M01035

October, 22-23th 2013

ISBN : 978-979-96595-4-5

OPTIMIZATION OF PRODUCTION PROCESS STEVIA BEVERAGES

WITH ANTIDIABETIC ACTIVITY

Yohanes Martono and Dewi K.A.K.H

Chemistry Department Faculty of Science and Math Satya Wacana Christian University e-mail: yohanes.martono@staff.uksw.edu

ABSTRACT

Stevia rebaudiana (Bert.) is an herb that contains stevioside that has hypoglycemic activity and can be used for diabetics. The purpose of this study was to optimize the method of making stevia beverages and to determine hypoglycemic activity with glucose tolerance test method. The pH adjustment and adsorption with adsorbent bentonite, kaolin, vermiculite and combinations were applied to clarify beverages solution. The optimization were concentration solution of honey starter, mass of stevia and fermentation period based on stevioside levels to determine the optimal stevia beverage to be used in vivo test. Stevioside content was determined by using High Performance Liquid Chromatography (HPLC). Quantitative data on blood glucose levels created curve relationship between blood glucose per unit of observation time. To determine the percentage of lowering blood glucose levels (% hypoglycemic activity), Area Under Curve-45-180 (AUC) was calculated by using Graph 4.3

program. The results showed that the most optimal stevia beverages was stevia beverages with 50 grams of stevia mass and fermentation period of 4 days with stevioside level of 0.99%. Hypoglycemic activity assay showed that 20% of stevia beverages had hypoglicemic activity greater than low-calorie syrup, which was 55.36%. Clarification technologies to maintain the optimal stevioside content in beverage was pH adjustment.

Keywords: optimization, stevia beverages, stevioside, antidiabetic

INTRODUCTION

The sweet taste is a very important sensory needs for the community. Most communities meet the needs of the sweetness of the sugar. Consumption of excessive amounts of sugar and a long period of time can lead to obesity, diabetes, and other degenerative diseases. In Indonesia, an overview of diabetes cases is not too encouraging. The World Health Organization (WHO) predicts rising living with type 2 diabetes mellitus of 8.4 million people in 2000, rising rapidly to 21.3 million in 2010. Based on the results of the Basic Health Research (2007), diabetes mellitus ranks second as a cause of death in the age group 45-54 years in urban areas. As in rural areas, diabetes mellitus was ranked sixth by the proportion of deaths by 5.8 percent (Anonymous, 2010).

Any type of low-calorie natural sweetener that does not have a negative impact on health is expected by society. Among the wide variety of sweeteners, these are glycoside compounds that can be extracted from the herb Stevia rebaudiana (Bert.). Steviol glycoside compounds called stevioside (figure 1) have the potential, functions and characteristics as sweeteners. Its sweetness are larger than other types sweetener which are 100 - 300 times higher than sugar or sucrose (Philip, 1987).

Health drink (beverages) is one of the products that the public interest due to the application of practical use. Stevia beverages contain stevioside compound as the active compound. This compound has pharmacologic hypoglycemic effect to lower blood glucose levels. The mechanism action of these compounds is increased secretion and insulin sensitivity, thereby reducing the accumulation of sugar in the blood (Chatsudthipong and Muanprasat, 2009).

ISBN : 978-979-96595-4-5

Fig.1 Chemical structures of stevioside

METHODS MATTERIALS

The sample used is Stevia rebaudiana (Bert.) obtained from the Tawangmangu, Karanganyar, Central Java. Mice used were obtained from the Faculty of Pharmacy, University of Muhammadiyah Surakarta, with specifications: swiss webster strain, male, aged between 2-3 months. Stevioside standard was purchased from WAKO, Japan with a purity of > 99%.

METHOD

Sample Preparation

All parts of the Stevia rebaudiana (Bert.) plant have been cleaned of soil, drained by cabynet drying for 24 hours at 50oC. Samples were dried and then pulverized with a grinder.

Optimization of Honey Starter Making

Honey solution concentration was varied 0, 2.5%, 5.0%, 7.5%, and 10% (w / v). Solution was sterilized for 15 minutes at 121oC and cooled untill reached room temperature. After solution at room temperature,

Saccharomyces cerevisiae and Rhizopus oligosporus were added each 1 gram. Solution was shaken with a shaker over a period of 0-25 hours.

Optimization of Stevia Beverages Fermentation

Stevia mass was varied between 50-300 grams and extracted by using 1 L hot distilled water. Stevia solution was filtered and then the residue was re-extracted with 1 L of hot distilled water. Extraction was carried out until the volume of solution to 2 L. Stevia solution sterilized with 100 ° C for 20 minutes. After it cooled down, solution was given 200 mL starter solution and then fermented for 0-6 days.

High Perfomance Liquid Chromatography (HPLC) conditions

The HPLC operating conditions as follows: stationary phase was Eurospher C-18 column (150 x 4.6 mm). Mobile phase was A = H2O: methanol (70: 20, v / v) (76%) and B = acetonitrile (24%). Mobile phase flow

rate was 1.50 ml / min and detected at a wavelength of 210 nm with a UV detector. Sample injection volume was 20 mL.

