a S Fearure/CHronique Sfevia Rebaudiana Leaves - A Low Calorie Source of Sweeteners The use of extracts of Stevia rebaudiana leaves as low calorie sweeteners are being explored in many countries. Shi Qiu Zhang, Oleh Kutowy, and Ashwani Kumar, MCIC. he sweetening properties of Stevia rebaudiana have been known for centuries, ‘expecially init natve land of Paraguay. The dred leaves of Stevia rebaudiana Contain about 42 per cent water soluble constituents. The sweet compounds represent about 14 percent constituents of dried leaves and are diterpene glycosides ‘based onthe kaurene skeleton. These are mainly compris of stevisldesteviaboside: rebaudloside A, B, D, E: dulcoside A: and dulceside B (1). These stevia sweeteners ae sitvilar in structure in that a stevil aglycone is connected at C-4 and C-13 to ‘mono. d- or trsaccharides consisting of glucose and/or chamose residues s shown in Figure 1. The most abundant glycoside issteviosde followed by rebaudioside. The rest of the sweet components are present in much smaller amounts. Sweetening potencies of varlous diterpenes found in Stevi rebaudiana are Usted in Table 1. The Fon sweet constituents mainly are labdane diterpenes, triterpenes, sterols and flavonoids. {ale 1. Swosteningpotncis of tava rebousinacarpane gyosies Al, Diterpene R, R ‘Sweetening potency (Gucrase~1) Stevioside hay Ge 250 300 Rebaudloside A ca) co) 350-450 6@ 300.350 Rebaudloside B B ce) 300-350 Rebaudioside C cw R,) 50.120 (Duleoside B) Rebaudioside D ce ce 200-300 Rebaudloside E Ce ce 250-300 Duleeside A a GQ) 50-120 RU) Stevioside H Ge 100125 T= GhuciaylR2= Rhamnosyl * Number of G or R groups Rebauiosie A Is considered to have a taste superior to stevisides. Normally, it ‘represents a much smaller percentage of the total weight of leaves. To alleviate this shortcoming chemical processes aired at producing higher yields of Rebaudioside ‘Ahave been reported [2] and new cultivars with higher concentration of Rebaudleside have been developed using genetic engineering techniques {3} Stevia sweetener is alow calorie, heat stable, intensely sweet (300 times sweeter than sucrose at 0.4% concentration), proven safe by years of use in Japan, and is approted in Europe. The consumption of steviesides in Japan has accounted for more than 20 percent of low calorie sweeteners [5). Interest inthis low calorie sweetener vas renewed in North America during the 1990s because of export opportunities and positive nutritional properties. Stevia based sweeteners are approved forsale in Japan. (Chin, Taiwan, South Korea, Viewam, Thailand, and a number of counties in South America. According fo an extensive marketing study commissioned by several independent companies, the potential market for Stevia sweeteners in the six Asian 22 UActus mai 1999 1 chimique canadienne, OR, Ey ‘ 7 Hs seh : oH od ; R,OOG. Figure 1. General chemical formula of set cterpenoid _slycosides - H,CHCH3gR,CHHR,OOC jy, (4) countries listed above would be in excess of $1.2 billion per year by the year 2005. This assumes @ modest 4 percent 0 8 per cent market penetration indifferent countries, Extracting and Refining ‘There are hundreds of patents for stevia extraction proceses ‘on worldwide basis (the Japanese alone have over 150). The processing steps involved inthe production of sweeteners are ‘extraction. pretreatment, separation, and refining ‘Waterss the mos popula solvent for extraction, Methanol or mistures of methanol and water are also used. The pH, temperature, mode of extraction. and solvent type affect the Impurities (protein, pigments, pectins, and lavoro) in the extract The impurities were reduced by precipitation with (CaCO, at pH of 10. Itwasalso reported thal recovery increased with use of ultrasonic waves during extraction [6]. Researchers at NRC {7 found thatthe pigment concentration at 25°C eatraction temperature was nearly doubled at 50°C with a column extraction. The impurities were reduced by 30 per cent by column extraction compared with batch extraction, In the case of column extraction, the impurities a 4°C were about 70 percent ofthe values a 25°C. The yield of sweeteners was higher at higher extraction temperatures; however, the crude extract was of lower quality with larger amount of impurities. Selection of the proper extraction methods to minimize the Impurities from the crude extract affect the product cost significanty,aa So Fearure/CHronique Pre-treatment can be done with inowganic salts or a membrane process. Calelurn hhydroxide is the most commonly use ofthe inorgante sats; however, sulfates of potassium. aluminum. and Iron have aso been used [8]. Aluminum salt removed pigment from the crude extract most effectively, but also resulted in lower ylelds and purty ‘Ceramic microfitration provides chemical fre pre-treatment to remove large suspended impurities from the extract. This unit operation with microfiltration membranes (0.02-20 jm) clarified the extract by removing pigments absorbing at 420 nm and other higher molecular weight materials. Pigments absorbing at 670 ‘am were not removed significantly (7). Similar results were reported with polymeric ‘membranes with higher depigmentation (93%), stovioside recovery (90%), and ‘sevioside purity ratio (469) as compared with the inorganic salt weatment at 71.97%, TO-86%, and 9.28%, respectively [8] Untrafltration is the mest common methad for separation of remaining