# Patent

## Title
Glucosyl stevia composition

## Abstract

Glucosyl stevia compositions are prepared from steviol glycosides of Stevia rebaudiana Bertoni. The glucosylation was performed by cyclodextrin glucanotransferase using starch as the source of glucose residues. The short-chain glucosyl stevia compositions were purified to >95% content of total steviol glycosides. The compositions can be used as sweetness enhancers, flavor enhancers and sweeteners in foods, beverages, cosmetics and pharmaceuticals.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part application of and claims the benefit of priority from U.S. patent application Ser. No. 13/029,263, filed on Feb. 17, 2011 and U.S. patent application Ser. No. 13/074,179, filed on Mar. 29, 2011.


BACKGROUND OF THE INVENTION
1. Field of the Invention
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.
2. Description of the Related Art
Nowadays sugar alternatives are receiving increasing attention due to awareness of many diseases in conjunction with consumption of high-sugar foods and beverages. However many artificial 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 C13 and C19 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%).
Two major glycosides—Stevioside and Rebaudioside A, were extensively studied and characterized in terms of their suitability as commercial high intensity sweeteners. Stability studies in carbonated beverages confirmed their heat and pH stability (Chang S. S., Cook, J. M. (1983) Stability studies of stevioside and Rebaudioside A in carbonated beverages. J. Agric. Food Chem. 31: 409-412.)
Steviol glycosides differ from each other not only by molecular structure, but also by their taste properties. Usually stevioside is found to be 110-270 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.)
Methods for the extraction and purification of sweet glycosides from the Stevia rebaudiana plant using water or organic solvents are described in, for example, U.S. Pat. Nos. 4,361,697; 4,082,858; 4,892,938; 5,972,120; 5,962,678; 7,838,044 and 7,862,845.
However, even in a highly purified state, steviol glycosides still possess undesirable taste attributes such as bitterness, sweet aftertaste, licorice flavor, etc. One of the main obstacles for the successful commercialization of stevia sweeteners are these undesirable taste attributes. It was shown that these flavor notes become more prominent as the concentration of steviol glycosides increases (Prakash I., DuBois G. E., Clos J. F., Wilkens K. L., Fosdick L. E. (2008) Development of rebiana, a natural, non-caloric sweetener. Food Chem. Toxicol., 46, S75-S82.)
Some of these undesirable properties can be reduced or eliminated by subjecting steviol glycosides to the reaction of intermolecular transglycosylation, when new carbohydrate residues are attached to initial molecule at C13 and C19 positions. Depending on the number of carbohydrate residues in these positions the quality and potency of the compounds taste will vary.
Pullulanase, isomaltase (Lobov S. V., Jasai R., Ohtani K., Tanaka O. Yamasaki K. (1991) Enzymatic production of sweet stevioside derivatives: transglycosylation by glucosidases. Agric. Biol. Chem. 55: 2959-2965), β-galactosidase (Kitahata S., Ishikawa S., Miyata T., Tanaka O. (1989) Production of rubusoside derivatives by transglycosylation of various β-galactosidase. Agric. Biol. Chem. 53: 2923-2928), and dextran saccharase (Yamamoto K., Yoshikawa K., Okada S. (1994) Effective production of glucosyl-stevioside by α-1,6-transglucosylation of dextran dextranase. Biosci. Biotech. Biochem. 58: 1657-1661) have been used as transglycosylating enzymes, together with pullulan, maltose, lactose, and partially hydrolyzed starch, respectively, as donors of glycosidic residues.
The transglucosylation of steviol glycosides was also performed by action of cyclodextrin glucanotransferases (CGTase) produced by Bacillus stearotherinophilus (U.S. Pat. Nos. 4,219,571, and 7,807,206) as a result α-1,4-glucosyl derivatives were formed with degree of polymerization up to 10.
It was shown (Tanaka O. (1987) Improvement of taste of natural sweetness. Pure Appl. Chem. 69:675-683) that the taste profile and sweetness power of glucosyl derivatives are largely dependent on number of additional α-1,4-glucosyl derivatives, i.e. the degree of polymerization of the α-1,4-glucosyl chain. However, in most of transgucosylated stevia products the degree of polymerization is usually below nine. As with any reaction, the transglucosylation reaction is inhibited by its products, and further increase of α-1,4-glucosyl residues is inhibited by reaction products, particularly short-chain maltooligosaccharides.
Therefore it is necessary to develop simple process of preparation of high purity glucosyl stevia products with greater α-1,4-glucosyl chain length and better taste profile.
SUMMARY OF THE INVENTION
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 rebaudidana Bertoni plant 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 glycodsides are selected from the group consisting of stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, 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, dulcoside A, steviolbioside, rubusoside, as well as other steviol glycosides found in Stevia rebaudiana Bertoni plant. The process can be an enzymatic transglucosylating process using CGTases produced by cultures of Bacillus stearothermophilus. The process may include 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 macroporuos polymeric resin.
In the invention, 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. Alternatively stevia extracts with different ratio of steviol glycosides as well as highly purified steviol glycosides such as Rebaudioside A, stevioside, Rebaudioside D, rubusoside etc, may be used as starting materials.
The starting material was subjected to enzymatic transglucosylation by action of cyclodextrin glycosyltransferase (CGTase) in the presence of starch as a glucose donor. As a result α-1,4-glucosyl derivatives were formed with a degree of polymerization up to 9. Then the maltooligosaccharides from obtained reaction mixture were removed by Amberlite XAD7 HP resin. The resulting mixture of α-1,4-glucosyl derivatives (with a degree of polymerization up to 9) was subjected to the second enzymatic transglucosylation by CGTase in the presence of starch as a glucose donor. As a result of the second glucosylation, α-1,4-glucosyl derivatives with a degree of polymerization up to 20 were formed. The maltooligosaccharides obtained during second glucosylation were removed by Amberlite XAD7 HP resin. Then the obtained mixture of α-1,4-glucosyl derivatives (with degree of polymerization up to 20) was decolorized, deionized, concentrated and spray dried.
The obtained products were applied in various foods and beverages as sweeteners, sweetener enhancers and flavor modifiers, including ice cream, cookies, bread, fruit juices, milk products, baked goods and confectionary products.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.



BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings are included to provide a further understanding of the invention. The drawings illustrate embodiments of the invention and together with the description serve to explain the principles of the embodiments of the invention.
FIG. 1 shows a high-performance liquid chromatogram of transglucosylated Stevia extract containing α-1,4-glucosyl-derivatives with up to nine α-1,4-glucosyl residues;
FIG. 2 shows a high-performance liquid chromatogram of transglucosylated Stevia extract containing α-1,4-glucosyl-derivatives with up to twenty α-1,4-glucosyl residues.



DETAILED DESCRIPTION OF THE INVENTION
Advantages of the present invention will become more apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
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 (hereinafter collectively, “steviol glycosides”) amounting to total steviol glycosides content of at least 95%, was used as a starting material. Alternatively stevia extracts with different ratio of steviol glycosides as well as highly purified steviol glycosides such as Rebaudioside A, stevioside, Rebaudioside D, rubusoside etc, may be used as starting materials.
The HPLC analysis of the raw materials and products was performed on Agilent Technologies 1200 Series (USA) liquid chromarograph, equipped with Zorbax-NH2 (4.6×250 mm) 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.
The transglucosylation was accomplished by cyclomaltodextrin glucanotransferases (CGTases; EC 2.4.1.19) produced by Bacillus stearothermophilus St-100 (PureCircle Sdn Bhd Collection of Industrial Microorganisms—Malaysia). However, any other CGTase or enzyme possessing intermolecular transglucosylation activity may be applied as well. 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 transglycosylation 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 α-amylases, β-amylases etc. In one embodiment, CGTase and α-amylase mixtures as liquefying enzymes are preferred.
α-Amylase activity is expressed in Kilo Novo α-amylase Units (KNU). One KNU is the amount of α-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 α-amylase per one unit of CGTase.
The use of α-amylase in liquefaction allows achieving higher throughputs in further activated carbon filtration. When the CGTase is used as the only liquefying enzyme the filtration rate is approximately 10-15 L/hr per 1 m2 of filter surface. In case of liquefaction enzyme mixture (comprising α-amylase and CGTase) the filtration rate is twice as fast—approximately 20-30 L/hr per 1 m2 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. during about 0.5-5 hours, preferably about 1-2 hours.
After liquefaction, the reaction mixture is subjected to thermal inactivation of α-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.
After the inactivation, 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 first transglucosylation reaction is conducted at about 65° C. for about 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.
Upon completion of transglucosylation the reaction was stopped by heating at about 95° C. for about 15 minutes to inactivate the enzyme. As a result a mixture of α-1,4-glucosyl derivatives with a degree of polymerization up to 9 was obtained. In order to remove the short chain maltoologisaccharides, which inhibit the further elongation of α-1,4-glucosydic chain, the reaction mixture was passed through a column packed with Amberlite XAD7 HP macroporous adsorbent resin. The steviol glycosides and their glucosylated derivatives were adsorbed on the resin and subsequently eluted by aqueous ethanol. The resulting aqueous ethanol eluate, containing glucosyl steviol glycosides, was evaporated, concentrated and spray dried to obtain transglucosylated Stevia extract containing glucosyl derivatives with up to nine α-1,4-glucosyl residues.
A second portion of starch was subjected to partial hydrolysis (liquefaction) as described above.
