extraction conditions on product responses 51
4.18 Statistics of various parameters 52
4.19 ANOVA for stevioside content 52
4.20 ANOVA for color (A420) of stevia extract 55
4.21 ANOVA for sugar content 57
4.22 ANOVA for TSS 59
4.23 ANOVA for pH 61
4.24 Optimization of the microwave assisted extraction conditions for
stevioside using water
63
4.25 Effect of extraction conditions on product responses 64
4.26 Statistics of various parameters 65
4.27 ANOVA for stevioside content 65
4.28 ANOVA for color (A420) of stevia extract 67
4.29 ANOVA for sugar content 69
4.30 ANOVA for TSS 71
4.31 ANOVA for pH 73
4.32 Optimization of the microwave assisted extraction conditions for
stevioside using methanol
75
4.33 Optimized conditions for different methods of extraction 77
-
LIST OF FIGURES
Figure
No.
Title Page
No.
4.1 Effect of different process parameters on stevioside content using water
extraction
27
4.2 Effect of different process parameters on color (A420) of stevia extract
using water extraction
30
4.3 Effect of different process parameters on sugar content using water
extraction
32
4.4 Effect of different process parameters on TSS using water extraction 34
4.5 Effect of different process parameters on pH values using water
extraction
36
4.6 Optimization of process parameters for extraction of stevioside with
water
37
4.7 Effect of different process parameters on stevioside content using
methanol extraction
41
4.8 Effect of different process parameters on color (A420) of stevia extract
using methanol extraction
43
4.9 Effect of different process parameters on sugar content using methanol
extraction
45
4.10 Effect of different process parameters on TSS using methanol extraction 47
4.11 Effect of different process parameters on pH values using methanol
extraction
49
4.12 Optimization of process parameters for extraction of stevioside using
methanol
50
4.13 Effect of different process parameters on stevioside content using MAE
with water
54
4.14 Effect of different process parameters on color (A420) of stevia extract
using MAE with water
56
4.15 Effect of different process parameters on sugar content using MAE with
water
58
4.16 Effect of different process parameters on TSS using MAE with water 60
4.17 Effect of different process parameters on pH values using MAE with
water
62
4.18 Optimization of process parameters for extraction of stevioside with
MAE using water
64
-
Figure
No.
Title Page
No.
4.19 Effect of different process parameters on stevioside content using MAE
with methanol
66
4.20 Effect of different process parameters on color (A420) of stevia extract
using MAE with methanol
68
4.21 Effect of different process parameters on sugar content using MAE with
methanol
70
4.22 Effect of different process parameters on TSS using MAE with methanol 72
4.23 Effect of different process parameters on pH values using MAE with
methanol
74
4.24 Optimization of process parameters for extraction of stevioside with
MAE using methanol
76
-
LIST OF PLATES
Plate
No.
Title Page
No.
3.1 Dried stevia powder and leaves 15
3.2 Heating mantle 20
3.3 Microwave oven 21
3.4 UV-Visible spectrophotometer 22
3.5 Erma hand refractometer 23
3.6 pH meter 23
-
CHAPTER I
INTRODUCTION
Stevia, botanically known as Stevia rebaudiana Bertoni (Family- Asteraceae) is a
sweet herb. The leaves are mild green and intensely sweet. The compounds in the leaves are
called stevioside and rebaudioside and they are more than 200 times sweeter than sugar
(Anon, 2004). Its leaves contain approximately 10% of stevioside which are intensely sweet
compound. The leaves have been traditionally used for hundreds of years in Paraguay and
Brazil to sweeten local teas, medicines and as a „sweet treat‟. There are now more than 150
species of Stevia grown in world. Stevia is sweet in nature and a native of Paraguay, so it is
called the “sweet herb of Paraguay.” It is also known as honey leaf, sweet leaf, sweet herb,
candy leaf, and honey yerba (Carakostas et al, 2008). Even though there are more than 200
species of the genus Stevia, only S. rebaudiana gives the sweetest essence (Savita et al, 2004).
Stevia has a four-year life span and yields three to four crops per year, with an initial
investment of ` 3.7 lakh ha-1, a farmer can earn about ` 2 lakh per annum, for four years. The
yearly yields can be in the range of 7.41-9.88 tons ha-1
. Stevia leaves can be sold at ` 200 kg-1,
so the dried leaves are thus economically beneficial to growers (Sharma and Chattopadhya,
2007). Stevia has been approved for several years in Brazil, Argentina, and Paraguay, as well
as in China, Korea, and Japan to sweeten soft drinks, soy sauce, yogurt, and other foods,
whereas in the United States they are used as dietary supplements since 1995.Although Stevia
has been in use in Asia and Europe for years, it was only in the past couple of years that it has
really started to capture attention in the Indian market as a healthy alternative sweetener to
sugar.
Stevia has no calcium cyclamate, no saccharin, no aspartame and no calories. It is
safe for diabetics, as it does not affect blood sugar levels; it does not have the neurological or
renal side effects associated with some of the artificial sweeteners. The main advantage of
stevioside over other sweeteners is that it is stable at 100°C (Buckenhuskers and Omran,
1997). Apart from this, stevia is nutrient rich, containing substantial amount of Protein,
Magnesium, Miocene, Riboflavin, Zinc, Chromium, Selenium, Calcium & Phosphorus,
besides stevia can also be used as a household sweetener in preparation of most Indian sweets
(Anon, 2004). Stevia products has ample amount of medicinal usage and advantages for
diabetic and blood pressure patients. Stevia sweetener extractives have been suggested to
exert beneficial effects on human health, including antihypertensive (Lee et al, 2001),
antioxidant (Xi et al, 1998), anti-human rotavirus activities (Takahashi et al, 2001).
Extraordinary antimicrobial activity of Stevia has presented it as a potent non antibiotic
pharmaceutical and an efficient food preservative (Ghosh et al, 2008). Reports have proved
-
2
that it is safe for consumption without any health risks. Thereafter, Stevia emerged as one of
the best alternative sources of sweeteners (Savita et al, 2004).
Stevioside gives the impression of slightly bitter taste, while rebaudioside A contributes
to the typical sweet taste (similar to sucrose) (Singh and Rao, 2005). Stevia is used in form of
fresh leaves, powder and liquid extract. Traditionally, it is used as dried leaf or fresh leaves
directly, but it leaves the sediments in domestic cooking and wastage is more. So, the extraction
of stevioside from stevia leaves is more useful and economical. The main sweet component in
the leaves of Stevia rebaudiana is stevioside and different technologies are available for
extraction of stevioside. Some of them are extraction with hot water followed by separation,
filtration, crystallization and drying. Extraction with hot water includes the boiling the leaves in
hot water to dissolve glycoside and filtering the liquid by precipitation. This filtered liquid is
then concentrated and resin exchange is used to separate the glycoside into high and low R-A
fractions and crystallization and drying is done to get stevia crystals. Boiling water extraction
can achieve 93-98% extraction of stevioside (Midmore and Rank, 2006). Hot-water treatment
has been used as a classical extraction method (Dacome et al, 2005). However, it should be
noted that hot-water extraction is associated with long extraction time and high temperature.
Yoda et al, (2003) studied the supercritical fluid extraction and the kinetics of the glycosides
from stevia leaves. The results showed the yield was approximately 1.6%. Zhang et al, (2000)
discussed the use of membrane separation technology to produce Stevia extracts without
residual taste. However, all these processes have complex steps and there was no information
about the effects of extraction method on the contents of stevioside and rebaudioside A in the
extracts obtained. In solvent extraction, different solvents are used to dissolve glycoside from
leaves and this process is repeated till we get miscella of high glycoside content. Then this
miscella is desolventized, purified and separated to get clear glycoside. For crystals and powder,
this liquid is crystallized and dehydrated (Nikolai et al, 2001).
