JP2010100583A - Lipid metabolism improver - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lipid metabolism improver which is safe even when ingested for a long period. <P>SOLUTION: This lipid metabolism improver, being α-glucan which comprises glucose as sugar of constituent, contains branched α-glucan having the following characteristics in a methylation analysis: (1) a ratio of 2,3,6-trimethyl-1,4,5-triacetyl glucitol to 2,3,4-trimethyl-1,5,6-triacetyl glucitol is in the range of 1:0.6 to 1:4; (2) the sum total of 2,3,6-trimethyl-1,4,5-triacetyl glucitol and 2,3,4-trimethyl-1,5,6-triacetyl glucitol accounts for 60% or more of partially methylated glucitol acetate; (3) 2,4,6-trimethyl-1,3,5-triacetyl glucitol is in the range of from more than 0.5% to less than 10% of partially methylated glucitol acetate; and (4) 2,4-dimethyl-1,3,5,6-tetraacetyl glucitol accounts for 0.5% or more of partially methylated glucitol acetate. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to an agent for improving lipid metabolism comprising a branched α-glucan as an active ingredient, and in particular, α-glucan containing glucose as a constituent sugar,
(1) The ratio of 2,3,6-trimethyl-1,4,5-triacetylglucitol to 2,3,4-trimethyl-1,5,6-triacetylglucitol is 1: 0.6 To 1: 4.
(2) The sum of 2,3,6-trimethyl-1,4,5-triacetylglucitol and 2,3,4-trimethyl-1,5,6-triacetylglucitol is a partially methylated group. Occupies more than 60% of the citrate acetate;
(3) 2,4,6-trimethyl-1,3,5-triacetylglucitol is 0.5% or more and less than 10% of partially methylated glucitol acetate; and (4) 2,4- Dimethyl-1,3,5,6-tetraacetylglucitol is 0.5% or more of partially methylated glucitol acetate;
A branched α-glucan that acts on an α-1,4 glucan having a degree of polymerization of maltose and glucose of 3 or more and undergoes α-glucosyl transfer to produce the branched α-glucan. The present invention relates to a lipid metabolism improving agent comprising glucan.

  In recent years, with the westernization of dietary habits and lifestyle habits, the chances of ingesting high-calorie, high-fat diets have increased, and hyperlipidemia, and overweight people who have exceeded obese or overweight have increased. Yes. In addition to obesity and overweight, when there are factors of lifestyle-related diseases such as hypertension and hyperlipidemia, it is called metabolic syndrome, and if the condition continues, cardiovascular such as myocardial infarction, stroke, type II diabetes It is said that there is an increased risk of developing sexually transmitted diseases, and it is increasingly important to control lipid metabolism to avoid these. Low calorie or low fat regimens are known as methods generally proposed by dietitians to improve lipid metabolism and prevent and / or prevent overweight. In addition, foods appealing for lipid metabolism improvement are also commercially available (see, for example, Patent Document 1). However, a low-calorie diet may be rejected by the person over a long period of time due to its monotonous flavor. In addition, even if there are health supplements, there are problems with their taste and texture, and when these cannot be continued for a long period of time, the rebound will occur, rather than before starting these therapies. It can even be observed if lipid metabolism is aggravated or health is impaired.

  In recent years, dietary fiber has attracted attention as a food material for preventing or improving hyperlipidemia and metabolic syndrome. Dietary fiber originally means plant cell components such as cellulose, lignin, hemicellulose, pectin that are difficult for animals to digest, but in a broad sense also includes indigestible water-soluble polysaccharides that are not digested by amylase. It is called water-soluble dietary fiber. In recent years, dietary fiber has been attracting attention as a prebiotic function for improving intestinal flora, in addition to the intestinal regulating action, blood cholesterol lowering action, blood sugar regulating action and the like as its original functions. However, dietary fiber is said to be a nutrient lacking in the Japanese diet along with calcium, and the current average dietary fiber intake of Japanese is the 5th issue published in 1994. It has been pointed out that the target intake of 20 to 25 g / day of dietary fiber shown in the revised “Japanese nutritional requirements” has reached only 50 to 80% of the target (for example, non-patent literature) 1 etc.). Under such circumstances, various indigestible polysaccharides that can be used as raw materials for various foods and drinks and are useful as water-soluble dietary fibers have been proposed.

  For example, water-soluble dietary fibers that are made from naturally occurring polysaccharides such as resistant starch (wet heat-treated high amylose corn starch), guar gum degradation products, glucomannan, and low-molecular alginic acid are on the market. However, since these are all highly viscous and have disadvantages such as the loss of flavor and texture when added to foods, their use is limited to a part. On the other hand, as a low-viscosity water-soluble dietary fiber, polydextrose (developed by Pfizer, USA) and indigestible dextrin are widely used in the food field. Polydextrose is a synthetic polysaccharide obtained by heating glucose, sorbitol and citric acid under high vacuum and polymerizing them by chemical reaction, and glucose is 1, 2, 1, 3, 1, 4, 1, 6 , 1, 2, 6, 1, 4, 6 and the like are known to have complex branches with glucoside bonds. Indigestible dextrin decomposes starch by a chemical reaction, and at the same time causes a transfer or reverse synthesis reaction. 1,2,1,3,1,2,4,1, It is a synthetic polysaccharide with reduced digestibility by introducing 3,4 glucoside bonds. This indigestible dextrin has a low viscosity obtained by adding a small amount of hydrochloric acid to starch, dissolving roasted dextrin obtained by heating in a powdered state in water, and adding α-amylase to hydrolyze. Manufactured by purifying, concentrating and spray drying the solution. For indigestible dextrin, for the purpose of further reducing digestibility, glucoamylase is added to break down the digestible part into glucose, and glucose is separated and removed in the same way. is there. The new glucoside bond introduced into these polydextrose and indigestible dextrin includes both α- and β-anomeric forms, and the reducing terminal glucose residue is partially changed to 1,6-anhydro-glucose. (For example, refer nonpatent literature 2 etc.). However, indigestible dextrin has a low yield from the raw material starch and, in addition, is easily colored, which is a major drawback in industrial production. In addition, polydextrose and indigestible dextrin may not be able to obtain a sufficient lipid metabolism improving effect by themselves.

  Of the glucoside bonds (hereinafter referred to as “glucoside bonds” in the present specification, which are simply referred to as “bonds”), the α-1,6 bonds are compared to the α-1,4 bonds. Since it is difficult to be decomposed by amylase, a glucan containing a lot of α-1,6 bonds can be expected to be used as a water-soluble dietary fiber. For example, dextran produced from sucrose as a raw material by dextransclase (EC 2.4.1.5) derived from Leuconostoc mesenteroides belonging to lactic acid bacteria is mainly composed of α-1,6 glucose. It may be a polymerized glucan and may have α-1,2 bond and α-1,3 bond branches. When the dextransclase derived from Leuconostoc mesenteroides B-512F strain is used, the content of α-1,6 linkages in the obtained dextran is 90% or more, which is expected to be indigestible. . However, since dextran has a low yield from sucrose and has a high viscosity, the purification operation is complicated and the cost is high, so that attempts to use it as a water-soluble dietary fiber have hardly been made.

  Attempts have also been made to prepare water-soluble dietary fiber by increasing the content of α-1,6 bonds by allowing amylase to act on inexpensive starch and mainly breaking down α-1,4 bonds. In Patent Document 2, a mixture of α-amylase and β-amylase is allowed to act on a starch liquefaction liquid, and then the remaining dextrin part is recovered, whereby α-1,6 bonds to α-1,4 bonds are recovered. A method for preparing a branched dextrin with a proportion increased to 10 to 20% has been proposed. However, this branched dextrin retains the original branch (α-1,6 bond) of starch while removing α-1,6 bond by removing the linear part where glucose is linked by α-1,4 bond. Since it is manufactured by a method of increasing the ratio, there are problems that the yield from the raw starch is low and a significant reduction in digestibility cannot be expected. In addition, dextrin dextranase (EC 2.1.1.2) is known as an enzyme that acts on a partially degraded starch (dextrin) and introduces α-1,6 bonds (for example, Non-Patent Document 3). See). Dextrin dextranase is an enzyme that acts on a partial degradation product of starch and generates a dextran structure (structure in which glucose is linked by α-1,6 bonds) mainly by catalyzing the α-1,6 glucosyl transfer reaction. However, conventionally known dextrin dextranase derived from Acetobacter capsultatum belonging to acetic acid bacteria has a low introduction ratio of α-1,6 bond (for example, Non-Patent Document 4) And the stability of the enzyme itself is low, and it has not been used in practice. Under such circumstances, a lipid metabolism-improving agent comprising a novel indigestible glucan (dietary fiber) that can solve the above-mentioned physical problems of conventional indigestible glucan and has an excellent lipid metabolism improving action. The provision of is strongly desired.

JP 2004-315379 A JP 2001-11101 A “Exploring dietary fiber market trends”, “Food and Development”, Vol. 34, No. 2, pp. 24 to 27 (1999) “Low-molecular water-soluble dietary fiber”, food ingredient series “Science of dietary fiber”, pages 116 to 131, Asakura Shoten (1997) Kazuya Yamamoto et al., “Bioscience / Biotechnology / Biochemistry”, Volume 56 (1992), pp. 169 to 173 Masayuki Suzuki et al., “Journal of Applied Glycoscience”, Vol. 48, No. 2, pages 143 to 151 (2001)

  An object of the present invention is to provide a lipid metabolism improving agent containing dietary fiber that is safe even if ingested for a long period of time.

  In order to solve the above problems, the present inventors use maltose and / or α-1,4 glucan having a degree of glucose polymerization of 3 or more as a raw material (substrate), and branch (in the present specification, “branch” means: A branched α-glucan having a relatively large number of glucose binding modes other than α-1,4 bonds among glucose binding modes in glucan was widely searched. As a result, when a novel α-glucosyltransferase produced by microorganisms isolated from soil, PP710 strain and PP349 strain is allowed to act on maltose and / or α-1,4 glucan having a glucose polymerization degree of 3 or more A branched α-glucan having α-1,4, α-1,6, α-1,3, α-1,4,6 and α-1,3,6 bonds having the following characteristics: Compared with α-1,4 glucan, the proportion of α-1,6 bonds is greatly increased, and α-1,3 and α-1,3,6 bonds are present. It has been found that a water-soluble dietary fiber exhibiting properties and a composition containing the same is useful as an agent for improving lipid metabolism. Furthermore, the branched α-glucan is unexpectedly taken in the form of a liquid composition rather than in the form of a solid composition, even if the blending ratio of the branched α-glucan is reduced, The present invention was completed by finding that the physiological function can be effectively exhibited.

Features of the branched α-glucan:
In methylation analysis,
(1) The ratio of 2,3,6-trimethyl-1,4,5-triacetylglucitol to 2,3,4-trimethyl-1,5,6-triacetylglucitol is 1: 0.6 To 1: 4.
(2) The sum of 2,3,6-trimethyl-1,4,5-triacetylglucitol and 2,3,4-trimethyl-1,5,6-triacetylglucitol is a partially methylated group. Occupies more than 60% of the citrate acetate;
(3) 2,4,6-trimethyl-1,3,5-triacetylglucitol is 0.5% or more and less than 10% of partially methylated glucitol acetate; and (4) 2,4- Dimethyl-1,3,5,6-tetraacetylglucitol is 0.5% or more of partially methylated glucitol acetate.

That is, the present invention is an α-glucan having glucose as a constituent sugar, and in methylation analysis,
(1) The ratio of 2,3,6-trimethyl-1,4,5-triacetylglucitol to 2,3,4-trimethyl-1,5,6-triacetylglucitol is 1: 0.6 To 1: 4.
(2) The sum of 2,3,6-trimethyl-1,4,5-triacetylglucitol and 2,3,4-trimethyl-1,5,6-triacetylglucitol is a partially methylated group. Occupies more than 60% of the citrate acetate;
(3) 2,4,6-trimethyl-1,3,5-triacetylglucitol is 0.5% or more and less than 10% of partially methylated glucitol acetate; and (4) 2,4- Dimethyl-1,3,5,6-tetraacetylglucitol is 0.5% or more of partially methylated glucitol acetate;
The above-mentioned problems are solved by providing a lipid metabolism improving agent containing a branched α-glucan characterized by the above, particularly a lipid metabolism improving agent in the form of a liquid.

  According to the present invention, lipid metabolism can be effectively improved, so that an increase in blood insulin, an increase in blood lipid, accumulation of visceral fat, and the like can be effectively suppressed or prevented. Moreover, the lipid metabolism improving agent of the present invention can be used safely without worrying about side effects even when taken for a long time.

The glucan referred to in the present invention means an oligosaccharide or a polysaccharide having a glucose polymerization degree of 3 or more with glucose as a constituent sugar. The branched α-glucan used in the present invention is an α-glucan having glucose as a constituent sugar, and in methylation analysis,
(1) The ratio of 2,3,6-trimethyl-1,4,5-triacetylglucitol to 2,3,4-trimethyl-1,5,6-triacetylglucitol is 1: 0.6 To 1: 4.
(2) The sum of 2,3,6-trimethyl-1,4,5-triacetylglucitol and 2,3,4-trimethyl-1,5,6-triacetylglucitol is a partially methylated group. Occupies more than 60% of the citrate acetate;
(3) 2,4,6-trimethyl-1,3,5-triacetylglucitol is 0.5% or more and less than 10% of partially methylated glucitol acetate; and (4) 2,4- Dimethyl-1,3,5,6-tetraacetylglucitol is 0.5% or more of partially methylated glucitol acetate;
It is characterized by that.

  The methylation analysis referred to in the present invention is a method generally known as a method for determining the binding mode of monosaccharides constituting the polysaccharide or oligosaccharide. When methylation analysis is used for analyzing the binding mode of glucose in glucan, first, all free hydroxyl groups in glucose residues constituting glucan are methylated, and then fully methylated glucan is hydrolyzed. Next, the methylated glucose obtained by hydrolysis is reduced to give methylated glucitol from which the anomeric form has been eliminated, and further, a free hydroxyl group in this methylated glucitol is acetylated to give partially methylated glucitol acetate (hereinafter referred to as “methylated glucitol acetate”). In this specification, acetylated sites in “partially methylated glucitol acetate” and “glucitol acetate” may be omitted and abbreviated as “partially methylated product” in some cases. . By analyzing the resulting partially methylated product by gas chromatography, various partially methylated products derived from glucose residues that have different binding modes in glucan have a peak area that occupies the peak area of all partially methylated products in the gas chromatogram. % (%). And the abundance ratio of glucose residues having different binding modes in the glucan, that is, the abundance ratio of each glucoside bond can be determined from the peak area%. In the present specification, “ratio” for a partially methylated product means a ratio of peak areas in the gas chromatogram of the methylation analysis, and “%” for the partially methylated product means “area% in the gas chromatogram of the methylated analysis”. ".

  In the above (1), 2,3,6-trimethyl-1,4,5-triacetylglucitol (hereinafter abbreviated as “2,3,6-trimethylated product”) is C-4 position. 1,3,4-trimethyl-1,5,6-triacetylglucitol (hereinafter abbreviated as “2,3,4-trimethylated product”) It means a glucose residue in which the C-6 position is associated with a 1,6 bond. “The ratio of 2,3,6-trimethylated product to 2,3,4-trimethylated product is in the range of 1: 0.6 to 1: 4” means that partially methylated glycids in methylation analysis are used. In the gas chromatogram of tall acetate, the branched α-glucan used in the present invention binds only to the C-6 position in addition to the glucose residue and the C-1 position in addition to the C-1 position in addition to the C-1 position. It means that the ratio of the glucose residue in which only the C-6 position is involved in the binding other than the C-1 position relative to the total of the glucose residues involved is in the range of 37.5 to 80.0%.

  In the above (2), “the total of 2,3,6-trimethylated product and 2,3,4-trimethylated product accounts for 60% or more of the partially methylated product” means that the branched α- used in the present invention. The glucan is composed of a total of glucose residues in which a total of glucose residues other than the C-1 position and only the C-4 position participates in binding and glucose residues other than the C-1 position that participate only in the C-6 position constitute a glucan. It means to occupy 60% or more of the group.

  Similarly, “2,4,6-trimethyl-1,3,5-triacetylglucitol” (hereinafter abbreviated as “2,4,6-trimethylated product”) in the above (3) , C-3 means a glucose residue that participates in a 1,3 bond, and “2,4,6-trimethylated product is 0.5% or more and less than 10% of partially methylated product” in the present invention. The branched α-glucan used means that there are 0.5% or more and less than 10% of all glucose residues constituting the glucan in which only the C-3 position other than the C-1 position participates in binding. .

  Similarly, “2,4-dimethyl-1,3,5,6-tetraacetylglucitol” in the above (4) (hereinafter abbreviated as “2,4-dimethylated product”) is C Both the −3 position and the C−6 position mean glucose residues that participate in the 1,3 bond and the 1,6 bond, respectively, and “2,4-dimethylated product is 0.5% or more of the partially methylated product”. The branched α-glucan used in the present invention is 0.5% of the total glucose residues in which glucose residues other than the C-1 position and the C-3 position and the C-6 position form a bond constitute the glucan. That means it exists.

  The branched α-glucan used in the present invention that satisfies all the above conditions (1) to (4) is a novel glucan that has not been known so far. In the branched α-glucan used in the present invention, the order of binding of glucose residues is not particularly limited as long as the conditions (1) to (4) are satisfied in the methylation analysis.

  The branched α-glucan used in the present invention is usually in the form of a mixture of branched α-glucan having various degrees of glucose polymerization, and the degree of glucose polymerization is usually 10 or more. In the branched α-glucan used in the present invention, the value (Mw / Mn) obtained by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn) is usually less than 20.

  Furthermore, the branched α-glucan used in the present invention has α-1,2, α-1,3, α-1,4 and α-1,6 bonds adjacent to the reducing end side of the isomaltose structure in glucan. When isomalt dextranase (EC 3.2.1.94), which has a characteristic of hydrolyzing any bond, isomaltose is usually 25% by mass to 50% by mass of the digested solid. Less than mass% is produced.

