CA1332123C - Enzyme sweetened cereal product - Google Patents
Enzyme sweetened cereal productInfo
- Publication number
- CA1332123C CA1332123C CA000578550A CA578550A CA1332123C CA 1332123 C CA1332123 C CA 1332123C CA 000578550 A CA000578550 A CA 000578550A CA 578550 A CA578550 A CA 578550A CA 1332123 C CA1332123 C CA 1332123C
- Authority
- CA
- Canada
- Prior art keywords
- cereal
- fructose
- enzymes
- flour
- cellulose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
- A23L7/107—Addition or treatment with enzymes not combined with fermentation with microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01001—Alpha-amylase (3.2.1.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01003—Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y503/00—Intramolecular oxidoreductases (5.3)
- C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
- C12Y503/01005—Xylose isomerase (5.3.1.5)
-
- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K1/00—Glucose; Glucose-containing syrups
- C13K1/06—Glucose; Glucose-containing syrups obtained by saccharification of starch or raw materials containing starch
-
- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K11/00—Fructose
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
- Y10S435/945—Trichoderma
Abstract
ENZYME SWEETENED CEREAL PRODUCT
Abstract of the Disclosure A process for producing fructose sweetened cereal products by enzymatically converting a portion of the cellulose fraction in a cereal comprising cereal fiber to fructose using cellulase and glucose isomerase is claimed. The process may be carried out at moisture contents exceeding 25% (w/w) and moisture contents between about 40% to 80% are preferred.
The sweetened cereal product produced by the process is also claimed.
Abstract of the Disclosure A process for producing fructose sweetened cereal products by enzymatically converting a portion of the cellulose fraction in a cereal comprising cereal fiber to fructose using cellulase and glucose isomerase is claimed. The process may be carried out at moisture contents exceeding 25% (w/w) and moisture contents between about 40% to 80% are preferred.
The sweetened cereal product produced by the process is also claimed.
Description
Z
ENZYME SWEETENED CEREAL PRODUCT
Field of the Invention The present invention relates to fructose sweetened cereal products and a process for producing such products by enzymatically converting a portion of the cellulose fraction in a cereal flour or meal to fructose using cellulase and glucose isomerase.
Backqround of the Invention Cereal grains are a dietary staple in almost every human community and are a source of protein and complex carbohydrates. Cereals contain a high content of starch and cellulosic fiber, and significant amounts of protein.
Throughout their long use as a food staple, cereal grains have been subjected to numerous enzymatic modifications to improve their functionality, primarily by converting starch contained in the cereal product to more easily assimilable or palatable forms. Conversion of fiber in cereals to different forms has received less, but significant, attention in the past because the major ; ; ~0 portion of fiber can be removed from grains in the grain .
milling process.
In those processes known in the art in which the cellulose content of the cereal grain is treated, the modifications are primarily directed to improving the accessibility of the starch fraction of the grain for further processing or for recovery of food value from a removed fiber fraction.
~ ,~
In Japanese Publication 53-62848, rice is subjected to digestion of cellulose or hemi-cellulose by a solution of enzymes, such as hemi-cellulase, pectinase and cellulase, and solid material is separated from the enzyme solution. The solid material is boiled with water to yield a liquid food~
Japanese Publication 57-47363 teaches the production of a cereal tea by treating cereal grains with heat between 100-200~C to puff the cereal, followed by treatment with an enzyme such as cellulase. The enzymatically treated cereal is heat dried and then roasted.
U.S. Patent 4,089,745 discloses the treatment of corn hull cellulose with alkali to obtain three fractions and the conversion of the treated cellulose fraction to glucose.
U.S. Patent 4,247,636 discloses treating a mixture of water and flour with ~-glucanase to produce a relatively low viscosity slurry which is treated with amylase, glucoamylase and glucose isomerase to produce a high fructose sweetener.
A process for silage treatment is described lin U.S.
Patent 4,292,331. Silaged vegetable matter is exposed to enzymes and bacteria or fungi that produce hemi-cellulases, amylase and amyloglucosidase. These enzymesmay be suspended on a starch support prior to adding to the vegetable matter.~ Fermentable sugars are produced which are in turn fermented to lactate by lactobacilli.
1 33~ 1 23 In U.S. Patent 4,378,432, vegetable matter is treated with phosphoric acid to hydrolyze cellulose and then enzymes capable of hydrolyzing cellulose and hemi cellulose to produce reducing sugars, whereby a sweetened aqueous solution is obtained.
U.S. Patent 3,395,019 discloses the production of animal feed from oat hulls by treatment of oat hulls, mixed with 50 to 70% water by weight, with alkali, yeast, or enzymes such as catalase, cellulase or amylase. The treatment increases absorbency of the oat hulls for liquid nutrients such as molasses.
U.S. Patents 4,431,674 and 4,435,430 disclose enzyme saccharlfied cereals made from whole grain cereal. In this process, the whole grain is milled and the germ, bran and endcsperm fractions are separated. The bran fraction is treated to increase its functionality and the endosperm fraction is separately treated enzymatically with ~-amylase and glucoamylase. The fractions are then recombined. Bran treatment is disclosed as critical to the process, but simultaneously treating all the cereal fractions enzymatically is taught as decreasing the quality of the dough.
~ . ~
U.S. Patent 2,555,235 teaches the treatment of grain endosperm by steeping vacuum-treated grain in a grain 25 extract at a temperature and pressure to promote ;~
enzymatic action. Among the enzymes present in the grains are cellulase and several amylases and glucosidases.
1 332t 23 :
'' 63227-228 In the present invention, the cellulose component of : ~:
a flour or meal comprising starch and cellulose fiber, is enzymatically altered to saccharify the cellulose and to produce ~ ~;
fructose in the treated flour or meal under conditions in which : ~-substantially all of the fructose so produced and all, or a;~
substantial portion, of the starch are retained.
Brief Description of the Invention In one aspect, the present invention relates to a process for producing a sweetened cereal product comprising~
a) moistening a cereal grain by adding an aqueous so1ution to a cereal grain containing cellulose and starch, in an amount sufficient to support conversion by enzymes of the cellulose and/or starch to fructose and in an amount such that substantially ~`
all of the solution is absorbed by the cereal;
b) disrupting the moistened cereal produced in step a) .;
to an extent effective to promote the enzymatic conversion of the ce}lulose and/or starch to fructose by heating under pressure or '~
~ treating with alkali;
;~ ~ c) adding to the disrupted, moistened cereal produced ~-20~ Ln step b) enzymss effective to convert cellulose and/or starch to fructose to produce a moistened cereal and enzyme mixture; . .
'~, ' i' ~; d) incubating said moistened cereal and enzyme mixture '- ' for a sufficient period of time to convert the cellulose and/or ';.'' starch in said cereal to fructose at a temperature at which the ''i'~;""
enzymes are active; and '`'~' e) collecting a cereal product enriched in fructose. ',~' '"' :.' "` `'"' '"' ~, ,,, ., , , , , ,, ,; , ~ j, i ,:,, ,; ' - 4a In another aspect, the invention relates to a process for producing a fructose-sweetened cereal product comprising the steps of:
providing a cereal comprising cereal fiber, moistening the cereal comprising cereal fiber, contacting said moistened cereal comprising cereal fiber with a series of enzymes comprising cellulase and glucose isomerase, incubating said moistened cereal comprising cereal fiber and enzymes for a sufficient period of ;
.
time to produce fructose and collecting a sweetened cereal product comprising fructose. A cereal flour or meal is preferred as the cereal comprises cereal fiber. ;
In another aspect of the invention, the process is carried out contacting the moistened cereal flour or meal with cellulase, saccharifying enzymes such as ~-amylase and amyloglucosidase and glucose isomerase.
In another aspect, the invention relates to a process for producing a sweetened cereal product in which the collected sweetened cereal product is baked.
