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Publication numberUS3701714 A
Publication typeGrant
Publication dateOct 31, 1972
Filing dateFeb 23, 1971
Priority dateFeb 24, 1970
Also published asCA947217A1, DE2108748A1, DE2108748B2, DE2108748C3
Publication numberUS 3701714 A, US 3701714A, US-A-3701714, US3701714 A, US3701714A
InventorsShigetaka Okada, Naoto Tsuyama
Original AssigneeHayashibara Co
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Processes for the production of oligosaccharides having fructose molecules on their reducing ends
US 3701714 A
Abstract  available in
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Description  (OCR text may contain errors)

United States Patent Oflice 3,701,714 Patented Oct. 31, 1972 US. Cl. 195-31 R 6 Claims ABSTRACT OF THE DISCLOSURE Process for the production of oligosaccharide mixtures having fructose molecules on their reducing ends (oligosyl fructose) by subjecting mixtures of starch, sucrose or fructose to the actions of specific alpha-amylases, and thus with the utilization of the alpha-amylases, attaining simultaneous hydrolysis of starch and transfer of the formed oligosaccharides into sucrose or fructose.

The invention relates to processes for the production of oligosaccharide mixtures having fructose molecules on their reducing ends (oligosyl fructose) by subjecting mixtures of starch, sucrose or fructose to the actions of specific alpha-amylases, and thus with the utilization of the alpha-amylases, attaining simultaneous hydrolysis of starch and transfer of the formed oligosaccharides into sucrose or fructose.

Generally alpha-amylases are conceived as enzymes that hydrolyse starch and glycogen to form reducing sugars, such as glucose, maltose and maltotriose. However the progress of studies on the actions of amylases has proved that alpha-amylases do not merely exhibit hydrolysis, but it has been found that in the presence of suitable substrates (sugar acceptors) it exhibits transferring actions. Both the bonds that are effected by hydrolysis and bonds that are formed by the transfer actions are alpha-1,4-glucosidic linkages, which are frequently detected by employing oligosaccharides that contain radioactive carbon atoms, i.e. labelled carbon atoms. Thus the precise actions of transferring has not been exactly determined.

The inventors reviewed extensively the previous studies on the actions and purification of amylases, which led to the findings that under certain conditions several alphaamylases exhibit considerable sugar transferring actions. In addition, the inventors found that sucrose, fructose, cellobiose, xylose and the likes may be employed as sugar acceptors besides glucose, maltose and oligosaccharides. The oligosaccharides obtained by the employment of sucrose and fructose as sugar acceptors are oligosyl fructose that contain fructose on their reducing ends, and are superior and high quality sweeteners.

Moreover, the processes according to the present invention are characterized in the employment of cheaper alpha-amylases, without the necessity of the employment of an expensive enzyme derived from Bacillus macerans.

In some cases liquefaction and saccharification of starch, and transferring action may be effected with the employment of a single enzyme. Thus the present invention provides advantageous processes that can be utilized for industrial production.

Employable enzymes in the invention includes saccharogenic alpha-amylase derived from Bacillus subtilis (BSA), alpha-amylases secreted generally from fungi, such as Aspergillus niger, Rhizopus niveus, and Taka-Diastase, alpha-amylase of Endomycapsis and pancreatic or salivary alpha-amylase. According to the results of studies conducted by the inventors, these enzymes are of the type that split or hydrolyse the second, third and fourth glucosidic' bonds from the non-reducing ends upon subjecting on oligosaccharides. On the other hand, when any of the enzyme of bacterial liquefying alpha-amylase (B'LA), rnalt, or alpha-amylase of Bacillus stearothcrmophilus is subjected on oligosaccharides, the enzymes hydrolyse the fifth and sixth linkages from the non-reducing ends, but hardly effect sugar transfer. Thus the latter enzymes are not applicable in the invention.

The enzymes employable in the invention are the ones that belong to the first group. However, for industrial practice 'BSA (bacterial saccharogenic alpha-amylase), fungal alpha-amylases or pancreatic alpha-enzymes are preferable.

The term BSA is defined in the invention as the enzymes which effect higher degree of starch hydrolysis among the alpha-amylases secreted from Bacillus swbtilis. Preparation of the enzymes may be carried out by the methods described in prior references or patent gazettes. Usually the strains are inoculated on medium containing soy bean cake, ammonium phosphate and inorganic salts, and then the mixture is cultivated by shaking or submerged culture. The filtered culture broth is used per se in the invention. Of course, salted out enzymes obtained by salting-out with sodium sulfate, or precipitated preparations obtained by precipitation with alcohol and the like are also employable.