  The branched α-glucan used in the present invention is the nutrition labeling standard of the Ministry of Health, Labor and Welfare Notification No. 146, “Analytical Methods for Nutritional Components, etc. The water-soluble dietary fiber content is determined according to the “high-performance liquid chromatographic method (enzyme-HPLC method)” described in item 8, “dietary fiber” in “ Contains at least mass%. If the outline of the high performance liquid chromatographic method (hereinafter abbreviated as “enzyme-HPLC method”) is described, a sample is subjected to a series of heat stable α-amylase, protease and amyloglucosidase (glucoamylase). A sample solution for high-performance liquid chromatography (HPLC) is prepared by decomposing by enzyme treatment and removing proteins, organic acids, and inorganic salts from the treatment solution with an ion exchange resin. Next, it is subjected to gel filtration HPLC, and the peak areas of undigested glucan and glucose in the chromatogram are obtained, and the respective peak areas and the glucose amount in the sample solution obtained separately by the usual glucose oxidase method are calculated. This is a method for calculating the water-soluble dietary fiber content of a sample. Details of this method will be described in the experimental section below.

  The branched α-glucan used in the present invention, as shown in Experiments 19-2 to 19-4 described later, is saliva α-amylase, pancreatic juice α-amylase or small intestinal mucosal enzyme (small intestinal mucosal α-glucosidase) even when taken orally. ), It is difficult to be digested and absorbed, and can be used as a low-calorie water-soluble dietary fiber that hardly raises blood sugar or stimulates insulin secretion. In addition, the branched α-glucan is less prone to acid fermentation by microorganisms in the oral cavity and has the effect of suppressing the production of insoluble dextran that causes plaque even when used in combination with sucrose. It can also be advantageously used as a carious or anti-cariogenic carbohydrate. Furthermore, as shown in Experiment 15-5, the branched α-glucan is a glucan that exhibits no toxicity in an acute toxicity test using mice.

  Furthermore, as shown in Experiments 20 and 21, which will be described later, the branched α-glucan has a physiological effect of suppressing an increase in blood sugar level and insulin when ingested together with normal starch as compared with the case of ingesting only starch. Since it has, it can also be used as a blood glucose rise inhibitor.

  Furthermore, in addition to the physiological action, the branched α-glucan has an action of suppressing the increase of neutral fat and cholesterol in the blood by ingestion as shown in Experiment 22 described later. Therefore, it can be used as an agent for improving lipid metabolism. Furthermore, since the branched α-glucan has an action of suppressing the accumulation of visceral fat, it is used as an inhibitor of visceral fat accumulation, further an inhibitor of increase in abdominal circumference, a preventive agent or a therapeutic agent of metabolic syndrome. Can also be used advantageously.

  When the branched α-glucan is used as an active ingredient of a lipid metabolism improving agent, the higher the water-soluble dietary fiber content, the better the action and effect of the branched α-glucan. Those containing at least 50% by mass, preferably at least 50% by mass, more preferably at least 60% by mass can be suitably used.

  The α-glucosyltransferase used in the preparation of the branched α-glucan used in the present invention acts on α-1,4 glucan having a maltose and / or glucose polymerization degree of 3 or more and substantially hydrolyzing. It means an enzyme that produces a branched α-glucan used in the present invention by catalyzing α-glucosyl transfer. The α-glucosyltransferase has a weak hydrolytic activity, has an efficient transfer activity independent of the concentration of the substrate solution from a low concentration to a high concentration, and α-1,3 and α-1, It is an enzyme different from conventionally known fungal-derived α-glucosidase and acetic acid-derived dextrin dextranase in that it also produces 3,6 bonds.

  The enzyme activity of α-glucosyltransferase can be measured as follows. Maltose is dissolved in 20 mM acetate buffer (pH 6.0) to a final concentration of 1 w / v% to form a substrate solution. 0.5 ml of the enzyme solution is added to 5 ml of the substrate solution, and the enzyme reaction is carried out at 40 ° C. for 30 minutes. Then, 0.5 ml of the reaction solution was mixed with 5 ml of 20 mM phosphate buffer (pH 7.0), and the reaction was stopped by heating in a boiling water bath for 10 minutes. According to the method, the glucose oxidase method is used to calculate the amount of glucose produced by the reaction. One unit of α-glucosyltransferase activity is defined as the amount of enzyme that produces 1 μmol of glucose per minute under the above conditions.

As a specific example of the α-glucosyltransferase used in the present invention, for example, α-glucosyltransferase having the following physicochemical properties can be mentioned.
(1) Molecular weight In SDS-polyacrylamide gel electrophoresis, 90,000 ± 10,000 daltons;
(2) Optimal temperature 50 to 55 ° C. under reaction conditions of pH 6.0 and 30 minutes;
(3) Optimum pH
5.0-6.3 under conditions of reaction at 40 ° C. for 30 minutes;
(4) Temperature stability pH 6.0, stable to 40 ° C under 60 minutes holding condition; and (5) pH stability, stable at pH 3.5 to 8.4 under 4 ° C, 24 hour holding condition. ;

Another specific example of this α-glucosyltransferase is α-glucosyltransferase having the following physicochemical properties.
(1) Molecular weight In SDS-polyacrylamide gel electrophoresis, 90,000 ± 10,000 daltons;
(2) Optimum temperature pH of about 50 ° C. under reaction conditions for 30 minutes;
(3) Optimum pH
About 6.0 under reaction conditions at 40 ° C. for 30 minutes;
(4) Temperature stability pH 6.0, stable to 40 ° C under 60 minutes holding condition; and (5) pH stability, pH 4.0 to 8.0 stable under 24 ° C holding condition. ;

  Although these α-glucosyltransferases are not limited by their sources, microorganisms may be mentioned as preferred sources, and in particular, microorganisms PP710 or PP349 strains isolated from soil by the present inventors are preferably used. Tables 1 and 2 show the mycological properties found in the identification test of the microorganisms PP710 and PP349 having the ability to produce α-glucosyltransferase used in the present invention. The identification test was conducted according to “Classification and Identification of Microorganisms” (Takeharu Hasegawa, Society Publishing Center, 1985).

  Based on the above bacteriological properties, “Bergey's Manual of Systematic Bacteriology”, Volume 2 (1986), and “Rebosomal Database” (URL: http://rdp.cme.msu.edu/index.jsp), the differences between the microorganisms PP710 and PP349 and known bacteria were examined. As a result, the microorganism PP710 strain was found to be a microorganism belonging to Bacillus circulans, and the microorganism PP349 strain was found to be a microorganism belonging to Arthrobacter globiformis. The present inventors named these two strains, respectively, as the novel microorganisms Bacillus circulans PP710 and Arthrobacter globiformis PP349, both of which are as of 1 February 2006, 1st East, Tsukuba, Ibaraki, Japan 1 Deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (AIST) located in the sixth central location, and were deposited under the accession numbers FERM BP-10771 and FERM BP-10770, respectively. The microorganisms having the ability to produce α-glucosyltransferase used in the present invention include not only the above strains but also high enzyme-producing mutant strains obtained by inducing mutations in the strains and selecting them.

  The medium used for culturing microorganisms capable of producing α-glucosyltransferase may be any nutrient medium that can grow microorganisms and can produce α-glucosyltransferase used in the present invention. But you can. Any carbon source may be used as long as microorganisms can be used for growth. For example, plant-derived starch and phytoglycogen, animal and microorganism-derived glycogen and pullulan, and partial decomposition products thereof, glucose, fructose, lactose Carbohydrates such as sucrose, mannitol, sorbitol and molasses, and organic acids such as citric acid and succinic acid can also be used. The concentration of these carbon sources in the medium can be appropriately selected depending on the type of carbon source. As the nitrogen source, for example, inorganic nitrogen compounds such as ammonium salts and nitrates and organic nitrogen-containing materials such as urea, corn steep liquor, casein, peptone, yeast extract and meat extract can be used as appropriate. In addition, as the inorganic component, for example, salts such as calcium salt, magnesium salt, potassium salt, sodium salt, phosphate, manganese salt, zinc salt, iron salt, copper salt, molybdenum salt, and cobalt salt are appropriately used. it can. Furthermore, amino acids, vitamins, and the like can be used as necessary.

  Culture of microorganisms capable of producing α-glucosyltransferase is usually selected from the range of pH 5.5 to 10 at a temperature of 15 to 37 ° C., preferably from the range of pH 5.5 to 8.5 at a temperature of 20 to 34 ° C. Done aerobically under conditions. The culture time may be a time during which the microorganism can grow, and is preferably 10 hours to 150 hours. Further, the dissolved oxygen concentration of the culture solution under the culture conditions is not particularly limited, but usually 0.5 to 20 ppm is preferable. For this purpose, means such as adjusting the air flow rate and stirring are appropriately employed. The culture method may be any of batch culture, semi-continuous culture, or continuous culture.

  After culturing a microorganism having the ability to produce α-glucosyltransferase in this manner, a culture containing α-glucosyltransferase is recovered. α-Glucosyltransferase activity is mainly observed in the sterilization solution of the culture when the microorganism is a Bacillus circulans PP710 (FERM BP-10771) or an Arthrobacter globiformis PP349 (FERM BP-10770). The sterilization solution can be collected as a crude enzyme solution, or the entire culture can be used as a crude enzyme solution. In order to remove the cells from the culture, a conventional solid-liquid separation method is employed. For example, a method of centrifuging the culture itself, a method of separating by filtration using a precoat filter or the like, a method of separating by membrane filtration such as a flat membrane or a hollow fiber membrane, etc. are appropriately employed. Although the sterilization solution can be used as a crude enzyme solution as it is, it is generally used after being concentrated. As the concentration method, an ammonium sulfate salting-out method, an acetone and alcohol precipitation method, a membrane concentration method using a flat membrane, a hollow membrane, or the like can be employed.

  Furthermore, α-glucosyltransferase can be immobilized by an appropriate method commonly used in the art, using a sterilizing solution having α-glucosyltransferase activity and a concentrated solution thereof. As the immobilization method, for example, a method of binding to an ion exchanger, a method of covalent bonding / adsorption with a resin and a membrane, a comprehensive method using a polymer substance, and the like can be appropriately employed.

  As described above, the α-glucosyltransferase used in the present invention can be used as it is or after concentrating the crude enzyme solution, but if necessary, an appropriate method commonly used in the art, for example, salting out. Further, separation and purification can be used by methods such as ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, affinity chromatography, and preparative electrophoresis.

  α-1,4 glucan having a degree of glucose polymerization of 3 or more, which is a substrate for α-glucosyltransferase, can be obtained by partially hydrolyzing starch, amylose, amylopectin, glycogen, etc., or partially with amylase or acid, etc. Examples include partial degradation products of starch such as amylodextrin, maltodextrin, and malto-oligosaccharides higher than maltose. Examples of the partially decomposed product decomposed with amylase are described in, for example, “Handbook of Amylases and Related Enzymes” (1988) Pergamon Press (Tokyo), α-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), maltotetraose producing amylase (EC 3.2.1.60), maltopentaose producing amylase, An amylase such as maltohexaose-producing amylase (EC 3.2.1.98) or cyclomaltodextrin glucanotransferase (EC 2.4.1.19, hereinafter abbreviated as “CGTase”) is used. Starch, amylose, amylopectin, Or the like can be used partially decomposed product obtained by decomposing Rikogen. Furthermore, when preparing a partial degradation product, it is also optional to allow starch debranching enzymes such as pullulanase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68) to act. The starch may be, for example, ground starch derived from corn, wheat, rice or the like, or may be ground starch derived from potato, sweet potato, tapioca or the like. In producing the branched α-glucan used in the present invention from starch, it is preferable to use the above-mentioned raw material starch after gelatinization and / or liquefaction. A known method can be adopted as the starch gelatinization / liquefaction method itself. Furthermore, the substrate of α-glucosyltransferase used in the present invention includes etherified starch (hydroxypropyl starch, carboxymethyl starch, acetate starch, etc.), esterified starch (phosphorylated starch, octenyl succinate starch, etc.) and crosslinked starch ( It may be a modified starch obtained by derivatizing a part of starch by a chemical method, such as acetylated adipic acid cross-linked starch, phosphoric acid cross-linked starch, and hydroxypropylated phosphoric acid cross-linked starch.

  When α-glucosyltransferase is allowed to act on a substrate, the substrate concentration is not particularly limited. For example, even when a relatively low concentration solution having a substrate concentration of 0.5% (w / v) is used, The reaction of the α-glucosyltransferase used proceeds to produce branched α-glucan. Industrially, the substrate concentration is preferably 1% (w / v) or higher, preferably 5 to 60% (w / v), more preferably 10 to 50% (w / v). Under these conditions, the branched α-glucan used in the present invention can be advantageously produced. The reaction temperature may be a temperature at which the reaction proceeds, that is, a temperature up to around 60 ° C. Preferably a temperature around 30 to 50 ° C. is used. The reaction pH is usually adjusted to a range of 4 to 8, preferably a pH of 5 to 7. The amount of enzyme used and the reaction time are closely related and may be appropriately selected depending on the progress of the target enzyme reaction.

For example, the production mechanism of branched α-glucan when α-glucosyltransferase is allowed to act on an aqueous solution of starch or a partially decomposed product thereof or amylose is presumed as follows.
1) This enzyme acts as a substrate on maltose and / or α-1,4 glucan having a degree of polymerization of glucose of 3 or more, and converts non-reducing terminal glucose residues to non-reducing terminal glucose residues of other α-1,4 glucans. Α-1,4 glucan (glucose polymerization degree) in which glucose is α-bonded to the 4-position or 6-position hydroxyl group of the non-reducing terminal glucose residue mainly by α-1,4 or α-1,6 glucosyl transfer to the group. Is α-glucan increased by 1), and α-1,4-glucan having a glucose polymerization degree decreased by 1.
2) The enzyme further acts on α-1,4 glucan having a 1-decrease degree of glucose polymerization generated in 1), and 1) in contrast to α-glucan having an increased glucose polymerization degree in 1) of 1). 4 or 6-position hydroxyl group of the non-reducing terminal glucose residue of α-glucan having an increased glucose polymerization degree of 1 produced in 1) by the intermolecular α-1,4 or α-1,6 glucosyl transfer as in Glucose is further transferred to and the chain length is extended.
3) By repeating the reactions 1) and 2) above, a glucan having α-1,4 and α-1,6 bonds is generated from maltose and / or α-1,4 glucan having a glucose polymerization degree of 3 or more. .
4) This enzyme is also less frequent, α-1,3 or α-1,3 glucosyl for α-1,3 glucosyl transfer and α-1,6 linked glucose residues in the glucan. By catalyzing the transfer, a glucan having α-1,3 bonds, α-1,4,6 bonds and α-1,3,6 bonds is also produced.
5) As a result of the above reactions 1) to 4) being repeated, glucose is bound mainly by α-1,4 bonds and α-1,6 bonds, and slightly α-1,3 bonds, α-1,4. , 6 bonds and α-1,3,6 bonds are used to produce branched α-glucans used in the present invention.

  Further, Bacillus circulans PP710 (FERM BP-10771) having the ability to produce α-glucosyltransferase simultaneously contains not only α-glucosyltransferase used in the present invention but also certain amylases in the culture. And, surprisingly, when α-glucosyltransferase and this amylase are used in combination to act on maltose and / or α-1,4 glucan having a glucose polymerization degree of 3 or more, α-glucosyltransferase acts alone. It was found that a branched α-glucan having a higher water-soluble dietary fiber content than that produced by the method can be produced.

Examples of the amylase produced by Bacillus circulans PP710 (FERM BP-10771) include those having the following physicochemical properties.
(1) Action It hydrolyzes starch and catalyzes the transfer of glycosyl groups to produce cyclodextrin. Hydrolyze pullulan to produce panose;
(2) Molecular weight In SDS-polyacrylamide gel electrophoresis, 58,000 ± 10,000 daltons;
(3) Optimal temperature 55 ° C. under reaction conditions of pH 6.0 for 30 minutes;
(4) Optimum pH
6-7 under conditions of reaction at 35 ° C. for 30 minutes;
(5) Temperature stability Stable up to 40 ° C. under conditions of pH 6.0 and holding for 60 minutes,
stable at pH 6.0 in the presence of 1 mM Ca ²⁺ ions up to 50 ° C .; and (6) pH stability stable at pH 6.0-8.0 at 4 ° C. for 24 hours;

  When the branched α-glucan used in the present invention is prepared, the water-soluble dietary fiber content of the branched α-glucan obtained when α-glucosyltransferase and this amylase are used in combination with a partially degraded starch is obtained. The reason for this is higher than that of the branched α-glucan prepared using only α-glucosyltransferase, and amylase further transfers the glycosyl group to the branched α-glucan produced by the action of α-glucosyltransferase. It is presumed that the degree (frequency) of branching in glucan was further increased.

  In addition, when preparing the branched α-glucan by an enzymatic reaction, adjusting the molecular weight distribution of the branched α-glucan by reacting with other known amylases also reduces digestibility. A branched α-glucan can also be advantageously implemented, and furthermore, the reducing power can be reduced. For example, together with α-glucosyltransferase, an enzyme that hydrolyzes α-1,4 bonds inside starch, such as α-glucosyltransferase, and generates a new non-reducing terminal glucose residue is used in the starch liquefaction solution. In this way, the molecular weight distribution is narrowed, the viscosity is reduced, and the proportion of α-1,3, α-1,6 and α-1,3,6 bonds contributing to the reduction of digestibility is further increased. It can also be advantageously carried out. In addition, by using a starch debranching enzyme such as isoamylase in combination, the molecular weight distribution is narrowed to reduce the viscosity, or a non-reducing saccharide-forming enzyme (also known as Japanese Patent Application Laid-Open No. 7-143876) or the like. : Malto-oligosyl trehalose-producing enzyme (EC 5.4.99.15)) can also be advantageously used to reduce the reducing power by partially converting the reducing end portion into a trehalose structure.

  The branched α-glucan used in the present invention is a nutrient medium containing maltose and / or α-1,4 glucan having a glucose polymerization degree of 3 or more, and a microorganism having α-glucosyltransferase production ability is cultured. It can also be produced by collecting branched α-glucan produced in the culture solution.