.~ .
1 332 1 23 : ~
In yet another aspect, the invention relates to a process for producing a sweetened cereal product in which the cereal flour or meal is moistened with a sucrose solution prior to incubation with the series of enzymes to produce fructose.
In still another aspect, the invention relates to a process for producing a sweetened cereal comprising hydrolyzing a portion of or substantially all of the cellulose in said cereal with cellulase and converting the products of said hydrolysis to fructose with glucose isomerase.
In yet another aspect, the invention relates to a sweetened cereal product containing fructose produced by the method of the invention.
,, , ,. :,~
Detailed Description of the Invention The present invention also concerns a process for making a swieetened cereal product in which a portion of the cellulose fraction, or both the starch and cellulose components of cereal flour or meal are converted into fructose. Prior to milling, cereals have high contents of fiber and starch. Such cereals include a varjiety,of large and small grains including corn, rye, rice, wheat and barley. Other food crops, including soy beans and peanuts, have significant starch and fiber content that may be converted into fructose by the general method according to the invention. Cereals are particularly high in fiber or cellulose as well as containing a significant amount of starch. For example, the pericap, nucellus and tegmen of wheat, all of which together comprise bran, are high in fiber and comprise a significant portion of graham and whole wheat flour.
Bran is separated from the endo-sperm of wheat kernels in the milling process to form bread flour and the bran may be collected and separately milled to produce a bran flour. The fiber components of these flours contain significant amounts of cellulose.
10In t~e process according to the invention a moistened milled cereal comprising cereal fiber, or whole cereal grain, preferably a cereal flour or meal comprising cellulose and starch is optionally disrupted and contacted with a series of enzymes which comprises cellulase and glucose isomerase and optionally will further include saccharifying enzymes; the moistened cereal fIour or meal with the series of enzymes is incubated for a sufficient period of time to produce ~ fructose, and the cereal product containing fructose is -~ 20 collected.
In the process according to the invention, a substantial portion of the cellulose content lof bhe cereal flour or meal may be enzymatically converted to fructose. The conversion of the cellulose portion of the cereal flour or meal into glucose, and the production of fructose therefrom, leaves intact a significant amount of the starch content of the cereal flour or meal which can be used to maintain texture and formability of the cereal product ultimately produced from the treated flour or meal. Alternatively, a portion of the starch may also be saccharified, in addition to producing glucose or other sugars or with the enzymatic action of glucose isomerase, additional fructose, while still maintaining a significant amount of starch and high molecular weight dextrins which may be desirable for texturing and forming operations of the treated cereal.
Fructose is known to have a higher degree of 10 sweetness than glucose or sucrose. Thus, high fiber ,, cereals may be produced having a desired degree of sweetness while retaining a relatively low digestible calorie content since it is not necessary to saccharify starch in one aspect of the process according to the invention. Accordingly, one product of the process of the invention is an enzymatically sweetened-low-calorie-high fiber cereal product. -~
In greater detail, the process according to the invention entails the production in a moistened cereal of fructose by enzymatic treatment. The cereal will generally be milled or subjected to some process whereby the cereal grain is fractured, crushed, or disrupt;ed into small pieces. Such milled cereal grain is preferably in the form of a coarse or fine flour or meal produced from ~ `;
a starch and cellulose containing cereal grain. Such grains include large and small grains including corn, rice, wheat, oats, barley and rye. The various flours and meals that may be produced from these grains are the ~
~, , material used for the process according to the invention.
Flours and meals produced from these grains and which are appropriate for the production of breakfast cereals are generally known. With respect to flours, those produced from wheat are preferred, and in particular, bran flours, graham flour and whole wheat flour are appropriate. Bran flour is especially preferred.
In general, the flour will be supplied in dry form having a water or moisture content of 20% or less, in general about 10%. Prior to or concurrent with enzymatic treatment according to the invention, the moisture content of the flour is increased. In general, the moisture content is increased sufficiently to permit the activity of the series of enzymes mentioned above, all of which in general function in an aqueous or hydrated environment. Moisture content is determined as the weight percent H2O of the total mixture. The moisture content will range from about 25% and up. A moisture ~ .
content of between about 40% and about 80~ is preferred for sustaining the activity of the series of enzymes ~; mentioned above.
The moisture content may be varied depending upon 1' . ' ! I :::
the type of flour used, the texture of the final cereal ~;
product desired, and the speed of the saccharification i~
and fructose formation reactions desired. However, in general, the moisture content will not exceed the amount -~
of water that the flour or meal can absorb. At a ... ` ~-~
g moisture content between about 55 and 60% using a bran flour, a friable sweetened cereal product is obtained.
The moisture content of the flour or meal is ad~usted by the addition to or removal of water from the flour. If the flour or meal is presoaked in an aqueous solution or in water prior to the addition to the enzyme series, it may be desirable to remove water by conventional means such as decanting, blotting, pressing, straining, and heating or a combination of these conventional means. If a dry flour or meal is used, moisture content may be adjusted by the addition of solutions containing water. Such a solution may include aqueous enzymes, including the cellulases and glucose isomerase mentioned above, or optionally, in addition, the amylases and amyloglucosidases.
~; Alternatively, or in addition, the flour may be moistened by aqueous flavoring syrup. Such flavoring syrups may contain sucrose, fruit extracts, and the like.
;~;The addition of such aqueous sucrose solutions, however, is not required in the process according to the ~ ~ .
~ invention, as substantial amounts of glucose and fructose : . ~
are developed from the flour by enzymatic action.
It is preferred in the process according to the invention to conduct the enzymatic treatment of the 25 cereal flour or meal with a moisture content sufficient ;
to sustain the activity of the series of enzymes.
However, in general, it is not desirable to add so much water to the cereal grain or flour that excess water may ~:
.', be recovered from the moistened grain or flour by draining after the enzymatic treatment. Moisture contents so great as to permit drainage of excess water will generally lead to a loss of soluble sugars produced as a result of the enzymatic treatment. Therefore, it is desirable to moisten the cereal grain or flour with no more solution of water than the cereal grain or flour can completely absorb.
In preparing flour or meal for further processing, the flour or meal is disrupted to partially alter the starch and cellulose contained in the flour or meal. In general, the disruption process involves the application of heat, and preferably moist heat. Thus, disruption may be carried out by steaming, steam injection, pressure cooking in a closed vessel or, less preferably, by the application of dry heat. Chemical disruption processes such as treatment with mild alkali are also known.
The addition of the enzyme series to the whole cereal grain, cereal meal or flour can occur at various , points in the process for producing the sweetened cereal product according to the invention. In general, the ~ enzyme may be added before, after, or during disruption :~ of the flour or meal. It is generally preferred to contact the cereal grains, flour or meal with the series of enzymes after disruption. Without being bound by this theory, the disruption of the cereal grains, flour or meal is believed to increase the microaccessability of enzyme to substrate in the disrupted flour and thus ~ ;~` '~
maximizes both the fructose yield and the availability of sugars and smaller saccharides for browning reactions if the sweetened cereal product is eventually cooked.
The series of enzymes may alternately be added prior to disruption. If the series of enzymes is added at this point in the process, the subsequent disruption step can be adjusted to maintain enzyme activity for a portion of the disruption cycle by adjusting the rate at which the temperature of the enzyme-cereal grain, flour or meal mixture is raised. Of course, the disruption step may be used to eventually substantially or completely inactivate the series of enzymes added to the whole cereal grain, flour or meal if the series of enzymes is added prior to the disruption step.