After the liquefaction the transglucosylated Stevia extract obtained during the first glucosylation (with up to nine α-1,4-glucosyl residues) was added. The preferred ratio of transglucosylated Stevia extract to starch (kg of transglucosylated Stevia extract per 1 kg of starch) is about 0.5-1.5, preferably about 0.8-1.2.
Another portion of the CGTase preparation was added and the second transglucosylation reaction was conducted at about 65° C. for about 24-48 hours. The amount of this 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.
Upon completion of transglucosylation the reaction was stopped by heating at about 95° C. for about 15 minutes to inactivate the enzyme. As a result a mixture of α-1,4-glucosyl derivatives with a degree of polymerization up to 20 was obtained.
The maltooligosaccharides were removed from reaction mixture using Amberlite XAD7 HP resin as described above. The steviol glycosides and their glucosylated derivatives were adsorbed on the resin and subsequently eluted by aqueous ethanol. The resulted aqueous ethanol eluate, containing glucosyl steviol glycosides, 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. The decolorized solution was further desalted by passing through 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 resulting product was transglucosylated Stevia extract containing α-1,4-glucosyl-derivatives with up to twenty α-1,4-glucosyl residues (Sample 2).
The transglucosylated Stevia extract can optionally be further purified by removing unreacted steviol glycosides. The dried transglucosylated Stevia extract powder is suspended in aqueous alcohol. The powder to aqueous alcohol ratio (wt/vol) can range from 1:1 to 1:20, preferably 1:3 to 1:10. The aqueous alcohol contains 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 t in the art may be used as well. Alternatively the separated solids may be dissolved in water, evaporated from traces of alcohol and spray dried.
The alcohols employed in this optional step 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.
The resulting product contains a low level of non-modified glycosides, even without the optional steviol glycoside removal step. As used herein, the expressions “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 less than about 12%, less than about 10% or even lower can be attained using this method. Performing the optional step of steviol glycoside removal results in even lower levels of unreacted steviol glycosides in the final product.
A small part of purified transglucosylated Stevia extract containing α-1,4-glucosyl-derivatives with up to nine α-1,4-glucosyl residues (obtained as described above) was separated and further subjected to decolorizing and desalting treatment (similar to Sample 2) to produce Sample 1.
The process used for preparing Sample 2 was repeated without removal of maltooligosaccharides from first transglucosylation mixture (containing α-1,4-glucosyl-derivatives with up to nine α-1,4-glucosyl residues). This process yielded Sample 3.
The analysis of each Sample's composition (Table 1) shows a similar composition for Samples 1 and 3, whereas Sample 2 had larger concentration of high glucosyl derivatives (containing up to 20 α-1,4-glucosyl residues).







TABLE 1







Composition of glucosyl steviol glycosides samples









Content, %










Compounds
Sample 1
Sample 2
Sample 3













Stevioside
3.1
1.6
2.9


Rebaudioside C
1.0
0.4
1.0


Rebaudioside A
6.1
2.8
5.8


Monoglucosyl-stevioside (StevG1)
7.4
3.7
7.5


Monoglucosyl-Rebaudioside A
11.1
4.5
11.5


(RebAG1)


Diglucosyl-stevioside (StevG2)
8.4
4.8
8.8


Diglucosyl-Rebaudioside A
9.6
5.3
9.7


(RebAG2)


Higher Glucosylated derivatives
48.4
57.5
49.3


up to G9


Higher Glucosylated derivatives
—
15.6
—


from G9 to G20


Total content of unreacted
10.2
4.8
9.7


glycosides


Total content of glycosides
95.8
96.2
96.5









The sensory assessment of samples was carried using aqueous solutions, with 20 panelists. Based on overall acceptance the most desirable and most undesirable samples were chosen. The results are shown in Table 2.







