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  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
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  • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
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  • C12P19/00—Preparation of compounds containing saccharide radicals
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  • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
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  • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof

Definitions

• the invention relates to a process for producing a highly purified food ingredient from the extract of the Stevia rebaudiana Bertoni plant and its use in various food products and beverages.
• sweeteners such as dulcin, sodium cyclamate and saccharin were banned or restricted in some countries due to concerns on their safety. Therefore non- caloric sweeteners of natural origin are becoming increasingly popular.
• the sweet herb Stevia rebaudiana Bertoni produces a number of diterpene glycosides which feature high intensity sweetness and sensory properties superior to those of many other high potency sweeteners.
• the above-mentioned sweet glycosides have a common aglycon, steviol, and differ by the number and type of carbohydrate residues at the C I 3 and C I 9 positions.
• the leaves of Stevia are able to accumulate up to 10-20% (on dry weight basis) steviol glycosides.
• the major glycosides found in Stevia leaves are Rebaudioside A (2-10%), Stevioside (2-10%), and Rebaudioside C (1 -2%).
• Other glycosides such as Rebaudioside B, D, E, and F, Steviolbioside and Rubusoside are found at much lower levels (approx. 0- 0.2%).
• Steviol glycosides differ from each other not only by molecular structure, but also by their taste properties. Usually stevioside is found to be 1 10-270 times sweeter than sucrose, Rebaudioside A between 150 and 320 times, and Rebaudioside C between 40-60 times sweeter than sucrose. Dulcoside A is 30 times sweeter than sucrose. Rebaudioside A has the least astringent, the least bitter, and the least persistent aftertaste thus possessing the most favorable sensory attributes in major steviol glycosides (Tanaka O. (1987) Improvement of taste of natural sweetners. Pure Appl. Chem. 69:675-683; Phillips K.C. (1989) Stevia: steps in developing a new sweetener. In: Grenby T.H. ed. Developments in sweeteners, vol. 3. Elsevier Applied Science, London. 1 -43.)
• the transglucosylation of steviol glycosides was also performed by action of cyclodextrin glucanotransferases (CGTase) produced by Bacillus stearothermophilus (U.S. Patent Numbers 4,219,571, and 7,807,206) as a result a- l ,4-glucosyl derivatives were formed with degree of polymerization up to 10.
• CCTase cyclodextrin glucanotransferases
• glucosyl derivatives are largely dependent on number of additional glucosyl derivatives, i.e. the degree of polymerization of the -l ,4-glucosyl chain.
• the increase in number of a-l,4-glucosyl residues improved the taste quality but at the same time reduced the sweetness level (Tanaka, 1987).
• the treatment of transglucosylated stevioside with / ⁇ -amylase resulted in a product consisting of mono- or di-a-l ,4-glucosyl derivatives (Tanaka, 1987).
• the resulting product contains a high level of initial unreacted (unmodified) glycosides (generally >20%) which makes it not compliant with regulatory requirements of less than 15% unreacted glycosides (a-Glucosyltransferase Treated Stevia, Japan 's Specifications and Standards for Food Additives, VIII edition, 2009, p.257). Therefore additional steps for chromatographic separation of unreacted steviol glycosides are used to reduce initial unreacted (unmodified) glycosides' content.
• chromatographic separation techniques generally involve high cost and are not suitable for large scale production.
• glucosyl stevia products contain up to 20% residual dextrins which do not possess significant functional properties and reduce the content of steviol glycosides in the product.
• the present invention is aimed to overcome the disadvantages of existing Stevia sweeteners.
• the invention describes a process for producing a high purity food ingredient from the extract of the Stevia rebaudiana Bertoni plant and use thereof in various food products and beverages as a sweetness and flavor modifier.
• the invention in part, pertains to an ingredient comprising glucosylated derivatives of steviol glycosides of Stevia rebaudiana Bertoni plant.
• the steviol glycosides are selected from the group consisting of stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside X, dulcoside A, steviolbioside, rubusoside, as well as other steviol glycosides found in Stevia rebaudiana Bertoni plant and mixtures thereof.
• the invention in part, pertains to a process for producing an ingredient containing glucosylated forms of stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside X, dulcoside A, steviolbioside, rubusoside, as well as other steviol glycosides found in Stevia rebaudiana Bertoni plant.
• the process can be an enzymatic transglucosylation process using CGTases produced by cultures of Bacillus stearothermophilus.