The series of enzymes may alternately be added during the disruption step, the amount of each enzyme added and the rate of heating may be adjusted to obtain the desired level of fructose formation in the final product. In each case, when the series of enzymes is added before or during the heat disruption step, and the heat of the disruption step is used to increase the activity of the series of enzymes, it may be desirable to 1,, , i I , ! .
use thermostable enzymes. Such thermostable enzymes are generally known and may be obtained from microorganisms 25 that are found in high thermal environments. A ;
thermostable glucose isomerase and glucoamylase has been ;~
described, for example, in U.S. Patent Nos. 4,532,208 and 4,53~,477, respectively. Furthermore, the use of thermo-,.
' ~'.
stable enzymes permits the process of the invention to be carried out at temperatures generally higher than 75C
and thus may be useful in shortening the time necessary for producing fructose in the sweetened cereal product.
The order in which the members of the series of enzymes is added to the moistened cereal flour or meal is also significant in the process according to the invention. In general, the enzymes may be added to the disrupted or non-disrupted grains, flour or meal all at lo one time or in an order wherein the glucose isomerase enzyme is added last. It is desirable to achieve a steady state of glucose isomerase activity similar to that achieved by a slow fe~d of glucose for conversion into fructose. The concentration of fructose produced under these conditions i5 not so great as to inhibit the formation of fructose from glucose. In general, when the ;~ flour substrate is in a moisture condition of 75% water or less, it is desirable to add all the enzymes at once.
Under these conditions, glucose produced by cellulase or by the combination of cellulase and saccharifying enzymes su~h as ~-amylase and amyloglucosidase is converted to fructose by glucose isomerase at a rate essentially equivalent to the rate at which glucose is produced. In general, it is desirable to add an excess of glucose ~ 25 isomerase.
;~ Glucose isomerase activity is expressed as IGIU
units. IGIU is the abbreviation for International Glucose Isomerase Unit and is that amount of enzyme '`',' "-"'~' 13 l 33~ 1 23 which will convert 1 micromole of glucose to fructose per minute in a solution initially containing 2 moles of glucose per liter, 0.02 moles of MgSO4 and 0.001 mole of CoC12 per liter at a pH of 6.84 to 6.85 (0.2 M sodium maleate) and at a temperature of 60C. See, N. E. Lloyd, et al., Cereal Chem., 49, No. 5, pp. 544-553 (1972).
The invention will be better understood in connection with the follcwing examples, which are intended by the inventors to be merely exemplary and non-lO limiting: ~ `
..
'',;-, ;';
~,, ::
White wheat bran was soaked in distilled deionized water for approximately 60 hours at 5OC, was squeeze dried through cheese cloth, and ground to a fine mush.
0~25 grams of the wetted bran was placed in a 50 ml closed vial and enzyme was added as shown below. Total volume was 10 ml and the final pH was 5 or 6 adjusted with 1 NHCl. The reaction was run in duplicate at 40C
with shaking for 3.0 hours. At the end of the reaction, the sample was analyzed for carbohydrate content using a Boehringer-Mannheim food analysis kit (catalogue No.
139106). The enzyme preparations were:
A cellulase enzyme preparation (referred to as CEL
in some of the following tables) from Trichode~ma reseii in 50 mM sodium acetate, 3 mM sodium azide buffer at pH
5.5 with a total specific activity of approximately 104 un1ts/ml was used. Shoemaker, S. et al, "Characterization and Properties of Cellulases Purified From Trichoderma reseii Strain L27," Bio/Technoloqy (Oct.
. " ., 20~ 1983). This enzyme preparation contains the following `--;
types and amounts of enzymes: cellobiohydrolase , tCBHI), 60%; CBHII, 10~; endoglucanse I (EGI) 3.6%; EGII, 3.0%; b~ta-glucosidase I (BGI), 0~05%; and BGII, 2.0%.
Enzyme activity was determined by incubating the enzyme preparation with the following substrates: 1% Avicel, ;phosphoric acid swollen cellulose (PSC), xylan ~Sigma) ~-and mannan in 50 mM sodium acetate buffer, pH 4.5 at 45C
for 30 minutes. Soluble reducing sugar was measured as i~ ``
. ~ .
~`~
- 1332123 ~
sugar equivalents. Units of activity were expressed as umole glucose (or xylose) equivalents per minute.
Activity on carboxymethylcellulose (CMC, Hercules) was determined by incubating enzymes with 0.27% CMC in 50 mM
acetate buffer, pH 4.5 for 30 minutes and measuring the decrease in viscosity versus time with a rotating viscometer (Brookfield). Units of CMC activity were arbitrarily expressed as change in fluidity per minute.
Cellobiose and paranitrophenyl-D-glucoside (PNPG) activities were determined according to Emert, Doctoral Thesis, Va. Polytech. Inst. and State U., Blacksburg, Va.
(1973), with units of activity expressed as umole cellobiose consumed per minute and umole PNP released per minute, respectively. The cellulase enzyme had the following total activity distribution (U/mg): Avicel (crystallized cellulose) 0.3; phosphoric acid swollen cellulose (PSC) 3.6; carboxymethyl cellulose 3.6;
~; cellobiose 0.3; paranitrophenyl-D-glucoside (PNPG) 0.3;
Xylan 1.2; Mannan 0.3.
Glucose isomerase (Spezyme) preparation in 50 mM
sodium phosphate, 10 mM magnesium sulfate, lmM CoCl2, pH
6.0 having an activity of 110 IGIU/ml was used. Results are shown in Table I~
' ~
I~BLE I -~
% l~
Avq. Yield (cm~L) Weight Reaction ~(q) CEL GI Buffer D-Glucose D-Fructose uctose 1 0.25 -- -- 10 ml O O
ENZYME SWEETENED CEREAL PRODUCT
Field of the Invention The present invention relates to fructose sweetened cereal products and a process for producing such products by enzymatically converting a portion of the cellulose fraction in a cereal flour or meal to fructose using cellulase and glucose isomerase.
Backqround of the Invention Cereal grains are a dietary staple in almost every human community and are a source of protein and complex carbohydrates. Cereals contain a high content of starch and cellulosic fiber, and significant amounts of protein.
Throughout their long use as a food staple, cereal grains have been subjected to numerous enzymatic modifications to improve their functionality, primarily by converting starch contained in the cereal product to more easily assimilable or palatable forms. Conversion of fiber in cereals to different forms has received less, but significant, attention in the past because the major ; ; ~0 portion of fiber can be removed from grains in the grain .
milling process.
In those processes known in the art in which the cellulose content of the cereal grain is treated, the modifications are primarily directed to improving the accessibility of the starch fraction of the grain for further processing or for recovery of food value from a removed fiber fraction.
~ ,~
In Japanese Publication 53-62848, rice is subjected to digestion of cellulose or hemi-cellulose by a solution of enzymes, such as hemi-cellulase, pectinase and cellulase, and solid material is separated from the enzyme solution. The solid material is boiled with water to yield a liquid food~
Japanese Publication 57-47363 teaches the production of a cereal tea by treating cereal grains with heat between 100-200~C to puff the cereal, followed by treatment with an enzyme such as cellulase. The enzymatically treated cereal is heat dried and then roasted.
U.S. Patent 4,089,745 discloses the treatment of corn hull cellulose with alkali to obtain three fractions and the conversion of the treated cellulose fraction to glucose.
U.S. Patent 4,247,636 discloses treating a mixture of water and flour with ~-glucanase to produce a relatively low viscosity slurry which is treated with amylase, glucoamylase and glucose isomerase to produce a high fructose sweetener.
A process for silage treatment is described lin U.S.
Patent 4,292,331. Silaged vegetable matter is exposed to enzymes and bacteria or fungi that produce hemi-cellulases, amylase and amyloglucosidase. These enzymesmay be suspended on a starch support prior to adding to the vegetable matter.~ Fermentable sugars are produced which are in turn fermented to lactate by lactobacilli.
1 33~ 1 23 In U.S. Patent 4,378,432, vegetable matter is treated with phosphoric acid to hydrolyze cellulose and then enzymes capable of hydrolyzing cellulose and hemi cellulose to produce reducing sugars, whereby a sweetened aqueous solution is obtained.
