OPTIMIZATION OF AQUEOUS EXTRACT OF STEVIA LEAVES AND ISOLATION OF STEVIOSIDE FROM THE EXTRACT

Abstract

A sweet component of the stevia plant (Stevia rebaudiana), stevioside, is used as a natural sweetener in the world. Stevia leaf contains well-characterized steviol glycosides such as stevioside which is 250 to 300 times sweeter than sucrose. Stevia has a negligible effect on blood glucose. The long-term use of artificial sweeteners such as cyclamate and saccharine have deleterious side effect but stevia is the safest alternative and is being preferred by the diabetic patients all over the world. In developed countries, pure stevioside is used as a food additive. The preparation of pure stevioside is based on complex chemical extraction process which is costly. Stevioside is water soluble and it can be extracted by water. Therefore, here we aimed to prepare a suitable aqueous extract of stevia leaf for using in the foods. Aqueous extraction of stevia is a non-chemical and economically feasible method. To do this, we selected aqueous extract based on different extraction conditions. In general, stevia extract gives a bitter taste due to the presence of pigments in the extracts. The release of stevioside and the release of pigments in the extract depend on the forms of leaf, time, temperature, and leaf volume etc. Firstly, we prepared stevia aqueous extract from dry whole leaf, leaf powder, and fresh leaf. We measured the pigment contents (total chlorophyll and carotenoid) of the extract and tested the sweetness sensitization on tongue of the extracts. Results showed that dry whole leaf released the less pigments in the extract. We also prepared the stevia extract to different time exposures such as 20 min, 40 min, 60 min, 80 min and 100 min. We determined the pigment contents of the extract and tested the sweetness sensitization on tongue of the extracts. We found that leaf extract prepared at 40 min contained less pigments and more sweetness on tongue. Next, we prepared the stevia aqueous extract exposed to different temperatures such as 200C, 400C, 600C, 800C and 1000C. We measured the pigment contents of the extract and tested the sweetness sensitization on tongue. We found that leaf extract prepared at 600C contained less pigments and more sweetness on tongue. We also prepared the stevia extract using different solvent volumes such as 20 ml, 40 ml, 60 ml, 80 ml and 100 ml. The results of pigment analysis and sweetness sensitization on tongue test revealed that leaf and solvent ratio 1:40 was the best. Based on the above results we concluded that water extraction of stevia dry whole leaf at 600C for 40 min using leaf: solvent (1:40) were the best aqueous extraction method. In a second attempt, we isolated stevioside from our optimized-aqueous extract. Then we use isobutanol to reduce the impurities of the extracts and we found white powder at the end. To confirm the presence of stevioside in the extracts we conducted FTIR analysis of white powder. FTIR analysis revealed the similar peaks of our white powder and the standard. However, some impurities were present in isolated stevioside. Therefore, further purification is needed to get more purified stevioside.