• the process may include the step of additional enzymatic treatment by / ⁇ -amylase or other enzymes.
• the process can also have the steps of decolorizing, desalting and removing maltooligosaccharides.
• the decolorizing can be performed using activated carbon.
• the desalting can be performed by passing through ion exchange resins and/or membrane filters. Removing the maltooligosaccharides can be performed by passing through macroporous polymeric resin.
• Stevia extract commercialized by PureCircle (JiangXi) Co., Ltd. (China), containing stevioside (28-30%), Rebaudioside A (50-55%), Rebaudioside C (9- 12%), Rebaudioside F (1-3%) and other glycosides amounting to total steviol glycosides' content of at least 95%, was used as a starting material.
• stevia extracts with different ratio of steviol glycosides as well as highly purified steviol glycosides such as Rebaudioside A, stevioside, Rebaudioside D, Rebaudioside X, rubusoside etc, may be used as starting materials.
• the starting material was subjected to the enzymatic transglucosylation by action of cyclodextrin glycosyltransferase (CGTase) in the presence of starch as a glucose donor.
• CGTase cyclodextrin glycosyltransferase
• a-l ,4-glucosyl derivatives were formed, in some embodiments with degree of polymerization up to 20.
• the formed derivatives were optionally subjected to treatment with / ⁇ -amylase or other enzymes to produce a-l ,4-glucosyl derivatives possessing a specific degree of polymerization.
• the obtained products were applied in various foods and beverages as sweeteners, sweetener enhancers, flavors and flavor modifiers, including soft drinks, ice cream, cookies, bread, fruit juices, milk products, baked goods and confectionary products.
• FIG. 1 shows a high-performance liquid chromatographic chromatogram of purified transglucosylated Stevia extract, without ⁇ -amylase treatment containing long- chain a-l ,4-glucosyl-derivatives with up to nine -l,4-glucosyl residues;
• FIG. 2 shows a high-performance liquid chromatographic chromatogram of purified transglucosylated Stevia extract after / ⁇ -amylase treatment with short-chain (containing four or less - l ,4-glucosyl residues) derivatives of stevioside and Rebaudioside A.
• FIG. 3 shows a high-performance liquid chromatographic (HPLC) chromatogram of / ⁇ -amylase treated product containing mono- and di-cc- 1 ,4-glucosyl-derivatives of steviol glycosides, as well as high level of unreacted steviol glycoside;
• HPLC high-performance liquid chromatographic
• FIG. 4 shows a high-performance liquid chromatographic (HPLC) chromatogram of / ⁇ -amylase treated product containing mono- and di- -l ,4-glucosyl-derivatives of steviol glycosides, as well as low level of unreacted steviol glycosides.
• stevia extracts with different ratio of steviol glycosides as well as highly purified steviol glycosides such as Rebaudioside A, stevioside, Rebaudioside D, Rebaudioside X, rubusoside etc, may be used as starting materials.
• the steviol glycosides may be replaced, partially or completely, by compounds from the group consisting of Luo Han Guo extract, Siraitia grosvenorii extract, mogrosides, mogroside HE, mogroside III, mogroside IV, mogroside V, mogroside VI, 1 1-oxo-mogroside V, siamenoside I, grosmomoside I, as well as other mogrol or oxo-mogrol glycosides found in Siraitia grosvenorii plant and mixtures thereof.
• the HPLC analysis of the raw materials and products was performed on Agilent Technologies 1200 Series (USA) liquid chromatograph, equipped with Zorbax-NH 2 (4.6X250mm) column.
• the mobile phase was acetonitrile-water gradient from 80:20, v/v (0-2 min) to 50:50, v/v (2-70 min).
• a diode array detector set at 210 nm was used as the detector.
• CGTases cyclomaltodextrin glucanotransferases
• Bacillus stear other mophilus St- 88 PureCircle Sdn Bhd Collection of Industrial Microorganisms - Malaysia.
• the enzyme can be in a form of cell-free culture broth, concentrated liquid cell-free culture broth, spray dried or freeze dried cell-free culture broth, or high purity protein. Free and immobilized enzyme preparations can be used.
• the activity of CGTase preparations was determined according to the procedure described in Hale W.S., Rawlins L.C. (1951) Amylase of Bacillus macerans. Cereal Chem. 28, 49-58.