Of thet fungal alpha-amylases, for example, in the submerged culture broth of Aspergillus niger, NRRL 337 alpha-amylases predominate and relatively low contents of glucoamylase or transglucosidase, and thus are employable per se in the invention. Compared with these culture broths, filtered culture broths of Rhizopus niveus contain relatively high content of glucoamylase, therefore it is preferable to separate in advance the glucoamylase and to use culture broths prepared to contain merely alphaamylases as far as possible. Since there is a variation in the content ratio of alpha-amplases according to the variety of strain employed, it is practical for industrial purposes to use the filtered culture broth of the selected strain which have stronger starch-hydrolysing activity and weaker or less maltose-hydrolysing activity. Pancreatic enzyme is a preferable enzyme preparation in view of the fact that it has alpha-amylase predominantly and scarcely has other amplases. However comparison tests on the actions of various enzymes on starch and sucrose mixture solutions showed that pancreatic enzyme has a tendency of exhibiting lower transferring ratio than BSA and fungal alpha-amplase. (Transferring ratio: the amount of fructose that reacted to the total amount of fructose.)

Employable starches include potato, sweet potato, tapioca, corn, waxy-maize, wheat, and rice starches, or short chain amylase obtained by hydrolysing amylopectin with isoamylases. One or more than one variety of the starches are admixed with sucrose or fructose, and then to the mixture is added the above described BSA, or fungal alpha-amplase or pancreatic alpha-amylase. The mixture is then liquefied at pH 5.07.0, 6090 C. continuously or by batch method, then allowed to stand at 40-60 C. If necessary, enzyme may be further added subsequent to the liquefaction.

A method to perform simultaneously liquefaction saccharification and transferring action Since these enzymes possess lower heat stability than BLA (bacterial liquefying alpha-amylase) or malt, and their liquefaction activity is lower than the saccharification activity, liquefaction of starch may be carried out with the employement of acid or commercialized liquefying enzyme. Then the above described alpha-amylases, and sucrose or fructose may be added.

A method to perform only staccharification and transferring action In this case, when the degree of hydrolysis of the liquefied starch solution is relatively high, the starch syrup will tend to contain higher amounts of glucose or fructose with the absence of sucrose.

Usually the employable starch concentration is 20-45% (dry substance, by weight; all parts and percentages hereinafter are given by weight unless stated otherwise). The amount of sucrose or fructose to be added as /s to 5 times based on the amount of starch. In practice the ratio is /3 to 3 times.

The transferring action of alpha-amylases depend substantially on the concentration of starch solution and sucrose, the variety of enzyme employed and the reaction conditions.

The activity of the enzyme to be employed is defined as follows. One unit is defined as the activity that forms 10 mg. of reducing sugar obtained by the reaction of 10 ml. of 0.5% soluble starch solution with enzyme solution at C. for 30 minutes. 1-10 units/gram starch, usually 3 units, are added and incubated at 55 C. for 3 days.

In case BSA or pancreatic amylase is used as the enzyme preparation, there is a jeopardy of causing a redecomposition of the once formed fructose-containing oligosaccharides into sucrose or fructose and malto-oligosaccharides. However, in case fungal enzyme is employed G2F accumulates without formation into sucrose or fructose caused by redecomposition.

The saccharified and transferred sugar solution obtained as above is decolorized with 1% of pulverized active carbon, filtered, and then deionized and decolored by strong acidic and weak basic ion exchangers. Subsequent to the treatment with ion exchangers, the liquid sugar like starch syrup may be prepared by condensing the product to moisture content of 15-25%. The product is colorless, transparent and possesses a relatively low viscosity. If preferable, the product can be pulverized by spray drying or by drying in vacuo.

The advantages of the invention are as follows.

(1) Since liquefaction and saccharification may be performed simultaneously with a single enzyme, the processes described herein are extremely more effective in simplifying the subsequent treating procedures than the method in which amylase derived from Bacillus macerans is employed. In some cases it is possible to produce transferred when the concentration of the substrate is 0.1-0: 5% hardly any transferring action is noted. However, transfer is effected in case the concentration exceeds 5%, and over 20% extremely remarkable transfer is observed. In addition it is preferable to perform transfer at over 20% in respect of theevaporation procedure.

