U.S. patent application number 13/029263 was filed with the patent office on 2012-08-23 for glucosyl stevia composition.
Invention is credited to Avetik MARKOSYAN.
Application Number | 20120214751 13/029263 |
Document ID | / |
Family ID | 46653252 |
Filed Date | 2012-08-23 |
United States Patent
Application |
20120214751 |
Kind Code |
A1 |
MARKOSYAN; Avetik |
August 23, 2012 |
Glucosyl Stevia Composition
Abstract
Glucosyl stevia compositions are prepared from steviol
glycosides of Stevia rebaudiana Bertoni. The glucosylation was
performed by cyclodextrin glucanotransferase using the starch as
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.
Inventors: |
MARKOSYAN; Avetik; (Kuala
Lumpur, MY) |
Family ID: |
46653252 |
Appl. No.: |
13/029263 |
Filed: |
February 17, 2011 |
Current U.S.
Class: |
514/23 ; 426/655;
435/105 |
Current CPC
Class: |
A61K 36/28 20130101;
A61K 8/9789 20170801; A23L 27/36 20160801; A61Q 19/00 20130101;
C12P 19/18 20130101; A61K 8/9794 20170801; A21D 2/36 20130101; A23L
2/60 20130101; A61K 2800/10 20130101; A61K 2800/805 20130101; A61Q
11/00 20130101; C12P 19/56 20130101; A21D 2/18 20130101; A23C
9/1307 20130101; A23L 27/33 20160801; A61K 8/602 20130101; C12P
33/00 20130101; A23V 2002/00 20130101 |
Class at
Publication: |
514/23 ; 435/105;
426/655 |
International
Class: |
A61K 31/70 20060101
A61K031/70; A23L 1/28 20060101 A23L001/28; C12P 19/02 20060101
C12P019/02 |
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 a-amylase and CGTase
into the starch suspension and incubating for about 0.5 to 2 hours
at about 75-80.degree. C., resulting in a liquefied starch
suspension; inactivating the a-amylase by low pH heat treatment;
cooling the liquefied starch suspension and adjusting the pH to
about 5.5 to 7.0; adding steviol glycosides into the liquefied
starch suspension, resulting in a reaction mixture; adding a second
batch of CGTase into the reaction mixture and incubating for about
12 to 48 hours at about 55-75.degree. C.; adding .beta.-amylase to
reaction mixture and incubating for about 12-24 hours at about
35-55.degree. C.; inactivating the enzymes in the reaction mixture
by heat treatment; decolorizing the reaction mixture; removing
non-diterpene compounds by contacting the decolorized reaction
mixture with macroporous adsorbent resin and subsequently eluting
adsorbed diterpene glycosides with aqueous ethanol to result in a
glycoside-containing aqueous ethanol eluate; desalting the
glycoside-containing aqueous ethanol eluate with ion-exchange
resins; removing ethanol from the aqueous ethanol eluate, resulting
in an aqueous eluate; and concentrating and drying the aqueous
eluate to obtain the highly purified glucosyl stevia composition,
wherein the highly purified glucosyl stevia composition comprises
short-chain steviol glycoside derivatives having four or less
.alpha.-1,4-glucosyl residues, and unmodified steviol
glycosides.
2. The process according to claim 1, wherein the mixture of
.alpha.-amylase and CGTase contains about 0.001-0.2 KNU of
.alpha.-amylase per one unit of CGTase.
3. The process of claim 2, wherein the mixture of .alpha.-amylase
and CGTase contains about 0.05-0.1 KNU of .alpha.-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.
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 CGTase is produced
by cultures of Bacillus stearothemophilus.
7. The process according to claim 1, wherein the second batch of
CGTase has about 0.2-4 units of CGTase per gram of solids.
8. The process according to claim 1, wherein the second batch of
CGTase has about 0.5-1.2 units of CGTase per gram of solids.
9. The process according to claim 1, wherein the .beta.-amylase is
produced from a source selected from the group consisting of
soybeans and barley.
