WO2016121885A1 - Procédé d'élimination du vieillissement de l'amidon - Google Patents

Procédé d'élimination du vieillissement de l'amidon Download PDF

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WO2016121885A1
WO2016121885A1 PCT/JP2016/052529 JP2016052529W WO2016121885A1 WO 2016121885 A1 WO2016121885 A1 WO 2016121885A1 JP 2016052529 W JP2016052529 W JP 2016052529W WO 2016121885 A1 WO2016121885 A1 WO 2016121885A1
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Prior art keywords
starch
aging
trehalose
amylose
reducing end
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PCT/JP2016/052529
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English (en)
Japanese (ja)
Inventor
尚樹 工藤
学 宮田
正浩 藤本
山本　拓生
西本　友之
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株式会社林原
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Priority to JP2016572153A priority Critical patent/JP6723167B2/ja
Publication of WO2016121885A1 publication Critical patent/WO2016121885A1/fr

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    • 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
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/206—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of vegetable origin
    • A23L29/212—Starch; Modified starch; Starch derivatives, e.g. esters or ethers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • 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

Definitions

  • the present invention relates to a method for inhibiting starch aging, a method for producing starch having reduced aging properties, and an aging inhibitor.
  • Starch is a high-molecular glucan stored mainly in the cells of higher plant seeds and rhizomes, and is generally a mixture of amylose and amylopectin.
  • Amylose is an ⁇ -1,4 glucan having a structure in which glucose is linearly bonded with ⁇ -1,4 bonds.
  • amylopectin has a structure in which ⁇ -1,4 glucan having a degree of polymerization of glucose of 6 or more is branched by ⁇ -1,6 linkages in the linear portion of ⁇ -1,4 glucan.
  • starch Since starch has excellent processability and storability and is inexpensive, it can be used as a so-called starchy substance including starch or a partially decomposed starch obtained by hydrolysis of starch with acid or enzyme.
  • starch for example, as a thickening stabilizer, colloid stabilizer, etc., for the purpose of improving the physical properties and maintaining the quality of food, and as a raw material for glucose, isomerized sugar, maltooligosaccharide, syrup, etc. Widely used industrially.
  • starchy substance swells and gelatinizes when the aqueous dispersion is heated, but has the property of insolubilization, that is, aging over time, and when the starchy substance ages, Not only the texture and flavor are impaired, but also inconveniences such as difficulty in digestion in the human body. For this reason, the range of use of starch in food and other industries is naturally limited. In view of such circumstances, conventionally, many attempts have been made to modify the structure of molecules constituting starchy materials for the purpose of imparting aging resistance to starchy materials. Since it is expected that there will be no influence on the original safety of starch, studies have been conducted energetically.
  • Patent Document 1 discloses a method for inhibiting starch aging by causing a branching enzyme (EC 2.4.1.18) to act on starch and introducing a branched structure into the starch. Is disclosed.
  • Patent Document 2 discloses that a branching enzyme, 4- ⁇ -glucanotransferase (EC 2.4.4.15) or cyclomaltodextrin glucanotransferase (EC 2.4.1.19) is starched.
  • a method is disclosed in which aging of starch is suppressed by acting and introducing an inner branched ring structure and an outer branched structure into the starch.
  • Patent Document 3 discloses cyclo ⁇ 6) - ⁇ -D-glucopyranosyl- (1 ⁇ 4) - ⁇ -D-glucopyranosyl- (1 ⁇ 6) - ⁇ -D-glucopyranosyl- (1 ⁇ 4)- Inhibits starch aging by acting on starch with an enzyme that produces a cyclic tetrasaccharide having the structure ⁇ -D-glucopyranosyl- (1 ⁇ ) and introducing a branched structure at the non-reducing end of the starch. A method is disclosed.
  • the starch material having reduced aging property by introducing a branched structure is advantageous in that aging property is reduced, but when this is simply used instead of existing starch material, Even the physical properties of the resulting product have changed, and while using a starch substance whose aging property has been reduced by the introduction of a branched structure, at the same time, a product having physical properties equivalent to those obtained using an existing starch substance is obtained. It was difficult. Under such circumstances, the aging of starch is suppressed without changing the physical properties other than aging of starch as much as possible, that is, without significantly changing the structure of starch and the molecular weight distribution of the whole starch. It is desirable to provide a method for doing this.
  • the present invention provides a method for suppressing aging of starch without significantly changing the structure of starch or the molecular weight distribution of the whole starch, that is, without significantly changing physical properties other than aging of starch. Is an issue. Moreover, this invention makes it a subject to provide the manufacturing method of the starch material by which aging was suppressed, and an aging inhibitor.
  • the inventors of the present invention in the process of conducting intensive research focusing on various enzymes that act on starch, surprisingly, a glycosyl trehalose-producing enzyme is allowed to act on aging starch. It was found that when a trehalose structure was introduced at the reducing end, aging was remarkably suppressed without greatly changing the structure of individual molecules or the molecular weight distribution of the whole starch, and the present invention was completed.