Glucose Tolerance Test (Yulinah et al., 2001 modified)

Glucose tolerance test was performed according to the method of Varley and Gowenblock. The test group consisted of three (3) male mices and as a whole consists of 5 treatment groups as follows: negative control (only given distilled water), a low calorie syrup test group "X" with concentration of 12.5%, stevia beverage groups with concentration 5%, 12.5% and 20%. Test subjects were fasted (12-18jam) prior to treatment with fixed given ad libitium drink. Each group received 50% glucose loading it orally in the 45 minute after administration of treatment. Determined blood glucose -45, 45, 90 and 180 min after glucose administration.

Blood Glucose Level Determination

Glucose levels determined by enzymatic methods with reagents Glucose Oxidase-Phenol-4-Aminoantipirin (GOD-PAP) followed by spectrophotometry measurement. Blood samples were taken from the lateral tail vein approximately 100 µL of rat blood then centrifuged at 3000 rpm for 10 minutes. At 100 µL of serum was added 100 µ L of deproteinase agent and centrifuged at 3000 rpm for 10 minutes. At 100 µL supernattan was added by 1 mL color reagent GOD-PAP then incubated for 30 min and measured at a wavelength of 546 nm using spectrophotometer .

Optimization of Color Removal (Dechlorophyillation)

Twenty grams of finely ground Stevia leaves macerated with hot water in the ratio 1: 5 (w / v). The filtrate was filtered and the pH adjusted with 50% citric acid to a pH of 4.00. Then, the solution was filtered and the filtrate was taken. Subsequently, the filtrate pH was raised to 10.00 with CaCO3 and filtered. Filtrate pH was

adjusted to pH 6.00 to 7.00 with 50% citric acid and filtered. The filtrate then clarified with bentonite and combination of bentonite - Vermiculite - Caoilin (by maceration and columns). Amount of filtrate collected and scanned between wavelengths 200-700 nm by using spectrophotometer.

October, 22-23th 2013

ISBN : 978-979-96595-4-5

The filtrate absobance of crude extract (CE), adjustment of pH-neutral (N), adsorption bentonite (B), a combination of bentonite - vermiculite - kaolin adsorbent adsorbed by maceration (Ms BVC) and a combination of bentonite - vermiculite - kaolin adsorbent adsorbed in columns (BVC Cl) was measured at a wavelength 665 nm (corresponding to the green pigment) and 410 nm (corresponding to the yellow pigment). Dechlorophyillation effectiveness was calculated as a percentage by comparing the absorbance of dechlorophyillation crude extract and the results of pH adjustment and clarification using adsorbents.

Result and Discussion

Optimization of Honey Starter

Production process of stevia beverages in this research was using fermentation process with the starter of honey. To determine the most effective microbial growth, optimization is done first, with various concentrations (2.5%, 5%, 7.5%, 10%) solution stater. Results optimization honey starter at different concentrations fermentation process of stevia beverages are presented in Figure 2.

(a) (b)

Fig. 2 Optimization Curves (a) Starter Honey From Different Concentration, (b) 7.5% Honey Stater

In Figure 2, the phase lag is shown at the time of 0-4 hours. While at the time of 4-10 hours is the exponential phase showed the addition of an increasing number of colonies. At the time of 10-22 hours a stationary phase which shows a balance between living and dead colonies. While at times above 22 hours a stationary phase which followed the death of colony growth phase begins to decrease and eventually will decline drastically. Incubation period of starter solution, 7.5% until the start of the exponential phase was 4 hours. This starter solution was used in the fermentation process.

Optimization of Fermentation Stevia Beverages

Based on the selection of the various mass stevia beverages and fermentation time were analyzed by HPLC, obtained stevia beverage that have the highest levels of stevioside was stevia beverage with a mass of 50 grams and fermentation time for 4 days. This is due to the mass ratio and the extraction of large volumes (1:40) so that the extracted compounds are also becoming more widely. Stevioside levels for each treatment is shown in Table 1.

Table 1. Stevioside content in various fermented stevia beverages

No Mass (Gram) Fermentation Period (day) Stevioside Content (%)

1 50 0 0.45

2 50 4 0.99 (4.51)*

3 100 2 0.51

4 200 4 0.26

5 300 6 0.23

Description: * stevioside highest levels after repeatedly extracted

The results of the HPLC measurements of stevioside content, found that beverage with stevia 50 grams of mass and fermentation time 4 days (D-4; 50gram) have the highest stevioside content, 0.99%. Optimal beverage stevia to be used for in vivo testing has stevioside content by 4.51%. This difference arises because optimal stevia beverages was repeatedly extracted using hot water so stevioside was dissolved into more. Stevioside standard and optimal stevia beverage chromatogram profile can be seen in Figure 3.