  The branched α-glucan obtained by the above reaction or culture can be directly used as a branched α-glucan product. If necessary, the reaction solution is allowed to act on one or more selected from α-amylase, β-amylase, glucoamylase and α-glucosidase to hydrolyze the digestible part, and then non-digested A branched α-glucan with further reduced digestibility can be prepared by recovering a portion by fractionation or removing a degradation product such as glucose by fermentation using yeast or the like. In general, a reaction solution containing a branched α-glucan is used after further purification. As a purification method, a normal method used for sugar purification may be appropriately employed. For example, decolorization with activated carbon, desalting with H-type or OH-type ion exchange resin, fractionation with an organic solvent such as alcohol and acetone, and the like. One or more purification methods such as separation by a membrane having separation performance can be appropriately employed.

  When α-glucosyltransferase is acted on gelatinized starch or a partially degraded starch having a relatively low DE (Dextrose Equivalent), preferably less than DE20, to prepare a branched α-glucan used in the present invention, glucose or maltose is used. The resulting reaction product need not be purified by a purification means such as column chromatography, but may be further fractionated according to the purpose such as use. When ion exchange chromatography is employed for this fractionation, for example, column chromatography using a strongly acidic cation exchange resin disclosed in JP-A-58-23799 and JP-A-58-72598 is advantageous. Can be used. At this time, it is optional to adopt any of a fixed floor method, a moving floor method, and a simulated moving floor method.

  In addition, the branched α-glucan can also be prepared by allowing α-glucosyltransferase to act on α-1,4 glucan having a maltose and / or glucose polymerization degree of 3 or more. Increasing the content of dietary fiber components can also be advantageously performed by digesting with isomalt dextranase (EC 3.2.1.9). Incidentally, to this branched α-glucan prepared by allowing α-glucosyltransferase to act on α-1,4 glucan having a maltose and / or glucose polymerization degree of 3 or more, isomaltdextranase (EC 3.2.1). .9) produces isomaltose in an amount of 25% by mass or more and 50% by mass or less per substrate solid.

  Although the branched α-glucan used in the present invention thus obtained can be used as it is in a solution, it is desirable to dry it into a powder product so that it is advantageous for storage and easy to use depending on the application. . For drying, freeze drying, spray drying, drum drying, or the like can be used. If necessary, the dried product can be pulverized and powdered, or sieved or granulated to adjust to a specific particle size range.

  In addition, the branched α-glucan is indigestible and, when taken orally, can exert an excellent lipid metabolism improving action due to the action of dietary fiber. Therefore, it is not only a lipid metabolism improving agent, but also blood insulin and medium. It can also be advantageously used as an inhibitor or preventive agent for the increase in sex fat, an inhibitor for visceral fat accumulation, a preventive agent, an inhibitor for increase in abdominal circumference, a preventive agent or a therapeutic agent for metabolic syndrome, and the like. In addition, the lipid metabolism improving agent of the present invention formulated with the branched α-glucan as an active ingredient has an excellent lipid metabolism improving action in a liquid form rather than a solid form even if the blending ratio in the composition is low. It has the feature of exerting its physiological function.

  As the dosage form of the lipid metabolism improving agent of the present invention, various forms can be selected according to the purpose of use, and representative examples thereof include tablets, pills, granules, capsules, powders, syrups and the like. And parenterals such as suppositories, suppositories, solutions, suspensions, and emulsions.

  As the lipid metabolism improving agent of the present invention, branched α-glucan may be used alone as a preparation as it is, but if necessary, pharmaceutically acceptable food materials, food additives, pharmaceuticals, Oral preparations or high-calorie infusions combined with additives such as pharmaceutical additives and quasi-drug additives can be used, or can be added to infusion preparations to make parenteral preparations. Specifically, for example, surfactants, preservatives (antibacterial agents), moisturizers, thickeners, water-soluble polymers, antioxidants, chelating agents, dyes, fragrances, pH adjusters, vitamins, various amino acids In combination with electrolytes, water, alcohols, organic solvents, animal or plant extracts, etc., they may be formulated by known methods. More specifically, in the production of oral preparations, for example, commonly used excipients such as lactose, sucrose, maltose, trehalose, cyclic tetrasaccharide, dextrin, starch, crystalline cellulose, corn starch, carboxymethylcellulose, agar, gelatin A disintegrating agent such as powder, a binder such as polyvinyl alcohol, methyl cellulose, and hydroxypropyl cellulose, a lubricant such as silica, magnesium stearate, and talc, and a coating agent such as hydroxypropyl methylcellulose and sucrose may be used. Also, in the production of oral solutions such as solutions, suspensions, syrups, and injections, they can be dissolved or suspended in distilled water for injection, physiological saline, etc., for example, inorganic or organic acids or bases, etc. What is necessary is just to add a pH adjuster, an isotonic agent, a stabilizer, a diluent, etc. as needed.

  Further, the lipid metabolism improving agent of the present invention can be used as it is or mixed with other saccharides, sweeteners, bulking agents, excipients, binders and blended with existing foods and drinks. Is voluntary. Specifically, for example, flour, glucose, fructose, isomerized sugar, sugar, maltose, trehalose, honey, maple sugar, sorbitol, maltitol, dihydrochalcone, stevioside, α-glycosyl stevioside, lacanka sweet, glycyrrhizin, Mixed with sweeteners such as thaumatin, sucralose, L-aspartylphenylalanine methyl ester, saccharin, glycine, alanine, etc., and with bulking agents such as dextrin, starch, dextran, lactose, etc. Further, it can be formed into various shapes such as a short bar shape, a plate shape, and a cube, and can be blended in food and drink.

  Moreover, even when the lipid metabolism improving agent of the present invention is blended in a food or drink, the branched α-glucan, which is an active ingredient, is difficult to digest, so that it can exert an effect of improving lipid metabolism. Specifically, for example, soy sauce, powdered soy sauce, miso, powdered miso, moromi, horsetail, fried potato, mayonnaise, dressing, vinegar, three cups of vinegar, powdered sushi vinegar, Chinese soup, tempura soup, noodle soup, sauce, ketchup, grilled meat It can also be advantageously used as a quality improver for various seasonings such as sauces, curry roux, stew ingredients, soup ingredients, dashi ingredients, compound seasonings, mirin, new mirin, table sugar, coffee sugar, etc. . Also, for example, various Japanese sweets such as rice crackers, hail, rice cakes, fertilizers, potatoes, manju, owls, cocoons, sheepskin, water sheepskin, brocade, jelly, castella, candy balls, bread, biscuits, crackers, cookies, pie , Pudding, butter cream, custard cream, cream puff, waffle, sponge cake, donut, chocolate, chewing gum, caramel, nougat, candy and other Western confectionery, ice cream, sorbet and other ice confectionery, fruit syrup pickles, syrup Fruit, paste such as peanut paste, fruit paste, jam, marmalade, syrup pickles, fruits such as sugar cane, processed foods of vegetables, pickles such as Fukujin pickles, bedara pickles, thousand pickles, takuwan pickles, Pickles such as Chinese cabbage Livestock products such as mums and sausages, fish meat ham, fish sausages, fish products such as sea cucumbers, chikuwa and tempura, various delicacies such as sea urchin, salted squid, vinegar comb, sakisume, cod, Thai and shrimp Foods such as seafood, seaweed, wild vegetables, sea bream, small fish, shellfish, boiled beans, potato salad, side dish foods such as kombu rolls, dairy products, fish meat, livestock meat, fruit, vegetable bottling, canned foods, synthetic sake , Brewed sake, sake, fruit wine, sparkling liquor, beer and other alcoholic beverages, coffee, cocoa, juice, mineral water, carbonated beverages, lactic acid beverages, lactic acid bacteria beverages, tea beverages and other soft drinks, pudding mix, hot cake mix, Instant foods such as instant juice, instant coffee, instant juice powder, and instant soup, as well as various foods such as baby food, therapeutic foods, drinks, peptide foods, and frozen foods Bayoi. In addition, the lipid metabolism improving agent of the present invention can effectively exert physiological functions such as lipid metabolism improving effect even if the liquid form is solid rather than solid, and even if the blending ratio to the composition is low. It is particularly desirable to use it in a liquid form such as beverages, soups and drinks.

  Moreover, the lipid metabolism improving agent of the present invention can be used for the purpose of improving lipid metabolism as a feed, a feed or the like for domestic animals such as domestic animals and poultry.

  As described above, as a method for containing the active ingredient branched α-glucan in the lipid metabolism improving agent of the present invention, it may be contained in the process until the product is completed, for example, mixing, kneading, dissolution, melting, immersion Well-known methods such as permeation, spraying, coating, coating, spraying, pouring and solidification are appropriately selected. The amount is usually 0.1% by mass or more, preferably 1% by mass or more of the composition. When the composition is solid, it is desirable to blend 1% by mass or more, and particularly desirably 2% or more. In the case of a liquid, 0.5% by mass or more is desirably blended, and 1% or more is particularly desirable. If the blending amount is less than 0.1% by mass, the effect of improving lipid metabolism may not be exhibited.

  Hereinafter, the branched α-glucan used in the present invention, α-glucosyltransferase used for the preparation of the branched α-glucan, and physiological functions of the branched α-glucan will be described in detail by experiments.

<Experiment 1: Preparation of glucan using α-glucosyltransferase derived from Bacillus circulans PP710 (FERM BP-10771)>
<Experiment 1-1: Preparation of α-glucosyltransferase derived from Bacillus circulans PP710 (FERM BP-10771)>
Partially decomposed starch (trade name “Paindex # 4”, sold by Matsutani Chemical Co., Ltd.) 1.5 w / v%, yeast extract (trade name “Polypeptone”, sold by Nippon Pharmaceutical Co., Ltd.) 0.5 w / v% Yeast extract (trade name “Yeast Extract S”, sold by Nippon Pharmaceutical Co., Ltd.) 0.1 w / v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate dihydrate 0.06 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, manganese sulfate pentahydrate 0.001 w / v%, ferrous sulfate heptahydrate 0.001 w / v% and water. 100 ml of liquid medium is put into one 500 ml Erlenmeyer flask, sterilized by autoclave at 121 ° C. for 20 minutes, cooled, inoculated with Bacillus circulans PP710 (FERM BP-10771), and incubated at 27 ° C. and 230 rpm for 48 hours. A material obtained by rolling shaking culture was a seed culture.

  100 ml each of liquid culture medium with the same composition as the seed culture is put into 12 500 ml Erlenmeyer flasks, heat-sterilized and cooled to a temperature of 27 ° C., then inoculated with about 1 ml of the seed culture solution at a temperature of 27 ° C. for 24 hours. Rotating and shaking culture. After culturing, the culture solution was extracted from the Erlenmeyer flask, centrifuged (8,000 rpm, 20 minutes) to remove the cells, and the α-glucosyltransferase activity of the obtained culture supernatant was measured. Unit / ml. To about 1 L of the culture supernatant, ammonium sulfate was added and dissolved so as to be 80% saturation, and salted out by leaving it at 4 ° C. for 24 hours. The precipitated salted-out product was collected by centrifugation (11,000 rpm, 30 minutes), dissolved in 20 mM acetate buffer (pH 4.5), dialyzed against the same buffer, and about 20 ml of crude enzyme solution. Got. This crude enzyme solution was subjected to cation exchange column chromatography (gel capacity 20 ml) using a “CM-Toyopearl 650S” gel manufactured by Tosoh Corporation equilibrated with 20 mM acetate buffer (pH 4.5). After elution of non-adsorbed protein, it was eluted with a linear gradient from 0 M to 0.5 M salt concentration, and the fraction eluted from about 0.18 M salt concentration to about 0.45 M salt was collected, and 20 mM acetate buffer (pH 6.0) was collected. ) Was dialyzed against. The obtained dialysate was used as an α-glucosyltransferase preparation.

<Experiment 1-2: Preparation of branched α-glucan using α-glucosyltransferase>
Using 100 ml of the α-glucosyltransferase preparation obtained in Experiment 1-1 as the enzyme solution, a partial starch degradation product (trade name “Paindex # 100”, Matsutani Chemical Co., Ltd.) so that the final concentration is 30 w / v%. Kogyo Co., Ltd.) was added and reacted at 40 ° C. for 72 hours, and then the reaction was stopped by heat treatment at about 100 ° C. for 10 minutes. After removing the insoluble matter by filtration, it was decolored and desalted using the ion exchange resins “Diaion SK-1B” and “Diaion WA30” manufactured by Mitsubishi Chemical and the anion exchange resin “IRA411” manufactured by Organo, followed by precise filtration. Thereafter, the resultant was concentrated by an evaporator, and a glucan solution having a solid content concentration of 30% by mass was obtained in a yield of 85.8% per solid from the partially decomposed starch used as a substrate.

<Experiment 2: Preparation of glucan using α-glucosyltransferase derived from Arthrobacter globiformis PP349 (FERM BP-10770)>

<Experiment 2-1: Preparation of α-glucosyltransferase derived from Arthrobacter globiformis PP349 (FERM BP-10770)>
A seed culture was used in the same manner as in Experiment 1-1 except that Arthrobacter globiformis PP349 (FERM BP-10770) was inoculated instead of Bacillus circulans PP710 (FERM BP-10771).

  100 ml each of liquid culture medium with the same composition as the seed culture is put into 12 500 ml Erlenmeyer flasks, heat-sterilized and cooled to a temperature of 27 ° C., then inoculated with about 1 ml of the seed culture solution at a temperature of 27 ° C. for 24 hours. Rotating and shaking culture. After culturing, the culture solution was extracted from the Erlenmeyer flask, centrifuged (8,000 rpm, 20 minutes) to remove the cells, and the α-glucosyltransferase activity of the obtained culture supernatant was measured to find 0.53 units. / Ml. To about 1 L of the culture supernatant, ammonium sulfate was added and dissolved so as to be 80% saturation, and salted out by leaving it at 4 ° C. for 24 hours. The precipitated salted-out product was collected by centrifugation (11,000 rpm, 30 minutes), dissolved in 20 mM acetate buffer (pH 6.0), and dialyzed against the same buffer. This crude enzyme solution was subjected to anion exchange column chromatography (gel volume: 20 ml) using “DEAE-Toyopearl 650S” gel manufactured by Tosoh Corporation equilibrated with 20 mM acetate buffer (pH 6.0). After elution of the non-adsorbed protein, it was eluted with a linear gradient with a salt concentration of 0 to 0.5 M, and the fraction eluted with a salt concentration of about 0.05 M to about 0.2 M was collected, and 20 mM acetate buffer (pH 6.0) was collected. ) Was dialyzed against. The obtained dialysate was used as an α-glucosyltransferase preparation.

<Experiment 2-2: Preparation of glucan using α-glucosyltransferase>
Using 20 ml of the α-glucosyltransferase preparation obtained in Experiment 2-1 as an enzyme solution, a partial starch degradation product (trade name “Paindex # 100”, Matsutani Chemical Co., Ltd.) to a final concentration of 30 w / v%. Kogyo Co., Ltd.) was added and reacted at 40 ° C. for 72 hours, and then the reaction was stopped by heat treatment at about 100 ° C. for 10 minutes. After insoluble matter was removed by filtration, it was decolorized and desalted using ion exchange resins “Diaion SK-1B” and “Diaion WA30” made by Mitsubishi Chemical and anion exchange resin “IRA411” made by Organo, and then finely filtered. Thereafter, the resultant was concentrated by an evaporator, and a glucan solution having a solid content of 30% by mass was obtained in a yield of 83.6% per solid from the partially decomposed starch used as a substrate.

  In the following Experiments 3 and 4, the glucan obtained in Experiment 1-2 and the glucan obtained in Experiment 2-2 are referred to as “glucan A” and “glucan B”, respectively, for the purpose of distinguishing them.

<Experiment 3: Evaluation of Glucan A and B as Water-soluble Dietary Fiber>
Analyzing the nutritional components in the nutrition labeling standards (Ministry of Health and Welfare Notification No. 146, May 1996), etc. of the water-soluble dietary fiber content of the glucan obtained (methods listed in the third column of the separate table of the nutrition labeling standards) , 8. According to the method described in Dietary fiber, (2) High-performance liquid chromatographic method (enzyme-HPLC method), the following method was used. As a kit for enzyme treatment, a total dietary fiber measurement kit (Dietary Fiber, Total, Assay, Control Kit, manufactured by Sigma) was used. In addition, a starch partially decomposed product (trade name “Paindex # 100”, sold by Matsutani Chemical Industry Co., Ltd.), which is a substrate used for the preparation of glucan A and B, was used as control 1, and commercially available indigestible glucan (trade name “ Pine fiber ”, sold by Matsutani Chemical Co., Ltd.) was used as a control 2, and the water-soluble dietary fiber content was similarly examined.

<Preparation of sample solution for analysis>
As a test sample, glucan having a solid content of 0.1 g was placed in a test tube, and 5 ml of 0.08 M phosphate buffer was added to adjust the pH to 6.0. To this was added 0.01 ml of a heat stable α-amylase (heat-resistant α-amylase derived from Bacillus licheniformis, Sigma) attached to the dietary fiber measurement kit, covered with aluminum foil, and stirred in a boiling water bath every 5 minutes. The reaction was allowed to proceed for 30 minutes while cooling. After adding about 1 ml of 0.275M sodium hydroxide solution to the obtained reaction solution and adjusting pH to 7.5, 0.01 ml of protease solution (derived from Bacillus licheniformis, Sigma) attached to the kit was added, It covered with the aluminum foil, it was made to react for 30 minutes, shaking in a 60 degreeC water bath, and it cooled. About 1 ml of 0.325M hydrochloric acid was added to the resulting protease treatment solution to adjust the pH to 4.3, and then 0.01 ml of an amyloglucosidase (derived from Aspergillus niger, Sigma) solution attached to the kit was added. Covered with foil, reacted for 30 minutes with shaking in a 60 ° C. water bath and cooled. Next, about 7 ml of the obtained reaction solution was passed through an ion exchange resin (mixed 1: 1 with Amberlite IRA-67 (OH type) and Amberlite 200CT (H type) sold by Organo Corporation) at SV 1.0. The solution was desalted by leaching, and further eluted with about 3 times the amount of deionized water to make the total amount of the eluate about 28 ml. The obtained eluate was concentrated by an evaporator, filtered through a membrane filter having a pore size of 0.45 μm, and then the volume was adjusted to 25 ml with a volumetric flask was used as a sample solution for analysis.