  • the present invention reduces the aging property of the starch than before introducing the trehalose structure by allowing a glycosyl trehalose-forming enzyme to act on the aging starch and introducing a trehalose structure to the reducing end of the starch.
  • the present invention includes a step of allowing a glycosyl trehalose-producing enzyme to act on aging starch, and a step of purifying the obtained starch having a trehalose structure introduced at the reducing end, the purification step comprising
  • the present invention solves the above-mentioned problems by providing a method for producing starchy material, which does not include a step of recovering the starchy precipitate, and has a reduced aging property compared with that before the introduction of the trehalose structure.
  • the present invention solves the above-mentioned problems by providing a starchy aging inhibitor containing a glycosyl trehalose-producing enzyme as an active ingredient.
  • the method for inhibiting aging of starchy material according to the present invention can impart aging resistance to starchy material without significantly changing the structure of individual molecules constituting the starchy material or the molecular weight distribution of the whole starchy material. Even if aging is imparted, the physical properties of the starch can be maintained, and the use of starch in various fields including the food field is expected to be expanded.
  • the present invention reduces the aging property of the starchy substance before the introduction of the trehalose structure by allowing a glycosyl trehalose producing enzyme to act on the aging starchy substance and introducing a trehalose structure at the reducing end of the starchy substance.
  • the present invention provides a method for inhibiting aging of starchy materials.
  • aging in the present specification means a phenomenon in which starch that has been gelatinized or dissolved by heating with water changes into a water-insoluble state over time. Starch aging is observed as cloudiness or solidification of the solution.
  • starch is starch, amylose or amylopectin that constitutes starch, and partially hydrolyzed starch that is partially hydrolyzed by acting an acid or a hydrolase such as ⁇ -amylase or isoamylase on starch, Furthermore, it means a starch partial degradation product in which a branched structure is introduced by allowing a branching enzyme or a glycosyltransferase such as ⁇ -glucosyltransferase to act on the partial starch degradation product thus obtained, or a mixture thereof.
  • the starch quality is not particularly limited depending on its origin and origin, and examples thereof include corn flour, rice flour, wheat flour, barley flour, rye flour, tef flour, millet flour, soybean flour, chickpea flour, pea flour, and mung bean flour.
  • Ground starch such as buckwheat flour, amaranth flour, chestnut flour, acorn flour, banana starch, and underground starch such as tapioca flour, potato flour, potato starch, sweet potato starch, kuzu flour, bracken flour, or the like
  • the starch may be artificially prepared based on one or more starches selected from the following.
  • the starchy substance that is the target of the method for inhibiting aging of starchy substances of the present invention is a starchy substance having an aging property, and the starchy substance having an aging property is introduced with a trehalose structure at its reducing end to some extent.
  • the aging property is improved, that is, the aging property is reduced more than before the introduction of the trehalose structure to the reducing end.
  • amylose has a weight average molecular weight of 3,000 to 3,000.
  • amylopectin Those having a weight average molecular weight of 12,000 to 120,000 are more preferred as amylopectin.
  • amylose and amylopectin are mixed together so that the weight average molecular weight of the whole starch is 3 1 to 120,000 is more suitable.
  • amylose having a weight average molecular weight of less than 3,000 or more than 5,000 the antiaging effect by introduction of a trehalose structure at the reducing end is inferior, but it is not exhibited at all.
  • the weight average molecular weight is 12
  • amylopectin of less than 12,000 and more than 120,000 has an inferior aging inhibitory effect due to the introduction of a trehalose structure at the reducing end, but is not exerted at all. Therefore, an amylose or amylopectin having a weight average molecular weight outside the above range is also obtained. It can be suitably used when the effect of inhibiting aging to be achieved may be relatively low.
  • the aging inhibitory effect by introducing a trehalose structure at the reducing end even when the weight average molecular weight is less than 3,000 or more than 120,000. Is not exhibited at all, but can be suitably used when the required aging inhibitory effect may be relatively low.
  • the aging starch substance targeted by the present invention is usually a starch substance in which the ratio of maltose per solid of ⁇ -amylase digest is 30% by mass or more.
  • ⁇ -amylase is an exo-type enzyme that hydrolyzes starch in maltose units from the non-reducing end, and the hydrolysis reaction stops before the ⁇ -1,6-glycosidically linked branch of starch. . Therefore, the ratio of maltose per solid of ⁇ -amylase digest can be used as an index of the content of the straight chain structure from the non-reducing end of the starch to the branched portion, and starch having a larger value is more linear. It can be used as one index of starch aging because of its high structure content and easy aging.
  • starch having a maltose content of 30% by mass or more per solid of ⁇ -amylase digest is a starch having aging properties, and when a trehalose structure is introduced at the reducing end of such starch, Since the aging inhibitory action is more remarkably exhibited, starch having a maltose content of 30% by mass or more per solid matter of ⁇ -amylase digest is suitable as an object of the aging inhibition method of the present invention. On the other hand, even if the starch content is less than 30% by mass of maltose per solid of ⁇ -amylase digested product, the antiaging effect due to the introduction of the trehalose structure at the reducing end is not exhibited at all. There is no problem in making it an object of the aging inhibiting method of the invention.