Hypoglicemics Activity Assay

ISBN : 978-979-96595-4-5

Figure 4 shows a pattern in which blood glucose levels are always increased in the 45minute and then declined in the 90th minute and 180. This increase was caused by glucose loading. Decrease in glucose levels in the hypoglycemic effects caused by treatment of stevia beverage containing stevioside while lowering glucose levels in the control (+) was influenced by the content of artificial sweeteners in low calorie syrup. Results of blood glucose levels followed by calculation of area under the curve (AUC-45-180) and a decrease in blood glucose levels (DBGL) can be seen in Table 2. Based on Table 2, result demonstrated that the higher the concentration of stevia drinks, bigger in decreased blood glucose levels. Lowering blood glucose levels Stevia beverages 12.5% and 20% are higher than syrup "X" which is one of the low-calorie syrups availabled in the market. These results indicate that stevia drinks in concentrations of 12.5% can lower blood glucose levels greater than the low-calorie syrups that contain lots of artificial sweeteners such as aspartame and cyclamate.

Fig. 3 Chromatogram profile [a] Stevioside standard (stevioside peak is no.1 with tR = 14.317 min) [b] Optimal Stevia beverage (peak is no.6 with tR = 13.650 min)

Figure 4. Curve blood glucose levels (mg / dL) vs. time (minutes)

Both stevioside and stevia extract can be used to lower blood sugar (Chatsudthipong and Muanprasat, 2009). Highest lowering blood glucose levels shown in beverage stevia 20%, ie 55.36 ± 33.71 (%). This result is very different from the stevia beverage 5% and 12.5% which can only reduce the blood sugar not more than 15%. From the results of our other studies, stevia beverages 20% that use a sugar starter has only decrease in blood sugar levels by 25.70%. This considerable difference is due to the interaction between the compounds in the honey, which is used as a starter, with the active compounds stevioside. Mechanism action of stevioside compound in lowering blood sugar levels is to increase insulin secretion and sensitivity, thereby reducing the amount of glucose in the blood. In addition, stevioside can also inhibit glucose absorption in the intestine and the formation of glucose in the liver by altering the activity of key enzymes involved in the synthesis of glucose, thus reducing the buildup of glucose in the blood plasma (Chatsudthipong and Muanprasat, 2009).

(b)

(a)

(a)

October, 22-23th 2013

ISBN : 978-979-96595-4-5

Table 2. Area Under Curve (AUC) and Lowering Blood Glucose Levels (% LBGL)

(N) Treatments

Nilai Purata AUC ± SE

(mg.min/dL) % (LBGL ± SE)

3 Aqua 23463,67 ± 6301,09 -

3 Syrup “X” 22576,33 ± 3020,98 5.84 ± 5,35

3 Stevia beverage 5% 22201,33 ± 6814,84 5.49 ± 5,49

3 Stevia beverage 12.5% 20103,67 ± 9493,92 14.86 ± 19,27

3 Stevia beverage 20% 9908,00 ± 15639,66 55.36 ± 83,76

Fructose is a major constituent of honey appeared to have hypoglycemic effects (Bogdanov et al., 2008). The mechanism of action of fructose is by activating glucokinase which is a key enzyme in intracellular glucose metabolism. Then the glucose is converted to glucose-6-phosphate, thereby reducing glucose levels in the blood (Watford, 2002). In addition, honey also contains elements such as zinc, selenium, copper, calcium, potassium, chromium, manganese, etc. (Bogdanov et al., 2008). Some minerals reportedly plays an important role in the maintenance of glucose and insulin secretion (Anderson et al., 1997 and Kar et al., 1999).

Optimization of Color Removal (Dechlorophyillation)

In our previous studies indicated that pH adjustment of acidic (pH = 3) with citric acid to alkaline pH (pH = 10) with CaCO3, then into the neutral solution (pH = 7) with citric acid can fade the color extraction of the

resulting solution. In addition, the next step is to perform adsorption using caolin and bentonite was able to reduce color and odor solution stevia. Therefore, the development of beverage production process can be pursued through the adjustment of pH and adsorption technologies. Color removal results with pH adjustment and clarification with the adsorbent can be seen in figure 5.

(a) (b)

Description: CE = crude extract; N = neutral solution (pH adjustment); B =bentonite; BVC = bentonite – vermiculite – caolin combination for clarifying by maseration (Ms) and column (Cl).

Fig. 5 (a) Color Removal efectiveness by dechlorophyllation of yellow pigments (A410) and green pigments

(A665); (b) comparison of solution visualization before (top left), pH adjustment (top right) and column

adsorption (bottom).

ISBN : 978-979-96595-4-5

(a) (b)

Fig. 6 Chromatogram profile of (a) crude extract and (b) neutral (pH adjustment) solution

CONCLUSION

The results showed that the most optimal stevia beverages was stevia beverages with 50 grams of stevia mass and fermentation period of 4 days with stevioside level of 0.99%. Hypoglycemic activity assay showed that 20% of stevia beverages had hypoglicemic activity greater than low-calorie syrup, which was 55.36%. The most effisient colour removal technologies to maintain the optimal stevioside content in beverage was pH adjustment.

ACKNOWLEDGEMENT

We thank you to Indonesian Directorate General of Higher Education (DIKTI), Indonesian Ministry of Education that has funded this research by Hibah Bersaing Scheme Desentalisasi Program in 2013.

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