Modern extraction techniques such as pressurized fluid extraction, pressurized hot
water extraction, supercritical fluid extraction and microwave assisted extraction have been
used for extracting stevioside content. Microwave assisted extraction is gaining popularity
because it allows faster extraction, reduced solvent, increases recovery, saves time and energy
consumption in comparison to conventional methods of extraction. Bondarev et al, (2003)
studied steviol glycoside content in different organs of Stevia rebaudiana and its dynamics
during ontogeny with HPLC. Kolb et al, (2001) developed an improved HPLC method for
quality control of stevioside and rebaudioside A contents in dried leaves of Stevia
rebaudiana. These methods study one variable at a time, but in extraction process there are
multiple independent variables affecting the extraction process. Therefore, it is necessary to
use optimization techniques for optimization of different process parameters involved in the
extraction of stevioside from Stevia leaves.
-
3
Optimization is done in order to improve the efficiency of the system in terms of
system performance, product yield and operating cost. Numerical optimization studies have
been used using response surface methodology (RSM). RSM is a collection of statistical and
mathematical techniques useful for the developing, improving, and optimizing process. RSM
has been widely used to evaluate and understand the interactions between different process
parameters. RSM consists of various stages such as determination of various independent
parameters and their effective levels, selection of the experimental design, prediction and
verification of model equations, generating response surfaces and contour plots, and at last the
determination of optimum points. In this process, the experimental design stage leads to an
efficient experimentation for evaluation of the process in a short period of time for laboratory
level tests. The main advantage of using RSM is that it takes into account the interactive
effects of independent variables on the process and the number of experiments reduces. In
recent years, RSM has been successfully used by various researchers for optimizing many of
the fruit based product development processes and in many other unit operations involved in
processing of liquid samples such as extraction and clarification.
Not much work has been reported on optimization of different process parameters for
extraction of stevioside using different methods. Taking the above discussions into
consideration the present work has been planned with the following objectives:
i. To study the effect of different extraction methods on the quality of the extract.
ii. Optimization of different process parameters for extraction of stevioside using different
methods.
-
CHAPTER II
REVIEW OF LITERATURE
The literature related to review of the present study has been reviewed as under:
The most favoured process for isolation of glycosides involve four steps; aqueous or
solvent extraction, ion exchange precipitation or coagulation with filtration, then
crystallization and drying. Ahmed and Dobberstein (1982) extracted stevioside and
rebaudioside A and C from the dried leaves of S.rebaudiana in a microsoxhlet apparatus.
They observed that choloform/methanol provided best results, compared to choloform.
Potential sweetening agents of plant origin and field search for sweet-tasting Stevia
species were studied by Soejarto et al, (1983). Field work in Paraguay, Peru, Colombia and
Mexico, including field organoleptic tests and interviews, was carried out in search of sweet-
tasting Stevia species. The data obtained showed that leaves of no other Stevia species studied
possessed a potent sweet taste comparable to that of Stevia rebaudiana leaves. Since only
15% of the approximately 200 known species in this genus were investigated in this study, it
is quite possible that further research may reveal additional sweet-tasting Stevia species.
Nishiyama et al, (1992) used the Near Infrared reflectance spectroscopy (NIRS) for
the analysis of stevioside in Stevia rebaudiana leaves with same accuracy as obtained by
HPLC. The leaves were extracted with near boiling water and the subjected to HPLC
analysis. For NIRS analysis, leaves were ground using a cyclone mill fitted with 1.0 mm
screen, NIRS calibration was developed from 64 samples covering the range of stevioside
normally found in Stevia rebaudiana leaves (4-13%). The result suggested that NIRS was a
precise and simple method for routine stevioside determinations in Stevia rebaudiana.
Extraction of stevioside, rebaudioside A and C and dulcoside was also performed by
supercritical fluid extraction method using CO2 and methanol as modifier by Liu et al,
(1997). The extraction conditions were optimized and extraction efficiency of more than 88%
was obtained. Such an extraction technique has been gaining popularity as an analytical tool
because it is rapid, simple and less expensive in terms of solvent.
The stevioside content in plant material and food samples was determined HPLC by
Bovanova et al, (1998). An HPLC method determination of sweet-tasting stevioside in the
leaves of the plant for the Stevia rebaudiana and in some beverages (e.g. tea, orange juice)
was developed. The pre-separation procedure consisted of extraction of sweet-tasting
stevioside from the plant material using boiling water and a solid-phase extraction (SPE).
Recovery rates of the SPE for the analyzed matrices ranged from 92.8% to 97.8% (for
concentrations of STS of 105, 210 and 300 μg/ml; Relative Standard Deviation (RSD) ≤
3.3%). The chromatographic separations were realized. The limits of determination of STS
were 5 μg/ml for leaf extract and tea sample whereas it was 8 μg/ml for the juice sample.
http://www.springerlink.com/content/?Author=D.+D.+Soejarto
-
5
Selectivity of polymer adsorbent in adsorptive separations of stevia diterpene
glycosides was studied by Chen et al, (1998). Some hydrophobic (including both the non-
polar and polar) and hydrophilic polymer adsorbents were designed and synthesized, and their
adsorption properties and adsorption mechanism toward stevia glycosides were studied in
great detail. The skeleton structure and polarity of the resins had effect on the adsorption
capacity and the selectivity properties during the adsorption of stevioside and rebaudioside A.
A sweetener with high rebaudioside A content was isolated by using the adsorption selectivity
of the polar resins.
Stevia glycosides were extracted by super critical fluid extraction (SCFE) method
using CO2 as solvent and water/ethanol as co-solvent. The mean total yield for SCFE
treatment was 3.0%. The yields of stevia glycosides for SCFE with co-solvent were below
0.50%, except at 120 bar, 16°C. Under this condition, total yield was 3.4%. The quality of the
glycoside fraction with respect to its capacity as sweetener was better for SCFE extract as
compared to extract obtained by conventional process. The overall extraction curves were
well described by Lack extended model (Pasquel et al, 2000).
Zhang et al (2000) studied the process of extraction and refining of sweeteners with
reduced number of unit operations and minimization or elimination of chemical usage
including organic solvents. Water was very effective for extracting glycosides at selected pH
and temperature. It was also shown that a multistage membrane process was successfully able
to concentrate glycoside sweeteners and bitter tasting components were washed out from the
sweetener concentrate in the nanofiltration process.
Supercritical fluid extraction and liquid chromatographic-electrospray mass
spectrometric analysis of stevioside from Stevia rebaudiana leaves was studied by Choi et al,
(2002). In developing an alternative extraction method for stevioside using SCFE, the effect
of temperature, pressure, and percentage of modifier was evaluated on the extraction yield.
Although sufficient extractability was not obtained by pure CO2 under any conditions of
temperature and pressure, the addition of a modifier dramatically improved the extraction
yield of stevioside, making it comparable to organic solvent extraction. Among the modifiers
evaluated, the mixture of methanol and water showed greater extraction efficiency than the
others. The extraction yield by CO2-methanol-water (80:16:4) was found to be 150% of
conventional organic extraction. In addition to improving the extraction yield, SFE obviously
provided a higher purity of stevioside in the final extract
The estimation of glycosides from Stevia rebaudiana was studied by Kovylyaeva et
al, (2007). A new laboratory method for isolating the glycosides stevioside and rebaudiosides
A and C from leaves of Stevia rebaudiana was proposed. According to HPLC, the glycoside
contents in plants grown in Russia (Voronezh Oblast) and Ukraine (Crimea) were 5–6%
(stevioside) and 0.3–1.3% (Rebaudiosides A and C).
http://www.springerlink.com/content/?Author=Tianhong+Chenhttp://www.springerlink.com/content/?Author=Young+Hae+Choihttp://www.springerlink.com/content/?Author=G.+I.+Kovylyaeva
-
6
The comparison of two different solvents methanol versus water was studied by Pol
et al, (2007). They studied that the pressurized fluid extraction using water or methanol was
employed for the extraction of stevioside from Stevia rebaudiana Bertoni. The extraction
method was optimized in terms of temperature and duration. Extracts were analyzed by liquid
chromatography followed by ultraviolet (UV) and mass-spectrometric (MS) detections.
