African Journal of Food Science Vol. 4(5) pp. 269- 281, May 2010 Available online http://www.academicjournals.org/ajfs ISSN 1996-0794 © 2010 Academic Journals Full Length Research paper Physico-chemical assessment of natural sweeteners steviosides produced from Stevia rebaudiana bertoni plant A. Esmat Abou-Arab*, A. Azza Abou-Arab and M. Ferial Abu-Salem Department of Food Technology, National Research Centre, Dokki, Cairo, Egypt. Accepted 14 May, 2010 Stevia plants are a good source of carbohydrates (61.93% d.w.), protein (11.41% d.w.), crude fiber (15.52% d.w.), minerals (K, 21.15; Ca, 17.7; Na, 14.93 and Mg, 3.26 mg/100 g d.w. and Cu, 0.73; Mn, 2.89; Fe, 5.89 and Zn, 1.26 mg/100 g d.w.) also essential amino acids were found in amounts higher than those recommended by FAO and WHO for adults as well as non- essential amino acids. Stevioside was extracted and purified from the dried Stevia leaves by three methods. The first one, extraction by hot water (65°C) at different ratios of leaves to water (1:15 - 1:75). The optimum ratio was 1:35 in which the maximum stevioside content was obtained (7.53%), recovery of stevioside was 80.21% and purity related to depigmentation was 97.56%. The second method, extraction by methanol at ratio 4:1 methanol/leaves and purification, recovery of stevioside was 94.90% and purity was 81.53%. The third method,extraction by mixture of methanol/water (4:1) and purification, recovery of stevioside and purity were 92.34 and 79.81%, respectively. Physical and chemical characteristics of the extracted stevioside were studied. Stevioside extractions were analyzed by HPLC in which the highest amount of stevioside (23.20%) was obtained in methanol extract. Also, organolyptic characteristics of extractions indicate that this sweetener has been applied as substitutes for sucrose in different drinks and baking products. Key words: Stevia, stevioside, physico-chemical properties, extraction, natural sweeteners. INTRODUCTION The plant Stevia rebaudiana Bertoni (compositae) has powder of the Stevia sugar into consideration; these 400 been widely cultivated in the world for the sweet Kg of Stevia sugar are equivalent to about 80,000 diterpene glycosides that are mainly contained in its sweetening units. Note that one feddan of “Sugar cane” leaves. In Egypt, the gap between sugar production produces about 5,000 sweetening units and one feddan (1.757 million tons) and consumption (2.6 million tons) of “Sugar beet” produces about 3,500 sweetening units. represents a serious problem, since it was estimated to A sweetening unit is equivalent to the sweetness of one be 0.843 millioin tons (Alaam, 2007). Nowadays, kilogram of sucrose (Alaam, 2007). Stevia sweeteners attention is concentrated upon using Stevia in food and extracted products which are commonly used as industries, in order to close the gap between the non-nutritive and high-intensity sweeteners in beverages, production and consumption. foods and medicines. The leaves naturally contain a The Stevia plant was recently introduced to Egyptian complex mixture of eight sweet diterpene glycosides, agriculture in order to produce a natural sweetener than including stevioside, steviolbioside, rebaudiosides (A, B, can cover some of the lack of sugar production in Egypt C, D, E) and dulcoside A (Dossier, 1999). (Alaam, 2007). Stevia cultivation in different places of the The increasing consumption of sugar (sucrose) has world; it is expected that in the Egyptian agricultural resulted in several nutritional and medical problems, such environment; one feddan of Stevia may produce up to as obesity. Therefore, low caloric sweeteners have been 400 kg of Stevia sugar, annually. Taking the sweetening investigated to substitute sugar. An important class of low caloric sugar substitutes is known as high intensity sweetener, this is at least 50 - 100 times sweeter than sucrose (Jaroslav et al., 2006). Nowadays, the most *Corresponding author. E-mail: eabouarab @yahoo.com. common high intensity sweeteners in the world market Afr. J. Food Sci. 270 are made of synthetic compounds. A frequent metallic was developed and more than 88% extraction efficiency aftertaste of such synthetic sweeteners does not provide was obtained by using methanol as a modifier. the realistic taste of sugar as well as some types of In Egypt there is a large interest on the strategical synthetic sweeteners such as saccharin is associated agriculture plan for new crops which can be suitable for with the potential risk of cancer of bladder when they are cultivation on new reclaimed areas. It may reduce food used heavily (Jaroslav et al., 2006). gap especially in the area of sugar and sweeteners. Stevioside is a diterpene glycoside present in S. Recently there is a great effort for natural sweeteners rebaudiana Bertoni. Stevia has been used by the Guarani production. Some trials are in the beginning for Stevia Paraguayan Indians and Mestizos as a general cultivation in Egypt. This study was conducted to