EFFECTS OF STEEPING CONDITIONS (TEMPERATURE, PARTICLE SIZE AND DURATION) USING WATER ON STEVIOL GLYCOSIDES CONTENT FROM Stevia rebaudiana AND SENSORY EVALUATION OF OPTIMISED SAMPLE

A. H. Nur Sabreena 1 , * Y. Nor Azma 1 , A. Asmah 1 and O. Mohamad 2 . 1. Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia. 2. Faculty of Agricultural, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5 (5), 1626-1631 1627 To reduce the usage of organic solvents, water is a feasible green solvent extraction. Hence, steeping method using water has become an efficient and low cost method of extraction for less-polar organic components from environmental soil, sediments and plant materials. Water is a highly polar solvent with a high dielectric constant (ε) at room temperature and atmospheric pressure due to the presence of extensive hydrogen-bonded structure. Hence, traditionally water is not considered as a suitable extraction fluid for non-polar or organic compounds at room temperature. When the temperature of water is raised, there is a steady decrease in its permittivity, viscosity and surface tension but an increase in its diffusivity characteristics. Under these conditions, water behaves like certain organic solvents which can dissolve a wide range of medium and low polarity analytes (Teo, Tan, Yong, Hew, & Ong, 2010).
The optimised samples was obtained and verified using RSM will be tested via sensory evaluation. The sample was evaluated using the performance of a hedonic scale for product acceptability. The most common scale for acceptance testing is the 9-point hedonic scale that was developed in the United States (US) by Peryam andPilgrim in 1957 (Yeh et al., 1998). Hedonic scales have been used for a number of years. The 9-point hedonic scale is a balanced bipolar scale around neutral at the center with four positive and four negative categories on each side. The categories are labeled with phrases representing various degrees of affect and those labels are arranged successively to suggest a single continuum of likes and dislikes (Peryam & Pilgrim, 1957).

Materials and Method:-
Materials:-Dried leaves of Stevia bought from China through the Stevia Sugar Coorporation in Distilled water which has been filtered using filteration set, water bath from Lab Tech. Refractometer was purchased from Surechem Sdn Bhd (Sri Petaling, Malaysia).

Methods:-Extraction of Stevia:-
The Stevia was extracted by following three parameters that has been chosed which were immersion temperature (°C), Particle size (mm), and Immersion duration (min). An amount of 1 g of dried leaves was extracted with 100 mL distilled water using steeping method. Water bath and thermometer are used to control the temperature of extract. The crude extract was then filtered using Whatman filter paper No. 1.Distilled water act as a control in this experiment.

Determination using Refractometer (% Brix):-
After the extraction process, the crude extract will be analyzed using refractometer to detect the total soluble solid. The range of refractometer that has been used was from 0% to 56 %. The refractometer need to be calibrated after reading first sample to avoid error in reading.
ICP-Analysis:-0.5000 g of Stevia leaves (China and MS012) were weighed and then put in a crucible silica. The samples were then baked in a muffle furnace at 550 o C for 6 to 7 hours and let them to be cooled overnight. After that, the samples were moved onto sandbath and 2 mL of HCl was added. Let them to be dried and 10 mL of 20 % HNO 3 was added. After 1/3 of the acid solution was dried, hence the remaining acid was filtered into a 50 mL volumetric flask.

Sensory evaluation via Hedonic:-
In order to analyze the optimised sample in the aspect of taste and human preference, human panel taste has to be conducted by using the hedonic scale rating. This is the most obvious choice, as it directly measures the effect one is trying to achieve, and consequently is the method most commonly used in product development. The panel testing is a psychophysical rating of gustatory stimuli. In this method, a group of about 20 to 30 human volunteers were required. The 9-point hedonic scale is a balanced bipolar scale around neutral at the center with four positive and four negative categories on each side.