U.S. Patent 3,395,019 discloses the production of animal feed from oat hulls by treatment of oat hulls, mixed with 50 to 70% water by weight, with alkali, yeast, or enzymes such as catalase, cellulase or amylase. The treatment increases absorbency of the oat hulls for liquid nutrients such as molasses.
U.S. Patents 4,431,674 and 4,435,430 disclose enzyme saccharlfied cereals made from whole grain cereal. In this process, the whole grain is milled and the germ, bran and endcsperm fractions are separated. The bran fraction is treated to increase its functionality and the endosperm fraction is separately treated enzymatically with ~-amylase and glucoamylase. The fractions are then recombined. Bran treatment is disclosed as critical to the process, but simultaneously treating all the cereal fractions enzymatically is taught as decreasing the quality of the dough.
~ . ~
U.S. Patent 2,555,235 teaches the treatment of grain endosperm by steeping vacuum-treated grain in a grain 25 extract at a temperature and pressure to promote ;~
enzymatic action. Among the enzymes present in the grains are cellulase and several amylases and glucosidases.
1 332t 23 :
'' 63227-228 In the present invention, the cellulose component of : ~:
a flour or meal comprising starch and cellulose fiber, is enzymatically altered to saccharify the cellulose and to produce ~ ~;
fructose in the treated flour or meal under conditions in which : ~-substantially all of the fructose so produced and all, or a;~
substantial portion, of the starch are retained.
Brief Description of the Invention In one aspect, the present invention relates to a process for producing a sweetened cereal product comprising~
a) moistening a cereal grain by adding an aqueous so1ution to a cereal grain containing cellulose and starch, in an amount sufficient to support conversion by enzymes of the cellulose and/or starch to fructose and in an amount such that substantially ~`
all of the solution is absorbed by the cereal;
b) disrupting the moistened cereal produced in step a) .;
to an extent effective to promote the enzymatic conversion of the ce}lulose and/or starch to fructose by heating under pressure or '~
~ treating with alkali;
;~ ~ c) adding to the disrupted, moistened cereal produced ~-20~ Ln step b) enzymss effective to convert cellulose and/or starch to fructose to produce a moistened cereal and enzyme mixture; . .
'~, ' i' ~; d) incubating said moistened cereal and enzyme mixture '- ' for a sufficient period of time to convert the cellulose and/or ';.'' starch in said cereal to fructose at a temperature at which the ''i'~;""
enzymes are active; and '`'~' e) collecting a cereal product enriched in fructose. ',~' '"' :.' "` `'"' '"' ~, ,,, ., , , , , ,, ,; , ~ j, i ,:,, ,; ' - 4a In another aspect, the invention relates to a process for producing a fructose-sweetened cereal product comprising the steps of:
providing a cereal comprising cereal fiber, moistening the cereal comprising cereal fiber, contacting said moistened cereal comprising cereal fiber with a series of enzymes comprising cellulase and glucose isomerase, incubating said moistened cereal comprising cereal fiber and enzymes for a sufficient period of ;
.
time to produce fructose and collecting a sweetened cereal product comprising fructose. A cereal flour or meal is preferred as the cereal comprises cereal fiber. ;
In another aspect of the invention, the process is carried out contacting the moistened cereal flour or meal with cellulase, saccharifying enzymes such as ~-amylase and amyloglucosidase and glucose isomerase.
In another aspect, the invention relates to a process for producing a sweetened cereal product in which the collected sweetened cereal product is baked.
.~ .
1 332 1 23 : ~
In yet another aspect, the invention relates to a process for producing a sweetened cereal product in which the cereal flour or meal is moistened with a sucrose solution prior to incubation with the series of enzymes to produce fructose.
In still another aspect, the invention relates to a process for producing a sweetened cereal comprising hydrolyzing a portion of or substantially all of the cellulose in said cereal with cellulase and converting the products of said hydrolysis to fructose with glucose isomerase.
In yet another aspect, the invention relates to a sweetened cereal product containing fructose produced by the method of the invention.
,, , ,. :,~
Detailed Description of the Invention The present invention also concerns a process for making a swieetened cereal product in which a portion of the cellulose fraction, or both the starch and cellulose components of cereal flour or meal are converted into fructose. Prior to milling, cereals have high contents of fiber and starch. Such cereals include a varjiety,of large and small grains including corn, rye, rice, wheat and barley. Other food crops, including soy beans and peanuts, have significant starch and fiber content that may be converted into fructose by the general method according to the invention. Cereals are particularly high in fiber or cellulose as well as containing a significant amount of starch. For example, the pericap, nucellus and tegmen of wheat, all of which together comprise bran, are high in fiber and comprise a significant portion of graham and whole wheat flour.
Bran is separated from the endo-sperm of wheat kernels in the milling process to form bread flour and the bran may be collected and separately milled to produce a bran flour. The fiber components of these flours contain significant amounts of cellulose.
10In t~e process according to the invention a moistened milled cereal comprising cereal fiber, or whole cereal grain, preferably a cereal flour or meal comprising cellulose and starch is optionally disrupted and contacted with a series of enzymes which comprises cellulase and glucose isomerase and optionally will further include saccharifying enzymes; the moistened cereal fIour or meal with the series of enzymes is incubated for a sufficient period of time to produce ~ fructose, and the cereal product containing fructose is -~ 20 collected.
In the process according to the invention, a substantial portion of the cellulose content lof bhe cereal flour or meal may be enzymatically converted to fructose. The conversion of the cellulose portion of the cereal flour or meal into glucose, and the production of fructose therefrom, leaves intact a significant amount of the starch content of the cereal flour or meal which can be used to maintain texture and formability of the cereal product ultimately produced from the treated flour or meal. Alternatively, a portion of the starch may also be saccharified, in addition to producing glucose or other sugars or with the enzymatic action of glucose isomerase, additional fructose, while still maintaining a significant amount of starch and high molecular weight dextrins which may be desirable for texturing and forming operations of the treated cereal.
Fructose is known to have a higher degree of 10 sweetness than glucose or sucrose. Thus, high fiber ,, cereals may be produced having a desired degree of sweetness while retaining a relatively low digestible calorie content since it is not necessary to saccharify starch in one aspect of the process according to the invention. Accordingly, one product of the process of the invention is an enzymatically sweetened-low-calorie-high fiber cereal product. -~
In greater detail, the process according to the invention entails the production in a moistened cereal of fructose by enzymatic treatment. The cereal will generally be milled or subjected to some process whereby the cereal grain is fractured, crushed, or disrupt;ed into small pieces. Such milled cereal grain is preferably in the form of a coarse or fine flour or meal produced from ~ `;
a starch and cellulose containing cereal grain. Such grains include large and small grains including corn, rice, wheat, oats, barley and rye. The various flours and meals that may be produced from these grains are the ~
~, , material used for the process according to the invention.
Flours and meals produced from these grains and which are appropriate for the production of breakfast cereals are generally known. With respect to flours, those produced from wheat are preferred, and in particular, bran flours, graham flour and whole wheat flour are appropriate. Bran flour is especially preferred.
In general, the flour will be supplied in dry form having a water or moisture content of 20% or less, in general about 10%. Prior to or concurrent with enzymatic treatment according to the invention, the moisture content of the flour is increased. In general, the moisture content is increased sufficiently to permit the activity of the series of enzymes mentioned above, all of which in general function in an aqueous or hydrated environment. Moisture content is determined as the weight percent H2O of the total mixture. The moisture content will range from about 25% and up. A moisture ~ .
content of between about 40% and about 80~ is preferred for sustaining the activity of the series of enzymes ~; mentioned above.