In case the concentration of starch is higher, the transferring ratio of the sucrose and fructose (the ratio of the mole number of sucrose or fructose reacted as a sugar acceptor to the mole number of the total sucrose or fructose) increases. However since a higher amount of residue of untransferred oligosaccharides is present the viscosity and the amount of reducing sugars of the resulting product increases, whereas in the case when the mole number of sucrose or fructose used as acceptors is higher the tansferring ratio decreases. However the absolute quantity of transferred vsaccharides increases, and the resulting product will be mixtures containing sucrose-or fructose which are sweeter and possess unique quality of sweetness, in addition to a tendency of less glucose.

These oligosyl fructoses and oligosaccharides were quantitively analysed by paper chromatography or identified by visualizing the paperchromatogram using coloring reagents, resulting in'the findings that the products contain molecules of around 64F (combined products of four glucose residues and a fructose molecule) or, molecules lower than GSF. Moreover the fact that oligosaccharides transfer to sucrose was identified by the reaction of oligosaccharides containing labelled carbon C on their reducing ends and sucrose, and then by examining the radio activity of the paperchromatogram spots.

In addition, oligosaccharides that colorate by silver nitrate is present in the starch syrup, therefore said starch syrup comprises, more particularly unreacted sucrose or fructose, oligosaccharides combined with frucose, and oligosaccharides combined with fructose, and oligosaccharides (a component of common starch syrup). Moreover the composition of the three components may be standardized by variations of the amount of starch and starch syrups from starch and sucrose or fructose without the employment of liquefying enzymes.

(2) The enzymes employed in the invention do not form cyclodextrins that are hard to dissolve as in the case of Bacillus macerans, therefore clearer and more highly concentrated sugar solutions are obtainable.

(3) Since the enzymes employed in the invention form higher amounts of lower molecular sugars, such as glucose and maltose, the sweetness of the products may be intensified. Moreover the products exhibit milder and more palatable sweetness owing to their compositions of sucrose, fructose, oligosyl-frnctose, glucose and maltose.

(4) The enzymes employed in the invention do not produce cyclodextrins that are diflicult to dissolve as in the case Bacillus macerans enzyme is employed. The facts that there is entirely no necessity to hydrolyse the starch used as starting material to high degrees and that sugar acceptors, such as sucrose, may be added at stages of lower hydrolysis degrees, i.e. when high or macro-molecular dextrins are formed, accelerate the reaction velocity as well as elevating the transferring degrees.

(5) Since the formed transferred sugar syrups consist mainly of colecules with degrees of polymerization ('D.P.) of 2-5, the viscosities of the products are relatively low. Thus it is attainable to obtain sweeteners which may be used as liquid sugars in extensive areas.

(6) Owing to the fact that said sweeteners comprise sucrose, transferred sugar, glucose, and fructose, as well as oligosaccharides, such as maltotriose, the products do not crystallize even at 70-80% concentrations, and thus the products may be employed as transparent liquid sugars.

(7) In addition the products possess extremely desirable hygroscopicities which make the products an effective sweetener for bakeries. Fermenting gradually, incorporation of said sweeteners results in bakery products with excellent flavor, and fine and spongy textures.

Before illustrating some examples of the invention, which are given as some methods of producing said sweeteners of the invention, and not given as the sole methods of the invention, specific examples to regulate the viscosities of liquid sugar by promoting the decomposition and transfer will be described.

Subsequent to the passing of 'the starch, isoamylase (enzymes that split the alpha-1,6-glucosidic linkages of starch, i.e. the branched linkages of amylopectin is added. The transferring degree is elevated by the hydrolysis of the alpha-1,6-glucosidic linkages.

By the above treatment, lower viscosities of the liquid sugar product are attained and in addition filtration may be performed with ease. Thus the invention provides advantageous processes for industrial practice.

Of course invert sugar is employable as a sugar acceptor similarly to fructose. However this case results in higher production of reducing sugars.

The invention will be illustrated by the following examples.

EXAMPLE 1 Strain of Bacillus subtilis var. amylosacchariticus (K2) was cultivated on a medium comprising of soybean cake, 1% of ammonium phosphate, and 3% of starch for three days in a shaking incubator. The filtered culture broth (enzyme activity 40 units/ml.) was used as an enzyme preparation (BSA).