larger molecular weight impurities from the clarified extract [7-9]. Selecting the proper pre- tteatment scheme can reduce fouling propensities of the ultrafiltration membrane, Diafiltration is used to wash out remaining sweet compounds in the retentate and increase recovery. Permeate from this tage is then subjected to further refining Jon exchange and adsorption column chrornatography have been widely used for refining sweeteners. The flow rate, pH, and eluents greatly affect both the separation ficiency and the treatment capaci ofthe columns. Strongly acide cation-exchange ‘sin, strongly basic anion exchange resin, and mixed bed columns of weakly acidic cation exchange resin and weakly basic anion exchange resin were used with @ Stevidosice (purty 80-87%) showing recovery of about 79% [10]. A patented styrene- divinylberzene-methylacrylate copolymer adsorbent was claimed to achieve higher recovery than the commercial resin with stable and exslr regeneration of capacity [11]. Adsorption of stevioside was also successfully done by active carbon. Optimal ethanol concentration for desorption of stevosides was 60-65%. Interestingly, 2 significantly higher amount of Rebaudloside A was recovered by active carbon from fan aqueous solution (12) Alternatively, permeate from the ultrafiltration stage could be refined and concentrated by nanolluaton. NRC researchers reported [7 that higher temperature ranofiltation was an effective process to remove certain low molecular weight ‘compounds that might contribute to the biter taste ofthe final product. Flavonoids such as Apigenin-4o glucoside, quercitrin, and others are present in Stevia water ‘aract (13), They are yellowish in colour witha biter taste and with molecular weights smaller than Stevia glycoside. Therefore, these compounds would permeate the nanofiltration membranes. The permeate stream from ultrafllwation treatment, heated to about 80°C, was treated by a commercial nanofiltration membrane ese for high temperature operation. At this temperature, the arent of impurities In the retentate was reduced by ebout 55% under the conditions employed, while no sweeteners were detected in the permeate stream. Additional impurities could ‘be removed by using nanofltation in dafration mode, Applications and Outlook ‘There ae two distinct markets for high potency, low calore sweeteners one & specfically forthe low calorelettic segment, while the other is as an alternative to sugar and other nuttive sweeteners (Le. com syrup, fructse, glucose). Stevia sweeteners used in beverages, tabletop sweeteners and processed food, and personal !aygene products are popular in Japan. There is potential fr additional applications withthe development of new proceses to remove the biter aerate (7 Another interesting area comes from studies of effects of Stevia sweeteners on carbohydrate metabolism. Based on the research on the hypoglycemic activity of Stevia, etracts, leaves, flowers, and stems are now used in popular medicine in Paragu as remedy for diabetes [14]. Itis aso an approved herbal medicine for diabetic in China, Although Stevi has just achieved the status ofa dletay supplement by the FDA in North America, this could also be a nutraceutical product with great potential References 1. Leung, A.Y. and S. Foster, Encyclopedia of Common Natural Ingredients Used {in Food, Drugs and Cosmetics, 2nd Edition John Wiley and Sons, Inc., New York, 478, 1996, 2. Dobberein, RH. and F.K. Suzuki, Composition and Process for Moding or Enhancing Favors, European Patent EPOLS4 235 BI 2. Anonymous, “High power Sweeteners rom the Stevia Plan, Chemical Week, November 14, pp. 4546, 1985 4. Cramer, Band R Ikan, ‘Sweet Glyosies from the Seva Plant, Chemis in Britain, 22915-916, 1986, 5. Kikuchi, H., "Food Chemicals’, Food Selence par), 85(3)52, 1985, 6. Shoji; MU, Yasuaki, and . Masao, Japanese PatentJP 77 95407, 7. Kumar, A... Kutowy, SQ. Zhang, ‘Extraction of Sweet Compounds From Stevia Rebaudiana Berton’, United States Patent Allowed Application No. 09/116.925 8. Fuh, W. and B. Chiang, ‘Purification of Steviosides by ‘Membrane and ion Exchange Poces Jounal af Food Scene, 55(5):1453-1457, 1900. 9. Liu, ¥..¥.Chen etal, Study of Stevioside Preparation by Membrane Separation Process, Desalination, 88:375-382, 191. 10. Cheng, TF. C.Y. Chang, and WH. Chang,’ Study on the Purification of Stevisides With the Use of on Exchange Resins. Journal ofthe Chinese Agricultural Chemical Sete, 23(12):178-190 1985. 11. He, B.,¥. Liu, and Z. Sh, “Application of an Adsorbent ‘ABS in Extraction and Separation of Steviosde’, Huasue ¥Yingyong.11():16:18, 1994 12. Chang, W.H, and S.H. Huang. ‘A Simple Method for Stevosides Purification With Active Carbon’, Recent Advances ‘in Foad Science and Technology, pp. 484-496, 1980, 13, Rajbhandar, A. and M, Rober, ‘The flavanotdsof Stevia Rebautiana’,joumal of Natral Products, 42UarvFeb) 196-195, 1979 14. Sogjarto, D.D., and A.D. Kinghorn, ‘Current Status of Using Stevia Rebauiana As Sweeter. Bconoic and Medicinal Plant Research 1:26, 1985. @ Shi Qiu Zhang, Oleh Kutowy, and Aswan Kamar, MCTC ae research ofews atthe National Research Counc’ Instat for Chemical Process and Enomental Technolgy in Otawa, ON, Let's celebrate! Canada’s 11% National Chemistry Week October 17-23, 1999 La tt Semaine nationale de la chimie au Canada Soyons de la partie! ’ May 1999 Canadian Chemical News 23