The moisture content may be varied depending upon 1' . ' ! I :::
the type of flour used, the texture of the final cereal ~;
product desired, and the speed of the saccharification i~
and fructose formation reactions desired. However, in general, the moisture content will not exceed the amount -~
of water that the flour or meal can absorb. At a ... ` ~-~
g moisture content between about 55 and 60% using a bran flour, a friable sweetened cereal product is obtained.
The moisture content of the flour or meal is ad~usted by the addition to or removal of water from the flour. If the flour or meal is presoaked in an aqueous solution or in water prior to the addition to the enzyme series, it may be desirable to remove water by conventional means such as decanting, blotting, pressing, straining, and heating or a combination of these conventional means. If a dry flour or meal is used, moisture content may be adjusted by the addition of solutions containing water. Such a solution may include aqueous enzymes, including the cellulases and glucose isomerase mentioned above, or optionally, in addition, the amylases and amyloglucosidases.
~; Alternatively, or in addition, the flour may be moistened by aqueous flavoring syrup. Such flavoring syrups may contain sucrose, fruit extracts, and the like.
;~;The addition of such aqueous sucrose solutions, however, is not required in the process according to the ~ ~ .
~ invention, as substantial amounts of glucose and fructose : . ~
are developed from the flour by enzymatic action.
It is preferred in the process according to the invention to conduct the enzymatic treatment of the 25 cereal flour or meal with a moisture content sufficient ;
to sustain the activity of the series of enzymes.
However, in general, it is not desirable to add so much water to the cereal grain or flour that excess water may ~:
.', be recovered from the moistened grain or flour by draining after the enzymatic treatment. Moisture contents so great as to permit drainage of excess water will generally lead to a loss of soluble sugars produced as a result of the enzymatic treatment. Therefore, it is desirable to moisten the cereal grain or flour with no more solution of water than the cereal grain or flour can completely absorb.
In preparing flour or meal for further processing, the flour or meal is disrupted to partially alter the starch and cellulose contained in the flour or meal. In general, the disruption process involves the application of heat, and preferably moist heat. Thus, disruption may be carried out by steaming, steam injection, pressure cooking in a closed vessel or, less preferably, by the application of dry heat. Chemical disruption processes such as treatment with mild alkali are also known.
The addition of the enzyme series to the whole cereal grain, cereal meal or flour can occur at various , points in the process for producing the sweetened cereal product according to the invention. In general, the ~ enzyme may be added before, after, or during disruption :~ of the flour or meal. It is generally preferred to contact the cereal grains, flour or meal with the series of enzymes after disruption. Without being bound by this theory, the disruption of the cereal grains, flour or meal is believed to increase the microaccessability of enzyme to substrate in the disrupted flour and thus ~ ;~` '~
maximizes both the fructose yield and the availability of sugars and smaller saccharides for browning reactions if the sweetened cereal product is eventually cooked.
The series of enzymes may alternately be added prior to disruption. If the series of enzymes is added at this point in the process, the subsequent disruption step can be adjusted to maintain enzyme activity for a portion of the disruption cycle by adjusting the rate at which the temperature of the enzyme-cereal grain, flour or meal mixture is raised. Of course, the disruption step may be used to eventually substantially or completely inactivate the series of enzymes added to the whole cereal grain, flour or meal if the series of enzymes is added prior to the disruption step.
The series of enzymes may alternately be added during the disruption step, the amount of each enzyme added and the rate of heating may be adjusted to obtain the desired level of fructose formation in the final product. In each case, when the series of enzymes is added before or during the heat disruption step, and the heat of the disruption step is used to increase the activity of the series of enzymes, it may be desirable to 1,, , i I , ! .
use thermostable enzymes. Such thermostable enzymes are generally known and may be obtained from microorganisms 25 that are found in high thermal environments. A ;
thermostable glucose isomerase and glucoamylase has been ;~
described, for example, in U.S. Patent Nos. 4,532,208 and 4,53~,477, respectively. Furthermore, the use of thermo-,.
' ~'.
stable enzymes permits the process of the invention to be carried out at temperatures generally higher than 75C
and thus may be useful in shortening the time necessary for producing fructose in the sweetened cereal product.
The order in which the members of the series of enzymes is added to the moistened cereal flour or meal is also significant in the process according to the invention. In general, the enzymes may be added to the disrupted or non-disrupted grains, flour or meal all at lo one time or in an order wherein the glucose isomerase enzyme is added last. It is desirable to achieve a steady state of glucose isomerase activity similar to that achieved by a slow fe~d of glucose for conversion into fructose. The concentration of fructose produced under these conditions i5 not so great as to inhibit the formation of fructose from glucose. In general, when the ;~ flour substrate is in a moisture condition of 75% water or less, it is desirable to add all the enzymes at once.
Under these conditions, glucose produced by cellulase or by the combination of cellulase and saccharifying enzymes su~h as ~-amylase and amyloglucosidase is converted to fructose by glucose isomerase at a rate essentially equivalent to the rate at which glucose is produced. In general, it is desirable to add an excess of glucose ~ 25 isomerase.
;~ Glucose isomerase activity is expressed as IGIU
units. IGIU is the abbreviation for International Glucose Isomerase Unit and is that amount of enzyme '`',' "-"'~' 13 l 33~ 1 23 which will convert 1 micromole of glucose to fructose per minute in a solution initially containing 2 moles of glucose per liter, 0.02 moles of MgSO4 and 0.001 mole of CoC12 per liter at a pH of 6.84 to 6.85 (0.2 M sodium maleate) and at a temperature of 60C. See, N. E. Lloyd, et al., Cereal Chem., 49, No. 5, pp. 544-553 (1972).
The invention will be better understood in connection with the follcwing examples, which are intended by the inventors to be merely exemplary and non-lO limiting: ~ `
..
'',;-, ;';
~,, ::
White wheat bran was soaked in distilled deionized water for approximately 60 hours at 5OC, was squeeze dried through cheese cloth, and ground to a fine mush.
0~25 grams of the wetted bran was placed in a 50 ml closed vial and enzyme was added as shown below. Total volume was 10 ml and the final pH was 5 or 6 adjusted with 1 NHCl. The reaction was run in duplicate at 40C
with shaking for 3.0 hours. At the end of the reaction, the sample was analyzed for carbohydrate content using a Boehringer-Mannheim food analysis kit (catalogue No.
139106). The enzyme preparations were:
A cellulase enzyme preparation (referred to as CEL
in some of the following tables) from Trichode~ma reseii in 50 mM sodium acetate, 3 mM sodium azide buffer at pH
5.5 with a total specific activity of approximately 104 un1ts/ml was used. Shoemaker, S. et al, "Characterization and Properties of Cellulases Purified From Trichoderma reseii Strain L27," Bio/Technoloqy (Oct.
. " ., 20~ 1983). This enzyme preparation contains the following `--;
types and amounts of enzymes: cellobiohydrolase , tCBHI), 60%; CBHII, 10~; endoglucanse I (EGI) 3.6%; EGII, 3.0%; b~ta-glucosidase I (BGI), 0~05%; and BGII, 2.0%.
Enzyme activity was determined by incubating the enzyme preparation with the following substrates: 1% Avicel, ;phosphoric acid swollen cellulose (PSC), xylan ~Sigma) ~-and mannan in 50 mM sodium acetate buffer, pH 4.5 at 45C
for 30 minutes. Soluble reducing sugar was measured as i~ ``
. ~ .
~`~
- 1332123 ~
sugar equivalents. Units of activity were expressed as umole glucose (or xylose) equivalents per minute.
Activity on carboxymethylcellulose (CMC, Hercules) was determined by incubating enzymes with 0.27% CMC in 50 mM
acetate buffer, pH 4.5 for 30 minutes and measuring the decrease in viscosity versus time with a rotating viscometer (Brookfield). Units of CMC activity were arbitrarily expressed as change in fluidity per minute.