<High performance liquid chromatography conditions>
The analytical sample solution obtained above was subjected to high performance liquid chromatography under the following conditions.
Column: TGKgel G2500PWXL (inner diameter 7.8 mm × length 300 mm, manufactured by Tosoh Corporation) connected in series Eluent: deionized water Sample sugar concentration: 0.8% by mass
Column temperature: 80 ° C
Flow rate: 0.5 ml / min Detection: Differential refractometer Injection volume: 20 μl
Analysis time: 50 minutes

<Calculation of water-soluble dietary fiber content of test sample>
In the chromatogram obtained above, the undigested glucan remaining without being decomposed by the enzyme treatment was used as water-soluble dietary fiber. The peak areas of the water-soluble dietary fiber and the glucose produced by decomposition are determined, respectively, and separately, using the amount of glucose in the sample solution for analysis quantified by a conventional glucose oxidase method, The amount of sexual dietary fiber was determined. Furthermore, the water-soluble dietary fiber content of the test sample was determined by the following formula 2.

Formula 1:

Formula 2:

  The water-soluble dietary fiber contents of glucan A and glucan B determined by the enzyme-HPLC method were 42.1% by mass and 41.8% by mass, respectively. On the other hand, the partially decomposed starch of Control 1 was completely degraded to glucose by the enzyme treatment, and the water-soluble dietary fiber content was evaluated as 0% by mass. Further, it was 48.7% by mass of Control 2 commercially available indigestible dextrin (trade name “Pine Fiber”, sold by Matsutani Chemical Co., Ltd.). These results show that glucan that has almost the same water-soluble dietary fiber content as commercially available indigestible dextrins can be easily obtained by using α-glucosyltransferase to act on a partially degraded starch that does not contain water-soluble dietary fiber as a substrate. It is shown that it can be prepared.

<Experiment 4: Structural analysis of glucan A and B>
<Experiment 4-1: Methylation analysis>
About glucan A and B obtained by the method of Experiment 1-2 and 2-2, methylation analysis was performed according to the conventional method, and the partial methylation thing was investigated by the gas chromatography method by the following conditions. The results are summarized in Table 3.
<Gas chromatography conditions>
Column: DB-5 capillary column (inner diameter 0.25 mm × length 30 m × film thickness 1 μm, manufactured by J & W Scientific)
Carrier gas: helium Column temperature: held at 130 ° C for 2 minutes, then heated to 250 ° C at 5 ° C / minute and held at 250 ° C for 20 minutes Flow rate: 1.0 ml / minute Detection: FID
Injection volume: 3 μl (split 1/30)
Analysis time: 46 minutes

As is clear from the results in Table 3, the results of methylation analysis of glucan A and B prepared by α-glucosyltransferase derived from Bacillus circulans PP710 and Arthrobacter globiformis PP349, respectively. Compared to that of the degradation product, 2,3,6-trimethylated product was significantly decreased in all glucans, while 2,3,4-trimethylated product was significantly increased to 30% or more. This is because a partially decomposed starch having a structure in which glucose is polymerized mainly by α-1,4 bonds by the reaction of α-glucosyltransferase derived from Bacillus circulans PP710 and Arthrobacter globiformis PP349, α-1 , 6 has been converted to branched α-glucan containing 30% or more. Further, since 2,3,4,6-tetramethylated product, 2,4,6-trimethylated product and 2,4-dimethylated product are increased, non-reducing terminal glucose, α-1,3 bond and α It was also found that -1,3,6 bonds were newly generated. The contents of 2,4,6-trimethylated product and 2,4-dimethylated product in the partially methylated product of glucan A are 1.1% and 0.8%, respectively, and 2,4,4 in the partially methylated product of glucan B The contents of 6-trimethylated product and 2,4-dimethylated product were 0.9% and 1.1%, respectively. Furthermore, in glucan A and B, since the abundance ratio of 2,3-dimethylated compound is almost the same as that of the substrate, it is considered that there is no significant change in the α-1,4,6 bond that is a branch originally present in the substrate. It was. From this result, glucan A and B are significantly different from the partial starch degradation product as a substrate, and the glucose binding mode is mainly α-1,4 bond and α-1,6 bond, and α-1,3 bond. And a branched glucan having a slight amount of α-1,3,6 bonds (branched α-glucan). The 1-position anomeric form of glucose composing the branched α-glucans A and B was all found to be α-form from ¹ H-NMR spectrum in nuclear magnetic resonance (NMR) analysis.

<Experiment 4-2: Isomaltdextranase Digestion Test of Branched α-Glucans A and B>
In order to characterize the structures of branched α-glucans A and B, an isomalt dextranase digestion test was performed. Isomaltodextranase derived from Arthrobacter globiformis (prepared in Hayashibara Biochemical Laboratories Co., Ltd.) in an aqueous solution of branched α-glucan A and B (final concentration 1 w / v%) per 100 g of solid substrate After adding the unit and acting at 50 ° C. and pH 5.0 for 16 hours and holding at 100 ° C. for 10 minutes to stop the reaction, the sugar composition in the reaction solution is abbreviated as high performance liquid chromatography (hereinafter “HPLC”). And gas chromatography (hereinafter abbreviated as “GC”). HPLC is performed using two “MCI GEL CK04SS” (manufactured by Mitsubishi Chemical Co., Ltd.) as the column, using water as the eluent, at a column temperature of 80 ° C., and a flow rate of 0.4 ml / min. The total RID-10A (manufactured by Shimadzu Corporation) was used. After GC trimethylsilylation (TMS conversion) according to a conventional method, GC uses “2% Silicon OV-17 Chromosorb W / AW-DMS” (manufactured by GL Sciences Inc.) per minute. The temperature was raised from 160 ° C. to 320 ° C. at a temperature raising rate of 7.5 ° C. Nitrogen gas was used as a carrier gas, and detection was performed by the FID method. In the digestion with isomalt dextranase, no isomaltose was produced from the partially decomposed starch as a substrate used for the preparation of the branched α-glucan, whereas from the branched α-glucan A, the sugar composition was 28. 4% by mass of isomaltose was produced, and 27.2% by mass of isomaltose was produced from the branched α-glucan B as a sugar composition. This result shows that branched α-glucans A and B contain isomaltose structures at least about 28.4% by mass and 27.2% by mass, respectively. In branched α-glucan, α-1,6 This supports the result of methylation analysis in Experiment 4-1, which showed that the proportion of binding increased. In addition, if isomaltdextranase is an α-glucoside bond adjacent to the reducing end of the isomaltose structure in glucan, it is an α-1,3, α-1,4 and α-1,6 bond. In any case, since it has the specificity of hydrolysis, the details of how the obtained isomaltose is bound in the branched α-glucans A and B are unknown. .

<Experiment 4-3: Digestion test of α-glucosidase and glucoamylase of branched α-glucan A and B>
Aspergillus niger-derived α-glucosidase (trade name “Transglucosidase Amano L”, manufactured by Amano Enzyme) and Rhizopus sp. ) Origin glucoamylase was allowed to act simultaneously to conduct a digestion test. After adding 5,000 units of α-glucosidase and 100 units of glucoamylase per gram of substrate solids, reacting at 50 ° C. and pH 5.0 for 16 hours, holding at 100 ° C. for 10 minutes to stop the reaction, the enzyme The sugar composition of the reaction solution was examined using HPLC under the same conditions as in Experiment 4-2. As a result, both glucan A and B were substantially completely decomposed into glucose as in the case of the partially decomposed starch as a substrate. This result indicates that both branched α-glucans A and B are α-glucans having glucose as a constituent sugar.

<Experiment 4-4: Molecular Weight Distribution Analysis>
For branched α-glucans A and B, the molecular weight distribution was analyzed by a conventional gel filtration HPLC method. Gel filtration HPLC uses a column in which two “TSK GEL α-M” (manufactured by Tosoh Corporation) are connected, and 10 mM phosphate buffer (pH 7.0) is used as an eluent, and column temperature is 40 ° C. The detection was carried out using a differential refractometer RID-10A (manufactured by Shimadzu Corporation). The molecular weight of glucan in the sample was calculated based on a molecular weight calibration curve prepared by subjecting a pullulan standard for molecular weight measurement (sales by Hayashibara Biochemical Laboratories, Inc.) to gel filtration analysis. FIG. 1 shows the gel filtration HPLC chromatogram (symbols b and c in FIG. 1) of branched α-glucans A and B, which is a partially decomposed starch (trade name “Paindex # 100”) used as a substrate (FIG. 1). Each of them is shown in comparison with the symbol a). In FIG. 1, symbols a, b, c, d, and ho mean elution positions corresponding to molecular weights of 1,000,000, 100,000, 10,000, 1,000, and 100 daltons, respectively (described later). The same applies to FIGS. 15 to 19). Table 4 shows the results of analyzing the molecular weight distribution of each sample based on the chromatogram obtained by gel filtration HPLC.

  The partially decomposed starch used as a substrate is a carbohydrate mixture having two peaks (reference numerals 1 and 2 in chromatogram a in FIG. 1) at positions corresponding to glucose polymerization degrees 499 and 6.3 in the molecular weight distribution analysis. On the other hand, branched α-glucan A has four peaks at the positions corresponding to glucose polymerization degrees 384, 22.2, 10.9 and 1 (reference numerals 3, 4, 5 and 6 in chromatogram b in FIG. 1). ), And branched α-glucan B has four peaks at positions corresponding to glucose polymerization degrees 433, 22.8, 10.9, and 1 (symbol 7, in chromatogram c in FIG. 1). It was a mixture of carbohydrates with 8, 9 and 10). Although the peaks at 6 and 10 correspond to glucose, but the content thereof is very small, it can be seen that the hydrolysis action of Bacillus circulans PP710 and Arthrobacter globiformis PP349-derived enzymes is slight. As apparent from Table 4, the number average molecular weight and the weight average molecular weight of glucan A and B were both reduced to about 60% as compared with the partially decomposed starch as a substrate, and the molecular weight was lowered as a whole. In addition, since the value (Mw / Mn) obtained by dividing the weight average molecular weight, which is an index of the spread of the molecular weight distribution, by the number average molecular weight does not change so much between the partially decomposed starch and glucan A and B, PP710 and Arthrobacter globiformis PP349-derived α-glucosyltransferase were considered to act only on the non-reducing end of the partially degraded starch.

  From the above results, in the partially decomposed starch used as a substrate, about 90% of the glucose binding mode is α-1,4 bonds, and has only α-1,4,6 bonds. On the other hand, glucans A and B have a very high ratio of α-1,6 bonds to α-1,4 bonds, and in addition to α-1,4,6 bonds, α-1,3 bonds and α-1,4 bonds It was found to be a glucan having a branch also having 3,6 bonds. A branched α-glucan having such a structure has never been known.

  The structure of the branched α-glucan was estimated based on the result of the methylation analysis, and a diagram schematically showing the structure is shown in FIG. 2 together with that of the partially decomposed starch as a substrate. In FIG. 2, the codes | symbols 1 and 2 are the schematic diagrams of a raw material starch partial decomposition product and branched alpha-glucan, respectively. In FIG. 2, the symbols a, b, c, d, e, and f represent the non-reducing terminal glucose residue and the α-1,3-linked glucose residue in the partially decomposed starch or branched α-glucan, respectively. Groups, α-1,4 linked glucose residues, α-1,6 linked glucose residues, α-1,3,6 linked glucose residues and α-1,4,6 Means a bound glucose residue. In addition, the oblique broken line, the horizontal solid line, and the vertical solid line between glucose in the schematic diagram mean α-1,3 bond, α-1,4 bond, and α-1,6 bond, respectively.

<Experiment 5: Production of α-glucosyltransferase derived from Bacillus circulans PP710 strain>
Partially decomposed starch (trade name “Paindex # 4”, manufactured by Matsutani Chemical Co., Ltd.) 1.5 w / v%, yeast extract (trade name “Polypeptone”, manufactured by Nippon Pharmaceutical Co., Ltd.) 0.5 w / v% Yeast extract (trade name “Yeast Extract S”, manufactured by Nippon Pharmaceutical Co., Ltd.) 0.1 w / v%, dipotassium phosphate 0.1 w / v%, monosodium phosphate dihydrate 0.06 w / v%, magnesium sulfate heptahydrate 0.05 w / v%, manganese sulfate pentahydrate 0.001 w / v%, ferrous sulfate heptahydrate 0.001 w / v% and water. 100 ml each of the liquid medium was put into two 500 ml Erlenmeyer flasks, sterilized by autoclaving at 121 ° C. for 20 minutes, cooled, inoculated with Bacillus circulans PP710 (FERM BP-10771), 48 at 27 ° C. and 230 rpm. What was between rotary shaking culture was a seed culture.

  About 20 L of a liquid medium having the same composition as the seed culture is placed in a fermenter having a capacity of 30 L, heat-sterilized and cooled to a temperature of 27 ° C. The culture was aerated and stirred for 24 hours while maintaining 8.0. After culturing, the culture solution was extracted from the fermenter, centrifuged (8,000 rpm, 20 minutes) to remove the cells, and about 18 L of culture supernatant was obtained. When the α-glucosyltransferase activity was measured for the culture broth and the culture supernatant, the enzyme activity of the culture broth was about 2.7 units / ml, and the enzyme activity of the culture supernatant was about 2.6 units / ml. there were. It has been found that most of the glucosyltransferase produced by Bacillus circulans PP710 exists outside the cells.

<Experiment 6: Purification of α-glucosyltransferase derived from Bacillus circulans PP710>
Of the culture supernatant obtained in Experiment 5, about 4 L (total activity of about 10,400 units) was added with ammonium sulfate so as to be 80% saturated, dissolved, and left to stand at 4 ° C. for 24 hours for salting out. . The precipitated salted-out product was collected by centrifugation (11,000 rpm, 30 minutes), dissolved in 20 mM acetate buffer (pH 4.5), dialyzed against the same buffer, and about 65 ml of crude enzyme solution. Got. The α-glucosyltransferase activity in the crude enzyme solution was about 74 units / ml (total activity about 4,780 units). This crude enzyme solution was subjected to cation exchange column chromatography (gel capacity: 70 ml) using “CM-Toyopearl 650S” gel manufactured by Tosoh Corporation. The α-glucosyltransferase activity was adsorbed on a “CM-Toyopearl 650S” gel equilibrated with 20 mM acetate buffer (pH 4.5) and eluted with a linear gradient from 0 M to 0.5 M at a salt concentration. It eluted at around 0.4M. This active fraction was collected, ammonium sulfate was added to a final concentration of 1 M, and the mixture was allowed to stand at 4 ° C. for 24 hours, and then centrifuged to remove insoluble materials. It used for the used hydrophobic column chromatography (gel volume 9ml). The α-glucosyltransferase activity was adsorbed on a “butyl-toyopearl 650M” gel equilibrated with 20 mM acetate buffer (pH 6.0) containing 1M ammonium sulfate, and eluted with a linear gradient from 1M to 0M ammonium sulfate. It eluted at an ammonium sulfate concentration of about 0.2M. This active fraction was collected, dialyzed against 20 mM acetate buffer (pH 4.5), and then subjected to cation exchange column chromatography (gel volume: 3.3 ml) using a “CM-5PW” gel manufactured by Tosoh Corporation. ). The α-glucosyltransferase activity was adsorbed on a “CM-5PW” gel equilibrated with 20 mM acetate buffer (pH 4.5) and eluted with a linear gradient from 0 M to 0.5 M. It eluted at around 0.4M. This active fraction was collected and dialyzed against 20 mM acetate buffer (pH 6.0). Table 5 shows the α-glucosyltransferase activity, the specific activity and yield of α-glucosyltransferase in each step of each purification.

  When the purity of the enzyme preparation was tested by 5 to 20 w / v% concentration gradient polyacrylamide gel electrophoresis of the purified α-glucosyltransferase preparation, the protein band was single and the purity was high. .

<Experiment 7: Properties of Bacillus circulans PP710-derived α-glucosyltransferase>
<Experiment 7-1: Molecular weight>
The purified molecular weight marker (5-20 w / v% concentration gradient) of the α-glucosyltransferase purified from Bacillus circulans PP710 obtained by the method of Experiment 6 was subjected to SDS-polyacrylamide gel electrophoresis (5 to 20 w / v% concentration gradient). The molecular weight of this α-glucosyltransferase was found to be 90,000 ± 10,000 daltons when measured for molecular weight in comparison with Japan Bio-Rad Laboratories Co., Ltd.).

<Experiment 7-2: Optimum temperature and pH of enzyme reaction>
Using the Bacillus circulans PP710-derived α-glucosyltransferase purified preparation obtained by the method of Experiment 6, the effects of temperature and pH on enzyme activity were examined according to the activity measurement method. These results are shown in FIG. 3 (optimum temperature) and FIG. 4 (optimum pH). The optimum temperature of the α-glucosyltransferase is 50 to 55 ° C. under a reaction condition of pH 6.0 for 30 minutes, and the optimum pH is 5.0 to 6.3 under a reaction condition of 40 ° C. for 30 minutes. It turned out to be.

<Experiment 7-3: Temperature stability and pH stability of enzyme>
Using the α-glucosyltransferase purified preparation obtained by the method of Experiment 6, the temperature stability and pH stability of the enzyme were examined. The temperature stability was determined by measuring the remaining enzyme activity after holding the enzyme solution (20 mM acetate buffer, pH 6.0) at each temperature for 60 minutes, cooling with water. The pH stability was determined by maintaining the enzyme in 20 mM buffer at each pH at 4 ° C. for 24 hours, adjusting the pH to 6.0, and measuring the remaining enzyme activity. These results are shown in FIG. 5 (temperature stability) and FIG. 6 (pH stability). As is apparent from FIGS. 5 and 6, the α-glucosyltransferase is stable up to 40 ° C. and stable in the range of pH 3.5 to 8.4.