Thermal degradation of stevioside was the same in both solvents within the range 70–160°C.
Methanol showed better extraction ability for isolation of stevioside from Stevia rebaudiana
leaves than water within the range 110–160°C.
Stevia rebaudiana Bertoni plants grown in vitro and ex vitro were investigated by
Rajasekaran et al, (2007) for variation in the profile of stevioside in their leaves, shoots, root
and flower. Stevioside was extracted by hydrolysis and esterification, evaporation to dryness
and dissolved in methanol for quantitative analysis by HPLC. The HPLC analysis and
separation profiles indicated the presence of eight known sweet diterpene glycosides. The
highest stevioside content was recorded in one month old greenhouse leaves (64.80 g/kg dried
plant material) and in vitro (0.99g, Rebaudioside A /kg dried leaves plant material).
Wang et al (2007) applied Microwave-assisted extraction (MAE) for pectin
extraction from the dried apple pomace and response surface methodology (RSM) was used
to optimize the effects of processing parameters of extraction on the yield of pectin. Four
independent variables such as extraction time (min), pH of HCl solution, solid:liquid ratio and
microwave power (W). The optimal conditions were determined and tri dimensional response
surfaces were plotted from the mathematical models. The F-test and p-value indicated that
both the extraction time and pH of HCl solution had highly significant effects on the response
value and the quadratic of microwave power also displayed significant effect, followed by the
interaction effects of pH and solid:liquid ratio. Considering the efficiency, the economization
of energy and the feasibility of experiment, the optimum conditions of pectin extraction were
extraction time 20.8 min, pH 1.01, solid:liquid ratio 0.069, microwave power 499.4 W.
Application of MAE in the extraction from dried apple pomace dramatically reduced
extraction time. The optimal predicted pectin yield of 0.315 g from the dried apple pomace (2
g) was obtained. Close agreement between experimental and predicted yields was obtained.
The Chloroplast ultrastructure, photosynthetic apparatus activities and production of
steviol glycosides in Stevia rebaudiana in vivo and in vitro were studied by Ladygin et al,
(2008). The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni
both in vivo and in vitro was related to the extent of the development of the membrane system
of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants,
unlike those of the intact plants, had poorly developed membrane system. Leaves of in vivo
plants accumulated greater amount of the pigments than leaves of the in vitro plants. The
callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the
http://www.springerlink.com/content/?Author=Jaroslav+P%c3%b3lhttp://www.springerlink.com/content/?Author=V.+G.+Ladygin
-
7
intact and the in vitro plants did not exhibit any significant differences in photosynthetic O2
evolution rate. However, photosynthetic O2 evolution rate in the callus cells was much lower
than that in the differentiated plant cells. The in vitro cell cultures containing merely
proplastids did not practically produce SGs. However, after transferring these cultures in the
light, both the formation of chloroplasts and the production of SGs in them were detected.
Extraction by conventional, ultrasound and microwave-assisted extraction techniques
using methanol, ethanol and water as single solvents as well as in binary mixtures was studied
by Jaitak et al, (2009). Conventional cold extraction was performed at 25°C for 12 h while
ultrasound extraction was carried out at temperature of 35 ± 5°C for 30 min. Microwave-
assisted extraction (MAE) was carried out at a power level of 80 W for 1min at 50°C. MAE
yielded 8.64 and 2.34% of stevioside and rebaudioside A, respectively, while conventional
and ultrasound techniques yielded 6.54 and 1.20%, and 4.20 and 1.98% of stevioside and
rebaudioside-A respectively.
Extraction of stevia by three methods, first by hot water (65oC) at different ratios of
leaves to water (1:15 – 1:75) was studied by Abou-Arab et al, (2010). The optimum ratio was
1:35 in which the maximum stevioside content was obtained (7.53%), recovery of stevioside
was 80.21%. The second method, extraction by methanol at ratio of 4:1 methanol/leaves, the
recovery was 94.9%.The third method of extraction by mixture of methanol/water (4:1), the
recovery was 92.34%.
Inamake et al, (2010) attempted to isolate stevioside from the dried leaves of Stevia
in its purest form. Isolated stevioside was purified, analyzed & characterized by using various
chromatographic & analytical methods including Thin layer chromatography (TLC), UV,
Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance spectroscopy
(NMR) and HPLC methods. The Rf value for TLC was 0.32, λmax of UV spectra was
obtained at 333 nm and HPLC showed the sharp peak with 1.958 min retention time. The
isolated stevioside was also compared with standard stevioside with all analytical methods
The nutrient composition of cultivated stevia leaves and the influence of polyphenols
and plant pigments on sensory and antioxidant properties of leaf extracts was studied by
Kaushik et al, (2010).The leaf and its extract although sweet have a bitter after taste that
precludes commercial acceptability. The composition of the leaf reflected a high nutritive
value and polyphenol concentration averaging 4.15% by weight of dried leaf. Presence of
polyphenols influenced the acceptability of the sweeteners marginally, while chlorophyll was
found unacceptable in any of the extracts. The antioxidant activity of the extracts was
synergistic when it was mixed with coffee and lime juice. Complete purification of stevia leaf
extracts to obtain pure glycosides is not necessary for it to become a commercially acceptable
sweetener.
-
8
The stability of the natural sweetener stevioside during different processing and
storage conditions was studied by Kroyer (2010). Incubation of the solid sweetener stevioside
at elevated temperatures for 1 hour showed good stability up to 120°C, while at temperatures
exceeding 140°C forced decomposition was noticed. In aqueous solutions stevioside is
remarkable stable in a pH range 2–10 under thermal treatment up to 80°C. However, under
strong acidic conditions (pH= 1) a significant decrease in the stevioside concentration was
detected.
Liu et al (2010) maximized the yield of total carbohydrates from Stevia rebaudiana
Bertoni, response surface methodology (RSM) was employed to optimize the ultrasound-
assisted extraction condition. The results indicated the optimal extraction conditions were an
extraction temperature of 68 oC, a sonic power of 60 Watt and an extraction time of 32 min.
Using the ultrasound-assisted extraction, the yield of extracts increased by a factor of 1.5 at
the lower extraction temperature (68oC) and the extraction time (32 min) substantially
shortened compared with that of classical extraction. The components analysis of crude
extracts revealed that the relative amount of rebaudioside A increased in the ultrasound-
assisted extracts as compared with extracts obtained by classical process, and the ultrasound-
assisted extracts had better quality.
The improved HPLC method for the evaluation of the major steviol glycosides in
leaves of Stevia rebaudiana was studied by Rieck et al, (2010). A simple reversed-phase
high-performance liquid chromatographic method has been developed for the determination
of the major steviol glycosides, the diterpene sweeteners derived from Stevia rebaudiana. The
method is based on a water extraction step and a solid-phase extraction (SPE) clean-up. The
applicability of this method was demonstrated in the analysis of stevioside and rebaudioside
A from Stevia plants grown in two different areas in Germany. Stevioside and rebaudioside A
contents showed statistically significant differences (f and t-test) between the two harvests.
Nevertheless, the total concentrations (>12%) and the ratio of stevioside to rebaudioside A
(6:4) were similar to those found in the countries in which Stevia rebaudiana originates.