TABLE 2







Sensory assessment of samples in water system










Judgment
Sample 1
Sample 2
Sample 3





Most desirable
 1
18
 1


Sweetness power
120
80
120


Comments
Sweet, slightly bitter,
Sweet, light, soft, round,
Sweet, slightly bitter,



astringent, slight lingering
pleasant, similar to sucrose,
astringent, slight lingering



aftertaste, sweetness onset is
no lingering aftertaste,
aftertaste, sweetness onset is



slow
sweetness onset is rapid
slow









As apparent from the results in Table 2, the sweetness quality of the Sample 2 was rated as most superior.
The glucosyl stevia composition represented by Sample 2 shows comparable sweetness power (80 times sweeter compared to a 5% sucrose solution) with control Samples 1 and 3 (120 times); however its flavor profile was clearly superior to the control samples.
The composition can be used as sweetness enhancer, flavor enhancer and sweetener in various food and beverage products. Non-limiting examples of 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.
Additionally the composition 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.
The composition 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, 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, maltose, sucrose, lactose, aspartame, saccharin, sucralose, sugar alcohols.
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, stabilizers, thickeners, gelling agents.
The following examples illustrate various embodiments of the invention. It will be understood that the invention is not limited to the materials, proportions, conditions and procedures set forth in the examples, which are only illustrative.
EXAMPLE 1
Preparation of CGTase
A strain of Bacillus stearothermophilus St-100 was inoculated in 2,000 liters of sterilized culture medium containing 1.0% starch, 0.25% corn extract, 0.5% (NH4)2SO4, and 0.2% CaCO3 (pH 7.0-7.5) at 56° C. for 24 hrs with continuous aeration (2,000 L/min) and agitation (150 rpm). The obtained culture broth was filtered using Kerasep 0.1 μm ceramic membrane (Novasep, France) to separate the cells. The cell-free permeate was further concentrated 2-fold on Persep 10 kDa 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.
EXAMPLE 2
Preparation of Glucosyl Stevia Composition
100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNU of α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units of CGTase obtained according to EXAMPLE 1 were added, and the liquefaction of starch was carried out at 80° C. for about one hour to dextrose equivalent about 15. The pH of the reaction mixture was adjusted to pH 2.8 by hydrochloric acid and the mixture was boiled at 100° C. during 5 minutes to inactivate the enzymes. After cooling to 65° C., the pH was adjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extract produced by PureCircle (JiangXi) Co., Ltd. (China), containing stevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%, Rebaudioside F (1.7%) and other glycosides amounting to total steviol glycosides content of about 96.4% was added to the liquefied starch and stirred until a homogeneous solution was obtained. 200 units of CGTase was added to the solution and the mixture was held at a temperature of 65° C. for 24 hours under continuous agitation. 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. The 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 1).
EXAMPLE 3
Preparation of Glycosyl Stevia Composition
100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNU of α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units of CGTase obtained according to EXAMPLE 1 were added, and the liquefaction of starch was carried out at 80° C. for about one hour to dextrose equivalent about 15. The pH of reaction mixture was adjusted to pH 2.8 by hydrochloric acid and the mixture was boiled at 100° C. during 5 minutes to inactivate the enzymes. After cooling to 65° C., the pH was adjusted to pH 6.0 with sodium hydroxide solution. 100 g of transglucosylated stevia extract obtained according to EXAMPLE 2 was added to the liquefied starch and stirred until a homogeneous solution was obtained. 200 units of CGTase were added to the solution and the mixture was held at a temperature of 65° C. for 24 hours under continuous agitation. The obtained reaction mixture was heated at 95° C. for 15 minutes to inactivate the enzyme. 20 grams of activated carbon was added and the mixture was heated to 75° C. and held during 30 min. 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. The 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. 140 grams of product was obtained (Sample 2).
EXAMPLE 4
Preparation of Glucosyl Stevia Composition
100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNU of α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units of CGTase obtained according to EXAMPLE 1 were added, and the liquefaction of starch was carried out at 80° C. for about one hour to dextrose equivalent about 15. The pH of reaction mixture was adjusted to pH 2.8 by hydrochloric acid and the mixture was boiled at 100° C. during 5 minutes to inactivate the enzymes. After cooling to 65° C., the pH was adjusted to pH 6.0 with sodium hydroxide solution. 100 g stevia extract produced by PureCircle (JiangXi) Co., Ltd. (China), containing stevioside 29.2%, Rebaudioside A 54.3%, Rebaudioside C 9.0%, Rebaudioside F (1.7%) and other glycosides amounting to total steviol glycosides content of about 96.4% was added to liquefied starch and stirred until a homogeneous solution was obtained. 200 units of CGTase was added to the solution and the mixture was held at a temperature of 65° C. for 24 hours under continuous agitation. 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 passed through columns packed with Amberlite FPC23 (H+) and Amberlite FPA51 (OH−) ion exchange resins. 195 grams of product was obtained.
EXAMPLE 5
Preparation of Glucosyl Stevia Composition
100 g of tapioca starch was suspended in 300 mL of water (pH 6.5). 2 KNU of α-amylase (Termamyl Classic, Novozymes, Denmark) and 30 units of CGTase obtained according to EXAMPLE 1 were added, and the liquefaction of starch was carried out at 80° C. for about one hour to dextrose equivalent about 15. The pH of reaction mixture was adjusted to pH 2.8 by hydrochloric acid and the mixture was boiled at 100° C. during 5 minutes to inactivate the enzymes. After cooling to 65° C., the pH was adjusted to pH 6.0 with sodium hydroxide solution. 100 g of transglucosylated stevia extract obtained according to EXAMPLE 4 was added to liquefied starch and stirred until a homogeneous solution was obtained. 200 units of CGTase was added to the solution and the mixture was held at a temperature of 65° C. for 24 hours under continuous agitation. The obtained reaction mixture was heated at 95° C. for 15 minutes to inactivate the enzyme. 20 grams of activated carbon was added and the mixture was heated to 75° C. and held during 30 min. 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. The 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. 105 grams of product was obtained (Sample 3).
EXAMPLE 6
Low-Calorie Orange Juice Drink
Orange concentrate (35%), citric acid (0.35%), ascorbic acid (0.05%), orange red color (0.01%), orange flavor (0.20%), Rebaudioside A (0.003%) and different glucosyl stevia compositions (0.03% for Samples 1 and 3 and 0.04% for Sample 2) were blended and dissolved completely in water (up to 100%) and pasteurized. Glucosyl stevia compositions were represented by Samples 1, 2, and 3, obtained according to EXAMPLES 2, 3 and 5, respectively.
The sensory evaluations of the samples are summarized in Table 3. The data show that the best results can be obtained by using the high purity glucosyl stevia composition (containing up to 20 α-1,4-glucosyl residues) (Sample 2). Particularly the drinks prepared with Sample 2 exhibited a rounded and complete flavor profile and mouthfeel.