  • the glycosyl trehalose-producing enzyme referred to in the present specification is an enzyme to which the enzyme number EC 5.4.99.15 is assigned, and is a reducing end of a glucan having a polymerization degree of 3 or more linked via an ⁇ -1,4 bond. It means an enzyme that introduces a trehalose structure at the reducing end by acting on glucose and converting its binding mode from ⁇ -1,4 linkage to ⁇ , ⁇ -1,1 linkage.
  • the glycosyl trehalose-producing enzyme is not particularly limited as long as it has an activity of introducing a trehalose structure into the reducing end of starch, and for example, Actinoplanes, Arthrobacter (Arthrobacter) ), Bradyrhizobium, Brevibacterium, Corynebacterium, Curtobacterium, Flavobacteria, et al.
  • Mycoba It is selected from the group consisting of Thecobacterium, Rhizobium, Sulfolobus, Terrabacter, Anabena (Anabaena), and Nostock (Nostoc, Algae) It may be a glycosyl trehalose producing enzyme derived from the microorganism to which it belongs, or a variant thereof, or a glycosyl trehalose producing enzyme obtained by genetic recombination.
  • glycosyl derived from a microorganism belonging to any genus selected from Arthrobacter, Brevibacterium, Kurtobacterium, Flavobacterium, Micrococcus, Mycobacterium, Rhizobium, Sulfolobus, and Terrabacter Trehalose-producing enzyme or a variant thereof is preferred, and among them, Arthrobacter species S34-derived glycosyl trehalose-producing enzyme disclosed in Japanese Patent No. 3958884, Arthrobacter disclosed in Japanese Patent No. 4249055 ⁇ Species Q34, Rhizobium sp.
  • the activity of the glycosyl trehalose producing enzyme can be determined by, for example, the method described in paragraph 0026 of Japanese Patent No. 3958884, that is, introduction of a trehalose structure at the reducing end by the action of glycosyl trehalose producing enzyme using maltopentaose as a substrate. It can be measured by a method of measuring the reduction in reducing power accompanying the.
  • 1 unit of activity of the glycosyl trehalose-producing enzyme is the amount of enzyme that reduces the reducing power corresponding to 1 ⁇ mol of maltopentaose per minute in the above activity assay method.
  • the ratio of the trehalose structure introduced into the reducing end of the starch due to the action of the glycosyl trehalose producing enzyme (hereinafter referred to as “trehalose structure introduction rate”) can be estimated based on the following formula 1. That is, a starch substance having a trehalose structure introduced at the reducing end is analyzed by size exclusion chromatography or the like, its number average molecular weight is obtained, and separately, a glucoamylase digest obtained by completely acting glucoamylase on the starch substance Is analyzed by high performance liquid chromatography or the like, and the mass ratio of trehalose per solid content of the digested glucoamylase is determined.
  • the number average molecular weight of the starch having a trehalose structure introduced at the reducing end is multiplied by the ratio of trehalose per solid product of the glucoamylase digest, and the value obtained by dividing by 342 which is the molecular weight of trehalose is trehalose. It can be regarded as the structure introduction rate.
  • the trehalose structure introduction rate of the starchy substance on which the glycosyl trehalose-producing enzyme is acted is desirably 20% or more, more desirably 30% or more, and further desirably 40% or more.
  • the amount of action of the glycosyl trehalose producing enzyme is not particularly limited, and is usually selected appropriately within a range of 0.1 to 100 units per 1 g of starch solid.
  • the temperature and pH at which the glycosyl trehalose-producing enzyme is allowed to act can be appropriately selected within the range in which the enzymatic reaction proceeds.
  • the reaction temperature is preferably 10 to 90 ° C.
  • the reaction pH is preferably in the range of pH 3 to 9. It is.
  • the reaction time can be appropriately selected according to the progress of the enzyme reaction, and may be selected from a range of 0.1 to 100 hours, for example.
  • trehalose structure introduced at the reducing end is not lost, for the purpose of further reducing aging with the glycosyl trehalose synthase or after acting the glycosyl trehalose synthase, It is also optional to allow an enzyme that introduces a branched structure to act on starch such as ⁇ -glucosyltransferase.
  • the present invention includes a step of allowing a glycosyl trehalose-producing enzyme to act on aging starch, and a step of purifying the obtained starch having a trehalose structure introduced at the reducing end, the purification step comprising
  • the present invention provides a method for producing starchy material, which does not include a step of recovering the starchy precipitate, and has a reduced aging property compared with that before the introduction of the trehalose structure.
  • the step of purifying does not include the step of recovering the starchy precipitate means that the aging-reduced starchy material obtained by allowing glycosyl trehalose-forming enzyme to act on the agingy starchy product is purified. This means that it is not necessary to recover the starchy precipitate by aging or precipitating the object by adding an organic solvent.