Based on a comparison of yields from different harvests, we discussed whether Stevia
rebaudiana can be economically grown in the temperate zones of the northern European
hemisphere.
RP-HPLC method with UV array detection was established by Jadhao et al, (2011)
for the determination of stevioside, an extract of herbal Stevia rebaudiana plant. The
stevioside was separated using isocratic solvent system consisting of methanol and 0.1%
orthophosphoric acid (v/v) in water (70:30) at flow rate of 1.0 ml/min and the detection
wavelength of 219 nm. The method was validated for linearity, precision, accuracy, limit of
detection (LOD), and limit of quantitation (LOQ). The linearity of the proposed method was
obtained in the range of 5.0-75 μg/ml with regression coefficient of 0.9999. Intraday and
-
9
interday precision studies showed the relative standard deviation less than 2.5%. The
accuracy of the proposed method was determined by a recovery study conducted at 3 different
levels. The average recovery was 97-99%. The LOD and LOQ were 0.02 and 0.05 μg/ml,
respectively. The content of stevioside obtained in the dried leaves powder was within the
ranges of 6.83 – 7.91 % and 1.7 – 2.9 % w/w, respectively. The proposed method is simple,
sensitive, yet reproducible. It is therefore suitable for routine analysis of stevioside in Stevia
rebaudiana Bertoni.
Shirwaikar et al (2011) described a method for rapid identification and estimation of
stevioside in commercial samples using HPTLC and HPLC. Identification of stevioside in
samples was done by HPTLC. The separation was achieved on a precoated silica gel 60F254
plate with mobile phase; ethyl acetate: methanol: water (75:15:10 v/v/v). Densitometric
scanning was performed at 510 nm after visualization with a solution of acetic
anhydride:sulphuric acid:ethanol (1:1:10 v/v/v). The identity of the peak corresponding to
stevioside was further confirmed by spectral analysis. HPLC was used for the estimation of
stevioside content. The calibration curve was linear in the concentration range from 0.1 to 1
mg/ml. The detection limit for stevioside was 0.05 mg/ml (1 μg per injection). The percentage
stevioside content of the samples i.e. stevia powder and stevia leaf was found to be 8.859 %
and 3.703 % respectively. The method allowed rapid identification and quantification of
stevioside in different samples and could be used for routine analysis of stevioside in
commercial samples
Kullu et al (2012) found that maximum mangiferin content of 1.1156 mg/g is
obtained at microwave power of 550 W and extraction time of 50 s with 80 % ethanol as a
solvent and preleaching time of 20 min. The results indicate that microwave power and
ethanol concentration have the most significant effect on the yield of mangiferin content. The
presence of mangiferin in final Curcuma amada extract is confirmed through high-
performance liquid chromatography and the functional groups are identified through Fourier
transform infrared spectroscopy analyses using standard mangiferin. The experimental
profiles are fitted into a two parameter modified first-order kinetic model and a three-
parameter modified logistic model and checked using the goodness-of-fit criterion. The
Curcuma amada retained its antioxidant activity after MAE treatment and the antioxidant
activity of mangiferin obtained after extraction using DPPH free radical scavenging assay is
studied.
Enzymatic extraction of stevioside from Stevia rebaudiana leaves with cellulase,
pectinase and hemicellulase, using various parameters, such as concentration of enzyme,
incubation time and temperature were studied by Puri et al, (2012). Hemicellulase was
observed to give the highest stevioside yield (369.23 ± 0.11 μg) in 1 h in comparison to
cellulase (359 ± 0.30 μg) and pectinases (333 ± 0.55 μg). Extraction from leaves under
-
10
optimized conditions showed a remarkable increase in the yield (35 times) compared with a
control experiment. Based on response surface methodology (RSM) analysis, temperature of
51–54°C, time of 36–45 min and the cocktail of pectinase, cellulase and hemicellulase set at
2% each, gave the best results.
Hot water extraction process was used for the extraction of steviosides by Rai et al,
(2012) from dry stevia leaves. The independent variables were, leaf to water ratio (1:5 to
1:20), heating time (10 to 120 min), and temperature (30 to 90oC). The combined effects of
these independent variables on the extracted stevioside concentration and color of the extract
were studied. For optimizing the extraction process, central composite rotatable design in
combination with response surface methodology was used. Significant regression models with
coefficient of determination greater than 0.90 were established to study the effect of
independent variables on the responses. The optimum conditions are: temperature of water:
78oC, time of heating: 56 min and leaf to water ratio: 1:14 (g:mL).
A new improvised process of extraction of steviosides from the stevia leaves was
established Rao et al (2012) in which the dry treated leaves were grounded, defatted, and
extracted through pressurized hot water extractor (PHWE), followed by purification and
concentration of the sweet glycosides through ultra (UF) and nano (NF) membrane filtration
in obtaining high (98.2%) purity steviosides. This process established “green” method for
isolation of high quality steviol glycosides, with improved final yield is 10.1% from 11% of
crude leaf extract and observed the improved organoleptic and biological activity
(antioxidant). Thus the method confirms a simple, inexpensive and eco-friendly process in
obtaining pure steviosides. Simple extraction and membrane purification process in isolation
of steviosides with improved organoleptic activity.
Analysis of stevioside from Stevia rebaudiana was carried out by Talha et al, (2012)
using systematic procedures of various chromatographic techniques including column
chromatography yielding the crude stevioside in 12.5%. This procedure was followed by thin
layer chromatography and examination of the spots under UV light at 254 and 366 nm, and
spraying reagent followed by mixing of fractions on the basis of Rf values. Semi purified
fraction obtained from crude stevioside by column chromatography and further evaluation by
thin layer chromatography appeared as a mixture showing the presence of four major
components (Rf values 0.27, 0.34, 0.42 and 0.60). These finding were similar to reported
values for the constituents namely rebaudiosides A, stevioside, rebaudiosides B and
steviolbioside, and stevia glycoside reported to occur in Stevia rebaudiana.
Alberto et al (2013) incorporated naturally into honey the main sweet diterpene
glycosides found in Stevia rebaudiana, called stevioside and rebaudioside A, in order to
combine the sweetening properties of these two substances. To determine their degree of
incorporation, the diterpene glycosides were identified and quantified both in the prepared
-
11
syrups and in the honey obtained by the analytical technique of high performance liquid
chromatography (HPLC) using a NH2 column (Zorbax-Agilent) and, as a mobile phase, a
mixture of acetonitrile: water (70:30) with ultraviolet detection to 194 nm. Quantification was
performed by means of a calibration curve where high rates of incorporation, exceeding 97%
for stevioside and rebaudioside A, were found. For the standardization of the analytical
technique, parameters were determined such as linearity, analytical sensitivity, detection
limit, quantification limit and precision, showing that the method developed is simple, fast
and reliable within the established limits, and levels up to 0,08 ppm for stevioside and 0,09
ppm for rebaudioside.
Two clarification processes, namely, centrifugation and microfiltration were used for
primary clarification of crude stevia extract by Chhaya et al (2013). The optimized condition
(speed and time) of centrifugation was obtained using response surface methodology.
Microfiltration was carried out using a 0.2 lm pore size membrane at different operating
pressures (138, 207, 276 kPa) and stirring speeds (500, 1500, and 2500 rpm). Clarified stevia
extract was analyzed in terms of color, clarity, total solid, and stevioside content. The
performance of both of these methods was compared based on the quality of clarified extract.
89.5% stevioside was recovered at 5334 g centrifugation speed with 25.6 minutes of
centrifugation. During microfiltration, severe flux decline was observed. At 138 kPa and 500
rpm, the flux decline was 87% within 5 minutes of filtration. This was more severe at higher
transmembrane pressure drop and it was 89% at 276 kPa under same stirring. Stirring speed
increased the flux significantly. After 25 minutes, at 138 kPa, 2.4 times flux enhancement
occurred when the stirring speed increased from 500 to 2500 rpm. Recovery of stevioside was
more at lower operating pressure and about 89% recovery was attained at 138 kPa.