10. The process according to claim 1, wherein the .beta.-amylase is
added at about 30-50 units per gram of total solids, and the
treatment is carried out at a temperature of about 40-60.degree.
C., for a duration of about 3-16 hours.
11. The process according to claim 1, wherein after the
.beta.-amylase treatment, the short-chain glucosylated derivatives
of stevioside and Rebaudioside A have four or less .alpha.-glucosyl
residues.
12. The process according to claim 1, wherein the decolorizing is
performed using activated carbon.
13. 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.
14. The process of claim 1, wherein removing non-diterpene
compounds 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.
15. 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.
16. 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.
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, fruity, 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, stabilisers, 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.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] 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.
[0003] 2. Description of the Related Art
[0004] 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.
[0005] 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%).
[0006] 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.)
[0007] 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.)
[0008] 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. Numbers
4,361,697; 4,082,858; 4,892,938; 5,972,120; 5,962,678; 7,838,044
and 7,862,845.
[0009] 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.)
[0010] On the other hand, replacing large amounts of sugar in the
formulations brings up such problems as reduced mouthfeel,
incomplete flavor profile etc. Therefore the application of high
intensity low calorie sweeteners has to provide solutions to
address these problems.
[0011] Thus, if a single composition will be able to deliver not
only sweetness, but also possess flavor enhancing properties and
correct the incomplete mouthfeel associated with the elimination of
sucrose from food and beverage formulations, it will certainly be
advantageous compared to other high intensity sweeteners known in
the art.
[0012] 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.
[0013] 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), .beta.-galactosidase (Kitahata S.,
Ishikawa S., Miyata T., Tanaka O. (1989) Production of rubusoside
derivatives by transglycosylation of various .beta.-galactosidase.
Agric. Biol. Chem. 53: 2923-2928), and dextran saccharase (Yamamoto
K., Yoshikawa K., Okada S. (1994) Effective production of
glucosyl-stevioside by .alpha.-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.
[0014] The transglucosylation of steviol glycosides was also
performed by action of cyclodextrin glucanotransferases (CGTase)
produced by Bacillus stearothermophilus (U.S. Pat. Nos. 4,219,571,
and 7,807,206) as a result .alpha.-1,4-glucosyl derivatives were
formed with degree of polymerization up to 10.
[0015] The treatment of transglucosylated stevioside with
.beta.-amylase resulted in a product consisting of mono- or
di-.alpha.-1,4-glucosyl derivatives (Tanaka, 1987).
[0016] It was shown that the taste profile and sweetness power of
glucosyl derivatives are largely dependent on number of additional
glucosyl derivatives, i.e. the degree of polymerization of the
.alpha.-1,4-glucosyl chain. The increase in number of
.alpha.-1,4-glucosyl residues improved the taste quality but at the
same time reduced the sweetness level (Tanaka, 1987).
[0017] It is noted also that many 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.
[0018] Therefore it is necessary to develop high purity products
with optimal .alpha.-1,4-glucosyl chain length which will deliver
the best combination of sweetness potency and flavor profile.
SUMMARY OF THE INVENTION
[0019] 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.
[0020] 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.
[0021] 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 step of
shortening glucosyl chains by .beta.-amylase. 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
macroporuos polymeric resin.
[0022] 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.
[0023] The starting material was subjected to the enzymatic
transglucosylation by action of cyclodextrin glycosyltransferase
(CGTase) in the presence of starch as a glucose donor. As a result
.alpha.-1,4-glucosyl derivatives were formed with degree of
polymerization up to 10. Then the formed derivatives were subjected
to treatment with .beta.-amylase to produce .alpha.-1,4-glucosyl
derivatives possessing a specific degree of polymerization.
[0024] The oligosaccharides from obtained reaction mixture were
removed by Amberlite XAD7 HP resin, and then decolorized,
deionized, concentrated and spray dried.
[0025] 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.
[0026] 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
[0027] 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.