TABLE 3







Evaluation of orange juice drink samples









Comments










Sample
Flavor
Aftertaste
Mouthfeel





No. 1
Sweet, licorice notes
Slight bitterness and
Not




aftertaste
acceptable


No. 2
High quality sweetness,
Clean, no bitterness and
Full



pleasant taste similar to
no aftertaste



sucrose, rounded and



balanced flavor


No. 3
Sweet, licorice notes
Slight bitterness and
Not




aftertaste
acceptable









The same method can be used to prepare juices and juice drinks from other fruits, such as apples, lemons, apricots, cherries, pineapples, mangoes, etc.
EXAMPLE 7
Low-Calorie Carbonated Beverage
A carbonated beverage according to formula presented below was prepared.













Quantity, %










Ingredients
Sample 1
Sample 2
Sample 3













Sucrose
5.5
5.5
5.5


Cola flavor
0.340
0.340
0.340


ortho-Phosphoric acid
0.100
0.100
0.100


Sodium citrate
0.310
0.310
0.310


Sodium benzoate
0.018
0.018
0.018


Citric acid
0.018
0.018
0.018


Rebaudioside A
0.003
0.003
0.003


Glucosyl stevia composition
0.05
0.06
0.05


Carbonated water
to 100
to 100
to 100









The sensory properties were evaluated by 20 panelists. The results are summarized in Table 4.







