STUDIES ON EXTRACTION OF STEVIOSIDE FROM STEVIA (Stevia ... · Dr. (Mrs.) Gagandeep Kaur Sidhu Er. Navdeep Jindal Major Advisor External Examiner Assistant Professor Deptt. of Food

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

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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


content

39


(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


of stevioside using methanol

50

4.3 Microwave assisted extraction of stevioside with water 51


content

52


(A420) of stevia extract

55

4.3.3 Effect of process parameters on sugar content (%) 57


(TSS)

59



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


(TSS)

71



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


extraction with methanol

16


microwave assisted extraction with water

16


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


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




Table

No.

Title Page

No.




4.15 ANOVA for pH 48

4.16 Optimization of the extraction conditions for stevioside using

methanol

50








4.24 Optimization of the microwave assisted extraction conditions for

stevioside using water

63








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


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


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


MAE using water

64

Figure

No.

Title Page

No.

4.19 Effect of different process parameters on stevioside content using MAE

with methanol

66


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


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


-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



-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


-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)


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)


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)


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)






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)






9.3

5.4

X1 = B: Temperature


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



Color (A420)



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)



Color (A420)

1.389

1.011


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)



Color (A420)



1.389

1.011


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

Documents

STUDIES ON EXTRACTION OF STEVIOSIDE FROM STEVIA (Stevia ... · Dr. (Mrs.) Gagandeep Kaur Sidhu Er. Navdeep Jindal Major Advisor External Examiner Assistant Professor Deptt. of Food