[0028] FIG. 1 shows a high-performance liquid chromatographic
(HPLC) chart of commercial .beta.-amylase treated product
containing only mono- and di-.alpha.-1,4-glucosyl-derivatives of
steviol glycosides;
[0029] FIG. 2 shows a high-performance liquid chromatographic
chromatogram of purified transglucosylated Stevia extract without
.beta.-amylase treatment containing long-chain
.alpha.-1,4-glucosyl-derivatives with up to nine
.alpha.-1,4-glucosyl residues;
[0030] FIG. 3 shows a high-performance liquid chromatographic
chromatogram of purified transglucosylated Stevia extract after
.beta.-amylase treatment with short-chain (containing four or less
.alpha.-1,4-glucosyl residues) derivatives of stevioside and
Rebaudioside A.
DETAILED DESCRIPTION OF THE INVENTION
[0031] 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.
[0032] 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.
[0033] The HPLC analysis of the raw materials and products was
performed on Agilent Technologies 1200 Series (USA) liquid
chromarograph, equipped with Zorbax-NH.sub.2 (4.6.times.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.
[0034] The transglucosylation was accomplished by cyclomaltodextrin
glucanotransferases (CGTases; EC 2.4.1.19) produced by Bacillus
stearothermophilus St-88 (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.
[0035] 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.
[0036] Starches of different origin may be used as donors of
glucosyl units such as, derived from wheat, corn, potato, tapioca,
and sago.
[0037] 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 .alpha.-amylases,
.beta.-amylases etc. In one embodiment, CGTase and .alpha.-amylase
mixtures as liquefying enzymes are preferred.
[0038] .alpha.-Amylase activity is expressed in Kilo Novo
.alpha.-amylase Units (KNU). One KNU is the amount of
.alpha.-amylase which, under standard conditions (pH 7.1;
37.degree. C.), dextrinizes 5.26 g starch dry substance per
hour.
[0039] The liquefaction mixture contains about 0.001-0.2 KNU,
preferably about 0.05-0.1 KNU of .alpha.-amylase per one unit of
CGTase.
[0040] The use of .alpha.-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 m.sup.2 of filter surface. In case
of liquefaction enzyme mixture (comprising .alpha.-amylase and
CGTase) the filtration rate is twice as fast--approximately 20-30
L/hr per 1 m.sup.2 of filter surface.
[0041] 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.
[0042] The concentration of starch in liquefaction mixture is about
15-40% (wt/wt), preferably about 20-30%.
[0043] The liquefaction is conducted at about 70-90.degree. C.
during about 0.5-5 hours, preferably about 1-2 hours.
[0044] After liquefaction, the reaction mixture is subjected to
thermal inactivation of .alpha.-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.degree. C. The duration of
thermal inactivation is about 5-10 minutes.
[0045] 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.
[0046] A second portion of CGTase preparation is added and the
transglucosylation reaction is conducted at about 65.degree. 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.
[0047] Upon completion of transglucosylation reaction, about 30-50
units per gram of solids of .beta.-amylase was added and the
reaction was continued for about 12-16 hours at about 35-55.degree.
C., preferably about 45.degree. C. Soybean .beta.-amylase was used
in this stage. However .beta.-amylases derived from any other
source including barley, bacterial, fungal .beta.-amylases and
others may be used as well.
[0048] .beta.-Amylase activity unit (1 AUN) is defined as the
activity which liberates 100 .mu.g of reducing sugar (expressed by
dextrose equivalent) per minute under the following conditions: 1
mL of enzyme solution is mixed with 5 mL of 1.2% starch solution
(pH 5.5, M/20 Acetate Buffer) and kept for 20 min at 40.degree.
C.
[0049] The reaction was stopped by heating at about 95.degree. 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.
[0050] The decolorized reaction mixture was desalted by passing
through ion exchange resins, such as Amberlite FPC23 (H.sup.+ type)
and Amberlite FPA51 (OH.sup.- type). Other appropriate decolorizing
and desalting methods, such as membrane filtration, or other
methods known in the art can be used.
[0051] 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 contains non-modified glycosides,
short-chain (containing four or less .alpha.-1,4-glucosyl residues)
derivatives and a mixture of maltooligosaccharides (Sample 1).