  • the starchy substance obtained by introducing the trehalose structure at the reducing end obtained by the present invention is less susceptible to aging than ordinary starchy substance, but the other physical properties are the same as the starchy substance before modification, so that the aging property is reduced. It can be suitably used as a substitute for ordinary starch that is not used. In addition, it is optional to use the mixture by mixing with ordinary starch which has not been reduced in aging property. Furthermore, since the aging property of the starch substance introduced with a trehalose structure at the reducing end obtained in the present invention is reduced, it is suitably used even in products in which the use of the starch substance is restricted due to aging property. can do.
  • the starchy substance obtained by introducing the trehalose structure at the reducing end obtained by the present invention has higher aging resistance than the starchy substance before introducing the trehalose structure, and the starchy substance before introducing the trehalose structure originally has. Since the physical properties other than the aging properties are maintained, it can be used as a substitute for ordinary starch or mixed with ordinary starch. For example, energy supply, richness, and consistency Food (for example, retort foods, frozen foods) for the purpose of imparting properties, gloss, improving shape retention, preventing freezing denaturation, improving texture, improving taste quality, preventing water activity, preventing crystal precipitation, imparting moisture retention, etc.
  • energy supply, richness, and consistency Food for example, retort foods, frozen foods
  • starch having a trehalose structure introduced at the reducing end obtained by the present invention is more soluble than ordinary starch
  • various liquids for example, soup, soup, sauce, soy sauce, vinegar, miso, fats and oils
  • Amylose has the property of forming a helical structure in solution, and it is known to form inclusion complexes by incorporating various hydrophobic low-molecular substances into the helical cavity. Because it is easy to age, its range of use has been limited. Amylose introduced with a trehalose structure at the reducing end obtained by the present invention is not significantly different from ordinary amylose in structure, and therefore has a helical forming ability equivalent to that of ordinary amylose, and moreover than ordinary amylose.
  • insoluble or poorly water-soluble pharmaceutical ingredient functional food ingredient, solubilizers and stabilizers such as oils, pigments, and fragrances, masking agents, sustained-release agents, and powdered substrates. it can. Further, it is optional to use as a substitute for ordinary amylose or mixed with ordinary amylose. Therefore, according to the present invention, a stabilizer, masking agent, sustained-release agent, or powdered base material containing amylose having a trehalose structure introduced at the reducing end as an active ingredient is provided.
  • Amylose as a typical example of aging starch may be amylose obtained by any production method.
  • isoamylase (EC 3.2.1.68) or pullulanase (EC) is added to starch.
  • Amylose obtained by acting a starch debranching enzyme such as 3.2.1.41 amylose obtained by allowing cyclomaltodextrin glucanotransferase (EC 2.4.1.19) to act on cyclodextrin
  • the present invention provides a starch aging inhibitor containing glycosyl trehalose-producing enzyme as an active ingredient.
  • the glycosyl trehalose-generating enzyme acts on a glucan having a polymerization degree of 3 or more linked through ⁇ -1,4 bonds, thereby changing the binding mode of glucose residues at the reducing end from ⁇ -1,4 bonds to ⁇ , ⁇ . Since it can be converted into a 1,1 bond and a trehalose structure can be introduced at the reducing end, and the starch having the trehalose structure introduced at the reducing end is difficult to age, the enzyme itself inhibits starch aging. Can be used as
  • the starchy aging inhibitor of the present invention can be blended with other components according to the purpose within a range in which the activity of the active ingredient glycosyl trehalose producing enzyme is stably maintained.
  • acids, bases, salts, carbohydrates, amino acids, proteins, etc. may be added.
  • the form as an aging inhibitor may be powder, granules, tablets, liquids and the like.
  • Amylose having a trehalose structure introduced at the reducing end was prepared by allowing a glycosyl trehalose producing enzyme to act on amylose and introducing a trehalose structure at the reducing end.
  • a reagent grade short chain amylose powder having a weight average molecular weight of 3,200 (trade name “Amylose EX-I”, manufactured by Hayashibara Co., Ltd., hereinafter referred to as “amylose 1”), and a weight average molecular weight of 5,000.
  • Amylose powder (prepared by Hayashibara Co., Ltd., hereinafter referred to as “amylose 2”) was dissolved in pure water so as to have a solid concentration of 1% by mass, adjusted to pH 5.8, and then patent No. 3958888. 2 units of glycosyl trehalose-producing enzyme derived from Arthrobacter sp. S34 obtained by the method described in Example 2-2 of the publication No.
  • amylose 1 and 2 used as a raw material and amylose 1 and 2 having a trehalose structure introduced at the reducing end, the weight average molecular weight, reducing power, ratio of maltose per solid of ⁇ -amylase digest, and The solubility was measured respectively. The results are shown in Table 1. Various analyzes were performed by the following methods.
  • ⁇ Weight average molecular weight> Amylose or amylose having a trehalose structure introduced at the reducing end was dissolved so as to have a solid concentration of 1% by mass, adjusted to pH 7.0, and then subjected to size exclusion chromatography. The weight average molecular weight was calculated based on a calibration curve prepared by subjecting a pullulan standard product for molecular weight measurement (produced by Hayashibara Co., Ltd.) to the same analysis.