<Experiment 7-4: Effect of metal salt on enzyme activity>
Using the α-glucosyltransferase purified preparation obtained by the method of Experiment 6, the influence of the metal salt on the enzyme activity was examined in the presence of various metal salts at a concentration of 1 mM according to the method of activity measurement. The results are shown in Table 6.

As is apparent from the results in Table 6, it was found that the activity of the α-glucosyltransferase is significantly inhibited by Hg ²⁺ ions and inhibited by Cu ²⁺ ions.

<Experiment 8: Preparation of α-glucosyltransferase derived from Arthrobacter globiformis PP349 (FERM BP-10770)>
The culture was carried out in the same manner as in Experiment 5 except that it was inoculated with Arthrobacter globiformis PP349 (FERM BP-10770) instead of Bacillus circulans PP710.

  About 20 L of a liquid medium having the same composition as the seed culture is placed in a fermenter having a capacity of 30 L, heat-sterilized and cooled to a temperature of 27 ° C., then inoculated with about 200 ml of the seed culture solution, temperature 27 ° C., pH 5.5 to While maintaining 7.0, the culture was aerated and stirred for 24 hours. After culturing, the culture solution was extracted from the fermenter, centrifuged (8,000 rpm, 20 minutes) to remove the cells, and about 18 L of culture supernatant was obtained. When the α-glucosyltransferase activity was measured for the culture solution and the culture supernatant, the enzyme activity of the culture solution was about 0.36 unit / ml, and the enzyme activity of the culture supernatant was about 0.42 unit / ml. there were. It was found that most of the α-glucosyltransferase produced by Arthrobacter globiformis PP349 exists outside the cells.

<Experiment 9: Purification of α-glucosyltransferase derived from Arthrobacter globiformis PP349>
About 18 L of the culture supernatant obtained in Experiment 8 (total activity of about 7,560 units) was added with ammonium sulfate so as to be 80% saturated, dissolved, and left to stand at 4 ° C. for 24 hours for salting out. The precipitated salted-out product is collected by centrifugation (11,000 rpm, 30 minutes), dissolved in 20 mM acetate buffer (pH 6.0), dialyzed against the same buffer, centrifuged and insoluble. The product was removed to obtain about 500 ml of a crude enzyme solution. The α-glucosyltransferase activity in the crude enzyme solution was about 14 units / ml (total activity about 7,000 units). To this crude enzyme solution, ammonium sulfate was added to a final concentration of 2M, centrifuged to remove insoluble matters, and equilibrated with 20 mM acetate buffer (pH 6.0) containing 2M ammonium sulfate. The sample was subjected to hydrophobic column chromatography (gel volume: 300 ml) using Toyopearl 650M gel. The α-glucosyltransferase activity was adsorbed on the gel and eluted with a linear gradient from 2M to 0M ammonium sulfate concentration. This active fraction was collected, dialyzed against 20 mM acetate buffer (pH 6.0), and then subjected to anion exchange column chromatography (gel volume 100 ml) using “DEAE-Toyopearl 650S” gel manufactured by Tosoh Corporation. did. α-Glucosyltransferase activity was adsorbed on a “DEAE-Toyopearl 650S” gel equilibrated with 20 mM acetate buffer (pH 6.0) and eluted with a linear gradient from 0 M to 0.5 M in salt concentration. It eluted at around 0.1M. Table 7 shows α-glucosyltransferase activity, specific activity and yield of α-glucosyltransferase in each step of each purification.

  When the purity of the enzyme preparation was tested by 5 to 20 w / v% concentration gradient polyacrylamide gel electrophoresis of the purified α-glucosyltransferase preparation, the protein band was single and the purity was high. .

<Experiment 10: Properties of α-glucosyltransferase derived from Arthrobacter globiformis PP349>
<Experiment 10-1: Molecular weight>
Molecular weight marker that was subjected to SDS-polyacrylamide gel electrophoresis (concentration gradient of 5 to 20 w / v%) using the α-glucosyltransferase purified preparation derived from Arthrobacter globiformis PP349 obtained by the method of Experiment 9 and simultaneously migrated When the molecular weight was measured in comparison with (manufactured by Nippon Bio-Rad Laboratories Co., Ltd.), it was found that the molecular weight of the α-glucosyltransferase was 90,000 ± 10,000 daltons.

<Experiment 10-2: Optimum temperature and pH of enzyme reaction>
Using the α-glucosyltransferase purified preparation derived from Arthrobacter globiformis PP349 obtained by the method of Experiment 9, the effects of temperature and pH on enzyme activity were examined according to the method of activity measurement. These results are shown in FIG. 7 (optimum temperature) and FIG. 8 (optimum pH). The optimal temperature of the α-glucosyltransferase is about 6.0 ° C. under a reaction condition of pH 6.0 for 30 minutes, and the optimal pH is about 6.0 under a reaction condition of 40 ° C. for 30 minutes. found.

<Experiment 10-3: Temperature stability and pH stability of enzyme>
Using the α-glucosyltransferase purified preparation obtained in Experiment 9, the temperature stability and pH stability of the enzyme were examined. The temperature stability was determined by measuring the remaining enzyme activity after holding the enzyme solution (20 mM acetate buffer, pH 6.0) at each temperature for 60 minutes, cooling with water. The pH stability was determined by maintaining the enzyme in 20 mM buffer at each pH at 4 ° C. for 24 hours, adjusting the pH to 6.0, and measuring the remaining enzyme activity. These results are shown in FIG. 9 (temperature stability) and FIG. 10 (pH stability). As is clear from FIGS. 9 and 10, the Arthrobacter globiformis-derived α-glucosyltransferase of the present invention was stable up to 40 ° C. and stable in the range of pH 4.0 to 8.0.

<Experiment 10-4: Effect of metal salt on enzyme activity>
Using the α-glucosyltransferase purified preparation obtained by the method of Experiment 9, the influence of the metal salt on the enzyme activity was examined in the presence of various metal salts at a concentration of 1 mM according to the method of activity measurement. The results are shown in Table 8.

As is apparent from the results in Table 8, it was found that the activity of the α-glucosyltransferase is significantly inhibited by Hg ²⁺ ions and inhibited by Cu ²⁺ ions.

<Experiment 11: Effects on various carbohydrates>
The substrate specificity of α-glucosyltransferase was examined using various carbohydrates. Methyl-α-glucoside, methyl-β-glucoside, paranitrophenyl-α-glucoside, paranitrophenyl-β-glucoside, glucose, sucrose, maltose, isomaltose, trehalose, cordobiose, nigerose, neotrehalose, cellobiose, lactose, An aqueous solution containing maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, isomaltotriose or isopanose was prepared. The final concentration of 20 mM acetate buffer (pH 6.0) was added to these substrate solutions, and then the Bacillus circulans PP710-derived α-glucosyltransferase purified preparation obtained by the method of Experiment 6 was used per gram of substrate solids. 10 units each were added to prepare a substrate concentration of 1 w / v%, and this was allowed to act at 40 ° C. and pH 6.0 for 24 hours. In order to examine the sugar in the reaction solution before and after the enzyme reaction, a mixed solution of n-butanol, pyridine and water (volume ratio 6: 4: 1) was used as a developing solvent, and “Kieselgel 60” (aluminum) manufactured by Merck as a thin layer plate. Using a plate, 10 × 20 cm), silica gel thin layer chromatography (hereinafter abbreviated as TLC) developed twice was performed, and after spraying with a 10% sulfuric acid-methanol solution, saccharides were detected by heating. The presence or absence of the production of reaction products other than the substrate carbohydrate in TLC was examined, and the presence or absence of the enzyme action on each carbohydrate was confirmed. The results are shown in Table 9. The substrate specificity of the α-glucosyltransferase derived from Arthrobacter globiformis PP349 was also examined in the same manner. As a result, it was the same as that of the Bacillus circulans PP710-derived α-glucosyltransferase.

  As is apparent from the results in Table 9, this α-glucosyltransferase acts well on maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose among the tested carbohydrates. In addition, it acted on nigerose, isomaltose, neotrehalose, isomalttriose, and isopanose. In addition, it slightly affected Kojibiose. From the saccharides that acted, in addition to degradation products, glycosyl transfer products were also observed. On the other hand, this α-glucosyltransferase has an effect on methyl-α-glucoside, methyl-β-glucoside, paranitrophenyl-α-glucoside, paranitrophenyl-β-glucoside, trehalose, cellobiose, sucrose, lactose and the like. I was not able to admit. As a result of these results and the α-glucosyltransferase acting on the partial degradation product of starch to produce branched α-glucan, the enzyme is composed of maltose and α-1,4 glucan having a degree of glucose polymerization of 3 or more or glucose. It has been found to act widely on α-glucooligosaccharides.

<Experiment 12: Mechanism of Action>
In order to investigate the mechanism of action of α-glucosyltransferase, the structure of the produced sugar was examined when it was allowed to act on maltose, the smallest substrate. In addition, the result when using the α-glucosyltransferase derived from Bacillus circulans PP710 and the α-glucosyltransferase derived from Arthrobacter globiformis PP349 were the same. In this experiment, the result using the Bacillus circulans PP710-derived α-glucosyltransferase purified preparation is shown.

<Experiment 12-1: Product from Maltose>
After adding a final concentration of 10 mM acetate buffer (pH 6.0) to a maltose aqueous solution having a final concentration of 1 w / v%, the purified α-glucosyltransferase obtained by the method of Experiment 6 was added per gram of substrate solid content. Ten units were added, the mixture was allowed to act at 40 ° C. and pH 6.0, sampling was performed over time, and the reaction was stopped by holding at 100 ° C. for 10 minutes. The sugar composition of the enzyme reaction solution was measured using HPLC and GC. HPLC and GC were performed using the conditions described in Experiment 4-2. The results are shown in Table 10.

  As is apparent from the results in Table 10, mainly glucose, maltotriose and panose were produced from the substrate maltose by the action of α-glucosyltransferase in the initial reaction (1 hour of reaction). Furthermore, oligosaccharides having glucose polymerization degrees of 4 and 5 were produced from the reaction of 2 hours and 4 hours. As the reaction proceeds, maltotriose peaked at 2 hours (14.4%) and panose peaked at 4 hours (29.3%), and an increase in isomaltose was observed. It was. Further, isomaltose and oligosaccharides having a polymerization degree of 4 or more were observed until 48 hours after the reaction.

  Inferring from these results, α-glucosyltransferase acts on maltose, and catalyzes both glucosyl transfer of α-1,4 glucosyl transfer and α-1,6 glucosyl transfer at the initial stage of the reaction, thereby producing glucose, malto Isomaltose that produced triose and panose and α-1,6-glucosyl transferred to glucose as the reaction progressed, and α-1,4 glucosyl transfer and α-1,6 glucosyl transfer isopanose transferred to the isomaltose and It was found that isomaltotriose was formed. In this experiment, it is difficult to identify many kinds of oligosaccharides having a polymerization degree of 4 or more. Therefore, in Experiment 12-2, maltopentaose having a polymerization degree higher than that of maltose was used as a substrate, and the reaction mechanism was further improved. Analyzed.

<Experiment 12-2: Product from Maltopentaose>
After adding a final concentration of 10 mM acetate buffer (pH 6.0) to an aqueous maltopentaose solution having a final concentration of 1 w / v%, the α-glucosyltransferase purified preparation obtained by the method of Experiment 6 was treated with a substrate solid content of 1 10 units were added per gram, the mixture was allowed to act at 40 ° C. and pH 6.0, sampling was performed over time, and the reaction was stopped by holding at 100 ° C. for 10 minutes. The sugar composition of the enzyme reaction solution was measured using HPLC. HPLC was performed using the conditions described in Experiment 4-2. The enzyme reaction solution was subjected to methylation analysis according to a conventional method, and the partially methylated product was examined by gas chromatography. The abundance ratio of each glucoside bond in the glucose binding mode was determined from the composition of the resulting partially methylated product. In addition, an isomalt dextranase digestion test was also conducted as in Experiment 4-2. Table 11 shows changes in the sugar composition during the reaction, and Table 12 shows the results of methylation analysis of the reaction product and isomalt dextranase digestion test.

  As is clear from the results of Tables 11 and 12, in the initial reaction (1 hour of reaction), oligosaccharide having a glucose polymerization degree 1 smaller than that of the substrate and oligosaccharide having a glucose polymerization degree 1 higher than the substrate are preferentially selected from the substrate maltopentaose. From this, it was confirmed that this enzyme catalyzes glucosyl transfer. When the reaction further progressed, reaction products having various degrees of glucose polymerization were produced, and after 24 hours of reaction, glucans having a degree of glucose polymerization of 9 or more reached 21.1%. As a result of methylation analysis, as the reaction proceeds, 1,4-bonded glucose residues are decreased and 1,6-bonded glucose residues are significantly increased in the reaction product. It was found that bound glucose residues, 1,4,6 linked glucose residues and 1,3,6 linked glucose residues increased gradually. It was also found that the isomaltose content in the sugar composition after digestion with isomaltodextranase also increases markedly with the progress of the reaction. In addition, when the same test was performed using α-glucosyltransferase derived from Arthrobacter globiformis PP349, almost the same result was obtained.

From the results of Experiments 12-1 and 12-2, the reaction mechanism of α-glucosyltransferase was considered as follows.
1) This enzyme acts as a substrate on maltose and / or α-1,4 glucan having a degree of polymerization of glucose of 3 or more, and converts non-reducing terminal glucose residues to non-reducing terminal glucose residues of other α-1,4 glucans. Α-1,4 glucan (glucose polymerization degree) in which glucose is α-bonded to the 4-position or 6-position hydroxyl group of the non-reducing terminal glucose residue mainly by α-1,4 or α-1,6 glucosyl transfer to the group. Is α-glucan increased by 1), and α-1,4-glucan having a glucose polymerization degree decreased by 1.
2) The enzyme further acts on α-1,4 glucan having a 1-decrease degree of glucose polymerization generated in 1), and 1) in contrast to α-glucan having an increased glucose polymerization degree in 1) of 1). 4 or 6-position hydroxyl group of the non-reducing terminal glucose residue of α-glucan having an increased glucose polymerization degree of 1 produced in 1) by the intermolecular α-1,4 or α-1,6 glucosyl transfer as in Glucose is further transferred to and the chain length is extended.
3) By repeating the reactions 1) and 2) above, a glucan having α-1,4 and α-1,6 bonds is generated from maltose and / or α-1,4 glucan having a glucose polymerization degree of 3 or more. .
4) This enzyme is also α-1,3 glucosyl transfer or α-1,4 or α-1,3 glucosyl transfer to α-1,6 linked glucose residues in the glucan, although the frequency is low. To produce a glucan having α-1,3 bonds, α-1,4,6 bonds and α-1,3,6 bonds.
5) As a result of the above reactions 1) to 4) being repeated, glucose is bound mainly by α-1,4 bonds and α-1,6 bonds, and slightly α-1,3 bonds, α-1,4. , 6 bonds and α-1,3,6 bonds are produced.

<Experiment 13: α-Glucosyl transfer reaction and change in reducing power of reaction solution>
After adding a final concentration of 20 mM acetate buffer (pH 6.0) to an aqueous maltose solution (final concentration of 1 or 30 w / v%), a purified α-glucosyltransferase derived from Bacillus circulans PP710 obtained by the method of Experiment 6 The product was added 4 units per gram of substrate solids, allowed to act at 40 ° C., pH 6.0, sampled over time, and held at 100 ° C. for 10 minutes to stop the reaction. The amount of maltose remaining in the reaction solution was quantified by the HPLC method and the GC method described in Experiment 4-2. In addition, the amount of reducing sugar in the enzyme reaction solution was quantified by the Somogy-Nelson method and the amount of all sugars was quantified by the Anthrone method, and the reducing power was calculated by the following formula: reducing power = (reducing sugar content / total sugar content) × 100. The results are shown in Table 13.

  As is apparent from the results in Table 13, when this α-glucosyltransferase was allowed to act on maltose, a slight increase in reducing power was observed when the maltose concentration was relatively low at 1 w / v%. Further, when the maltose concentration was relatively high at 30 w / v%, almost no increase in the reducing power of the reaction solution was observed. This α-glucosyltransferase is essential in that even when the maltose concentration is relatively low at 1 w / v% and the residual amount of maltose is 10% or less, the reduction power of the reaction solution is only slightly increased. It is an enzyme that catalyzes a transfer reaction, and means that hydrolysis is hardly performed during the reaction. This α-glucosyltransferase was found to be an enzyme that efficiently performs α-glucosyltransferase. In addition, when the same test was performed using α-glucosyltransferase derived from Arthrobacter globiformis PP349, almost the same result was obtained.

<Experiment 14: Comparison of purified α-glucosyltransferase and crude enzyme in production of branched α-glucan>
In consideration of the industrial production of branched α-glucan, it is more preferable that a crude enzyme of α-glucosyltransferase can be used because it saves labor and labor for purifying the enzyme. Therefore, it was examined whether a branched α-glucan equivalent to glucan A prepared in Experiment 1-2 was obtained using a crude enzyme of α-glucosyltransferase derived from Bacillus circulans PP710. Bacillus circulans PP710 was cultured by the method described in Experiment 1-1, and the culture supernatant was salted out with ammonium sulfate and dialyzed against 20 mM acetate buffer (pH 4.5) to obtain a crude α-glucosyltransferase. The enzyme solution was used. This crude enzyme solution was allowed to act on a partially decomposed starch (trade name “Paindex # 100”, sold by Matsutani Chemical Industry Co., Ltd.) by the method described in Experiment 1-2, and a glucan solution with a solid concentration of 30% was used as a substrate. It was obtained in a yield of 88.2% per solid from the partially decomposed starch. The obtained branched α-glucan was named “glucan C”, and the result of methylation analysis was measured in Table 14 and the molecular weight distribution and water-soluble dietary fiber content were measured by the methods described in Experiment 4 and Experiment 3. Table 15 shows the results. In Tables 14 and 15, data of glucan A prepared using partially degraded starch and partially purified α-glucosyltransferase as raw materials prepared from Tables 3 and 4 are shown again for comparison.