reveal sweetening substance for centuries in which part of the and shed more light on the effect of some technological plant are used as consumption sweetener. Nowadays, process on the production of natural sweeteners Stevia plant and stevioside are being used as sweetener steviosides from S. rebaudiana Bretoni, besides physico- in South America, Asia, Japan, China, and some chemical and organolyptic properties of produced countries in Europe (Jaroslav et al., 2006). Toxicological steviosides as substitute of sugar (sucrose) were studied studies have shown that stevioside does not have mutagenic, teratogenic or carcinogenic effects and no allergic reaction have been observed when it is used as a MATERIALS AND METHODS sweetener. So, Stevia and stevioside have been applied Materials as substitutes for sucrose, for treatment of diabetes mellitus, obesity, hypertension, and caries prevention Stevia plant leaves (Jaroslav et al., 2006). Stevioside is extracted from the leaves of S. The Stevia leaves (S. rebaudiana Bretoni), were obtained from Stevia International Company from Agro-industry Product (SICAP), rebaudiana Bertoni, a plant from north-eastern Paraguay. Cairo, Egypt. It is a white, crystalline, odourless powder which is approximately 300 times sweeter than sucrose (Allam et al., 2001). Structurally, stevioside (13-[2-O-ß-D- Reagents glucopyranosyl--glucopyran-osyl) oxy] kaur-16-en-19- Ion exchange resin (Amberlite IR-4B), organic acids and solvents oic-acid ß-D- glucopyranosyl ester) is a glycoside with a were obtained from Sigma Chemical Co. (St. Louis, Mo). glucosyl and a sophorosyl residue attached to the aglycone steviol, which has a cyclopentanon hydrophenanthrene skeleton. Sugar standards Midmore and Rank (2006) classified the refining The standard of stevioside was obtained from M.B. Bio medical methods of stevioside to solvent partition extraction USA. mainly methanol or water extraction and solvent partition extraction incorporating a de-colonizing agent (mainly calcium hydroxide precipitation to remove impurities). Mineral standards They reported different methods of purification that is, Standard solution (1000 ppm) of macro elements; potassium (K), adsorption column chromatography ion-exchange or calcium (Ca), sodium (Na), and magnesium (Mg) as well as micro plasmide gel or adsorbed by the activated carbon; elements; copper (Cu), manganese (Mn), iron (Fe) and zinc (Zn) electrolytic technique; isolation procedures for were provided by Merck (Darmstadt, Germany). The standards rebaudioside A, rebaudioside C, and dulcoside A can be were prepared from the individual 1000 mg/L (Merck).Working removed. Hot water extraction appeared to be the standards were prepared from the previous stock solutions. preferred medium for extraction since the better-tasting rebaudioside A was more soluble than stevioside in Preparation of Stevia leaves water. However, some patents claimed the advantages from the use of solvents, such as ethanol, The Stevia plants that reached the maximum growth stage (mature methanol/chloroform or glycerin, sorbitol or propylene stage before flowering) were harvested by cutting the plant at 5 - 10 glycol. Liu et al. (1997) extracted stevioside from the cm from the ground. The brown and yellow leaves were removed dried leaves of S. rebaudiana with hot methanol for 7 h, from the plants then, washed in clean water and spread on trays covered with cheese-cloth to remove the excess of water. The and then concentrated it to dryness. A suspension of the plants were also dried in direct sunlight at temperature ranged from residue in water was washed with ether and then to 25 - 30°C for 24 - 48 h. extracted with butanol. The organic phase was evaporated, and the residue was re-crystallized from methanol, giving the stevioside. They also, studied the Methods extraction of steviol glycosides that is, rebaudioside A, The prepared Stevia leaves were analyzed chemically to determine rebaudioside C, and dulcoside A by suberitical fluid the gross composition, amino acids content and mineral levels as extraction (Sub FE). A simple efficient Sub FE method well as total chlorophylls and carotenoids as follows: Abou-Arab et al. 271 Chemical analysis of stevia plants Extraction with water The sample materials were blended to powder form with a high- The dried ground leaves of stevia plant were extracted by water speed blender (Braun KMM 30 mill), type 3045, CombiMax according to Nishiyama et al. (1992).The dried ground leaves were (Germany).This was kept in polyethylene bags and stored at 4 ± mixed with hot water (65°C) at different percentage of powder 1°C in a refrigerator until used. leaves/water ratio of 1:15, 1:25, 1:35, 1:45, 1:55, 1:65 and 1:75 The prepared samples were analyzed for moisture, protein, fat, (w/v). Stevia leaves were extracted by using hot water for 3 h as crude fiber and ash according to the methods described in the follow: The crude extract containing stevioside was filtered through AOAC (2000).