Dorta et al (2013) analyzed the extraction efficiency of antioxidants from mango peel
by comparing two techniques: microwave-assisted (MAE) and traditional solvent (TE)
extraction. The number of extraction steps, water content in the extractant, peel weight-to-
solvent volume ratio in extractions and extraction time all had an influence on obtaining
extracts with high antioxidant capacity, but the extraction technique and the water content in
the extractant were the factors with the greatest effect. Using three steps, a water content of
50 % in the ethanol:water extractant, an extraction time of 60 min and a weight-tovolume
ratio of 1:10 or 1:50 (w/v) led to the highest antioxidant activity and phytochemicals content
in extracts. The extraction time needed to extract phytochemicals from mango peel was
similar when MAE and TE were used. However, the antioxidant capacity and phytochemical
content were around 1.5–6.0 times higher in the extracts obtained by MAE.
Microwave-assisted extraction was applied for pectin extraction from the dried
orange peel and Box–Behnken response surface design was used to study and optimize the
effects of processing variables (microwave power, irradiation time, pH and solid–liquid ratio)
-
12
on the yield of pectin by Maran et al (2013). The amount of pectin extracted increased with
increasing microwave power, while it reduces as the time, pH and solid–liquid ratio
increased. From the results, second order polynomial model was developed and it adequately
explained the data variation and significantly represented the actual relationship between
independent variables and the response. An optimization study using Derringer‟s desired
function methodology was performed and optimal conditions based on both individual and
combinations of all independent variables (microwave power of 422 W, irradiation time of
169 s, pH of 1.4 and solid liquid ratio of 1:16.9 g/ml) were determined with maximum pectin
yield of 19.24%, which was confirmed through validation experiments.
Samah et al (2013) determined stevioside and rebaudioside A in Stevia rebaudiana
leaves qualitatively by using soxhlet extraction method analyzed via preparative HPLC. A
series of standard solution for both stevioside and rebaudioside A were analyzed. Samples
were sequentially extracted using methanol as the solvent and the column had been used in
this study was Waters XBridge C18 column (150 mm x 4.6 mm I.D., 5μm). The mobile phase
for C18 column performed in isocratic mode elution consisting of acetonitrile:water
(80:20,v/v). Stevioside were the most abundant steviol-glycosides (Rt = 4.23 min) found in
Stevia rebaudiana leaves samples followed by rebaudioside A (Rt = 4.28 min). The
objective of analysis was achieved and this leads to the suggestion of using the Capcell Pak
C18 – MGII Column (250 mm x 4.60 mm ID, 5 μm) as the replacement to the C18 column
that was used in this study..
Statistical experimental designs were applied to optimize microwave-assisted
extraction of puerarin from Radix Puerariae by Wu et al (2013). The most important factors
affecting the extraction procedure were determined using a Plackett-Burman design. Results
indicated that the concentration of ethanol, solvent-material ratio, extraction time, and
microwave power were the main factors affecting the extraction yield. These factors were
further optimized using a central composite design and response surface methodology. The
optimal extraction parameters were ethanol concentration of 52.36%, microwave irradiation
time of 60 s, microwave power of 184.8W and solvent-material ratio of 25:1(mL/g). The
average experimental puerarin yield under the optimum conditions was found to be 11.97
mg/g, which agreed with the predicted value of 11.8 mg/g. The proposed method showed high
degree of reproducibility.
Response surface methodology was used to optimize the influence of microwave
power (300–600 W), plant material-to-solvent ratio (0.05–0.2 g/cm3), and extraction time
(10–30 min) on the efficiency of microwave-assisted extraction of the cherry laurel (Prunus
laurocerasus L.) fruit by Karabegovic et al (2014). From experimental data, a quadratic
polynomial mathematical model (R2=0.9949) was developed to predict the extract yield. All
considered factors were statistically significant for extraction efficiency, while the most
-
13
important factor was extraction time. Microwave power of 550 W, plant material-to-solvent
ratio of 0.05 g/cm3, and time of 25 min were determined as optimal conditions with a
maximum yield of 9.36 g/100 g fresh plant material, which was confirmed through laboratory
experiments (9.12 g/100 g fresh plant material). An economic condition for simultaneous
maximum extract yield (7.58 g/100 g fresh plant material) with minimal energy and solvent
consumption was determined by the desirability function method (18.2 min, 300 W, and 0.2
g/cm3). Additionally, the total phenol and flavonoid quantification and antioxidant activity of
both extracts were tested. There is no statistically significant difference in the total flavonoid
content in the extracts obtained under both proposed conditions, while the total phenolic
content and antioxidant activity of the extract obtained under economic conditions were
slightly lower.
Lorenzo et al (2014) proposed an HPLC method for analysing major steviol
glycosides as well as to optimise the extraction and clarification conditions for obtaining these
compounds. The analytical method proposed was adequate in terms of selectivity, sensitivity
and accuracy. The methodology was safe and eco-friendly, as only water was used for
extraction and solid-phase extraction was not done, which requires solvents that are banned in
the food industry to condition the cartridge and elute the steviol glycosides. In addition the
methodology consumed little time as leaves are not ground and the filtration is faster, and the
peak resolution is better.
-
CHAPTER III
MATERIAL AND METHODS
The study was conducted in the laboratories of Department of Processing and Food
Engineering, Punjab Agricultural University, Ludhiana. The material and methods adopted
have been described as under:
3.1 Experimental design and equipment used
Table 3.1 defined the experimental setup for preparation of stevia extract.
Table 3.1: Design of Experiment
Heading Description
Crop Stevia Rebaudiana Bertoni
Extraction method
Extraction with water
Extraction with methanol
Microwave assisted extraction
i. With water
ii. With methanol
No. of replications 3
Design Layout Box-Behnken
Quality parameters Stevioside Content, Color, Sugar Content, TSS and pH
Statistical Analysis The statistical analysis of the data was done by using Design
Expert software (Version 9.0, Stat-Ease, Minneapolis, MN, USA)
A number of equipments have been used in the study. The list of important
equipments along with major specifications was shown in Table 3.2.
Table 3.2: Equipments used in the study
S.
No.
Equipment Model/Specification Test performed/Quality
measured
1. Heating mantle Sunbim, range=0-100o Celsius To prepare liquid stevia
extract.
2. Microwave Oven LG Microwave MG 607 APR Used for microwave
assisted extraction
3. Spectrophotometer Rayleigh UV-2601 Color (A420) , Stevioside
Content and Sugar
Content
4. Refractometer 0-32° Brix Erma hand Refractometer TSS (°Brix).
5. pH meter ELCO LI 614 pH
-
15
3.2 Preparation and Procurement of material
Dried stevia leaves were procured from Green Valley Farms, Garhshankar, Punjab.
The leaves were crushed with bare hands. Only the fine powder was kept for use during
extraction and coarse parts were discarded
Plate 3.1: Dried stevia powder and leaves
3.3 Experimental Design for Optimization of Process Parameters
The experimental plan was chosen from the family of three level designs, suggested
by Box and Behnken (1960). The design is three level incomplete factorial design for
estimation of parameters in a second order model. The extraction of stevioside was assumed
to be a system affected by three independent variables, also called inputs of factors, ξi (time
of extraction (min), temperature during extraction (°C) or power during microwave assisted
extraction (watts) and ratio of stevia powder:water/ methanol (g: ml) which were closely
controlled and accurately measured. The effects of the variables were studied on stevioside
content, color, sugar content, TSS and pH. The variables were standardized for ease in
computation and to reduce their relative effect on the responses. For the analysis of
experimental design by the response surface methodology (RSM), it was assumed that n-
mathematical functions, fk (where, k =1, 2,.....,n), Yk in terms of m independent factors ξi
(where, i= 1, 2,................,, m) existed for each response variable.