  As shown in Table 14, the glucan C prepared using a crude enzyme of α-glucosyltransferase derived from Bacillus circulans PP710 surprisingly has a 2, The contents of 3,4,6-tetramethylated product and 2,3,4-trimethylated product corresponding to α-1,6-linked glucose residues were increased compared to glucan A. This result indicates that glucan C has more non-reducing ends and more α-1,6 bonds than glucan A. Further, in glucan C, 2,4-dimethylated product was 4.8%, which was higher than 0.8% of glucan A. This result shows that the glucose residues responsible for both 1,3 and 1,6 bonds are increasing.

  Further, as is apparent from Table 15, glucan C had a number average molecular weight and a weight average molecular weight both smaller than that of glucan A (the degree of glucose polymerization was small), and showed a much higher water-soluble dietary fiber content. This result shows that the crude enzyme of Bacillus circulans PP710-derived α-glucosyltransferase is mixed with other enzymes different from α-glucosyltransferase, and the mixed enzyme is α-glycan in branched α-glucan. This suggests that it contributes to an increase in 1,6 bonds, an increase in glucose residues in both 1,3 bonds and 1,6 bonds, a decrease in molecular weight, and an increase in water-soluble dietary fiber content. is there.

<Experiment 15: Identification, Isolation, and Purification of Enzyme Mixed with Crude Enzyme of α-Glucosyltransferase Derived from Bacillus Circus PP710>
Increased α-1,6 bond in branched α-glucan, increased glucose residues in both 1,3 bond and 1,6 bond, mixed in crude enzyme of α-glucosyltransferase derived from Bacillus circulans PP710 Experiments were conducted to identify, isolate and purify enzymes involved in lowering the molecular weight and increasing the water-soluble dietary fiber content.

<Experiment 15-1: Identification of mixed enzyme and activity measurement>
Bacillus circulans PP710 (FERM BP-10771) was cultured in the same manner as in Experiment 1-1, and about 3 L of the obtained culture supernatant was salted out with ammonium sulfate, and then 20 mM Tris-HCl buffer containing 1 mM calcium chloride. About 40 ml of the dialyzed solution obtained by dialysis was used as a crude enzyme solution. This crude enzyme solution was allowed to act on a 2 w / v% soluble starch solution or 2 w / v% pullulan solution at pH 6.0, 40 ° C. for 16 hours, and the reaction was stopped by heating at 100 ° C. for 10 minutes. , And subjected to TLC using a 20 cm wide silica gel 60F ₂₅₄ (Merck) TLC plate. Development was performed twice using n-butanol: pyridine: water (volume ratio 6: 4: 1) as a developing solvent, and after spraying with a 20% sulfuric acid-methanol solution, the product was heated at 100 ° C. for 5 minutes. Spot detected. As a result, malto-oligosaccharides having a maltose and a glucose polymerization degree of 3 or more were produced from soluble starch, and slight isomaltose and panose were produced from pullulan. It was found that the crude enzyme of α-glucosyltransferase of Bacillus circulans PP710 (FERM BP-10771) is mixed with amylase that decomposes starch and also pullulan.

  The activity of mixed amylase was measured as follows. That is, short-chain amylose (trade name “Amylose EX-I”, sold by Hayashibara Biochemical Laboratories, Inc., average polymerization degree 17) is 50 mM acetate buffer containing 1 mM calcium chloride so that the final concentration is 1 w / v%. Dissolve in (pH 6.0) to make a substrate solution, add 0.2 ml of the enzyme solution to 2 ml of the substrate solution, and allow the enzyme reaction at 35 ° C. for 30 minutes, and then add 0.2 ml of the reaction solution to 8 ml of 0.02N sulfuric acid solution. After mixing and stopping the reaction, 0.2 ml of 0.1N iodine solution is added, and after maintaining at 25 ° C. for 15 minutes, the absorbance at 660 nm is measured. Separately, the reaction solution at 0 hour reaction is measured in the same manner, and the decrease in iodine coloration per reaction time is measured. One unit of amylase activity was defined as 10 times the amount of enzyme that reduces the absorbance (iodine coloration) at 660 nm of 20 mg of short-chain amylose by 10% under the above conditions.

<Experiment 15-2: Isolation and purification of mixed amylase>
The crude enzyme solution prepared in Experiment 15-1 was subjected to anion exchange column chromatography (gel capacity 70 ml) using “DEAE-Toyopearl 650S” gel manufactured by Tosoh Corporation. Amylase did not adsorb on the “DEAE-Toyopearl 650S” gel equilibrated with 1 mM calcium chloride 20 mM Tris-HCl buffer (pH 7.5), but eluted in the non-adsorbed fraction. Collect this active fraction, add ammonium sulfate to a final concentration of 1.5M, leave it at 4 ° C for 24 hours, centrifuge to remove insolubles, and use Pharmacia Biotech's "Resource PHE" gel. Was subjected to hydrophobic column chromatography (gel volume: 1 ml). The target amylase is adsorbed on the “Resource PHE” gel equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 1.5 M ammonium sulfate and 1 mM calcium chloride, and has a linear gradient from 1.5 M to 0 M ammonium sulfate concentration. When it was eluted, it was eluted at an ammonium sulfate concentration of about 0.3M. This active fraction was collected and concentrated, and then subjected to gel filtration column chromatography (gel capacity 118 ml) using “Superdex 200 pg” gel manufactured by Pharmacia Biotech, and 20 mM Tris-containing 0.2 M sodium chloride and 1 mM calcium chloride. Elution was performed with a hydrochloric acid buffer (pH 7.5). The active fraction was collected and subjected to anion exchange column chromatography (gel volume 1 ml) using “Resource Q” gel manufactured by Pharmacia Biotech. Amylase did not adsorb on the “Resource Q” gel equilibrated with 1 mM calcium chloride 20 mM Tris-HCl buffer (pH 7.5), but eluted in the non-adsorbed fraction. The obtained active fraction was used as an amylase purified sample. Table 16 shows the amylase activity, the specific activity of amylase and the yield in each step of each purification.

  When the purity of the enzyme preparation was tested by 5-20 w / v% concentration gradient polyacrylamide gel electrophoresis of the purified amylase preparation, the protein band was single and the purity was high.

<Experiment 16: Properties of amylase derived from Bacillus circulans PP710>
<Experiment 16-1: Molecular Weight>
The purified amylase sample obtained by the method of Experiment 15-2 was subjected to SDS-polyacrylamide gel electrophoresis (5 to 20 w / v% concentration gradient) and simultaneously migrated molecular weight marker (manufactured by Nippon Bio-Rad Laboratories, Inc.) ), The molecular weight of the amylase was found to be 58,000 ± 10,000 daltons.

<Experiment 16-2: Optimum temperature and pH of amylase>
Using the Bacillus circulans PP710-derived purified amylase preparation obtained by the method of Experiment 15-2, the effects of temperature and pH on amylase activity were examined according to the activity measurement method. These results are shown in FIG. 11 (optimum temperature) and FIG. 12 (optimum pH). The optimum temperature of the amylase was found to be 55 ° C. under the reaction condition of pH 6.0 for 30 minutes, and the optimum pH was 6.0 to 7.0 under the reaction condition of 35 ° C. for 30 minutes. .

<Experiment 16-3: Temperature stability and pH stability of amylase>
Using the amylase purified sample obtained by the method of Experiment 15-2, the temperature stability and pH stability were examined. The temperature stability was determined by measuring the remaining enzyme activity after holding the enzyme solution (20 mM acetate buffer, the same buffer containing pH 6.0 or 1 mM calcium chloride) at each temperature for 60 minutes, cooling with water. . The pH stability was determined by maintaining the enzyme in 20 mM buffer at each pH at 4 ° C. for 24 hours, adjusting the pH to 6.0, and measuring the remaining enzyme activity. These results are shown in FIG. 13 (temperature stability) and FIG. 14 (pH stability). As apparent from FIGS. 13 and 14, the amylase was stable up to 40 ° C. in the absence of calcium ions, and stable up to 50 ° C. in the presence of 1 mM calcium ions. The amylase was stable in the range of pH 6.0 to 8.0.

<Experiment 16-5: Substrate specificity of amylase>
Using the purified amylase preparation obtained by the method of Experiment 15-2, when the action of the amylase on various substrates was examined, the amylase was starch, maltose, and α-1,4 glucan having a glucose polymerization degree of 3 or more. Was hydrolyzed and also catalyzed glycosyl transfer. It has also been found that the amylase has the action of producing cyclodextrin from starch and hydrolyzing pullulan to produce panose.

<Experiment 17: Preparation of branched α-glucan using α-glucosyltransferase and amylase>
Using the Bacillus circulans PP710-derived α-glucosyltransferase purified preparation obtained by the method of Experiment 6 and the amylase purified preparation obtained by the method of Experiment 15-2, derived from the Bacillus circulans PP710 described in Experiment 14 It was investigated whether the preparation of glucan C using a crude enzyme of α-glucosyltransferase could be reproduced. That is, a starch partial decomposition product (trade name “Paindex # 100”, sold by Matsutani Chemical Co., Ltd.) was dissolved in water to a concentration of 30% by mass, adjusted to pH 6.0, and obtained by the method of Experiment 6. Bacillus circulans PP710-derived α-glucosyltransferase purified preparation was added at 10 units per gram of solid, and amylase obtained by the method of Experiment 15-2 was added at 0, 0.1, 0 per gram of solid. .2, 0.5, or 1.0 unit was added and allowed to act at 40 ° C. and pH 6.0 for 72 hours, and then the reaction solution was boiled for 10 minutes to stop the reaction. A chromatogram obtained by subjecting the branched α-glucan obtained under each reaction condition to the gel filtration HPLC method described in Experiment 4-4 is shown in FIG. 15 together with that of the partially decomposed starch used as a substrate. In FIG. 15, symbol a is a gel filtration HPLC chromatogram of a partially degraded starch as a substrate, and symbols b, c, d, and e are each 10 units of α-glucosyltransferase and 0 of amylase. . Gel filtration HPLC chromatogram of branched α-glucan obtained by acting 1 unit, 0.2 unit, 0.5 unit and 1.0 unit (branched α in which 10 units of α-glucosyltransferase were allowed to act) -The gel filtration HPLC chromatogram of glucan is almost the same as the chromatogram of glucan A shown in Fig. 1 and omitted in Fig. 15. The same applies to Figs. The results of molecular weight distribution analysis of each branched α-glucan based on the chromatogram obtained by gel filtration HPLC and the water-soluble dietary fiber content examined by the enzyme-HPLC method described in Experiment 3 are shown in Table 17. Table 17 also shows the results of molecular weight distribution analysis and water-soluble dietary fiber content (α-glucosyltransferase 0 unit, amylase 0 unit) examined for the partially degraded starch used as a substrate.

  As is clear from the results in Table 17, when amylase was used in combination with this α-glucosyltransferase, the molecular weight of the obtained branched α-glucan decreased and the water-soluble dietary fiber content increased dramatically. Further, as the amount of amylase acting increased, both the number average molecular weight and the weight average molecular weight of the obtained branched α-glucan decreased, and the value obtained by dividing the weight average molecular weight by the number average molecular weight (Mw / Mn) also decreased. From this, it was found that the width of the molecular weight distribution was narrowed. When the amount of amylase acting was 0.5 units per gram of solid, Mw / Mn was reduced to 2.1. Further, the content of water-soluble dietary fiber in the obtained branched α-glucan reached about 76% by mass when the amount of amylase acting was 0.5 units or more per gram of solid.

  From this result, it was found in Experiment 14 that the glucan C obtained by using the crude enzyme of α-glucosyltransferase derived from Bacillus circulans PP710 had a low molecular weight and a high dietary fiber content. It was confirmed that it was caused by amylase. The mixed amylase lowers the molecular weight of the branched α-glucan by partially hydrolyzing the branched starch-glucan that is a product of the partial starch degradation product and α-glucosyltransferase as a substrate. Further, it is presumed that by transferring to branched α-glucan, it acts to increase the dietary fiber content as a result. Furthermore, this result shows that a branched α-glucan having a low molecular weight and an increased water-soluble dietary fiber content can be produced by combining amylase with this α-glucosyltransferase.

<Experiment 18: Preparation of branched α-glucan using α-glucosyltransferase in combination with other known amylase>
Using the α-glucosyltransferase used in the present invention in combination with other known amylases to act on the partially degraded starch, a branched α-glucan is prepared, and its structural characteristics and water-soluble dietary fiber content are examined. did. In addition, in this experiment, the results when using the α-glucosyltransferase derived from Bacillus circulans PP710 and the α-glucosyltransferase derived from Arthrobacter globiformis PP349 were equivalent. The result using the Bacillus circulans PP710 origin alpha-glucosyltransferase purified sample is shown.

<Experiment 18-1: Preparation of Branched α-Glucan Combined with α-Glucosyltransferase and Isoamylase, Molecular Weight Distribution and Water-soluble Dietary Fiber Content of Branched α-Glucan Obtained>
Instead of Bacillus circulans PP710-derived amylase, Pseudomonas amyloderamosa-derived isoamylase (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) 0, 50, 200, 500 or 1,000 units per gram of solid The reaction was carried out in the same manner as in Experiment 17 except that it was added. A chromatogram obtained by subjecting the branched α-glucan obtained under each reaction condition to the gel filtration HPLC method described in Experiment 4-4 is shown in FIG. 16 together with that of the partially degraded starch used as a substrate. In FIG. 16, symbol a is a gel filtration HPLC chromatogram of a partially decomposed starch as a substrate, and symbols b, c, d, and e each have 10 units of α-glucosyltransferase and isoamylase. It is a gel filtration HPLC chromatogram of branched α-glucan obtained by acting 50 units, 200 units, 500 units and 1,000 units. Table 18 shows the results of molecular weight distribution analysis of each branched α-glucan based on the chromatogram obtained by gel filtration HPLC and the water-soluble dietary fiber content examined by the enzyme-HPLC method described in Experiment 3. .

  As is apparent from the results obtained when only the α-glucosyltransferase of Table 18 was allowed to act on the partial degradation product of starch, α-glucosyltransferase alone did not significantly affect the molecular weight distribution, but Table 18 and FIG. As is apparent from the results, the number average molecular weight and the weight average molecular weight of the resulting branched α-glucan both decrease as the amount of isoamylase acting increases, and the value obtained by dividing the weight average molecular weight by the number average molecular weight (Mw / Mn) also decreased, and it was found that the molecular weight distribution narrowed. When the acting amount of isoamylase was 1000 units per gram of the solid, Mw / Mn decreased to 1.9, and the branched α-glucan showed a molecular weight distribution having a peak at a glucose polymerization degree of 20.6. On the other hand, the content of water-soluble dietary fiber in the branched α-glucan obtained under each reaction condition was about 40 to 44% by mass without being significantly affected by the amount of isoamylase acting.

  From this result, branched α-glucan with a reduced molecular weight is produced with almost no change in the content of water-soluble dietary fiber by using this α-glucosyltransferase in combination with isoamylase and acting on the partially degraded starch. It turns out that you can.

<Experiment 18-2: Preparation of branched α-glucan using α-glucosyltransferase in combination with α-amylase and molecular weight distribution and water-soluble dietary fiber content of the obtained branched α-glucan>
Instead of Pseudomonas amyloderamosa-derived isoamylase, a commercially available α-amylase (trade name “Neospirase PK2”, manufactured by Nagase Seikagaku Corporation) is 0, 0.1, 0.2, 0.5 per gram of solid. Or it was made to react like experiment 18-1 except having added 1.0 unit. A chromatogram obtained by subjecting the branched α-glucan obtained under each reaction condition to the gel filtration HPLC method described in Experiment 4-4 is shown in FIG. 17 together with that of the partially decomposed starch used as a substrate. In FIG. 17, symbol a is a gel filtration HPLC chromatogram of a partially decomposed starch as a substrate, and symbols b, c, d, and e each have 10 units of α-glucosyltransferase and α-amylase. Is a gel filtration HPLC chromatogram of a branched α-glucan obtained by allowing 0.1 unit, 0.2 unit, 0.5 unit and 1.0 unit to act. The results of molecular weight distribution analysis of each branched α-glucan based on the chromatogram obtained by gel filtration HPLC and the water-soluble dietary fiber content examined by the enzyme-HPLC method described in Experiment 3 are shown in Table 19. .

  As apparent from the results of FIG. 17 and Table 19, both the number average molecular weight and the weight average molecular weight of the branched α-glucan obtained as the amount of action of α-amylase increases, and the weight average molecular weight is further reduced to the number average molecular weight. Since the value divided by (Mw / Mn) also decreased, it was found that the width of the molecular weight distribution was narrowed. When the amount of α-amylase acting is 1.0 unit per gram of solid (reference symbol e in FIG. 17), Mw / Mn decreases to 2.4, and the branched α-glucan has a glucose polymerization degree of 11.8. A molecular weight distribution with a peak was shown. On the other hand, the water-soluble dietary fiber content in the branched α-glucan obtained under each reaction condition tended to increase as the amount of α-amylase acting increased.

  From this result, it was found that by using α-amylase in combination with α-amylase and acting on a partially degraded starch, a branched α-glucan having an increased water-soluble dietary fiber content and a reduced molecular weight can be produced. did.

<Experiment 18-3: Preparation of branched α-glucan using α-glucosyltransferase and CGTase together, molecular weight distribution of the obtained branched α-glucan and water-soluble dietary fiber content>
CGTase derived from Bacillus thermophilus (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) instead of Pseudomonas amyloderamosa-derived isoamylase is 0, 0.1, 0.2, The reaction was conducted in the same manner as in Experiment 18-1, except that 0.5 or 1.0 unit was added. A chromatogram obtained by subjecting the branched α-glucan obtained under each reaction condition to the gel filtration HPLC method described in Experiment 4-4 is shown in FIG. 18 together with that of the partially degraded starch used as a substrate. In FIG. 18, symbol a is a gel filtration HPLC chromatogram of a partially degraded starch as a substrate, and symbols b, c, d and e each have 10 units of α-glucosyltransferase and 0 of CGTase. It is a gel filtration HPLC chromatogram of a branched α-glucan obtained by acting 1 unit, 0.2 unit, 0.5 unit and 1.0 unit. Table 20 shows the results of molecular weight distribution analysis of each branched α-glucan based on the chromatogram obtained by gel filtration HPLC and the water-soluble dietary fiber content examined by the enzyme-HPLC method described in Experiment 3. .