  • For size exclusion chromatography two columns of “TSK GEL ⁇ -M” (manufactured by Tosoh Corporation) were connected to the column, and 10 mM phosphate buffer (pH 7.0) was used as the eluent. Detection was performed using a differential refractometer “RID-10A” (manufactured by Shimadzu Corporation) under conditions of a temperature of 40 ° C. and a flow rate of 0.3 ml / min.
  • Amylose and amylose having a trehalose structure introduced at the reducing end were each subjected to the anthrone sulfate method, and the total amount of sugar was measured based on a calibration curve prepared using glucose as a standard carbohydrate. Subsequently, amylose and amylose having a trehalose structure introduced at the reducing end were each subjected to the Somogy-Nelson method, and the amount of reducing sugar was measured based on a calibration curve prepared using glucose as a standard carbohydrate. And the ratio of the amount of reducing sugars with respect to the total amount of sugars of amylose and the amylose in which the trehalose structure was introduce
  • ⁇ Ratio of maltose per solid of ⁇ -amylase digest After dissolving amylose to a solid concentration of 1% by mass and adjusting to pH 5.0, add 50 units of ⁇ -amylase (trade name “# 1500”, manufactured by Nagase ChemteX Corporation) per gram of solid. After acting at 50 ° C. for 20 hours, the enzyme reaction was stopped by heating at 100 ° C. for 10 minutes. Next, the obtained ⁇ -amylase digest was subjected to high-performance liquid chromatography, and the ratio of the maltose peak area to the total peak area of the chromatogram was determined to obtain the ratio of maltose per solid of the ⁇ -amylase digest.
  • the high performance liquid chromatography uses two MCI gel CK04SS (Mitsubishi Chemical Corporation) connected to the column, ultrapure water as the eluent, column temperature of 80 ° C., flow rate of 0.4 ml. Detection was performed using a differential refractometer “RID-10A” (manufactured by Shimadzu Corporation).
  • amylose 1 and 2 used as raw materials had a reducing power of 6.0% and 3.8%, respectively, whereas amylose 1 and trehalose structure introduced at the reducing end.
  • No. 2 had a reducing power of 0.1% and 0.2%, respectively, and showed substantially no reducing power. Therefore, amylose 1 and 2 in which a trehalose structure was introduced at the reducing end were introduced with a trehalose structure. The rate was confirmed to be substantially 100%.
  • amylose 1 and 2 had a solubility in 100 g of water of 4.5 g and 1.7 g, respectively, whereas amylose 1 and 2 in which a trehalose structure was introduced at the reducing end had a solubility in 100 g of water. Since they were 7.4 g and 4.6 g, respectively, the solubility of amylose having a trehalose structure introduced at the reducing end was improved 1.6 to 2.7 times that of amylose used as a raw material.
  • the trehalose structure was introduced at the reducing end.
  • the solubility of amylose 1 and 2 in 100 g of a 1% by mass trehalose aqueous solution was 4.8 g and 1.5 g, respectively, which was equivalent to the solubility in 100 g of water, respectively, so simply add free trehalose to amylose.
  • the effect of improving the solubility of amylose 1 and 2 was not observed. From the above, it was concluded that the solubility is improved by introducing a trehalose structure at the reducing end of amylose.
  • Example 1-2 Evaluation of aging ability of amylose having a trehalose structure introduced at the reducing end> The aging property of the amylose having a trehalose structure introduced at the reducing end obtained in Experiment 1-1 was examined, and the influence of the introduction of the trehalose structure at the reducing end of amylose on the aging property was evaluated.
  • Amylose 1 used in Experiment 1-1 Amylose 1 with a trehalose structure introduced at the reducing end obtained in Experiment 1-1, and the ratios of 20:80, 40:60, 60:40, and 80:20
  • Each of the mixtures mixed in (1) was suspended in pure water so as to have a solid concentration of 1% by mass, dissolved by heating at 100 ° C. for 10 minutes, and then stored at 4 ° C.
  • the turbidity (absorbance at a wavelength of 650 nm) of the solution was measured with a spectrophotometer (Shimadzu Corporation).
  • amylose 1 solution showed an increase in turbidity of more than 2.0 after 1 day, that is, 24 hours after storage, whereas amylose having a trehalose structure introduced at the reducing end. 1 took 4 days (96 hours) to show turbidity of more than 2.0, and the rate of aging had dropped to a maximum of 1/4. Further, even when the mixing ratio of amylose 1 having a trehalose structure introduced at the reducing end was 20%, the increase in turbidity was suppressed as seen in the measured values after 2 hours and 4 hours. As a whole, the higher the mixing ratio of amylose 1 having a trehalose structure introduced at the reducing end, the greater the decrease in aging rate was observed.
  • amylose 2 solution showed an increase in turbidity of more than 2.0 at 1 hour after storage, whereas amylose 2 having a trehalose structure introduced at the reducing end was turbid. It took one day (24 hours) to show a degree of over 2.0, and the rate of aging had dropped to 1/24 at the maximum.