Cellobiose and paranitrophenyl-D-glucoside (PNPG) activities were determined according to Emert, Doctoral Thesis, Va. Polytech. Inst. and State U., Blacksburg, Va.
(1973), with units of activity expressed as umole cellobiose consumed per minute and umole PNP released per minute, respectively. The cellulase enzyme had the following total activity distribution (U/mg): Avicel (crystallized cellulose) 0.3; phosphoric acid swollen cellulose (PSC) 3.6; carboxymethyl cellulose 3.6;
~; cellobiose 0.3; paranitrophenyl-D-glucoside (PNPG) 0.3;
Xylan 1.2; Mannan 0.3.
Glucose isomerase (Spezyme) preparation in 50 mM
sodium phosphate, 10 mM magnesium sulfate, lmM CoCl2, pH
6.0 having an activity of 110 IGIU/ml was used. Results are shown in Table I~
' ~
I~BLE I -~
% l~
Avq. Yield (cm~L) Weight Reaction ~(q) CEL GI Buffer D-Glucose D-Fructose uctose 1 0.25 -- -- 10 ml O O
2 0.25 5 ml -- 5 ml .534 o 3 0.25 5 ml 5 ml ~ .50 0.052 ~9%
An additional 5 ml aliquot of glucose isomerase was ~
added to Reaction vessel 3. Reaction vessels 2 and 3 -; -were incubated at 05C overnight without shaking. In addition, a duplicate set of flasks identical to Reaction 3 was run overnight at 05C without shaking. Yields were ;
as follows- :~:~
% Dry Weight 15 Re~5¢ion D-Fructose D-GlucoseFructose -`~
; 2 0 .534 0 --3 (~GI) .12 .47 -20%
3 (dupli~ate) 0.05~ .71 ~6.6%
~:: :;-" ' ,~ ~ `'.''''.`''''",'', The following reactions were run using the same enzyme preparation as in Example 1 at 45~C without shaking overnight one flask per preparation. Both white and red wheat bran flour, prepared as in Example 1 were used:
1. White bran flour :
%
Bran Weight : 10 Reaction Flour Cel GI Buffer Glucose Fructose Fructose 1 0.25 gm - 10 ml 0 0 2 ~ 5 ml 5 ml -- 0 0 3 0.25 gm 5 ml 5 ml .86 0 .
~: 4 0.25,gm 5 ml 5 ml .860.99 ~9.3%
15 2. Red Eran Flour . .~
~ ~ % ~ '~
: Eran Weight ~:
: : Rea~tion Flour Cel GI B~uffer Glucose Fruck~e Fructose 1 0.25 - 10 0 0 : 2 5 ml ~ 5 0 0 3 0.25 5 ml ~ 5 .66 0 4 0.25 5 ml 5 - .62 .08-11.4%
, ~ -, In both experiments the reaction products containing :~
gluco-e or glucose/fructose were darker than the reaction prod~cts without glucose or fructose.
... ...
1 332 1 23 ~:
EXAMPLE 3 ~ :
The enzymes used in the following reaction were indicated in Example 3. Reactions were run at a total ~;
volume of 20 ml in a 250 ml Erlenmeyer flask. The bran :
flour was not prewashed. Temperature was 45C for 20 hours then 60C for four hours; no shaking for 20 hours, then 75 rpm for four hours.
ML Yield g(L) 24 hr.
Reaction Bran (g) Cellulase GI Buffer Glucose Fructose . ~
1 5 5.5 1.1 13.4 11.0 10.8 ~-`-. .
3 -- 5.5 1.1 13.4 0 0 ~ Time course of yield for Reaction ~g/l) ;~ _ Hours lS 0 1 2 . 3 20 22_ 24 D-glucose 0.9 4.0 5.1 4.6 9.9 10.311.0 ;~
D-fructose 0.06 2.2 3.5 4.3 9.4 10.110.8 A9suming an approximate cellulose content of 11.3% ~-he percent conversîon of cellulose to glucose and , ~:
~ 20~ ~fructose lS 77%. - ~`
,~ . ` ` : ':' ' '` ' . ~' . .
19 1 33~ 1 23 Enzymes, buffers and bran flour were the same as in Example 2. In addition, the following enzymes were used:
~-amylase (1,4-~-D-glucan-glucanohydrolase E.C. 3.2.1.1) from B. lichen_formis (Sigma) (referred to herein as ~A), amyloglucosidase, exo 1,4 ~-glucosidase 1,4 ~-D-Glucan glucohydrolase E.C. 3.2.1.3 from As~erqillus oryzae ~Sigma) referred to here as GA). ~-Amylase concentration was 11040 U/ml, GA was 1400 U/ml, GI was 2250 IGIU/ml, and cellulase was 18.9-227 U/ml. Buffer was the same as that used for glucose isomerase: 50 mM NaPO4, pH 6.0, 10 mM MgS04, 1 mM CoC12. A flavoring syrup having the following composition was used in parts per hundred (ppC): liquid sucrose (67 brix) 34 ppC; NaCl 2.0: fig :
15 and prune concentrate 2 ppC; vitamin solution including ~ i niacinamide, Bl, B6, B2 and C .8 ppC; balance water.
The following protocol was used: Into a 1 liter beaker, bran (102.5 gm) and flavor syrups (46.6 gm (41 ml) were measured. The material was thoroughly mixed and : ~, ~ `20 loosely covered with aluminum foil. The mixture was ; ~ ~ autoclaved at 121C, 15 psi for 25 minutes and then ~
cooled to room temperature. The mixture was distributed ~ ~ -in 39 gm aliquots into 1 liter beakers. The aliquots `
were treated as follows:
,, ~. ~ .
. . .
1 3321 23 ~:
A
Cellulases 2500 U (7 ml) Glucose isomerase 2500 U (1.6 ml) Buffer, pH 5.5 11.4 ml ..
An additional 5 ml aliquot of glucose isomerase was ~
added to Reaction vessel 3. Reaction vessels 2 and 3 -; -were incubated at 05C overnight without shaking. In addition, a duplicate set of flasks identical to Reaction 3 was run overnight at 05C without shaking. Yields were ;
as follows- :~:~
% Dry Weight 15 Re~5¢ion D-Fructose D-GlucoseFructose -`~
; 2 0 .534 0 --3 (~GI) .12 .47 -20%
3 (dupli~ate) 0.05~ .71 ~6.6%
~:: :;-" ' ,~ ~ `'.''''.`''''",'', The following reactions were run using the same enzyme preparation as in Example 1 at 45~C without shaking overnight one flask per preparation. Both white and red wheat bran flour, prepared as in Example 1 were used:
1. White bran flour :
%
Bran Weight : 10 Reaction Flour Cel GI Buffer Glucose Fructose Fructose 1 0.25 gm - 10 ml 0 0 2 ~ 5 ml 5 ml -- 0 0 3 0.25 gm 5 ml 5 ml .86 0 .
~: 4 0.25,gm 5 ml 5 ml .860.99 ~9.3%
15 2. Red Eran Flour . .~
~ ~ % ~ '~
: Eran Weight ~:
: : Rea~tion Flour Cel GI B~uffer Glucose Fruck~e Fructose 1 0.25 - 10 0 0 : 2 5 ml ~ 5 0 0 3 0.25 5 ml ~ 5 .66 0 4 0.25 5 ml 5 - .62 .08-11.4%
, ~ -, In both experiments the reaction products containing :~
gluco-e or glucose/fructose were darker than the reaction prod~cts without glucose or fructose.
... ...
1 332 1 23 ~:
EXAMPLE 3 ~ :
The enzymes used in the following reaction were indicated in Example 3. Reactions were run at a total ~;
volume of 20 ml in a 250 ml Erlenmeyer flask. The bran :
flour was not prewashed. Temperature was 45C for 20 hours then 60C for four hours; no shaking for 20 hours, then 75 rpm for four hours.