  As apparent from the results of FIG. 18 and Table 20, as the acting amount of CGTase increases, both the number average molecular weight and the weight average molecular weight decrease, and the value obtained by dividing the weight average molecular weight by the number average molecular weight (Mw / Mn ) Also decreased, and the width of the molecular weight distribution was found to be narrow. When the amount of action of CGTase is 1.0 unit per gram of solids (reference symbol e in FIG. 18), Mw / Mn decreases to 3.2, and branched α-glucan peaks at a glucose polymerization degree of 79.1. The molecular weight distribution was shown. On the other hand, the content of the water-soluble dietary fiber in the branched α-glucan tended to increase as the amount of action of CGTase increased. When 1.0 unit per gram of solid was used, the water-soluble dietary fiber content reached 70.5% by mass.

  From this result, it was found that by using CGTase in combination with this α-glucosyltransferase and acting on a partially degraded starch, a branched α-glucan having a reduced molecular weight and a significantly increased water-soluble dietary fiber content can be prepared. did. CGTase generates a non-reducing terminal glucosyl residue without drastically reducing the molecular weight compared to α-amylase because CGTase has both a hydrolysis action of α-1,4 bonds and a transfer action. It is speculated that a branched α-glucan having a higher frequency of action of α-glucosyltransferase and having a higher water-soluble dietary fiber content than the addition of α-amylase was obtained.

<Experiment 18-4: Preparation of Branched α-Glucan Using α-Glucosyltransferase, Isoamylase, and CGTase, Molecular Weight Distribution of the Branched α-Glucan Obtained, and Water-soluble Dietary Fiber Content>
In the experiment described in Experiment 18-1, CGTase derived from Bacillus stearothermophilus was further reacted in the same manner as in Experiment 18-1, except that 0 or 1.0 unit was added per gram of the solid. A chromatogram obtained by subjecting the branched α-glucan obtained under each reaction condition to the gel filtration HPLC method described in Experiment 4-4 is shown in FIG. 19 together with that of the partially degraded starch used as a substrate. In FIG. 19, symbol a is a gel filtration HPLC chromatogram of a partially decomposed starch as a substrate, and symbols b, c, d and e are 10 units and 1 unit respectively for α-glucosyltransferase and CGTase. It is a gel filtration HPLC chromatogram of a branched α-glucan obtained by reacting isoamylase with 50 units, 200 units, 500 units and 1,000 units. The results of molecular weight distribution analysis of each branched α-glucan based on the chromatogram obtained by gel filtration HPLC and the water-soluble dietary fiber content examined by the enzyme-HPLC method described in Experiment 3 are shown in Table 21. .

  As is clear from the results of FIG. 19 and Table 21, the weight average molecular weight of the branched α-glucan prepared by using this α-glucosyltransferase and 1 unit of CGTase per gram of the solid was divided by the number average molecular weight. The value (Mw / Mn) decreased to 3.2, and further decreased to 1.9 when 1000 units of isoamylase was used in combination with 1 gram of the solid (sign e in FIG. 19). The water-soluble dietary fiber content of the branched α-glucan prepared by combining α-glucosyltransferase and CGTase is increased to about 70% by mass, and even when isoamylase is used in combination, it remains high without being reduced. Was.

  From this result, by using this α-glucosyltransferase, isoamylase and CGTase in combination and acting on a partially degraded starch, branched α-glucan having a markedly decreased molecular weight and a significantly increased water-soluble dietary fiber content. It was found that can be prepared.

<Experiment 19: Functionality of branched α-glucan>
As a branched α-glucan having the highest water-soluble dietary fiber content and relatively low molecular weight, in Experiment 18-4, 10 units of α-glucosyltransferase, 50 units of isoamylase, and 1 unit of CGTase were added per gram of solid. The branched α-glucan prepared in combination was selected, and the digestibility and structural characteristics and functionality of the branched α-glucan were examined.

<Experiment 19-1: Purification of branched α-glucan using α-glucosyltransferase, isoamylase and CGTase in combination>
Filter the branched α-glucan reaction solution obtained by reacting in the same manner as in Experiment 18-4 using 10 units of α-glucosyltransferase, 50 units of isoamylase, and 1 unit of CGTase per gram of solid matter. After removing insoluble matter, decolorizing and desalting using ion exchange resins “Diaion SK-1B” and “Diaion WA30” made by Mitsubishi Chemical and anion exchange resin “IRA411” made by Organo, followed by fine filtration Then, the solution was concentrated by an evaporator, and a branched α-glucan-containing liquid having a solid content concentration of 30% was obtained from the starch used in a yield of 85.8% per solid matter. Table 22 shows the results of methylation analysis of the obtained branched α-glucan performed by the method described in Experiment 4-1. Moreover, the molecular weight distribution analysis performed by the gel filtration HPLC method described in Experiment 4-4, the water-soluble dietary fiber content determined by the enzyme-HPLC method described in Experiment 3, and the isomaltdextranase digestion test described in Experiment 4-2 The results are summarized in Table 23.

  As is apparent from the results in Tables 22 and 23, the obtained branched α-glucan was obtained by converting the partially methylated 2,3,6-trimethylated and 2,3,4-trimethylated compounds to 1 in the methylation analysis. The ratio of 2,3,6-trimethylated product and 2,3,4-trimethylated product accounted for 77.5% of the partially methylated product. In this branched α-glucan, 2,4,6-trimethylated product and 2,4-dimethylated product showed 1.6% and 2.4% of partially methylated product, respectively. The branched α-glucan has a weight average molecular weight of 5,480 daltons, Mw / Mn 2.3, a water-soluble dietary fiber content determined by enzyme-HPLC method of 68.6% by mass, and isomalt-dextranase digestion. Thus, it was a glucan that produced 36.4% by mass of isomaltose per saccharide composition.

  For the purpose of evaluating the usefulness of the branched α-glucan, using the branched α-glucan prepared in Experiment 19-1, It investigated in experiment 19-2 thru | or 19-7.

<Experiment 19-2: Acid fermentability test by cariogenic bacteria of branched α-glucan>
Using the branched α-glucan obtained by the method of Experiment 19-1, according to the method of Oshima et al. Described in “Infection and Immunity”, Vol. 39, pages 43 to 49 (1983). Then, an acid fermentation test using cariogenic bacteria was performed. Two strains, Streptococcus sobrinus 6715 and Streptococcus mutans OMZ-176, were used as cariogenic bacteria. As a control, the same operation was performed using sucrose. The results are shown in Table 24.

  As is apparent from the results in Table 24, in the case of sucrose subjected to acid fermentation, the pH is lowered to about 4, whereas the branched α-glucan hardly causes acid fermentation by Streptococcus sobrinus and Streptococcus mutans. The pH was maintained at about 6, and was higher than 5.5, which is the critical pH at which tooth enamel is decalcified. The branched α-glucan was confirmed to have extremely low cariogenicity.

<Experiment 19-3: Digestibility test of branched α-glucan>
Using the branched α-glucan obtained by the method of Experiment 19-1, saliva was obtained in a test tube in accordance with the method of Okada et al. The digestibility of branched α-glucan by amylase, artificial gastric juice, pancreatic juice amylase and small intestinal mucosal enzyme was examined. As a control, commercially available indigestible dextrin (trade name “Pine Fiber”, manufactured by Matsutani Chemical Co., Ltd.) was used. The results are shown in Table 25.

  As is apparent from the results in Table 25, the branched α-glucan was not digested at all by salivary amylase and artificial gastric juice, but was slightly degraded by pancreatic juice amylase. The degradation rate of the indigestible dextrin of the control by the small intestinal mucosal enzyme is 41.1%, whereas the degradation rate of the branched α-glucan is as low as 16.4%, and the branched α-glucan is commercially available. It was found that it was less digestible than indigestible dextrin.

<Experiment 19-4: Effect of intake of branched α-glucan on blood glucose level and insulin amount>
Using the branched α-glucan obtained by the method of Experiment 19-1, an increase in blood glucose level and an increase in insulin were examined. A group of five 7-week-old male male Wistar rats was fasted for 1 day, and an aqueous solution of branched α-glucan was orally administered using a stomach tube. The dose was 1.5 g as a solid per kg body weight of the rat. Blood was collected from the tail vein immediately before oral administration, 15 minutes, 30 minutes, 60 minutes, and 120 minutes after oral administration. Each blood was collected in a heparinized blood collection tube and centrifuged (2,000 rpm, 10 minutes) to obtain plasma. The blood glucose level was measured by a glucose oxidase method, and the amount of insulin was measured using a rat insulin measurement kit (manufactured by Morinaga Bioscience Research Institute). Glucose was used as control 1, and commercially available indigestible dextrin (trade name “Pine Fiber” manufactured by Matsutani Chemical Industry Co., Ltd.) was used as control 2. The results of blood glucose level and insulin amount in each test system are shown in Table 26 and Table 27, respectively.

  As is clear from the results in Table 26 and Table 27, it was found that the branched α-glucan had lower blood glucose level and higher insulin level than glucose, as with commercially available indigestible dextrin.

<Experiment 19-5: Acute toxicity test>
Using mice, the branched α-glucan obtained by the method of Experiment 19-1 was orally administered to conduct an acute toxicity test. As a result, the branch α- glucan is non-toxic, death even in the maximum amount that can be administered is not observed, its LD ₅₀ value was 5 g / kg-mouse weight or higher.

<Experiment 20: Blood sugar elevation inhibitory action of branched α-glucan>
In Experiment 19-4, it was found that the increase in the blood glucose level and the amount of insulin was lower than that of glucose due to the intake of branched α-glucan. Thus, the effect of intake of branched α-glucan on the increase in blood glucose was examined in more detail.

<Experiment 20-1: Effect of ingestion of branched α-glucan on blood sugar level and insulin amount when ingesting partially decomposed starch>
The effect of branched α-glucan on the increase in blood glucose after ingestion of a partially decomposed starch (trade name “Paindex # 1”, sold by Matsutani Chemical Co., Ltd.) was examined. Feed for rearing rats (prepared by Hayashibara Biochemical Laboratories, Inc., “AIN-93G”; Journal of Nutrition, Vol. 123, pages 1939-951 (1993), hereinafter “Purification” It is referred to as “feed” (see the composition shown in Table 33 below), and a 7-week-old Wistar male rat (sold by Nihon Charles River Co., Ltd.), 3 weeks, preliminarily raised for 1 week, is used for 5 days in each group. After fasting, an aqueous solution in which a starch partial decomposition product (1.5 g / kg / body weight as a solid) and a branched α-glucan were dissolved was orally administered through a stomach tube. The dosage of branched α-glucan was 0.15 g, 0.30 g, or 0.75 g as a solid per kg body weight of the rat. Rat blood was collected from the tail vein immediately before oral administration, 15 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, and 240 minutes after oral administration. Each blood was collected in a heparinized blood collection tube and centrifuged (2,000 rpm, 10 minutes) to obtain plasma. The blood glucose level and the amount of insulin were measured by the same method as in Experiment 19-4. As a control 1, 1.5 g / kg / body weight was orally administered as a solid only to a partially degraded starch to 5 rats per group. In addition, as control 2, instead of branched α-glucan, commercially available indigestible dextrin (trade name “Fibersol 2”, manufactured by Matsutani Chemical Industry Co., Ltd.) was used together with partially decomposed products of starch, group 2 and group 5 each. One rat was orally administered either 0.15 g or 0.75 g as a solid per kg body weight. Table 28, Table 29, Table 30, and Table 31 show the blood glucose level change, blood glucose AUC value (area under the blood concentration-time curve), insulin amount, and AUC value of insulin amount in each test system, respectively. Shown in

  As is clear from the results of Tables 28 to 31, the branched α-glucan is dose-dependently subjected to carbohydrate (starch degradation product) in a dose-dependent manner, similar to the commercially available indigestible dextrin (Control 2). It was found that blood glucose level, AUC level of blood glucose level, insulin level, and increase in AUC level of insulin level were suppressed as compared with the case where the partial starch degradation product was ingested alone (control 1). Moreover, when comparing the degree of the inhibitory effect, the branched α-glucan showed a stronger inhibitory effect than the commercially available indigestible dextrin in any of the measured indices.

<Experiment 20-2: Influence of molecular weight of branched α-glucan on blood glucose level and insulin amount when ingesting partially decomposed starch>
In Experiment 20-1, it was found that the branched α-glucan suppresses the increase in blood sugar level, sugar level AUC value, insulin level, and insulin level AUC at the time of carbohydrate (partially decomposed starch) load. Therefore, the influence of the difference in the molecular weight of the branched α-glucan on the blood glucose level and the insulin level increase inhibitory action when the carbohydrate was loaded was examined. That is, a branched α-glucan having a weight average molecular weight shown in Table 32 was prepared by adjusting the amounts of raw material starch partial decomposition products, α-glucosyltransferase, and amylase. As in Experiment 20-1, 7 7-week-old male male Wistar rats (sold by Nippon Charles River Co., Ltd.) pre-bred for 1 week with purified feed were used for each group of 8 groups after 5 days of fasting. A group of 5 rats were orally administered with an aqueous solution in which either a partially decomposed starch (1.5 g / kg / body weight as a solid) or a branched α-glucan shown in Table 32 was dissolved in a stomach tube. The dosage of branched α-glucan was 0.75 g as a solid per kg body weight of the rat. The remaining 5 rats in 1 group were orally administered with 1.5 g of a starch partial degradation product as a solid per 1 kg of rat body weight, and used as a control group. Blood from these rats was collected from the tail vein immediately before oral administration and 30 minutes after administration. Each blood was collected in a heparinized blood collection tube and centrifuged (2,000 rpm, 10 minutes) to obtain plasma. The measurement results of blood glucose level and insulin amount in each group are also shown in Table 32. The blood collection time after administration was 30 minutes after administration when blood sugar level and blood insulin amount peaked when only the partial starch degradation product was administered.

  As is apparent from the results in Table 32, the branched α-glucan having a weight average molecular weight in the range of 1,168 to 200,000 is increased in blood glucose level and insulin amount after oral administration of a partially degraded starch. Was suppressed. Further, when compared with the degree of the blood glucose level and the strength of the increase in insulin, the molecular weight of the branched α-glucan is 1,168 to 60,000 (the water-soluble dietary fiber content is 58.1 to 80.4 mass). %), The inhibitory effect was remarkable, and a particularly strong inhibitory effect was observed in the case of 2,670 to 44,151 (water-soluble dietary fiber content was 64.2 to 80.4% by mass).

<Experiment 20-3: Effect of long-term intake of branched α-glucan on blood sugar level and insulin amount during partial starch decomposition product intake>
In Experiment 20-1, it was found that the intake of branched α-glucan suppressed the increase in blood glucose level at the time of carbohydrate loading, AUC value of blood glucose level, insulin amount, and AUC value of insulin amount. After long-term intake of α-glucan (8 weeks), the blood glucose level at the time of carbohydrate loading, the blood glucose level AUC value, the insulin amount, and the inhibitory effect on the AUC value of the insulin amount were examined. Seven 7-week-old Wistar male rats (sold by Nippon Charles River Co., Ltd.), 5 groups, 5 per group, preliminarily raised on purified feed for 1 week, were used. Three rats in each of the three groups were reared for 8 weeks in any of three kinds of test feeds containing 1, 2 or 5% by mass of branched α-glucan (converted to solid matter) shown in Table 33. Food and water were freely consumed during the breeding period. After 4 days and 8 weeks of breeding with the test feed, after fasting for one day, an aqueous solution of a partially degraded starch was orally administered as a solid to 1.5 g / kg · body weight with a stomach tube. Rat blood was collected from the tail vein immediately before administration, 15 minutes, 30 minutes, 60 minutes, 120 minutes, 180 minutes, and 240 minutes after administration. Each blood was collected in a heparinized blood collection tube and centrifuged (2,000 rpm, 10 minutes) to obtain plasma. The blood glucose level and the amount of insulin were measured by the same method as in Experiment 19-4. As a control 1, 5 rats per group were raised with purified feed alone. The remaining 5 rats in 1 group were replaced with a commercially available indigestible dextrin (trade name “Fibersol 2”, sold by Matsutani Chemical Industry Co., Ltd.) as a control 2 for a portion of the purified corn starch. The animals were reared with feed having the composition shown in Table 33. Since the results were almost the same for 4 weeks and 8 weeks of breeding, the measurement results of blood glucose level, blood glucose level AUC value, insulin level and insulin level AUC level in each test system for 4 weeks in the test feed They are shown in Table 34, Table 35, Table 36 and Table 37, respectively.

  As is apparent from the results in Tables 34 to 37, the branched α-glucan is similar to the commercially available indigestible dextrin (Control 2), and is a blood glucose level and a blood glucose level when loaded with a saccharide (partially degraded starch). It was found that the AUC value, the amount of insulin, and the increase in AUC of the amount of insulin were suppressed as compared with the case of breeding with purified feed alone (control 1). In addition, the suppression of these numerical increases depends on the concentration of the branched α-glucan blended in the feed, and the effect becomes remarkable when 2% by weight of the branched α-glucan is blended as a solid substance. % Showed a particularly strong suppression. In addition, the degree of suppression of these indices in rats fed with a diet containing 5% by mass of a commercially indigestible dextrin as a solid is the same as that of a diet containing 1% by mass of branched α-glucan as a solid content. Since the degree of inhibition was observed, the branched α-glucan was more blood sugar, AUC of blood sugar, insulin amount and insulin when loaded with carbohydrate (starch degradation product) than the indigestible dextrin. It was confirmed that the amount of AUC was excellent in an increase suppressing effect. Furthermore, when comparing the blood glucose level and the amount of insulin before administration of the partially degraded starch, in the group fed with 2% or 5% by weight of branched α-glucan as a solid, the purified feed alone or indigestible The branched α-glucan is lower than the group ingested feed containing 5% by mass of dextrin as a solid substance, and the branched α-glucan is more effective than the commercially resistant indigestible dextrin in fasting blood glucose level and blood insulin level by long-term intake. It also became clear that it has an inhibitory effect. In addition, when the body weights of the rats were compared between the test diet and the control diet for 4 weeks and 8 weeks, no difference in the average body weight was observed between any groups. Therefore, the branched α− confirmed in this experiment It was determined that the effect of suppressing the increase in blood glucose level and insulin level by glucan had no effect on the health of rats.