  • the mixing ratio of amylose 2 having a trehalose structure introduced at the reducing end is 20%, the increase in turbidity is suppressed as seen in the measured value after 1 hour, and the trehalose structure is present at the reducing end. The higher the mixing ratio of amylose 2 introduced, the greater the decrease in aging rate was observed.
  • the trehalose structure introduction rate is substantially 100%, when the mixing ratio of the two is at least 20% or more, aging is suppressed. This means that if a trehalose structure is introduced at the reducing end of amylose at the above trehalose structure introduction rate, aging of starch can be suppressed.
  • amyloses 1 and 2 having a trehalose structure introduced at the reducing end do not have a trehalose structure introduced at the reducing end, whereas starch having a larger weight average molecular weight is more likely to age.
  • the weight average molecular weights were slightly larger than the control amyloses 1 and 2, the aging rate was lowered. This result clearly shows that the remarkable aging resistance improvement effect according to the present invention is not brought about by the decrease in molecular weight, but is brought about by introducing a trehalose structure at the reducing end of amylose. Is.
  • Experiment 2 Effect of introduction of trehalose structure on reducing end of amylopectin with different weight average molecular weight on aging>
  • a trehalose structure was introduced into the reducing end of a partially decomposed product of waxy corn starch consisting essentially of amylopectin, Aging was evaluated.
  • each of the obtained solutions was stored at 4 ° C., and the turbidity (absorbance at a wavelength of 650 nm) of the solution was measured at the start of storage, 1 day, 2 days, 4 days, 8 days, 16 days, and 32 days. Each was measured with a spectrophotometer (manufactured by Shimadzu Corporation), and the amount of increase in turbidity from the start of storage was determined.
  • a spectrophotometer manufactured by Shimadzu Corporation
  • amylopectin 1 to 4 treated in the same manner except that a glycosyl trehalose producing enzyme was not added, The amount of increase in turbidity was determined.
  • Table 4 In the case where the amount of increase in turbidity exceeded 2.0, it was indicated as “over 2.0” in the table, and the turbidity was not measured after that, and indicated by hatching in the table.
  • amylopectin 4 which has a relatively low molecular weight of 2,660 and inherently low aging properties, the introduction of a trehalose structure at the reducing end is slight. Has been confirmed to exhibit an anti-aging effect.
  • amylopectin 1 having a weight average molecular weight of 118,000 showed aging that reached turbidity exceeding 2.0 when stored at 4 ° C. for 4 days, whereas amylopectin with glycosyl trehalose synthase acted on. It took 8 days for turbidity to reach over 2.0, and aging was clearly suppressed.
  • amylopectin 2 having a weight average molecular weight of 23,400 reached a turbidity of more than 2.0 when stored at 4 ° C. for 2 days
  • amylopectin 2 treated with glycosyl trehalose producing enzyme was cloudy. It took 8 days for the degree to reach over 2.0
  • amylopectin 3 having a weight average molecular weight of 12,100 showed a turbidity of 0.6 when stored at 4 ° C. for 32 days
  • Amylopectin 3 on which glycosyl trehalose-producing enzyme was allowed to act did not show an increase in turbidity even when stored for 32 days, and in each case, a remarkable aging inhibitory effect was confirmed.
  • Experiment 3 Effect of introduction of trehalose structure on reducing end of mixture of amylose and amylopectin on aging>
  • Experiments 1 and 2 it was found that aging can be suppressed by introducing a trehalose structure at the reducing end of amylose and amylopectin. Therefore, in Experiment 3, in order to investigate the influence of the introduction of the trehalose structure at the reducing end on the aging of the mixture of amylose and amylopectin, a partial degradation product of tapioca starch containing amylose and amylopectin in a ratio of about 1: 4 was used. The trehalose structure was introduced into the reducing end and the aging property was evaluated.
  • tapioca starch was suspended in pure water so as to have a solid concentration of 30% by mass, and calcium chloride was added thereto so as to have a final concentration of 1 mM, and then adjusted to pH 6.0 to prepare a starch suspension.
  • 10 units of ⁇ -amylase (trade name “Spitase HK”, manufactured by Nagase ChemteX Corporation) is added to the obtained starch suspension per gram of the solid, and the solution is passed through the continuous liquefier at a flow rate of 1 L / min.
  • the enzyme reaction was stopped by heating at 100 ° C. for 25 minutes and then at 140 ° C. for 5 minutes to obtain a solution of partially decomposed starch 1.
  • ⁇ -amylase (trade name “Chrytase E5C”, manufactured by Nagase ChemteX Corporation) was added to the resulting solution of partially decomposed starch 1 at 0.01, 0.02, 0.05 per gram of solid. , 0.1, 0.2, 0.5, or 1.0 unit was added and reacted at 50 ° C. for 22 hours, and then the enzyme reaction was stopped by heating at 100 ° C. for 20 minutes. 2 to 8 solutions were obtained. Thereafter, the obtained solutions of partially decomposed starches 1 to 8 were decolorized using activated carbon, desalted using an ion exchange resin, and then freeze-dried to obtain a powder. With respect to the obtained powders of partially decomposed starches 1 to 8, the weight average molecular weight and the ratio of maltose per ⁇ -amylase digested solid were determined in the same manner as in Experiment 1-1.