[0052] In order to prepare a product with higher content of total
sweet glycosides (the sum of glycosylated and non-glycosylated
glycosides), the maltooligosaccharides were removed using Amberlite
XAD7 HP prior to the desalting treatment. 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
subsequently decolorized and desalted as described above and the
glycosides solution, after the evaporation of eluting solvent, was
powdered by spray drying. The resulting product contains
non-modified glycosides, and short-chain (containing four or less
.alpha.-1,4-glucosyl residues) derivatives (Sample 2).
[0053] The embodiments of the invention exemplified by Samples 1
and 2 are free or substantially free of higher glucosylated
derivatives having more than 4 glucosyl residues. In accordance
with this invention, the highly purified glucosyl stevia
composition preferably comprises greater than about 25% by weight
di-, tri- and tetraglucosyl
[0054] Rebaudioside A, and greater than about 9% by weight tri- and
tetraglucosyl steviosides.
[0055] Using a similar process as for sample 2, with exclusion of
the .beta.-amylase treatment stage, a product containing
non-modified glycosides and long chain
.alpha.-1,4-glucosyl-derivatives (with up to nine
.alpha.-1,4-glucosyl residues) was prepared (Sample 3).
[0056] As a control, a commercial .beta.-amylase treated product
containing non-modified glycosides, and short-chain (containing two
or less .alpha.-1,4-glucosyl residues) derivatives was used (Sample
4). The composition of the samples is summarized in Table 1.
TABLE-US-00001 TABLE 1 Composition of glucosyl steviol glycosides
samples Content, % Sample Sample Sample Sample Compounds 1 2 3 4
Stevioside 2.5 3.0 3.1 9.5 Rebaudioside C 0.9 1.0 1.0 0.4
Rebaudioside A 5.2 6.1 6.0 2.8 Monoglucosyl-stevioside (StevG1)
11.0 13.2 7.4 34.9 Monoglucosyl-Rebaudioside A 14.6 17.5 11.1 6.3
(RebAG1) Diglucosyl-stevioside (StevG2) 10.4 12.4 8.4 26.4
Diglucosyl-Rebaudioside A 15.6 18.6 9.6 -- (RebAG2)
Triglucosyl-stevioside (StevG3) 5.8 7.0 6.3 --
Triglucosyl-Rebaudioside A 7.9 9.5 7.7 -- (RebAG3)
Tetraglucosyl-stevioside (StevG4) 3.7 4.4 5.6 --
Tetraglucosyl-Rebaudioside A 2.9 3.4 6.1 -- (RebAG4) Higher
glucosylated derivatives -- -- 22.7 -- Total content of glycosides
80.5 96.1 95.0 80.3
[0057] 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-US-00002 TABLE 2 Sensory assessment of samples in water
system Judgment Sample 1 Sample 2 Sample 3 Sample 4 Most desirable
5 11 1 2 Most undesirable 1 0 7 12 Sweetness power 150 160 120 150
Comments Sweet, light, Sweet, light, Sweet, Sweet, soft, round,
soft, round, slightly slightly pleasant, pleasant, bitter, bitter,
almost similar to astringent, astringent, similar to sucrose, no no
lingering slight sucrose, no lingering aftertaste, lingering
lingering aftertaste, sweetness aftertaste, aftertaste, sweetness
onset sweetness sweetness onset is is moderate onset onset rapid is
slow is rapid
[0058] As apparent from the results in Table 2, the sweetness
quality of the Samples 1 and 2 was rated as most superior. Overall
the samples with short-chain (containing four or less
.alpha.-1,4-glucosyl residues) derivatives (No 1, and No. 2)
possessed better taste profiles compared to samples with long-chain
glucosyl derivatives (No. 3) and two or less .alpha.-1,4-glucosyl
residues short-chain derivatives (No. 4).
[0059] Samples 1 and 2 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.
[0060] The compositions can be used as sweetness enhancers, flavor
enhancers and sweeteners 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.
[0061] Additionally the 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.