ML Yield g(L) 24 hr.
Reaction Bran (g) Cellulase GI Buffer Glucose Fructose . ~
1 5 5.5 1.1 13.4 11.0 10.8 ~-`-. .
3 -- 5.5 1.1 13.4 0 0 ~ Time course of yield for Reaction ~g/l) ;~ _ Hours lS 0 1 2 . 3 20 22_ 24 D-glucose 0.9 4.0 5.1 4.6 9.9 10.311.0 ;~
D-fructose 0.06 2.2 3.5 4.3 9.4 10.110.8 A9suming an approximate cellulose content of 11.3% ~-he percent conversîon of cellulose to glucose and , ~:
~ 20~ ~fructose lS 77%. - ~`
,~ . ` ` : ':' ' '` ' . ~' . .
19 1 33~ 1 23 Enzymes, buffers and bran flour were the same as in Example 2. In addition, the following enzymes were used:
~-amylase (1,4-~-D-glucan-glucanohydrolase E.C. 3.2.1.1) from B. lichen_formis (Sigma) (referred to herein as ~A), amyloglucosidase, exo 1,4 ~-glucosidase 1,4 ~-D-Glucan glucohydrolase E.C. 3.2.1.3 from As~erqillus oryzae ~Sigma) referred to here as GA). ~-Amylase concentration was 11040 U/ml, GA was 1400 U/ml, GI was 2250 IGIU/ml, and cellulase was 18.9-227 U/ml. Buffer was the same as that used for glucose isomerase: 50 mM NaPO4, pH 6.0, 10 mM MgS04, 1 mM CoC12. A flavoring syrup having the following composition was used in parts per hundred (ppC): liquid sucrose (67 brix) 34 ppC; NaCl 2.0: fig :
15 and prune concentrate 2 ppC; vitamin solution including ~ i niacinamide, Bl, B6, B2 and C .8 ppC; balance water.
The following protocol was used: Into a 1 liter beaker, bran (102.5 gm) and flavor syrups (46.6 gm (41 ml) were measured. The material was thoroughly mixed and : ~, ~ `20 loosely covered with aluminum foil. The mixture was ; ~ ~ autoclaved at 121C, 15 psi for 25 minutes and then ~
cooled to room temperature. The mixture was distributed ~ ~ -in 39 gm aliquots into 1 liter beakers. The aliquots `
were treated as follows:
,, ~. ~ .
. . .
1 3321 23 ~:
A
Cellulases 2500 U (7 ml) Glucose isomerase 2500 U (1.6 ml) Buffer, pH 5.5 11.4 ml ..
5 Incubated at 45C, no shaking, moisture saturated ;
..
atmosphere, tightly covered with foil.
B
~-amylase 2500 U (0.23 ml) ~:~
Amyloglucosidase 2500 U (1.8 ml) Glucose isomerase 2500 U ~1.6 ml) Cellulases 2500 U (7 ml) -,. . ~, , ~ Buffer, pH 5.5 9.37 ml , ~ C ~
; 15 Buffer, pH 6.0 20 ml ;~
~ Incubated at 60-C in a water bath, no shaking, tightly -~
; covered with foil. ~ -Samples were taken periodically and frozen. Assay ;-~ for D-glucose and D-fructose were carried out on 0.2 gm ~-~
20 of each samples as described above. Dry weights of ~i~
various samples were determined by heating to 150-C for two hours in aluminum pans using a drying oven.
` 1 3321 23 D-glucose/D-fructose analyses: These were done as described in Examples XVIII through XX. The bran used for the study had a moisture content -50-60%. Therefore, each reaction beaker contained 23.1 gm dry bran; and each sample analyzed was 0.1084 gm, the bulk of which was dry bran.
Results: Production of D-alucose/D-fructose:
1. Reaction A (cellulases/glucose isomerase) qm/L
1 hr. 2.hr. 5 hr. 22 hr. 24 hr~
D-glucose 12.4 12.0 8.3 12.2 15.0 D-fructose 1.2 1.6 2.2 4.2 5.1 '':
% conversion -72 -72 ~53 -86 -106 of cellulose 0.084 gm aliquot 2. Reaction B (amylases/cellulases/glucose isomerase) am/L
1 hr. 2.hr. 5 hr. 22 hr. 24 hr.
' -~:
D-glucose 19.4 19.9 13.3 18.8 21.6 ~;
, ~ D-fructose 2.4 4.1 4.5 4.9 9.6 `~
:, ' ' % conversion ~34 ~37 -28 ~43 -48 ;~ of cellulose *
0.084 gm aliguo~
.:
3. Reaction C (control) No D-glucose or D-fructose.
The mixtures in the enzyme-treated instances were darker and stickier than the control, due to the high ~ -5 monosaccharide content and browning reactions. They had ~ ~~
a pleasant aroma.
Samples of beakers of Reactions B and C contents were baked for 20 minutes at 150C. The resultant -~
products were very different in obvious properties. The ~-aromas of the enzyme-treated samples were much more intense and pleasant than that of the control. Also, the enzyme-treated samples were darker and particles adhered ~
to each other, whereas the control was lighter and;' particles were discrete. -~
EXAMPLES 5 throuah 8 Fructose sweetened cereal products may be produced as in Examples 1 through 4 except the sodium azide and CoCl2 are eliminated. ;
.
~: ~: ' '' '' .
''~', ' . '.;
....
! ~
.'', ~ . ~ ~ ,
..
atmosphere, tightly covered with foil.
B
~-amylase 2500 U (0.23 ml) ~:~
Amyloglucosidase 2500 U (1.8 ml) Glucose isomerase 2500 U ~1.6 ml) Cellulases 2500 U (7 ml) -,. . ~, , ~ Buffer, pH 5.5 9.37 ml , ~ C ~
; 15 Buffer, pH 6.0 20 ml ;~
~ Incubated at 60-C in a water bath, no shaking, tightly -~
; covered with foil. ~ -Samples were taken periodically and frozen. Assay ;-~ for D-glucose and D-fructose were carried out on 0.2 gm ~-~
20 of each samples as described above. Dry weights of ~i~
various samples were determined by heating to 150-C for two hours in aluminum pans using a drying oven.
` 1 3321 23 D-glucose/D-fructose analyses: These were done as described in Examples XVIII through XX. The bran used for the study had a moisture content -50-60%. Therefore, each reaction beaker contained 23.1 gm dry bran; and each sample analyzed was 0.1084 gm, the bulk of which was dry bran.
Results: Production of D-alucose/D-fructose:
1. Reaction A (cellulases/glucose isomerase) qm/L
1 hr. 2.hr. 5 hr. 22 hr. 24 hr~
D-glucose 12.4 12.0 8.3 12.2 15.0 D-fructose 1.2 1.6 2.2 4.2 5.1 '':
% conversion -72 -72 ~53 -86 -106 of cellulose 0.084 gm aliquot 2. Reaction B (amylases/cellulases/glucose isomerase) am/L
1 hr. 2.hr. 5 hr. 22 hr. 24 hr.
' -~:
D-glucose 19.4 19.9 13.3 18.8 21.6 ~;
, ~ D-fructose 2.4 4.1 4.5 4.9 9.6 `~
:, ' ' % conversion ~34 ~37 -28 ~43 -48 ;~ of cellulose *
0.084 gm aliguo~
.:
3. Reaction C (control) No D-glucose or D-fructose.
The mixtures in the enzyme-treated instances were darker and stickier than the control, due to the high ~ -5 monosaccharide content and browning reactions. They had ~ ~~
a pleasant aroma.