• Starches of different origin may be used as donors of glucosyl units such as, derived from wheat, corn, potato, tapioca, and sago.
• Starch was subjected to partial hydrolysis (liquefaction) prior to the transglucosylation reaction.
• the dextrose equivalent of the partially hydrolyzed starch can be in the range of about 10-25, preferably about 12-16.
• Any enzyme capable of starch hydrolysis may be used for liquefaction, such as a-amylases, ⁇ -amylases etc.
• CGTase and a-amylase mixtures as liquefying enzymes are preferred.
• a-Amylase activity is expressed in Kilo Novo ⁇ -amylase Units (KNU).
• KNU Kilo Novo ⁇ -amylase Units
• One KNU is the amount of ⁇ -amylase which, under standard conditions (pH 7.1 ; 37°C), dextrinizes 5.26 g starch dry substance per hour.
• the liquefaction mixture contains about 0.001-0.2 KNU, preferably about 0.05-0.1 KNU of ⁇ -amylase per one unit of CGTase.
• ⁇ -amylase in liquefaction allows achieving higher throughputs in further activated carbon filtration.
• CGTase is used as the only liquefying enzyme the filtration rate is approximately 10-15 L/hr per l m 2 of filter surface.
• liquefaction enzyme mixture comprising ⁇ -amylase and CGTase the filtration rate is twice as fast - approximately 20-30 L/hr per lm 2 of filter surface.
• the ratio of starch and CGTase in the liquefaction mixture is about 0.1-0.5 units per one gram of starch, preferably about 0.2-0.4 units per gram.
• the concentration of starch in liquefaction mixture is about 15-40% (wt/wt), preferably about 20-30%.
• the liquefaction is conducted at about 70-90°C, or 75-80°C, during about 0.5-5 hours, for example, about 0.5 to 2 hours, and preferably about 1 -2 hours.
• the reaction mixture is subjected to thermal inactivation of ⁇ -amylase at low pH conditions.
• the preferred pH range for inactivation is about pH 2.5 to pH 3.0 and preferred temperature is about 95-105°C.
• the duration of thermal inactivation is about 5-10 minutes.
• the pH of the reaction mixture is adjusted to about pH 5.5- 6.5 and the steviol glycosides are added to the mixture and dissolved.
• the preferred ratio of steviol glycosides to starch (kg of steviol glycosides per 1 kg of starch) is about 0.5-1.5, preferably about 0.8-1.2.
• a second portion of CGTase preparation is added and the transglucosylation reaction is conducted at temperature of between about 5-125°C, such as 65°C, for about 1 to 168 hours, such as 24-48 hours.
• the amount of the second portion of CGTase is about 0.2-4 units of CGTase per gram of solids, preferably about 0.5- 1.2 units per gram of solids.
• additional steps may include optionally inactivating the enzyme(s) in the reaction mixture; optionally decolorizing the reaction mixture; and optionally concentrating and drying the reaction mixture to obtain glucosyl stevia composition.
• the glucosyl stevia composition at this stage comprises steviol glycoside derivatives having twenty or less a-l,4-glucosyl residues.
• Further enzymatic treatment can include the addition of amylase, ⁇ -amylase, maltase, glucoamylase, fructofuranosidase, glucosidase, glucanase, ?-glucanase, transglucosidase, glucosyltransferase, fructosyltransferase, galactosyltransferase, lactase, galactosidase, cellulase, pullulanase, xylanase, mannanase, Maltogenase®, Fungamyl®, Novamyl®, Optimalt®, or mixtures thereof, along with the substrate or substrates for the respective enzyme or enzymes utilized.
• the reaction mixture can be incubated for a period of time ranging from 0.0001 to 168 hours, at a temperature ranging from 5-125°C.
• Additional steps may include inactivating the enzymes in the reaction mixture by heat treatment; optionally decolorizing the reaction mixture; optionally removing non- diterpene compounds by contacting the decolorized reaction mixture with macroporous adsorbent resin and subsequently eluting adsorbed diterpene glycosides with alcohol or aqueous alcohol to result in a glycoside-containing eluate; optionally desalting the glycoside-containing eluate with ion-exchange resins; optionally removing alcohol from the eluate, resulting in an aqueous eluate; optionally concentrating and drying the aqueous eluate to obtain the dried glucosyl stevia composition, and optionally suspending the dried glucosyl stevia composition in aqueous alcohol, separating the crystals from suspension and drying them to obtain the desired glucosyl stevia composition.