[0062] The compositions can be used "as-is" or in combination with
other sweeteners, flavors and food ingredients.
[0063] 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.
[0064] Non-limiting examples of flavors include lemon, orange,
fruity, banana, grape, pear, pineapple, bitter almond, cola,
cinnamon, sugar, cotton candy, vanilla flavors.
[0065] 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.
[0066] 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
[0067] 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.sub.4).sub.2SO.sub.4, and 0.2%
CaCO.sub.3 (pH 7.0-7.5) at 56.degree. 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 .mu.m 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 Short-Chain Glucosyl Stevia Composition
[0068] 100 g of tapioca starch was suspended in 300 mL of water (pH
6.5). 2 KNU of .alpha.-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.degree. 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.degree. C. during 5 minutes
to inactivate the enzymes. After cooling to 65.degree. 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.degree. C. for 24 hours under continuous
agitation. Then the temperature was reduced to 45.degree. C., and
8,000 units soybean .beta.-amylase (#1500S, Nagase Chemtex Corp.,
Japan) was added to reaction mixture. The reaction was continued
for another 12 hours. The obtained reaction mixture was heated at
95.degree. C. for 15 minutes to inactivate the enzymes. 20 grams of
activated carbon was added and the mixture was heated to 75.degree.
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.sup.+) and Amberlite
FPA51 (OH.sup.-) ion exchange resins. The desalted solution was
concentrated at 60.degree. C. under vacuum, and dried into a powder
form using laboratory spray dryer. 196 grams of product was
obtained (Sample 1).
EXAMPLE 3
Preparation of Highly Purified Short-Chain Glucosyl Stevia
Composition
[0069] 100 g of tapioca starch was suspended in 300 mL of water (pH
6.5). 2 KNU of .alpha.-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.degree. 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.degree. C. during 5 minutes
to inactivate the enzymes. After cooling to 65.degree. 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.degree. C. for 24 hours under continuous
agitation. Then the temperature was reduced to 45.degree. C., and
8,000 units soybean .beta.-amylase (#1500S, Nagase Chemtex Corp.,
Japan) was added to reaction mixture. The reaction was continued
for another 12 hours. The obtained reaction mixture was heated at
95.degree. C. for 15 minutes to inactivate the enzymes. 20 grams of
activated carbon was added and the mixture was heated to 75.degree.
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.sup.+) and Amberlite
FPA51 (OH.sup.-) ion exchange resins. The ethanol was evaporated
and the desalted and decolorized water solution was concentrated at
60.degree. C. under vacuum, then dried into a powder form using
laboratory spray dryer. 151 grams of product was obtained (Sample
2).
EXAMPLE 4
Preparation of Highly Purified Long-Chain Glucosyl Stevia
Composition
[0070] 100 g of tapioca starch was suspended in 300 mL of water (pH
6.5). 2 KNU of .alpha.-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.degree. 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.degree. C. during 5 minutes
to inactivate the enzymes. After cooling to 65.degree. 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.degree. C. for 24 hours under continuous
agitation. The obtained reaction mixture was heated at 95.degree.
C. for 15 minutes to inactivate the enzyme. 20 grams of activated
carbon was added and the mixture was heated to 75.degree. 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. Obtained eluate was passed through columns packed
with Amberlite FPC23 (H.sup.+) and Amberlite FPA51 (OH.sup.-) ion
exchange resins. The ethanol was evaporated and the desalted and
decolorized water solution was concentrated at 60.degree. C. under
vacuum, then dried into a powder form using laboratory spray dryer.
166 grams of product was obtained (Sample 3).
EXAMPLE 5
Low-Calorie Orange Juice Drink
[0071] 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%) 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 4,
respectively; and Sample 4 was a commercial .beta.-amylase treated
product (containing only mono- and
di-.alpha.-1,4-glucosyl-derivatives of steviol glycosides).
[0072] 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 short-chain glucosyl stevia compositions
(containing four or less .alpha.-1,4-glucosyl residues) derivatives
(Samples 1 and 2). Particularly the drinks prepared with Samples 1
and 2 exhibited a rounded and complete flavor profile and
mouthfeel.