TABLE 4







Evaluation of low-calorie carbonated beverage samples









Number of panelists detected the attribute










Taste attribute
Sample No. 1
Sample No. 2
Sample No. 3





Bitter taste
10
0
11


Astringent taste
12
0
10


Aftertaste
14
0
14


Comments


Quality of sweet taste
Bitter aftertaste
Clean
Bitter aftertaste



(10 of 20)
(20 of 20)
(12 of 20)


Overall evaluation
Satisfactory
Satisfactory
Satisfactory



(3 of 20)
(20 of 20)
(4 of 20)









The above results show that the beverages prepared using Sample 2 possessed the best organoleptic characteristics.
EXAMPLE 8
Diet Cookies
Flour (50.0%), margarine (30.0%) fructose (10.0%), maltitol (8.0%), whole milk (1.0%), salt (0.2%), baking powder (0.15%), vanillin (0.1%) and different glucosyl stevia compositions (0.03% for Samples 1 and 3, and 0.04% for Sample 2) were kneaded well in dough-mixing machine. The obtained dough was molded and baked in oven at 200° C. for 15 minutes. Glucosyl stevia compositions were represented by Samples 1, 2 and 3, obtained according to EXAMPLES 2, 3 and 5, respectively.
The sensory properties were evaluated by 20 panelists. The best results were obtained in samples prepared by high glucosyl stevia composition containing derivatives with up to 20 α-1,4-glucosyl residues (Sample 2). The panelists noted rounded and complete flavor profile and mouthfeel in cookies prepared with Sample 2.
EXAMPLE 9
Yoghurt
Different glucosyl stevia compositions (0.03% for Samples 1 and 3, and 0.04% for Sample 2) and sucrose (4%) were dissolved in low fat milk. Glucosyl stevia compositions were represented by Samples 1, 2 and 3, obtained according to EXAMPLES 2, 3 and 5, 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 sensory properties were evaluated by 20 panelists. The best results were obtained in samples prepared by high glucosyl stevia composition containing derivatives with up to 20 α-1,4-glucosyl residues (Sample 2). The panelists noted rounded and complete flavor profile and mouthfeel in samples prepared with Sample 2.
It is to be understood that the foregoing descriptions and specific embodiments shown herein are merely illustrative of the best mode of the invention and the principles thereof, and that modifications and additions may be easily made by those skilled in the art without departing for the spirit and scope of the invention, which is therefore understood to be limited only by the scope of the appended claims.

## Claims

1. A process for producing a highly purified glucosyl stevia composition, comprising the steps of:
adding starch into water to form a starch suspension;
adding a mixture of α-amylase and CGTase into the starch suspension and incubating for about 0.5 to 2 hours at about 75-80° C., resulting in a first liquefied starch suspension;
inactivating the α-amylase by low pH heat treatment;
cooling the first liquefied starch suspension and adjusting the pH to about 5.5 to 7.0;
adding steviol glycosides into the first liquefied starch suspension, resulting in a first reaction mixture;
adding CGTase into the first reaction mixture and incubating for about 12 to 48 hours at about 55-75° C.;
removing non-reactant maltoologosaccharides by contacting the first reaction mixture with macroporous adsorbent resin and subsequently eluting adsorbed diterpene glycosides with aqueous ethanol to result in a glycoside-containing first aqueous ethanol eluate;
removing ethanol from the first aqueous ethanol eluate, resulting in a first aqueous eluate;
concentrating and drying the first aqueous eluate to obtain the first dried glucosyl stevia composition;
preparing a second liquefied starch suspension in the same manner as the first liquefied starch suspension;
adding the first dried glucosyl stevia composition into the second liquefied starch suspension, resulting in the second reaction mixture;
adding CGTase into the second reaction mixture and incubating for about 12 to 48 hours at about 55-75° C.;
inactivating the enzyme in the second reaction mixture by heat treatment;
decolorizing the second reaction mixture;
removing non-diterpene compounds by contacting the decolorized second reaction mixture with macroporous adsorbent resin and subsequently eluting adsorbed diterpene glycosides with aqueous ethanol to result in a glycoside-containing second aqueous ethanol eluate;
desalting the glycoside-containing second aqueous ethanol eluate with ion-exchange resins;
removing ethanol from the second aqueous ethanol eluate, resulting in a second aqueous eluate; and
concentrating and drying the second aqueous eluate to obtain the highly purified glucosyl stevia composition;

wherein the highly purified glucosyl stevia composition comprises steviol glycosides and their derivatives having up to twenty α-1,4-glucosyl residues.