• any of these steps may be changed depending on a variety of factors.
• about 30- 50 units per gram of solids of / ⁇ -amylase was added and the reaction was continued for about 12-16 hours at about 35-55°C, preferably about 45°C.
• Soybean ⁇ -amylase was used in this stage for Samples la and 2a, while the ⁇ -amylase made in accordance with EXAMPLE 2 was used for Samples lb and 2b.
• ⁇ -amylases derived from any other source including barley, bacterial, fungal / ⁇ -amylases and others may be used as well.
• ⁇ - Amylase activity unit ( 1 AUN) is defined as the activity which liberates 100 ⁇ g of reducing sugar (expressed by dextrose equivalent) per minute under the following conditions: l mL of enzyme solution is mixed with 5mL of 1.2% starch solution (pH 5.5, M / 20 Acetate Buffer) and kept for 20 min at 40°C.
• the reaction was stopped by heating at about 95°C for about 15 minutes to inactivate the enzymes, and the solution was treated with activated carbon, to obtain decolorized reaction mixture.
• the amount of activated carbon was about 0.02-0.4 grams per gram of solids, preferably about 0.05-0.2 grams per gram of solids.
• Other appropriate decolorizing methods such as using ion exchange resins, membrane filtration using ultrafiltration, nanofiltration or reverse osmosis membranes, or other methods known in the art can be used.
• Non-diterpene compounds may optionally be removed using, for example, a plurality of sequentially connected columns packed with a macroporous adsorbent resin, followed by washing the columns with water, then washing with about 10-50% (v/v) ethanol, disconnecting the columns, and then eluting each column individually with 30- 100%) ethanol.
• the decolorized reaction mixture was desalted by passing through ion exchange resins, such as Amberlite FPC23 (H + type) and Amberlite FPA51 (OH " type).
• ion exchange resins such as Amberlite FPC23 (H + type) and Amberlite FPA51 (OH " type).
• Other appropriate decolorizing and desalting methods such as membrane filtration or other methods known in the art can be used.
• the desalted reaction mixture was further concentrated by vacuum evaporator and dried by means of a spray dryer.
• Other appropriate concentrating and drying methods such as membrane filtration, freeze drying, or other methods known to art can be used.
• the dried powder was suspended in aqueous alcohol.
• the powder to aqueous alcohol ratio (wt/vol) was 1 : 1 to 1 : 20, preferably 1 :3 to 1 : 10.
• the aqueous alcohol contained 0-50% (vol), preferably 1 -10% water.
• the suspension is agitated at 30-100°C, preferably 50-85°C during 1-24 hours, preferably 2-15 hours. Then the suspended solids are separated by means of filtration.
• any other technique known in the art suitable for separating suspended solids from liquid such as centrifugation, decanting, etc. can be used.
• the obtained solids are dried in rotary drum vacuum drier. Any other dryer known in the art may be used as well.
• the separated solids may be dissolved in water, evaporated from traces of alcohol and spray dried.
• the alcohols employed in the invention may be selected from the group consisting of alkanols, and are preferably selected from the group including methanol, ethanol, n- propanol, 2-propanol, 1-butanol, and 2-butanol, or mixtures thereof.
• the resulting product contains low level non-modified glycosides, short-chain (containing four or less, or two or less, a-l ,4-glucosyl residues) derivatives and a mixture of maltooligosaccharides (Samples la and lb).
• low level non-modified glycosides or “low level unreacted glycosides” shall refer to glycoside levels of less than about 20%, and preferably less than about 15%, on an anhydrous basis. In some embodiments, an unreacted glycoside level of about 12%, about 10% or even lower can be attained using this method.
• the dried powder was suspended in aqueous alcohol and processed as described above to remove unmodified (unreacted) steviol glycosides (Sample 2b).
• the resulting product contains low level non-modified glycosides, and short-chain (containing four or less, or two or less a-l,4-glucosyl residues) derivatives (Samples 2a and 2b).
• the embodiments of the invention exemplified by Samples la, lb, 2a and 2b are free or substantially free of higher glucosylated derivatives having more than 4 or more than 2 glucosyl residues.
• the highly purified glucosyl stevia composition preferably comprises greater than about 25% by weight di-, tri- and tetraglucosyl Rebaudioside A, and greater than about 9% by weight tri- and tetraglucosyl steviosides.