TABLE-US-00003 TABLE 3 Evaluation of orange juice drink samples
Comments Sample Flavor Aftertaste Mouthfeel No. 1 High quality
sweetness, Clean, almost no Full pleasant taste similar to
bitterness, no sucrose, rounded and aftertaste balanced flavor No.
2 High quality sweetness, Clean, no bitterness Full pleasant taste
similar to and no aftertaste sucrose, rounded and balanced flavor
No. 3 High quality sweetness, Clean, almost no Almost pleasant
taste almost bitterness, no acceptable similar to aftertaste
sucrose, rounded and balanced flavor No. 4 Sweet, licorice notes
Slight bitterness and Not acceptable aftertaste
[0073] 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 6
Low-Calorie Carbonated Beverage
[0074] A carbonated beverage according to formula presented below
was prepared.
TABLE-US-00004 Ingredients Quantity, % Sucrose 5.5 Cola flavor
0.340 ortho-Phosphoric acid 0.100 Sodium citrate 0.310 Sodium
benzoate 0.018 Citric acid 0.018 Rebaudioside A 0.003 Glucosyl
stevia composition 0.05 Carbonated water to 100
[0075] The sensory properties were evaluated by 20 panelists. The
results are summarized in Table 4.
TABLE-US-00005 TABLE 4 Evaluation of low-calorie carbonated
beverage samples Number of panelists detected the attribute Taste
Sample Sample Sample Sample attribute No. 1 No. 2 No. 3 No. 4
Bitter taste 0 0 2 20 Astringent 1 0 3 20 taste Aftertaste 1 0 2 20
Comments Quality of Clean Clean Clean Bitter aftertaste sweet taste
(19 of 20) (20 of 20) (17 of 20) (5 of 20) Overall Satisfactory
Satisfactory Satisfactory Satisfactory evaluation (18 of 20) (20 of
20) (15 of 20) (3 of 20)
[0076] The above results show that the beverages prepared using
Samples 1 and 2 possessed the best organoleptic
characteristics.
EXAMPLE 7
Diet Cookies
[0077] 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%)
were kneaded well in dough-mixing machine. The obtained dough was
molded and baked in oven at 200.degree. C. for 15 minutes. Glucosyl
stevia compositions were by represented by Samples 1, 2, and 3,
obtained according to EXAMPLES 2, 3, and 4, respectively; with
Sample 4 being a commercial .beta.-amylase treated product
(containing only mono- and di-.alpha.-1,4-glucosyl-derivatives of
steviol glycosides).
[0078] The sensory properties were evaluated by 20 panelists. The
best results were obtained in samples prepared by high purity
short-chain glucosyl stevia compositions (containing four or less
.alpha.-1,4-glucosyl residues) derivatives (Samples 1 and 2). The
panelists noted rounded and complete flavor profile and mouthfeel
in cookies prepared with Samples 1 and 2.
EXAMPLE 8
Yoghurt
[0079] Different glucosyl stevia compositions (0.03%) and sucrose
(4%) were dissolved in low fat milk. Glucosyl stevia compositions
were by represented by Samples 1, 2, and 3, obtained according to
EXAMPLES 2, 3, and 4, respectively; with Sample 4 being a
commercial .beta.-amylase treated product (containing only mono-
and di-.alpha.-1,4-glucosyl-derivatives of steviol glycosides).
After pasteurizing at 82.degree. C. for 20 minutes, the milk was
cooled to 37.degree. C. A starter culture (3%) was added and the
mixture was incubated at 37.degree. C. for 6 hours then at
5.degree. C. for 12 hours.
[0080] The sensory properties were evaluated by 20 panelists. The
best results were obtained in samples prepared by high purity
short-chain glucosyl stevia compositions (containing four or less
.alpha.-1,4-glucosyl residues) derivatives (Samples 1 and 2). The
panelists noted rounded and complete flavor profile and mouthfeel
in samples prepared with Samples 1 and 2.
[0081] 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.
* * * * *