To a mixture of 1 kg. of starch and 500 grs. of sucrose was added 4 liters of water and 1.25 ml. of the above mentioned enzyme solution (total activities, 5,000 units), and heated in a boiling water bath with stirring to 80 C. At the completion of liquefaction the solution was cooled to 55 C. and after being allowed to stand for 3 days the hydrolysis ratio became approximately 30%. To the reaction solution was added 1% of active carbon and boiled. Subsequent to filtration the solution was passed through ion exchanger layers, and then decolorized, deionized and concentrated in vacuo. Thus a starch syrup product was obtained.

1 gr. of the syrup was diluted with ml. of water. 0.01 ml. of the dilution was spotted on the end of a sheet of filter paper, developed by using n-butanol, pyridine and H 0 (6:4:3, in volume) as a solvent, and then visualized with the employment of phloroglucinol as a colorating reagent. Thus spots that correspond to GZF, 63F, 64F, etc., besides sucrose were obtained.

EXAMPLE 2 As described in Example 1, to a mixture comprising 1 kg. of starch, 500 grs. of sucrose and 4 liters of water was added 50 ml. of culture broth (2,000 units) and liquefied by heating. When the temperature of the solution declined to 60 C., the solution was further incubated with a further addition of ml. of enzyme. Thus an excellent starch syrup as the one described in Example 1 was obtained.

EXAMPLE 3 With the addition of 4 liters of water, 1 kg. of corn starch was heated in a boiling water bath with 0.5 gr. of commercialized liquefying enzyme with stirring to 90 C.

When a hydrolysis degree of about 5% was attained (when the iodine stain became bluish violet), the liquefied starch solution was boiled to stop the enzyme action. At the point that the temperature of the solution declined to 65 C., 1 kg. of fructose and commercialized bacterial saccharogenic amylase preparation (2,000 units/gr., a product of Daiwa Kasei Kabushiki Kaisha) was added, and then the mixture was kept at 55 C. for two days to attain approximately hydrolysis degree against starch. By treating the solution according to the method described in Example 1, a starch syrup containing fructose was obtained.

Determination of said syrup by paperchromatography showed that about 30% of the fructose was converted into oligosyl fructose. The product was colorless and transparent, and similar to honey exhibiting an intensive sweetness.

EXAMPLE 4 Strains of Aspergillus niger NRRL 337 were inoculated on a medium comprising 2% of corn steep liquor, 2% of soy bean cake, 3% of starch and 0.5% of ammonium phosphate in a shaking incubator at 30 C. for 3 days. Filtered solution of the culture broth had a saccharifying activity of 30 units/ml. Since when maltose was used as the substarate instead of starch the saccharifying activity was determined to be less than one unit, alpha-amylases were considered predominating in the filtered solution.

Using said enzyme solution as the amylase preparation described in Example 3 the hydrolysis degree was about 28%, and about 20% of the fructose was found converted into oligosyl fructose, determined by paperchromatography.

The obtained product had an intensive sweetness. It had a little lower viscosity compared with the product in Example 2.

EXAMPLE 5 1 kg. of starch was used to perform liquefaction as described in Example 2. Subsequent to the liquefaction, 2 kg. of sucrose was added and with the above exception the mixture was reacted under the same conditions described in Example 2.

The reacted product was decolorized and purified, thus a colorless and transparent starch syrup, exhibiting extremely intensive sweetness and possessing a desirable body, was obtained.

EXAMPLE 6 Under the same conditions described in Example 3, to a mixed solution comprising starch and invert sugar (1: 1) was added 5 units/gram starch of alpha-amylase of Rhizopus niger, from which glucoamylase was removed, and then incubated. The obtained product was desirously sweet and transparent.

EXAMPLE 7 A saccharified product, produced in accordance with the methods described in Example 3 with 3 units/gram starch of taka amylase A or pancreatic alpha-amylase, was converted into a hydrolysis degree of over 25%, and a desirously sweet and non-crystallizable starch syrup was obtained.