Yk = fk (ξ1, ξ2, ξ3) + ek
Where, ek is the experimental error associated with the kth response such that E (ek) =
0 and (ek) = σ2. Due to unknown and/or complex form of the function fk second order
polynomial equations are assumed to appropriate the true functions:
Where, β0 is the value of the fitted response at the centre point of the design, i.e. point (0,0)
and βi, βii and βij are the linear, quadratic and interactive regression terms, respectively. The
term xi denotes the coded independent variables and the coding of ξi into Xi is given by the
following equation
iiiid/) ( 2X
(3.11)
n
1i
n
1i
n
1j
1-n
1i
2
0
i
jiijiiiiik xxxxy
-
16
Where,
ξi = actual value in original units
i = mean of high and low levels of ξi and
di = spacing (difference) between two successive of ξi
Total number of experiments = (Number of variables)2 + Number of variables + Central points
For three variables, Total number of experiments = 32 + 3 +5 = 17
The experiment was conducted according to the requirement of response surface
methodology. The second order Box-Behnken design was conducted to work out the range of
independent process variables and their levels (Table 3.3 to 3.6). After the coding the
experimental region extended from -1 to 1 in terms of Xi. The three level three factor
experimental plans according to the Box and Behnken design (1960) design consists of 17
points of treatment combinations of the independent variables and are presented in Tables 3.7 to
3.10. For each extraction experiment, the known ratio of stevia leaf powder: water/methanol
was heated at known temperature for specified time. For the same combinations of stevia leaf
powder: water/methanol, microwave assisted extraction was also done for specified time.
Table 3.3: The level of variables chosen for the Box–Behnken design for extraction
with water
Independent variables Symbol Levels
-1 0 +1
Time (Minutes) X1 20 70 120
Temp (°C) X2 30 60 90
Ratio (Leaf:Water) (g:ml) X3 1:5 1:15 1:25
Table 3.4: The level of variables chosen for the Box–Behnken design for extraction
with methanol
Independent variables Symbol Levels
-1 0 +1
Time (Minutes) X1 10 25 40
Temp (°C) X2 30 45 60
Ratio (Leaf: Methanol) (g:ml) X3 1:5 1:7.5 1:10
Table 3.5: The level of variables chosen for the Box–Behnken design for microwave
assisted extraction with water
Independent variables Symbol Levels
-1 0 +1
Time (Minutes) X1 0.5 1.25 2
Power (Watts) X2 180 540 900
Ratio (Leaf: Water) (g:ml) X3 1:5 1:15 1:25
-
17
Table 3.6: The level of variables chosen for the Box–Behnken design for microwave
assisted extraction with methanol
Independent variables Symbol Levels
-1 0 +1
Time (Minutes) X1 0.5 1.25 2
Power (Watts) X2 180 540 900
Ratio (Leaf: Methanol) (g:ml) X3 1:5 1:7.5 1:10
3.3.1 Analysis of design
The data obtained were regressed using multiple regression technique. The
coefficients of second-order polynomial models obtained after multiple regressions of the
responses show the effect of a particular variable on the response. The adequacy of the model
was tested using F-ratio, coefficient of correlation (R2) and lack of fit test. The models were
considered adequate when the calculated F-ratio was more than the table F-value and lack of
fit test (LoF) was insignificant.
Table 3.7: Three-factor Box-Behnken experimental design for extraction with water
S.No. A:Time (Min) B:Temperature (°C) C:Ratio (Leaf: Water) (g:ml)
Actual Coded Actual Coded Actual Coded
1 70 0 90 1 1:5 -1
2 120 1 60 0 1:5 -1
3 20 -1 90 1 1:15 0
4 120 1 90 1 1:15 0
5 120 1 60 0 1:25 1
6 120 1 30 -1 1:15 0
7 70 0 60 0 1:15 0
8 20 -1 30 -1 1:15 0
9 70 0 60 0 1:15 0
10 20 -1 60 0 1:5 -1
11 70 0 30 -1 1:5 -1
12 70 0 90 1 1:25 1
13 70 0 60 0 1:15 0
14 20 -1 60 0 1:25 1
15 70 0 60 0 1:15 0
16 70 0 30 -1 1:25 1
17 70 0 60 0 1:15 0
-
18
Table 3.8: Three-factor Box-Behnken experimental design for extraction with methanol
S.No. A:Time (Min) B:Temperature (°C)
C:Ratio (Leaf: Methanol)
(g:ml)
Actual Coded Actual Coded Actual Coded
1 40 1 45 0 1:5 -1
2 25 0 45 0 1:7.5 0
3 10 -1 60 1 1:7.5 0
4 40 1 45 0 1:10 1
5 25 0 45 0 1:7.5 0
6 25 0 45 0 1:7.5 0
7 40 1 60 1 1:7.5 0
8 25 0 45 0 1:7.5 0
9 40 1 30 -1 1:7.5 0
10 25 0 60 1 1:5 -1
11 25 0 45 0 1:7.5 0
12 25 0 30 -1 1:5 -1
13 25 0 60 1 1:10 1
14 10 -1 45 0 1:5 -1
15 10 -1 30 -1 1:7.5 0
16 25 0 30 -1 1:10 1
17 10 -1 45 0 1:10 1
Table 3.9: Three-factor Box-Behnken experimental design for microwave assisted
extraction with water
S.No. A:Time (Min) B:Power (Watts) C:Ratio (Leaf: Water) (g:ml)
Actual Coded Actual Coded Actual Coded
1 0.50 -1 540 0 1:25 1
2 0.50 -1 180 -1 1:15 0
3 1.25 0 180 -1 1:5 -1
4 0.50 -1 540 0 1:5 -1
5 1.25 0 540 0 1:15 0
6 2.00 1 540 0 1:25 1
7 1.25 0 540 0 1:15 0
8 1.25 0 540 0 1:15 0
9 1.25 0 540 0 1:15 0
10 2.00 1 180 -1 1:15 0
11 1.25 0 900 1 1:5 -1
12 1.25 0 180 -1 1:25 1
13 1.25 0 900 1 1:25 1
14 0.50 -1 900 1 1:15 0
15 2.00 1 540 0 1:5 -1
16 1.25 0 540 0 1:15 0
17 2.00 1 900 1 1:15 0
-
19
Table 3.10: Three-factor Box-Behnken experimental design for microwave assisted
extraction with methanol
S.No. A:Time (Min) B:Power (Watts)
C:Ratio (Leaf: Methanol)
(g:ml)
Actual Coded Actual Coded Actual Coded
1 1.25 0 540 0 1:7.5 0
2 2.0 1 900 1 1:7.5 0
3 2.0 1 180 -1 1:7.5 0
4 2.0 1 540 0 1:5 -1
5 0.5 -1 900 1 1:7.5 0
6 1.25 0 540 0 1:7.5 0
7 1.25 0 540 0 1:7.5 0
8 1.25 0 180 -1 1:5 -1
9 1.25 0 540 0 1:7.5 0
10 0.5 -1 540 0 1:5 -1
11 0.5 -1 180 -1 1:7.5 0
12 1.25 0 180 -1 1:10 1
13 1.25 0 900 1 1:10 1
14 0.5 -1 540 0 1:10 1
15 1.25 0 540 0 1:7.5 0
16 1.25 0 900 1 1:5 -1
17 2.0 1 540 0 1:10 1
3.3.2 Optimization of stevioside extraction process
Optimization of fitted polynomials for the response variables was carried out through
nonlinear mathematical optimization method using Design Expert software (Version 9.0,
Statease, Minneapolis, MN, USA). Surface plots were generated from the fitted quadratic
polynomial regression equations in order to visualize the relationship among the variables and
responses and to obtain the numerical solution for optimum conditions for variables at desired
response levels. The mapping of the fitted response was achieved using the software. The
contour plots for the models were plotted as a function of the two variables, while keeping the
other one at optimum levels. The result showing highest desirability was selected and
considered for further processing.