  • the obtained partially decomposed starches 1 to 8 were dissolved in pure water so as to have a solid concentration of 30% by mass, and 2 units of glycosyl trehalose-forming enzyme used in Experiment 1 was added per 1 g of the solid, and 22 ° C. at 22 ° C. After acting for a period of time, the enzyme reaction was stopped by heat treatment at 100 ° C. for 10 minutes. Thereafter, the sample was stored at 4 ° C., and after 30 minutes, 1 hour, 2 hours, and 3 days, the aging property was visually evaluated based on the criteria shown below.
  • the weight average molecular weight is 45,300 to 49 by the action of glycosyl trehalose producing enzyme.
  • the starch partial degradation products 7 and 8 having a weight average molecular weight of less than 15,000 did not change even when stored for 3 days regardless of whether or not the glycosyl trehalose-producing enzyme was allowed to act, and no aging was observed.
  • the molecular weight distribution was confirmed by size exclusion chromatography in the same manner as in Experiment 1-1, there was no significant change in the molecular weight distribution of the partial starch degradation products 1 to 8 before and after the action of the glycosyl trehalose synthase.
  • the partially decomposed starches 1 to 6 in which aging was confirmed to be inhibited by introducing a trehalose structure at the reducing end by the action of a glycosyl trehalose producing enzyme are maltose per solid of ⁇ -amylase digested product.
  • the ratio was 56.5 to 59.2% by mass.
  • amylopectin 1 to 3 and starch partial degradation products 1 to 6 on which glycosyl trehalose-forming enzyme is acted are dissolved in deionized pure water so as to have a solid concentration of 1% by mass, subjected to size exclusion chromatography, and molecular weight measurement.
  • the number average molecular weight was calculated on the basis of a calibration curve prepared by subjecting pullulan standard products (manufactured by Hayashibara Co., Ltd.) to analysis in the same manner.
  • pullulan standard products manufactured by Hayashibara Co., Ltd.
  • For size exclusion chromatography two TSK GEL ⁇ -M (manufactured by Tosoh Corporation) were connected to the column, and 10 mM phosphate buffer (pH 7.0) was used as the eluent.
  • Detection was performed using a differential refractometer “RID-10A” (manufactured by Shimadzu Corporation) under conditions of a temperature of 40 ° C
  • glucoamylase (trade name “Glucoteam # 20000”, manufactured by Nagase ChemteX) is added to a solution having a solid concentration of 1% by mass of amylopectins 1 to 3 and starch partial degradation products 1 to 6 with glycosyl trehalose-producing enzyme.
  • Glucoamylase (trade name “Glucoteam # 20000”, manufactured by Nagase ChemteX) is added to a solution having a solid concentration of 1% by mass of amylopectins 1 to 3 and starch partial degradation products 1 to 6 with glycosyl trehalose-producing enzyme.
  • Glucoamylase (trade name “Glucoteam # 20000”, manufactured by Nagase ChemteX) is added to a solution having a solid concentration of 1% by mass of amylopectins 1 to 3 and starch partial degradation products 1 to 6 with glycosyl trehalose-producing enzyme.
  • the obtained glucoamylase digest was analyzed by high performance liquid chromatography, the ratio of the peak area of trehalose to the total peak area of the chromatogram was determined, and the ratio of trehalose per solid of the glucoamylase digest was determined.
  • the high performance liquid chromatography uses two MCI gel CK04SS (Mitsubishi Chemical Corporation) connected as a column, ultrapure water as the eluent, column temperature of 80 ° C., flow rate of 0.4 ml. Detection was performed using a differential refractometer “RID-10A” (manufactured by Shimadzu Corporation).
  • the trehalose structure introduction rate was determined according to the above formula 1. That is, the trehalose structure is obtained by multiplying the number average molecular weight of the partially decomposed starch obtained by acting the glycosyl trehalose-forming enzyme by the ratio of the mass of trehalose per solid product of the glucoamylase digested product and dividing by the molecular weight 342 of trehalose. The introduction rate was calculated. The results are shown in Table 6.
  • the starch partially decomposed products 6 to 8 prepared in Experiment 3 were each dissolved in pure water so that the solid concentration was 30% by mass, and then adjusted to pH 5.8, and Example 2-2 of Japanese Patent No. 3958888 was disclosed.
  • 2 units of glycosyl trehalose-producing enzyme derived from Arthrobacter sp. S34 obtained by the described method per gram of solid and 400 units of isoamylase (manufactured by Hayashibara Co., Ltd.) per gram of solid are added, and 48 at 50 ° C. It was allowed to act for hours.
  • the resulting reaction solution was heated at 100 ° C.
  • the obtained starch partially decomposed products A to C containing amylose having a trehalose structure introduced at the reducing end have a weight average molecular weight of 2,350 to 5,020, and have remarkable aging resistance. It was what had.