EXAMPLE 8 30% corn starch slurry was prepared, gelatinized at C., cooled rapidly to 50 C., and then incubated for 10 hours at pH 4.0 with the addition of 15 units/gram starch of isoamylase solution derived from strains of Pseudomonas wmyloderamosa (ATCC 21216). Then to the starch solution treated with isoamylase was added 5 units/ gram stanch of the enzyme solution, as described in Example 1 and derived from Bacillus subtilis, and equivalent amount of invert sugar against the amount of starch and incubated under the conditions of pH 6.0, temperature 55 C. for 3 days. The incubation was discontinued when a hydrolysis degree of approximately 40% was attained and the product was purified with active carbon and ion exchanger. The purified product was determined by paperchromatography, resulting in the findings that the product had a transferring degree of about 25%. Thus a liquid sugar with a more intensive sweetness than that of sucrose, and a heavy body was obtained.

What is claimed is:

1. A process for the production of oligosyl fructoses which possess fructose on their reducing ends, comprising admixing starch solution or starch 'hydrolysate solution having a concentration of 545% with a member selected from the group consisting of fructose, sucrose and a sugar mixture containing fructose and/or sucrose, and subjecting the mixture to the actions of a transferring enzyme selected from the group consisting of Bacillus subtilis saccharogenic alpha-amylase, fungal alpha-amylases, and pancreatic or salivary alpha-amylses.

2. The process for the production of oligosyl fructoses according to claim 1 wherein hydrolysis of the starch and sugar transferring are effected with the same enzyme.

8 3. Process according to claim 1 wherein the starch solu- References Cited tion is a starch slurry. I

4. Process according to claim 3 wherein the starch Palm B101 Chem" 217-25 slurry is gelatinized by heating and hydrolysed by alpha- 1952- amylase or a debranching isoamylase and then subjecting 5 Greenwood et al., Arch. Biochem. Biophys., vol. 126,

the resultant hydrolysate and sugar acceptor to the action PP 244 g 9 of Sald transfemng enzyme Dedonder, R., Meth. in Eny., vol. VIII, Academic Press,

5 Process for the production of ohgosyl fructoses according to claim 1 wherein the transferring enzyme is 500-505 1966' selected from the group consisting of Bacillus subtilis sac- 10 charogenic alpha-amylase and alpha-amylase from Asper- LOUIS MONACELL, Primary Examiner gillis niger NRRL 337 and pancreatic amylase. G. M. NATH Assistant Examiner 6. Process for the production of oligosyl fructoses according to claim 1 wherein the sugar is selected from the U S Cl X R group consisting of fructose, sucrose, invert sugar, par- 5 tially inverted sugar, honey and hydrol. 99-l4lR

Referenced by
Citing PatentFiling datePublication dateApplicantTitle
US3819484 *Dec 13, 1971Jun 25, 1974Hayashibara KenProcess for preparing sucrose-starch sweetner free from reducing sugar
US4254227 *Mar 8, 1979Mar 3, 1981Kabushiki Kaisha Hayashibara Seibutsu Kagaku KenkyujoUsing immobilized cyclodextrin glucanotransferase
US4649058 *Jun 5, 1985Mar 10, 1987Pfeifer & LangenGlucosyl transferase, glucose mono- or disaccharide, sucrose, artificial sweeteners, carriers
US4902674 *Oct 13, 1987Feb 20, 1990Coors Biotech, Inc.Method for inhibiting the growth of salmonella
US4987124 *Nov 8, 1989Jan 22, 1991Coors Biotech, Inc.Fermentation by other microflora to produce growth-inhibiting metabolites, followed later by administration of antibiotic
US5032579 *Jul 25, 1990Jul 16, 1991Coors Biotech, Inc.Method for inhibiting the growth of salmonella
US6193988Jul 12, 1996Feb 27, 2001Stoner, Ii Richard J.For controlling disease in growing plants
US8512739Sep 11, 2003Aug 20, 2013Cargill, IncorporatedUse of low-glycemic sweeteners in food and beverage compositions
WO1991007181A1 *Nov 6, 1990May 30, 1991Coors Biotech IncMethod for inhibiting the growth of salmonella
U.S. Classification435/97, 435/193, 435/99, 435/839, 435/202, 435/917, 435/203, 435/98
International ClassificationC13K1/00, C12N9/26, C12P19/18, C12P19/14, C12N9/10
Cooperative ClassificationY10S435/917, C12P19/18, C12P19/14, Y10S435/839
European ClassificationC12P19/18, C12P19/14