• the highly purified glucosyl stevia composition comprises greater than about 50% by weight mono-, and diglucosyl steviol glycosides.
• composition of the samples is summarized in Tables l a and lb, in which Samples la and 2a made using the processes described above contain four or less a- 1,4- glucosyl residues, and Samples lb and 2b made using the processes described above contain two or less -l,4-glucosyl residues.
• Samples la and 2a show comparable sweetness power (150-160 times sweeter compared to a 5% sucrose solution) with control Sample 4 (150 times); however their flavor profile was clearly superior to the control sample.
• Samples lb and 2b which contain two or fewer a- 1,4-glucosyl residues.
• Sample 5 was prepared in accordance with EXAMPLE 12. Table lb
• Samples l b and 2b show comparable sweetness power (150-160 times sweeter compared to a 5% sucrose solution) with control Sample 5 (160 times); however their flavor profile was clearly superior to the control Sample 5.
• compositions can be used as sweetness enhancers, flavors, flavor enhancers and sweeteners in various food and beverage products.
• food and beverage products include carbonated soft drinks, ready to drink beverages, energy drinks, isotonic drinks, low-calorie drinks, zero-calorie drinks, sports drinks, teas, fruit and vegetable juices, juice drinks, dairy drinks, yoghurt drinks, alcohol beverages, powdered beverages, bakery products, cookies, biscuits, baking mixes, cereals, confectioneries, candies, toffees, chewing gum, dairy products, flavored milk, yoghurts, flavored yoghurts, cultured milk, soy sauce and other soy base products, salad dressings, mayonnaise, vinegar, frozen-desserts, meat products, fish-meat products, bottled and canned foods, tabletop sweeteners, fruits and vegetables.
• compositions can be used in drug or pharmaceutical preparations and cosmetics, including but not limited to toothpaste, mouthwash, cough syrup, chewable tablets, lozenges, vitamin preparations, and the like.
• compositions can be used "as-is” or in combination with other sweeteners, flavors and food ingredients.
• Non-limiting examples of sweeteners include steviol glycosides, stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, Rebaudioside X, dulcoside A, steviolbioside, rubusoside, as well as other steviol glycosides found in Stevia rebaudiana Bertoni plant and mixtures thereof, stevia extract, Luo Han Guo extract, mogrosides, high-fructose corn syrup, corn syrup, invert sugar, fructooligosaccharides, inulin, inulooligosaccharides, coupling sugar, maltooligosaccharides, maltodextins, corn syrup solids, glucose, fructose, maltose, sucrose, lactose, aspartame, saccharin, sucralose, sugar alcohols, and combinations thereof.
• Non-limiting examples of flavors include lemon, orange, fruity, banana, grape, pear, pineapple, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla flavors.
• Non-limiting examples of other food ingredients include flavors, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilisers, thickeners, gelling agents, and combinations thereof.
• a strain of Bacillus stearothermophilus St-88 was inoculated in 2,000 liters of sterilized culture medium containing 1.0% starch, 0.25% corn extract, 0.5% (NH 4 ) 2 S0 4 , and 0.2% CaC0 3 (pH 7.0-7.5) at 56°C for 24 hrs with continuous aeration (2,000 L/min) and agitation ( 150rpm).
• the obtained culture broth was filtered using Kerasep 0.1 ⁇ ceramic membrane (Novasep, France) to separate the cells.
• the cell-free permeate was further concentrated 2-fold on Persep l OkDa ultrafilters (Orelis, France).
• the activity of the enzyme was determined according to Hale, Rawlins (1951). A crude enzyme preparation with activity of about 2 unit/mL was obtained.
• a strain of Bacillus polymyxa St-3504 was inoculated in 2,000 liters of sterilized culture medium containing 1.0% starch, 0.5% peptone, 0.5% corn extract, 0.5% NaCl, 0.02% MnS0 4 and 0.1% CaC0 3 (pH 7.0-7.5) at 32°C for 24 hrs with continuous aeration (2,000 L/min) and agitation (150rpm).
• the obtained culture broth was filtered using Kerasep 0.1 ⁇ ceramic membrane (Novasep, France) to separate the cells.
• ⁇ -Amylase activity unit (1 AUN) was defined as the activity which liberates 100 g of reducing sugar (expressed by dextrose equivalent) per minute under the following conditions: ImL of enzyme solution is mixed with 5mL of 1.2% starch solution (pH 5.5, M / 20 Acetate Buffer) and kept for 20 min at 40°C.