-
20
3.4 Extraction of stevia extract
The ground leaves of Stevia rebaudiana were mixed with water or methanol. The
mixture was heated. Subsequently, the aqueous extract was removed by draining. The extract
was allowed to cool to room temperature. In order to remove impurity particles, the extract
was allowed to rest while particulate matter settles out. The extract contains the sweetener
principles, the plant pigments and other water-soluble components.
3.4.1 Extraction with water
Hot water extraction method was used for preparing aqueous stevia extract. A
specific ratio (1:5, 1:15 and 1:25) of leaf powder to water (weight to volume) was measured
and the stevia leaves powder was mixed with water. This sample was exposed to a particular
temperature (30oC, 60
oC, and 90
oC) for a fixed duration (20, 70 and 120 min). After the
termination of the heating process, the stevia extract was allowed to cool and then filtered
using Whatman filter paper No.4. This extract was analyzed for quality parameters.
Thermostatic heater was used to control the temperature of the process (Plate 3.2).
Plate 3.2: Heating mantle
3.4.2 Extraction with methanol
Similar to the extraction using water, methanolic extract was prepared by mixing a
specific ratio (1:5, 1:7.5 and 1:10) of leaf powder to methanol (weight to volume). This
sample was exposed to a particular temperature (30oC, 45
oC, and 60
oC) for a fixed duration
(10min, 25 min, and 40min). After the termination of the heating process, the stevia extract
-
21
was allowed to cool and then filtered using Whatman filter paper No.4. This extract was
analyzed for quality parameters. Thermostatic heater was used to control the temperature of
the process.
3.4.3 Microwave assisted extraction
All the combinations of leaf powder: water (1:5, 1:15 and 1:25) and leaf powder:
methanol (1:5, 1:7.5 and 1:10) which were used for previous extractions were put in a conical
flask and extracted using microwave (Plate 3.3). The extracts obtained by MAE were filtered
using Whatman filter paper No.4 and the filtered extract was used for analyzing quality
parameters. Extraction was carried out at different power levels (180W, 540W and 900W)
with extraction time range between 0.5 min, 1.25 min and 2 min.
Plate 3.3: Microwave oven
3.5 Determination of different physico-chemical characteristics of stevia extract
The quality parameters of stevia extract i.e. stevioside content, color (A420), total
sugars, TSS and pH were estimated using standard methods described as under:
3.5.1 Estimation of stevioside content
The stevioside content in the stevia extract obtained in different combinations was
estimated by method described by Kaur G 2009.
Reagents:
a) 5N HCl: 42 ml of HCl was added in distilled water to make its volume 100 ml.
b) 5% phenol: 5 g phenol was added in distilled water to make its volume 100 ml.
c) 95% sulphuric acid (H2SO4): 95 ml of sulphuric acid was added to 5 ml of distilled
water.
-
22
Procedure
Steviol glycoside extracts was hydrolyzed with 5N HCl at 70oC for 1 hour. The glucose
units liberated from the stevioside upon hydrolysis took part in the Dubois reaction with 5%
phenol and 95% sulphuric acid (H2SO4). The intensity of orange brown color was read at
490nm. The concentration of glucose was measured against glucose standard and was
multiplied by a factor of 1.64 (on the basis of molecular weight) to calculate stevioside content.
Stevioside = 1.64 * Glucose
3.5.2 Determination of color (A420)
Color (A420) was measured in order to analyze the concentration of extract in different
samples. In crude stevia extract there are many kinds of pigments and it is difficult to
characterize the decolorization capacity for each pigment quantitatively. The visible
absorption spectrum of the transparent solution of crude extracts is usually tested by a
spectrophotometer (Plate 3.4). There are strong absorption peaks at 420 nm (Markosyan and
Yerevan, 2013). Therefore, color of sample was measured in terms of optical absorbance
(A420) at a wavelength of 420 nm using spectrophotometer instead of Hunter Colorimeter as
proposed earlier. The color of the extract is further denoted by A420 throughout the
manuscript.
Plate 3.4: UV-Visible Spectrophotometer
3.5.3 Estimation of total sugars
The total sugar content in the stevia extract was estimated by method given by
Dubois et al 1956
Reagents
a) 95% sulphuric acid (H2SO4): 95 ml of concentrated sulphuric acid were mixed with
distilled water to make its volume 100ml.
b) 5% phenol: It was prepared by dissolving 5g of phenol in 60ml distilled water and
volume was made to 100ml with distilled water.
-
23
Procedure
To extract (1 ml) 5% phenol (1 ml) was added followed by addition of 5ml of
sulphuric acid. The sulphuric acid was poured directly in the centre of the test tube to ensure
proper mixing. The test tubes were cooled at room temperature. After 20 minutes, the
intensity of brown color was measured at 490nm and the concentration of glucose was
measured against glucose standard.
3.5.4 Determination of total soluble solids
The total soluble solids were determined with the help of 0-32 ºBx Erma Hand
Refractometer (Plate 3.5). One or two drops of extract were put on the sample plate and read
the % total soluble solids on the scale.
Plate 3.5: Erma hand Refractometer
3.5.5 Determination of pH
pH of the different extract samples were evaluated by using the pH meter (Plate 3.6).
The pH meter was calibrated by using the standard buffer solution. After calibration, the
readings of pH of the different samples were taken.
Plate 3.6: pH meter
-
CHAPTER IV
RESULTS AND DISCUSSION
The present investigation entitled “Studies on extraction of stevioside from stevia
(Stevia rebaudiana Bertoni) leaves” was carried out in Department of Processing and Food
Engineering, PAU, Ludhiana in wake of commercial importance of Stevioside.
Stevia extract was extracted from grounded stevia leaves by using different methods
i.e. extraction with water, extraction with methanol and microwave assisted extraction with
water and methanol. Different process parameters viz. solvent to leaf ratio, temperature of
incubation, extraction time and power of microwave in microwave assisted methods was
optimized using response surface methodology on the basis of quality parameters.
Optimization consists of maximizing or minimizing a real function by systematically
choosing input values from within an allowed set and computing the value of the function.
The values of extraction conditions that produce the desired optimum value are termed
optimum conditions. Numerical optimization using Design expert software was carried out in
each experiment with following goals:
Stevioside content should be maximum
Color (A420) should be minimum
Minimum level of Sugar content should be 3%
Minimum level of TSS should be 6%
pH should be in range of 2 to 7
The results of the present study are discussed as under:
4.1 Extraction of stevioside with water
Stevia extract was extracted using hot water extraction method at different ratio of
leaf to water 1:5 to 1:25, temperature of incubation was kept between 30 to 90°C and time
was varied between 20 to 120 min. The effect of different extraction conditions on the
product responses is shown in Table 4.1. Regression analysis was carried out to fit the
mathematical models to the experimental data. Statistics of various parameters is presented in
Table 4.2. Stevioside content (%), Sugar content (%) and TSS was analysed using quadratic
model. A420 and pH was analysed using linear model.
-
25
Table 4.1: Effect of extraction conditions on product responses
Extraction Conditions Responses
S.
No.
