Classifications

• • 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
  • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
• • 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
  • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
• • 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
  • Y10S435/822—Microorganisms using bacteria or actinomycetales
  • Y10S435/832—Bacillus
  • Y10S435/839—Bacillus subtilis
• • 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
  • Y10S435/913—Aspergillus
  • Y10S435/917—Aspergillus niger

Definitions

• 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.
• alpha-amylases are conceived as enzymes that hydrolyse starch and glycogen to form reducing sugars, such as glucose, maltose and maltotriose.
• 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.
• substrates substrates
• 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.
• the precise actions of transferring has not been exactly determined.
• 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.
• 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.
• liquefaction and saccharification of starch, and transferring action may be effected with the employment of a single enzyme.
• 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.
• BSA Bacillus subtilis
• 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.
• these enzymes are of the type that split or hydroly
• 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.
• 'BSA bacterial saccharogenic alpha-amylase
• fungal alpha-amylases or pancreatic alpha-enzymes are preferable.
• 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.
• salted out enzymes obtained by salting-out with sodium sulfate, or precipitated preparations obtained by precipitation with alcohol and the like are also employable.
• NRRL 337 alpha-amylases predominate and relatively low contents of glucoamylase or transglucosidase, and thus are employable per se in the invention.
• 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.
• pancreatic enzyme is a preferable enzyme preparation in view of the fact that it has alpha-amylase predominantly and scarcely has other amplases.
• 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.
• 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.
• starch syrup will tend to contain higher amounts of glucose or fructose with the absence of sucrose.
• 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.
• 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.
• 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.
• 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 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• 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.
• the sweetness of the products may be intensified.
• the products exhibit milder and more palatable sweetness owing to their compositions of sucrose, fructose, oligosyl-frnctose, glucose and maltose.
• the enzymes employed in the invention do not produce cyclodextrins that are diflicult to dissolve as in the case Bacillus macerans enzyme is employed.
• sugar acceptors such as sucrose
• 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.
• 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.
• 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.
• 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.
• invert sugar is employable as a sugar acceptor similarly to fructose. However this case results in higher production of reducing sugars.
• 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).
• 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.
• 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.
• 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.
• 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.

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• Organic Chemistry (AREA)
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Cited By (16)

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• 1970
  • 1970-02-24 JP JP1569270A patent/JPS5622520B1/ja active Pending
• 1971
  • 1971-02-23 CA CA106063A patent/CA947217A/en not_active Expired
  • 1971-02-23 US US118193A patent/US3701714A/en not_active Expired - Lifetime
  • 1971-02-24 FR FR7106245A patent/FR2079003A5/fr not_active Expired
  • 1971-02-24 DE DE2108748A patent/DE2108748C3/de not_active Expired
  • 1971-04-19 GB GB2246771A patent/GB1352633A/en not_active Expired

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