2. The process according to claim 1, wherein the mixture of α-amylase and CGTase contains about 0.001-0.2 KNU of α-amylase per one unit of CGTase.

3. The process of claim 2, wherein the mixture of α-amylase and CGTase contains about 0.05-0.1 KNU of α-amylase per one unit of CGTase.

4. The process according to claim 1, wherein the weight of added steviol glycosides is about equal to that of the starch used to make the first liquefied starch suspension.

5. The process according to claim 1, wherein the added steviol glycosides are selected from the group consisting of stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, dulcoside A, steviolbioside, rubusoside, as well as other steviol glycosides found in Stevia rebaudiana Bertoni plant and mixtures thereof.

6. The process according to claim 1, wherein the weight of added first dried glucosyl stevia composition is about equal to that of the starch used to make the second liquefied starch suspension.

7. The process according to claim 1, wherein the first dried glucosyl stevia composition comprises stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, dulcoside A, steviolbioside, rubusoside, and α-1,4-glucosyl derivatives thereof with up to nine α-1,4-glucosyl residues.

8. The process according to claim 1, wherein the CGTase is produced by cultures of Bacillus stearothermophilus.

9. The process according to claim 1, wherein the CGTase added to the first or second reaction mixture_is added at amount of about 0.2-4 units per gram of solids.

10. The process according to claim 1, wherein the CGTase added to the first or second reaction mixture is added at amount of about 0.5-1.2 units per gram of solids.

11. The process according to claim 1, wherein the decolorizing is performed using activated carbon.

12. The process according to claim 1, wherein the decolorizing is performed using ion exchange resins or membranes, said membranes being selected from the group consisting of ultrafiltration, nanofiltration, and reverse osmosis membranes.

13. The process of claim 1, wherein removing maltooligosaccharides and non-diterpene compounds from the first or second reaction mixture is conducted with 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.

14. The process according to claim 1, wherein the desalting is performed by passing the eluate through columns packed with ion exchange resins or membranes, said membranes being selected from the group consisting of ultrafiltration, nanofiltration, and reverse osmosis membranes.

15. The process according to claim 1, wherein the highly purified glucosyl stevia composition has at least about 95% total steviol glycosides on an anhydrous basis.

16. The process according to claim 1, wherein the highly purified glucosyl stevia composition has less than 10% unreacted steviol glycosides on an anhydrous basis.

17. A sweetener composition comprising a highly purified glucosyl stevia composition made by the process of claim 1, and an additional sweetening agent selected from the group consisting of: stevia extract, steviol glycosides, stevioside, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rebaudioside D, Rebaudioside E, Rebaudioside F, dulcoside A, steviolbioside, rubusoside, other steviol glycosides found in Stevia rebaudiana Bertoni plant and mixtures thereof, Luo Han Guo extract, mogrosides, high-fructose corn syrup, corn syrup, invert sugar, fructooligosaccharides, inulin, inulooligosaccharides, coupling sugar, maltooligosaccharides, maltodextins, corn syrup solids, glucose, maltose, sucrose, lactose, aspartame, saccharin, sucralose, sugar alcohols, and a combination thereof.

18. A flavor composition comprising a highly purified glucosyl stevia composition made by the process of claim 1, and an additional flavoring agent selected from the group consisting of: lemon, orange, fruit, banana, grape, pear, pineapple, mango, bitter almond, cola, cinnamon, sugar, cotton candy, vanilla, and a combination thereof.

19. A food ingredient comprising a highly purified glucosyl stevia composition made by the process of claim 1, and an additional food ingredient selected from the group consisting of: acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, emulsifiers, stabilizers, thickeners, gelling agents, and a combination thereof.

20. A food, beverage, cosmetic or pharmaceutical product comprising a highly purified glucosyl stevia composition made by the process of claim 1.