Extraction of Stevia:-
The proposed methods, namely steeping showed that Stevia compound, could be extracted at elevated temperature using water without the addition of organic solvent. Table 1 shows the result of Stevia extraction using water which have been analysed via refractometer (% Brix) and HPLC. It involved three variables which were immersion 1628 temperature, particle size and immersion duration. The 20 samples was optimised using RSM from the previous paper (A.H. Nur Sabreena, 2017). Previously, a group of researchers conducted an experiment of Stevia extraction using hot water. The variables involved were water ratio, time and temperature. The experiment also conducted an optimisation processes to achieved favourable condition in order to obtained high content of stevioside. The optimum conditions were leaf to water ratio was 1:14 (g:mL), temperature of water at 78°C and time of heating was 56 min (Rai, Majumdar, & De, 2012). Figure 1 shows the effect of temperature on Stevia extraction. Elevation in temperature leads to increasing of the extraction yields of compounds. Although higher temperatures had positive effects on the extraction yields, temperature cannot be increased indefinitely, because of the instability of compounds. For example, partial hydrolysis of polysaccharides and denaturation of membranes may take place at certain temperatures (Cacace & Mazza, 2003).  Temperature is the main factor which could affect the extraction efficiency. It could influence the physicochemical properties of water and also subject thermally labile analytes to their decomposition or hydrolytic attack (Smith, 2002). Besides, the high temperatures have also changed the properties of water and thus making the polarity of water closer to those of non-polar compounds. This will enhance the solubility of less polar compounds in water for extraction (Teo et al., 2010). Figure 2 indicated the effect of particle size on Stevia extraction. Smaller size of the leaves lead to higher compound content of Stevia extract. The results of this study show that the particle size of the leaves influence the Stevia compositions. In 2003, a group of researchers conducted an experiment on the extraction of antioxidants from Balm (Melissa officinalis L.) leaves with ethanol. The study claimed that the total yield of extraction increases with the decrease of particle size. These results indicate that, in the latter stage of the extraction process, the diffusion of the solvent into the particle and the solvent-solute diffusion out of the particle are rate-governing steps of the process. (Herodež, Hadolin, Škerget, & Knez, 2003).

Sensory Evaluation:-
Before the sensory test was conducted, the ICP analysis and pH test was performed in order to ensure the safety of the extracts formed. ICP analysis was performed to check on the heavy metal content, the result obtained was 0.02 ppm for mercury, Hg and 0.03 ppm for lead, Pb. In addition, pH for the extract was conducted and the result was 5.59. Besides, the sensory test was conducted towards the optimised sample from previous research which was immersion temperature of 92 °C, particle size 0.7 mm and immersion duration of 4.   Table 3, by loking at the median column, the median engagement were higher for Stevia compared to fine sugar.The median difference between Stevia and fine sugar for those three attributes sweetness, bitterness and aftertaste were -0.000, 1.00 and 2.00 respectively with 95.8 % confidence interval. The Mann Whitney test did not show any statistically significant differences between Stevia and fine sugar for sweetness attribute. This proved from the p-value obtained ( Table 3) which is more than 0.05. This insignificant value implied that both sweeteners showing the sweetness attribute almost at the same level. Stevia act as a natural sweetener also well known as sugar leaf, candy leaf and sweet leaf due to its sweet taste that estimately 300 times sweeter than cane sugar (Balaswamy, Rao, Rao, Nagender, & Satyanarayana, 2014). Previous research has investigated that glycosides were purely bitter principles which commonly found in plants of the Genitiaceae family and though they are chemically unrelated but possess the common property of an intensely bitter taste (Doughari, 2012). Many studies have attempted to improve the taste of Stevia. However, Stevia has an incredibly sweet taste, but in its raw form it has a very significant liquorice essence to it. There was a report which ratified that Stevia is the sweetest plant in the world because the leaves part contain diterpene glycoside that has a sweet taste but it is not metabolized and contain no calories (Kumar, Jha, & Singh, 2007).

Conclusion:-
Based on the results obtained, it suggested that the Stevia extraction was affected by those three parameters which were immersion temperature, particle size and immersion duration. Besides, the sensory test indicated that the sweetness level between Stevia and cane sugar were almost equal due to insignificant of p-value (p>0.05), whereas for bitterness and aftertaste there were significant p-value between the sweeteners that can be referred in Table 2. Therefore, Stevia extraction required an improvement of taste in order to be chosen as a daily sweetener. However people that are concern about their health would chose Stevia for their drinks and foods as Stevia has a medicinal properties.