A:Time
(Min)
B:Temperature
(°C)
C:Ratio
(Leaf:Water)
(g:ml)
Stevioside
Content
(%)
A420 Sugar
Content
(%)
TSS
(°Brix)
pH
1 70 90 1:5 8.0 1.244 3.88 8.8 6.0
2 120 60 1:5 6.1 1.281 2.76 9.3 5.9
3 20 90 1:15 7.9 1.325 3.83 6.8 6.2
4 120 90 1:15 6.9 1.389 3.45 7.5 5.9
5 120 60 1:25 6.0 1.119 2.91 7.4 6.1
6 120 30 1:15 5.7 1.122 2.76 6.9 6.0
7 70 60 1:15 9.3 1.119 4.51 6.7 6.1
8 20 30 1:15 7.4 1.069 3.59 6.5 6.5
9 70 60 1:15 8.0 1.156 3.88 6.5 6.2
10 20 60 1:5 6.4 1.090 3.10 9.1 6.2
11 70 30 1:5 5.4 1.114 2.52 8.5 6.2
12 70 90 1:25 6.9 1.345 3.34 7.3 6.2
13 70 60 1:15 8.0 1.189 3.88 6.3 6.2
14 20 60 1:25 6.9 1.011 3.31 5.9 6.4
15 70 60 1:15 8.7 1.112 4.22 6.0 6.1
16 70 30 1:25 7.1 1.012 3.44 6.2 6.4
17 70 60 1:15 8.5 1.192 4.12 6.2 6.2
Table 4.2: Statistics of various parameters
Parameters Stevioside
Content (%)
A420 Sugar
Content (%)
TSS (°Brix) pH
Std. Dev. 0.47 0.063 0.21 0.29 0.051
Mean 7.25 1.17 3.50 7.17 6.16
C.V. % 6.45 5.40 6.10 4.01 0.83
PRESS 7.40 0.10 1.09 5.06 0.059
R-Squared 0.92 0.74 0.94 0.97 0.92
Adj R-Squared 0.82 0.68 0.86 0.93 0.90
Pred R-Squared 0.62 0.49 0.79 0.74 0.87
Adeq Precision 8.339 11.44 9.524 13.425 22.229
-
26
4.1.1 Effect of different process parameters on stevioside content
Regression analysis is a statistical process for estimating the relationships among
variables. More specifically, regression analysis helps one understand how the typical value
of the dependent variable changes when any one of the independent variables is varied, while
the other independent variables are held fixed. It was carried out to fit the quadratic model to
experimental data for stevioside content as suggested by Design expert software (Table 4.3).
The significance of coefficient of fitted quadratic model was evaluated by using F- test and P-
value.
Table 4.3: ANOVA for Stevioside Content
Factors Coefficient Sum of
squares
Df Mean
Square
F-
Value
Prob>F
Model 8.50 18.29 9 2.03 9.32 0.0038
A- Time -0.49* 1.90 1 1.90 8.71 0.0214
B- Temperature 0.51* 2.10 1 2.10 9.63 0.0172
C- Ratio (leaf:water) 0.13 0.13 1 0.13 0.57 0.4738
AB- (Time*Temperature) 0.17 0.12 1 0.12 0.56 0.4781
AC- (Time*Ratio) -0.15 0.090 1 0.090 0.41 0.5412
BC- (Temperature*Ratio) -0.70* 1.96 1 1.96 8.98 0.0200
A2- (Time
2) -1.01
** 4.32 1 4.32 19.78 0.0030
B2- (Temperature
2) -0.51
** 1.11 1 1.11 5.07 0.0591
C2- (Ratio
2) -1.14 5.45 1 5.45 24.97 0.0016
Lack of fit - 0.3475 3 0.11 0.39 0.76
* Significant at P
-
27
Design-Expert® Software
Factor Coding: Actual
Stevioside Content (%)
Design points above predicted value
Design points below predicted value
9.3
5.4
X1 = A: Time
X2 = B: Temperature
Actual Factor
C: Ratio (Leaf:Water) = 15
30
40
50
60
70
80
90
20
40
60
80
100
120
5
6
7
8
9
10
Ste
vio
sid
e C
on
ten
t (%
)
A: Time (Min)B: Temperature (Celsius)
Design-Expert® Software
Factor Coding: Actual
Stevioside Content (%)
Design points above predicted value
Design points below predicted value
9.3
5.4
X1 = A: Time
X2 = C: Ratio (Leaf:Water)
Actual Factor
B: Temperature = 60
5
10
15
20
25
20
40
60
80
100
120
5
6
7
8
9
10
Ste
vio
sid
e C
on
ten
t (%
)
A: Time (Min)C: Ratio (Leaf:Water) (g:ml)
Design-Expert® Software
Factor Coding: Actual
Stevioside Content (%)
Design points above predicted value
Design points below predicted value
9.3
5.4
X1 = B: Temperature
X2 = C: Ratio (Leaf:Water)
Actual Factor
A: Time = 70
5
10
15
20
25
30
40
50
60
70
80
90
5
6
7
8
9
10
Ste
vio
sid
e C
on
ten
t (%
)
B: Temperature (Celsius)C: Ratio (Leaf:Water) (g:ml)
Fig 4.1: Effect of different process parameters on stevioside content using water
extraction
-
28
Ratio of 8.339 indicates that this model can be used to navigate the design space
(Table 4.2). The stevioside content ranged from 5.4 to 9.3% (Table 4.1). Time and
temperature had significant effects on stevioside content (P
-
29
Table 4.4: ANOVA for color (A420) of stevia extract
Factors Coefficient Sum of
squares
Df Mean
Square
F-
Value
Prob>F
Model 1.17 0.15 3 0.050 12.58 0.0004
A- Time 0.052* 0.022 1 0.022 5.43 0.0366
B- Temperature 0.12**
0.12 1 0.12 30.48 < 0.0001
C- Ratio
(Leaf:Water)
-0.030 7.321E-003 1 7.321E-003 1.84 0.1985
* Significant at P
-
30
Design-Expert® Software
Factor Coding: Actual
Color (A420)
Design points above predicted value
Design points below predicted value
1.389
1.011
X1 = B: Temperature
X2 = A: Time
Actual Factor
C: Ratio (Leaf:Water) = 15
20
40
60
80
100
120
30
40
50
60
70
80
90
0.9
1
1.1
1.2
1.3
1.4
Co
lor (
A4
20
)
B: Temperature (Celsius)A: Time (Min)
Design-Expert® Software
Factor Coding: Actual
Color (A420)
1.389
1.011
X1 = C: Ratio (Leaf:Water)
X2 = A: Time
Actual Factor
B: Temperature = 74.5946
20
40
60
80
100
120
5
10
15
20
25
0.9
1
1.1
1.2
1.3
1.4
Co
lor (
A4
20
)
C: Ratio (Leaf:Water) (g:ml)A: Time (Min)
Design-Expert® Software
Factor Coding: Actual
Color (A420)
Design points above predicted value
Design points below predicted value
1.389
1.011
X1 = C: Ratio (Leaf:Water)
X2 = B: Temperature
Actual Factor
A: Time = 70
30
40
50
60
70
80
90
5
10
15
20
25
1
1.1
1.2
1.3
1.4
Co
lor (
A4
20
)
C: Ratio (Leaf:Water) (g:ml)B: Temperature (Celsius)
Fig 4.2: Effect of process parameters on color (A420) of stevia extract using water
extraction
-
31
4.1.3 Effect of process parameters on sugar content
Regression analysis was carried out to fit the quadratic model to experimental data
(Table 4.5).
Table 4.5: ANOVA for Sugar content
Factors Coefficient Sum of squares Df Mean Square F-Value Prob>F
Model 4.12 4.79 9 0.53 11.69 0.0019
A-Time -0.24* 0.48 1 0.48 10.44 0.0144
B-Temperature 0.27**
0.60 1 0.60 13.17 0.0084
C- Ratio (leaf:water) 0.093 0.068 1 0.068 1.50