  • the partially decomposed starches A to C containing amylose having a trehalose structure introduced at the reducing end did not become aged and insolubilized. There was no need to go through the steps, and it could be produced by an extremely simpler method than ordinary amylose.
  • the powders of the partially decomposed starches A to C containing amylose having a trehalose structure introduced at the reducing end thus obtained are less likely to age than ordinary amylose when dissolved in water, they are insolubilized by aging.
  • a solubilizer and stabilizer for compounds such as insoluble or poorly water-soluble drugs, functional food ingredients, oils, dyes, and fragrances, masking agents, sustained-release agents, and powdered base materials It can be suitably used. Further, it is optional to use as a substitute for ordinary amylose or mixed with ordinary amylose.
  • a method described in Example 2-2 of Japanese Patent No. 3958884 is prepared by dissolving the powder of partially decomposed starch 6 prepared in Experiment 3 in pure water so as to have a solid concentration of 30% by mass, and then adjusting the pH to 5.8. 2 units of the glycosyl trehalose-producing enzyme derived from Arthrobacter species S34 obtained in 1 above was added and allowed to act at 50 ° C. for 48 hours. The resulting reaction solution was heated at 100 ° C.
  • the powder of the partially decomposed starch obtained as described above has a trehalose structure introduced at the reducing end, the aging property is remarkably reduced, or a substitute for the partially decomposed starch, or In addition, it is optional to use it by mixing with a normal starch partial decomposition product.
  • ⁇ Starch aging inhibitor> The glycosyl trehalose-producing enzyme derived from Arthrobacter species S34 obtained by the method described in Example 2-2 of Japanese Patent No. 3958884 is dialyzed against a 2% by mass trehalose aqueous solution, and then to a protein concentration of 4 mg / ml. After concentration, freeze-drying was performed to obtain a glycosyl trehalose-producing enzyme powder.
  • This product has an enzyme activity of about 10,000 units per gram, and can be suitably used as a starch aging inhibitor.
  • This product has a trehalose structure at the reducing end of the starch due to the action of glycosyl trehalose synthase, which is an active ingredient of aging inhibitors. It is a high quality sashimi that does not generate any other problems and retains its softness immediately after preparation.
  • the structure of starch and the molecular weight distribution as a whole are increased by allowing a glycosyl trehalose producing enzyme to act on aging starch and introducing a trehalose structure at the reducing end.
  • the aging can be suppressed without changing.
  • the starch material into which the trehalose structure obtained in the present invention has been introduced can be used as a starch material with reduced aging properties, easily replacing the existing starch material.
  • the use of the starch material is expanded to products in which the use of the starch material is restricted due to aging property. can do.
  • the present invention is a truly significant invention that makes a great contribution to the world.

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Abstract

La présente invention vise à créer un procédé permettant d'éliminer le vieillissement de l'amidon, en ne modifiant aucune propriété physique autre que les propriétés de vieillissement de l'amidon, plus spécifiquement, sans modifier de manière significative la structure de l'amidon. La présente invention atteint cet objectif en créant un procédé d'élimination du vieillissement de l'amidon caractérisé par les étapes consistant à amener une enzyme produisant du glycosyl-tréhalose à agir sur un amidon qui présente des propriétés de vieillissement, et à introduire une structure de tréhalose dans une extrémité réductrice de l'amidon, ce qui permet de réduire les propriétés de vieillissement de l'amidon par comparaison avec l'état de ces propriétés avant l'introduction de la structure de tréhalose.
PCT/JP2016/052529 2015-01-30 2016-01-28 Procédé d'élimination du vieillissement de l'amidon WO2016121885A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07143876A (ja) * 1992-12-28 1995-06-06 Hayashibara Biochem Lab Inc 非還元性糖質生成酵素とその製造方法並びに用途
JPH08134104A (ja) * 1994-09-13 1996-05-28 Ezaki Glico Co Ltd 環状構造を有するグルカンおよびその製造方法
JP2006288218A (ja) * 2005-04-06 2006-10-26 Ajinomoto Co Inc 麺類の製造方法
JP2006312705A (ja) * 2005-04-08 2006-11-16 Hayashibara Biochem Lab Inc 分岐澱粉とその製造方法並びに用途

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CN100352920C (zh) * 1993-09-30 2007-12-05 株式会社林原生物化学研究所 非还原性糖类生成酶及其制备和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07143876A (ja) * 1992-12-28 1995-06-06 Hayashibara Biochem Lab Inc 非還元性糖質生成酵素とその製造方法並びに用途
JPH08134104A (ja) * 1994-09-13 1996-05-28 Ezaki Glico Co Ltd 環状構造を有するグルカンおよびその製造方法
JP2006288218A (ja) * 2005-04-06 2006-10-26 Ajinomoto Co Inc 麺類の製造方法
JP2006312705A (ja) * 2005-04-08 2006-11-16 Hayashibara Biochem Lab Inc 分岐澱粉とその製造方法並びに用途

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