Samples of beakers of Reactions B and C contents were baked for 20 minutes at 150C. The resultant -~
products were very different in obvious properties. The ~-aromas of the enzyme-treated samples were much more intense and pleasant than that of the control. Also, the enzyme-treated samples were darker and particles adhered ~
to each other, whereas the control was lighter and;' particles were discrete. -~
EXAMPLES 5 throuah 8 Fructose sweetened cereal products may be produced as in Examples 1 through 4 except the sodium azide and CoCl2 are eliminated. ;
.
~: ~: ' '' '' .
''~', ' . '.;
....
! ~
.'', ~ . ~ ~ ,
Claims (14)
1. A process for producing a sweetened cereal product comprising:
a) moistening a cereal grain by adding an aqueous solution to a cereal grain containing cellulose and starch, in an amount sufficient to support conversion by enzymes of the cellulose and/or starch to fructose and in an amount such that substantially all of the solution is absorbed by the cereal;
b) disrupting the moistened cereal produced in step a) to an extent effective to promote the enzymatic conversion of the cellulose and/or starch to fructose by heating under pressure or treating with alkali;
c) adding to the disrupted, moistened cereal produced in step b) enzymes effective to convert cellulose and/or starch to fructose to produce a moistened cereal and enzyme mixture;
d) incubating said moistened cereal and enzyme mixture for a sufficient period of time to convert the cellulose and/or starch in said cereal to fructose at a temperature at which the enzymes are active; and e) collecting a cereal product enriched in fructose.
a) moistening a cereal grain by adding an aqueous solution to a cereal grain containing cellulose and starch, in an amount sufficient to support conversion by enzymes of the cellulose and/or starch to fructose and in an amount such that substantially all of the solution is absorbed by the cereal;
b) disrupting the moistened cereal produced in step a) to an extent effective to promote the enzymatic conversion of the cellulose and/or starch to fructose by heating under pressure or treating with alkali;
c) adding to the disrupted, moistened cereal produced in step b) enzymes effective to convert cellulose and/or starch to fructose to produce a moistened cereal and enzyme mixture;
d) incubating said moistened cereal and enzyme mixture for a sufficient period of time to convert the cellulose and/or starch in said cereal to fructose at a temperature at which the enzymes are active; and e) collecting a cereal product enriched in fructose.
2. The process of claim 1 wherein the enzymes are selected from the group consisting of cellulases, glucose isomerases, alpha-amylases and amylo-glucosidases.
3. The process of claim 2 wherein the enzymes comprise cellulase and glucose isomerase and the cellulase enzyme is added to the disrupted, moistened cereal before the glucose isomerase.
4. The process of claim 2 wherein the enzymes are cellulase, alpha-amylase and amylo-glucosidase and glucose isomerase and said cellulase and alpha-amylase and amylo-glucosidase enzymes are added to the disrupted, moistened cereal before glucose isomerase.
5. The process of claim 1 further including a drying step after said step of collecting.
6. The process of claim 5 wherein said drying step is carried out at a temperature in the range of between about 250° and 450°F.
7. The process of claim 1 wherein said step of incubating is carried out at a temperature of between about 25° and 80°C.
8. The process of claim 1 wherein said step of moistening and said step of adding the enzymes are carried out concurrently.
9. The process of claim 1 wherein said moistened cereal has a moisture content of about 40% to 75%
weight volume.
weight volume.
10. A sweetened cereal product enriched in fructose content produced by the process of claim 1.
11. The process of claim 1 wherein said incubating step is carried out at a temperature of between 45 and 60 degrees C.
12. The process of claim 3 wherein said cellulases are selected from the group consisting of cellobiohydrolases and endoglucanases.
13. The process of claim 1 wherein said cereal grain is a cereal flour or meal comprising cereal fiber.
14. The process of claim 13 wherein said cereal flour or meal is selected from the group consisting of bran flour, bran meal, graham flour, graham meal, whole wheat flour, and whole wheat meal.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/101,564 US4859474A (en) | 1987-09-28 | 1987-09-28 | Method of making an enzyme sweetened cereal product |
| US101,564 | 1987-09-28 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1332123C true CA1332123C (en) | 1994-09-27 |
Family
ID=22285310
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000578550A Expired - Fee Related CA1332123C (en) | 1987-09-28 | 1988-09-27 | Enzyme sweetened cereal product |
Country Status (2)
| Country | Link |
|---|---|
| US (1) | US4859474A (en) |
| CA (1) | CA1332123C (en) |
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|---|---|---|---|---|
| US5066218A (en) * | 1987-05-13 | 1991-11-19 | Genencor International, Inc. | Composition of a steeped starched-containing grain and a cellulase enzyme |
| US5366755A (en) * | 1989-02-10 | 1994-11-22 | Maritta Timonen | Foodstuffs containing novel degraded cellulose derivatives |
| US5631032A (en) * | 1991-04-17 | 1997-05-20 | Union Industrial Y Agro-Ganadera, S.A. (Uniasa) | Process for the preparation of ground cereal based foods and foodstuffs obtained thereby |
| US5292537A (en) * | 1992-11-12 | 1994-03-08 | Bran Tec, Inc. | Method for stabilizing rice bran and rice bran products |
| ES2234067T5 (en) * | 1997-07-31 | 2009-05-01 | Dsm Ip Assets B.V. | COMPOSITION TO IMPROVE BREAD. |
| US20090258106A1 (en) * | 2005-07-20 | 2009-10-15 | Robert Jansen | Corn Wet Milling Process |
| US20070020375A1 (en) * | 2005-07-20 | 2007-01-25 | Robert Jansen | Corn wet milling process |
| EP2181194A1 (en) * | 2007-08-03 | 2010-05-05 | Tate & Lyle Ingredients Americas, Inc. | Cereal refining process |
| US20090047385A1 (en) * | 2007-08-15 | 2009-02-19 | The Quaker Oats Company | Natural oat-derived sweetener |
| WO2009094418A2 (en) * | 2008-01-24 | 2009-07-30 | Tate And Lyle Ingredients Americas, Inc. | Corn wet milling process |
| WO2017205337A1 (en) | 2016-05-23 | 2017-11-30 | Dupont Nutrition Biosciences Aps | Baking process and a method thereof |
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| US897854A (en) * | 1908-09-01 | Theodor Schlueter Jr | Process for the production of a digestible flour from bran. | |
| US1108555A (en) * | 1914-08-25 | Macey F Deming | Process for preparing food for infants, invalids, and old persons. | |
| US1543458A (en) * | 1922-09-14 | 1925-06-23 | Takamine Ferment Co | Starch-free bran and method of making the same |
| US2206619A (en) * | 1939-08-04 | 1940-07-02 | Konrad F Schreier | Wheat crisp |
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| US3395019A (en) * | 1965-10-18 | 1968-07-30 | Vylactos Lab Inc | Preparation of animal feed from oat hulls |
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| NL7400363A (en) * | 1973-01-30 | 1974-08-01 | ||
| US3922201A (en) * | 1973-07-12 | 1975-11-25 | Cpc International Inc | Preparation of levulose from granular starch |
| US3972775A (en) * | 1974-06-28 | 1976-08-03 | The United States Of America As Represented By The United States Energy Research And Development Administration | Conversion of cellulosic materials to sugar |
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| US4247636A (en) * | 1978-10-26 | 1981-01-27 | The Amalgamated Sugar Company | Process for producing a high fructose sweetener, high protein meal, and cereal germ oils |
| US4299847A (en) * | 1979-07-25 | 1981-11-10 | Morris William F | Process for treating cereal grains |
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| SE8505783D0 (en) * | 1985-12-06 | 1985-12-06 | Rolf Bergkvist | PROCEDURE FOR MANUFACTURING FOOD PRODUCTS AND ITS USE |
-
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- 1987-09-28 US US07/101,564 patent/US4859474A/en not_active Expired - Fee Related
-
1988
- 1988-09-27 CA CA000578550A patent/CA1332123C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US4859474A (en) | 1989-08-22 |
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