• reaction mixture was heated at 95°C for 15 minutes to inactivate the enzymes.
• 20 grams of activated carbon was added and the mixture was heated to 75°C and held for 30 minutes.
• the mixture was filtered and the filtrate was diluted with water to 5% solids content and passed through columns packed with Amberlite FPC23 (H + ) and Amberlite FPA51 (OH " ) ion exchange resins.
• the desalted solution was concentrated at 60°C under vacuum, and dried into a powder form using laboratory spray dryer. 196 grams of product was obtained (Sample la).
• the obtained reaction mixture was heated at 95°C for 15 minutes to inactivate the enzymes. 20 grams of activated carbon was added and the mixture was heated to 75°C and held for 30 minutes. The mixture was filtered and the filtrate was diluted with water to 5% solids content and passed through columns each packed with 4000 mL Amberlite XAD 7HP macroporous adsorbent resin. The columns were washed with 5 volumes of water and 2 volumes of 20% (v/v) ethanol. The adsorbed glycosides were eluted with 50% ethanol. Obtained eluate was passed through columns packed with Amberlite FPC23 (H + ) and Amberlite FPA51 (OH " ) ion exchange resins. The ethanol was evaporated and the desalted and decolorized water solution was concentrated at 60°C under vacuum, then dried into a powder form using laboratory spray dryer. 151 grams of product was obtained (Sample 2a).
• Glucosyl stevia compositions were blended and dissolved completely in water (up to 100%) and pasteurized.
• Glucosyl stevia compositions were represented by Samples la, 2a, and 3, obtained according to EXAMPLES 3, 4, and 5, respectively; and Sample 4 was a commercial /J-amylase treated product (containing only mono- and di- -l,4-glucosyl- derivatives of steviol glycosides).
• the same method can be used to prepare juices and juice drinks from other fruits, such as apples, lemons, apricots, cherries, pineapples, mangoes, etc.
• a carbonated beverage according to formula presented below was prepared.
• Glucosyl stevia compositions were by represented by Samples l a, 2a, and 3, obtained according to EXAMPLES 3, 4, and 5, respectively; with Sample 4 being a commercial / ⁇ -amylase treated product (containing only mono- and di-a-l ,4-glucosyl- derivatives of steviol glycosides).
• Sample 4 being a commercial / ⁇ -amylase treated product (containing only mono- and di-a-l ,4-glucosyl- derivatives of steviol glycosides).
• Glucosyl stevia compositions were blended and dissolved completely in water (up to 100%) and pasteurized.
• Glucosyl stevia compositions were represented by Samples lb, 2b, 3, 5 and 6, obtained according to EXAMPLES 10, 1 1, 5, 12, and 13 respectively.
• the same method can be used to prepare juices and juice drinks from other fruits, such as apples, lemons, apricots, cherries, pineapples, mangoes, etc.
• a carbonated beverage according to formula presented below was prepared.
• Glucosyl stevia compositions were represented by Samples lb, 2b, 3, and 5, obtained according to EXAMPLES 10, 1 1, 5, and 12, respectively. After pasteurizing at 82°C for 20 minutes, the milk was cooled to 37°C. A starter culture (3%) was added and the mixture was incubated at 37°C for 6 hours then at 5°C for 12 hours.
• the macroporous resin columns were washed with 5 volumes of water and 2 volumes of 20% (v/v) ethanol.
• the adsorbed glycosides were eluted with 50% ethanol.
• the ethanol of obtained eluate was evaporated and concentrated at 60°C under vacuum, then dried into a powder form using laboratory spray dryer. 1 14 grams of product was obtained (Sample 7).
• Sample 7 composition was analyzed using HPLC, and its sensory assessment along with other samples (Samples l a, 2a, 3 and 4) was carried out using aqueous solutions, with 20 trained panelists.
• the dried powder was suspended in 5 volumes of 95% aqueous ethanol. The suspension was agitated at 80°C, during 12 hours. Then the suspended solids were separated by filtration. The obtained solids were dried in vacuum dryer at 90°C during 5 hours. 67 grams of product was obtained (Sample 8).
• Sample 8 composition was analyzed using HPLC, and its sensory assessment along with other samples (Samples lb, 2b, 3, and 5 as described above) was carried out using aqueous solutions, with 20 trained panelists.

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