<Experiment 21: Effect of branched α-glucan intake on human blood glucose level and insulin level>
It has been clarified that when the branched α-glucan is ingested by the rat, the increase in blood glucose level and insulin amount is suppressed compared to the case where the partial starch degradation product is ingested. The test which investigates the influence which it has on the blood glucose level and the amount of insulin in blood was conducted. By using a commercially available starch partial decomposition product (Matsutani Chemical Industry Co., Ltd., trade name “Paindex # 1”) and a branched α-glucan prepared according to the method of Example 5 described later, The increase in insulin was examined. Twelve healthy volunteer males (age 26 to 54 years old, average 41 ± 7 years old) as subjects, food and drink other than water from 21:00 on the day before the test until the start of the next day (around 9:00) Was prohibited. First, a test preparation in which 50 g (converted to a solid substance) of a starch partial decomposition product was dissolved in water to make a total amount of 200 ml was ingested within 2 minutes from the start of ingestion. Blood was collected immediately before oral administration, 15 minutes, 30 minutes, 45 minutes, 60 minutes, 90 minutes, and 120 minutes after oral administration. Subsequently, the same test subject was used as in the case of the partially decomposed starch product except that a test sample was prepared by dissolving 50 g of branched α-glucan (in terms of solids) in water and making a total volume of 200 ml with an interval of one week or longer. Tested and collected blood. The blood glucose level and insulin level of each blood were measured by entrusting to a private clinical laboratory (Okayama City Medical Association General Medical Center). Changes in blood glucose level and insulin amount over time when the test sample was ingested, and AUC value (area under the blood concentration-time curve: AUC ^{0- 2hr} (mg · hr / dl)) and AUC ^0-2hr (μU · hr / dl) value of insulin amount, increase amount of AUC value of blood glucose level and increase amount of AUC value of insulin amount immediately after intake to 120 minutes (ΔAUC ^0-2hr ) is summarized in Table 38.

  As is apparent from the results of Table 38, when the branched α-glucan is ingested by humans, the blood glucose level, the AUC value of the blood glucose level, the amount of insulin, and insulin, as in the experiment using rats (Experiment 20) It was found that the amount of AUC value was significantly lower than when the starch partial decomposition product was ingested.

<Experiment 22: In vivo lipid reducing action of branched α-glucan>
In Experiment 19-4 and Experiment 21, it was found that the intake of branched α-glucan decreased the increase in blood glucose level and the amount of insulin in the blood. The effects were also examined.

<Experiment 22-1: Effect of intake of branched α-glucan on fat absorption>
Using the branched α-glucan prepared by the method of Example 5 described later, 7-week-old Wistar male rats (sold by Nippon Charles River Co., Ltd.) were randomly divided into 15 groups of 4 groups. Weekly, after preliminary breeding with the purified feed shown in Table 33, 15 mice in each of the 2 groups are feeds (test feeds) having a composition containing 5% by mass of the branched α-glucan shown in the same table as solids. Raised. Each of the remaining 2 groups, 15 animals, was raised on purified feed as a control in the same manner for 4 or 8 weeks. One group of 15 animals fed with a branched α-glucan mixed feed was cultivated for 4 weeks, and the remaining 1 group of 15 animals was collected for 8 weeks under ether anesthesia after blood collection from the inferior vena cava and sacrificed and dissected. Visceral accumulated fat mass, serum lipids, intestinal mucosal wet mass, caecal contents, etc. were examined. Control rats were treated similarly. The results are shown in Table 39. Rats were kept free of food and water throughout the breeding period as in Experiment 20-1, while measuring body weight and food intake every 2 or 3 days throughout the test period. In addition, overnight fasting was performed before dissection. Weight gain, food intake, feed utilization efficiency (weight gain / food intake) for 4 weeks and 8 weeks of feed fed with branched α-glucan, weight at the time of dissection, organ mass, intestinal mucosa mass, viscera Table 39 shows the fat mass, the mass of the contents in the cecum, moisture, and pH. Moreover, the measurement results of serum lipids are also shown in Table 39. Among serum lipids, neutral fat, total cholesterol, and HDL-cholesterol are measured by commercially available neutral fat triglyceride measurement kits (trade name “Triglyceride E-Test Wako”, sold by Wako Pure Chemical Industries, Ltd.). , Kit for measuring total cholesterol (trade name “Cholesterol E-Test Wako”, sold by Wako Pure Chemical Industries, Ltd.) and kit for measuring HDL-cholesterol (trade name “HDL-cholesterol E-Test Wako”, Wako Pure Chemical Industries, Ltd.) Used). Further, the HDL-cholesterol value was subtracted from the total cholesterol value to obtain an LDL-cholesterol value.

  As is apparent from the results in Table 39, when a diet containing 5% by mass of branched α-glucan as a solid substance was ingested, compared with the case where the diet was fed with purified feed alone, in the intake period of 4 weeks, The testicular fat mass showed a low value, and at 8 weeks of intake, both the perirenal and testicular fat showed a significantly low value, and the decrease was particularly remarkable in the testicular fat. Intestinal mucosa mass increased significantly at 4 weeks and 8 weeks after ingestion, and the increase became significant at 8 weeks after ingestion. In addition, the pH in the cecum significantly decreased after 8 weeks of ingestion. Further, regarding serum lipids, the neutral fat level was 8 weeks after ingestion, the total cholesterol was at 4 weeks and 8 weeks after ingestion, and both showed a tendency to decrease, and LDL-cholesterol also decreased. The measured values other than these were not different from the control. Although specific data is not shown, the organic acid concentration in the cecum was not different from the control. This result indicates that branched α-glucan has an action of reducing in vivo lipids. Moreover, since an increase in the mass of the intestinal mucosa was observed, the suppression of excessive lipid accumulation by branched α-glucan and the enhancement of glucose tolerance confirmed in Experiments 20 to 21 were accompanied by an increase in mucin secretion confirmed in this test. It is presumed that the reduced activity of digestive enzymes due to the thickening of the small intestinal mucosal layer accompanied by the action, inhibition of digestion and absorption of digested glucose and fat, and delay play important roles.

<Experiment 22-2: Effect of Molecular Weight of Branched α-Glucan on Suppression of Excess Accumulation of Lipids in Living Body>
In Experiment 22-1, since it was found that excessive accumulation of lipids in the living body was suppressed by ingestion of branched α-glucan, the effect of the difference in weight average molecular weight of the branched α-glucan was examined. That is, 8 types of test feeds were prepared by mixing 5% by mass of 8 types of branched α-glucans having different weight average molecular weights as those used in Experiment 20-2 with the purified feed as solids. Forty-five Wistar rats female 7-week-old (available from Nippon Charles River Co., Ltd.) were randomly divided into 9 groups and 5 animals in each group, and preliminarily raised on purified feed for 1 week. Among these rats, group 8 was reared for 8 weeks on test diets (test diets 1 to 8) containing any of the branched α-glucans having different weight average molecular weights shown in Table 40. The remaining 5 rats per group were kept on the purified feed for 8 weeks and used as a control group. Rats that had been ingested for 8 weeks after ingesting a diet containing a branched α-glucan and rats in a control group that had ingested purified diet for 8 weeks were blood-collected under ether anesthesia, and then sacrificed, and their mesentery The fat mass (wet mass) around the periphery, around the kidney, and around the testicles, and neutral fat and total cholesterol in the serum were measured in the same manner as in Experiment 22-1. The results are shown in Table 40.

  As is clear from the results of Table 40, when a branched α-glucan having a different weight average molecular weight was ingested, the visceral fat mass and the serum lipid amount were lower in both groups than in the control group, and the branched α -It was found that glucan suppresses increases in visceral fat mass and serum lipid content. Further, when compared with the degree of the suppression effect, the effect is remarkable when the weight-average molecular weight of the branched α-glucan is 2,670 to 44,151, and the particularly strong effect is recognized when it is 2,670 to 25,618. It was.

<Experiment 22-3: Effect of difference in intake of branched α-glucan on visceral accumulated fat>
Seven-week-old Wistar male rats (sold by Nippon Charles River Co., Ltd.) were randomly divided into 5 groups of 5 animals each, and after 1 week pre-breeding with commercially available solid feed, 5 animals of 3 groups were They were bred for 4 weeks on purified feed (Table Feed 1, Test Feed 2 or Test Feed 3) having the composition shown in Table 33 containing 1, 2 or 5% by weight of branched α-glucan. Of the remaining 2 groups, 5 animals each, 5 animals per group are 4 weeks with the purified feed shown in Table 33 containing 5% by mass of commercially available indigestible dextrin (Matsutani Chemical Sales, product “Fibersol II”). Reared (Control 1). In addition, the remaining 5 animals per group were bred for 4 weeks on the purified feed shown in Table 33 (Control 2). After raising for 4 weeks on a purified feed containing a branched α-glucan, each group of rats was sacrificed and dissected, and the visceral accumulated fat mass was measured. Control 1 and 2 rats were treated similarly. The results are shown in Table 41. Rats were kept free of food and water throughout the breeding period as in Experiment 22-1, while measuring body weight and food intake every two or three days throughout the test period. The rats before dissection were fasted overnight. In addition, the proportion of branched α-glucan or indigestible dextrin (%) is added to the total intake of the feed during the breeding period when it is raised for 4 weeks in a purified feed containing branched α-glucan or indigestible dextrin. Multiplied by 100 and calculated the total intake of branched α-glucan or indigestible dextrin during the period of breeding with the feed, and further divided by the breeding period (28 days). The intake amount of branched α-glucan or indigestible dextrin is calculated and shown together in Table 41.

  As is clear from the results in Table 41, when a purified feed (test feed 1) containing 1% by mass of a branched α-glucan as a solid was ingested for 4 weeks, when it was raised with only the purified feed (control 2) In comparison, the visceral fat mass around the testicles was low, and when the purified feed (test feed 2 or test feed 3) containing 2 or 5% by mass was ingested for 4 weeks, the fat mass around the kidneys and testicles was low. Value, and testicular fat mass was significantly lower. The mass around the mesentery fat is the case where the branched α-glucan is bred with a refined feed (test feed 1 or test feed 2) containing 1 or 2% by mass as a solid, when bred only with the purified feed ( When compared with the control 2), the difference was not observed between the case of breeding with the refined feed (test feed 3) containing 5% by mass and the case of breeding with the purified feed (control 2). In addition, when the purified feed containing 5% by mass of commercially indigestible dextrin was ingested for 4 weeks (control 1), the fat around the mesentery was compared with the case of raising only the purified feed (control 2). There was no difference in mass, and although the fat mass around the kidneys and testicles was low, the range of fat mass variation between individuals was large, and no significant difference was observed. Comparing the strength of the inhibitory effect on visceral fat accumulation when the branched α-glucan and the commercially indigestible dextrin are ingested, the branched α-glucan is stronger, and the effect is remarkable for the testicular periphery fat. there were. This result indicates that the branched α-glucan has an action of suppressing the accumulation of visceral fat, particularly, testicular fat. In addition, although specific data are not shown, there was no significant difference in body weight gain or food intake between groups.

<Experiment 22-4: Effect of difference in intake form of branched α-glucan on visceral accumulated fat>
Seven-week-old Wistar male rats (marketed by Nippon Charles River Co., Ltd.) were randomly divided into 11 groups of 5 groups, and were raised on a commercially available solid feed for 1 week. While giving deionized water (test drinking water 1, test drinking water 2 or test drinking water 3) containing 0.5, 1 or 2% by mass of the branched α-glucan prepared by the method of Example 5 as drinking water, The rats were bred for 4 weeks on rat chow. Of the remaining 2 groups of 11 animals, 1 group of 11 animals was marketed while giving deionized water containing 2% by mass of commercially available indigestible dextrin (Matsutani Chemical Sales, product “Fibersol II”) as drinking water. Of rats were fed for 4 weeks (control 1). The remaining 11 animals per group were similarly raised for 4 weeks on a commercial rat chow while giving deionized water as drinking water (Control 2). Each group of rats after 4 weeks of feeding with water containing branched α-glucan was sacrificed and dissected to measure visceral accumulated fat mass. Control 1 and 2 rats were treated similarly. The results are shown in Table 42. Rats are fed freely throughout the test period, while the body weight, food intake, and drinking water intake are measured every 2 or 3 days. It was. Moreover, when it was raised for 4 weeks in drinking water containing branched α-glucan or indigestible dextrin, the content of branched α-glucan or indigestible dextrin (%) was multiplied by the total intake of drinking water during the breeding period, Divide by 100, calculate the total intake of branched α-glucan or indigestible dextrin during the period of breeding with the drinking water, and further divided by the breeding period (28 days). -The intake of glucan or indigestible dextrin is calculated and shown together in Table 42.

  As is clear from the results of Table 42, when drinking water containing 0.5%, 1 or 2% by mass of branched α-glucan as a solid (test drinking water 1, test drinking water 2 or testing drinking water 3) and fed for 4 weeks, As compared with the case of breeding with only deionized water (control 2), the fat masses around the mesentery, around the kidney, and around the testicle were all significantly lower. In addition, even when fed with drinking water containing 5% by mass of commercially available indigestible dextrin and raised for 4 weeks (Control 1), the fat masses around the mesentery, around the kidney and around the testicle all showed significantly low values. Comparing the strength of the inhibitory effect on visceral fat accumulation when the branched α-glucan and the commercially indigestible dextrin were ingested with water, the branched α-glucan was stronger and the difference in the effect was It was prominent for any of the surrounding, peri-renal, and testicular fat. In addition, although the breeding conditions are different, when the visceral fat mass is compared between the case where the branched α-glucan is ingested with food (Experiment 22-3) and the case where the ingested water is dissolved in drinking water, for example, 2% by mass of branched α -When the glucan-containing purified feed (test feed 2) and 0.5% by weight of drinking water (test drinking water 1) were raised for 4 weeks, the branched α-glucan intake per day was 0.71 g and 0, respectively. .34 g and 0.5% by mass of branched α-glucan-containing drinking water, although the amount of daily intake of branched α-glucan is small, the mesentery, the kidney, and the testicles For any part of the fat, the suppression of the increase in fat mass was more pronounced when drinking water. Specifically, when the 2% by weight branched α-glucan-mixed purified feed (test feed 2) was bred, only the pericardial fat mass was significantly different from that of control 2, and the mesenteric fat was The mass increased compared to Control 2, whereas when bred with drinking water containing 0.5% by mass (test drinking water 1), the fat masses around the mesentery, around the kidney, and around the testicle were all The value was significantly low. This result indicates that the branched α-glucan has an excellent action of suppressing the accumulation of visceral fat. In addition, the branched α-glucan is more excellent in visceral fat accumulation-inhibiting effect when the amount of intake is the same when dissolved in drinking water than when mixed with food. In addition, although specific data are not shown, there was no significant difference in body weight gain, food intake, or water intake between the groups.

  From the results of the above experiments, it was found that the branched α-glucan can be used as a blood insulin increase inhibitor, a visceral fat accumulation inhibitor and a lipid metabolism improver after intake of carbohydrates and lipids. Furthermore, from the results of Experiment 22, branched α-glucan can effectively exert its physiological function when ingested in the form of a solution rather than ingested in a solid form when the intake is the same. It was also found that its physiological action is stronger than that of commercially available indigestible dextrin.

  Examples of production of α-glucosyltransferase used in the present invention are shown in Examples 1 and 2, and examples of production of branched α-glucan which is an active ingredient of the lipid metabolism improving agent of the present invention are shown in Examples 3 to 6. . Further, Example 7 illustrates the physicochemical properties of branched α-glucan which is an active ingredient of the lipid metabolism improving agent of the present invention. Examples 8 to 15 show lipid metabolism improving agents containing a branched α-glucan that is the lipid metabolism improving agent of the present invention.

  According to the method of Experiment 5, Bacillus circulans PP710 (FERM BP-10771) was cultured with a fermenter for about 24 hours. After culturing, the culture supernatant was collected by centrifugation, added with ammonium sulfate so as to be 80% saturated, and allowed to stand at 4 ° C. for 24 hours for salting out. The salted-out product was collected by centrifugation, dissolved in 20 mM acetate buffer (pH 6.0), dialyzed against the same buffer, and concentrated to prepare a concentrated crude enzyme solution. The α-glucosyltransferase activity of this concentrated crude enzyme solution was 200 units / ml. In addition, about 25 units / ml amylase activity was also observed in this concentrated enzyme solution. This product can be advantageously used for the production of α-glucan used in the present invention using a starchy substrate.

  Arthrobacter globiformis PP349 (FERM BP-10770) was cultured in a fermenter for about 24 hours according to the method of Experiment 8. After culturing, the culture supernatant was collected by centrifugation, added with ammonium sulfate so as to be 80% saturated, and allowed to stand at 4 ° C. for 24 hours for salting out. The salted-out product was collected by centrifugation, dissolved in 20 mM acetate buffer (pH 6.0), dialyzed against the same buffer, and concentrated to prepare a concentrated enzyme solution. The α-glucosyltransferase activity of this concentrated enzyme solution was 50 units / ml. This product can be advantageously used for the production of branched α-glucan used in the present invention using a starchy substrate.

  A starch partial decomposition product (trade name “Paindex # 100”, sold by Matsutani Chemical Co., Ltd.) was dissolved in water to a concentration of 30% by mass, adjusted to pH 6.0, and obtained by the method of Example 1. The concentrated crude enzyme solution was added as an α-glucosyltransferase activity at 10 units per gram of substrate solid, and allowed to act at 40 ° C. for 48 hours. After completion of the reaction, the reaction solution is heated to 95 ° C., kept for 10 minutes, then cooled, and the filtrate obtained by filtration is decolorized with activated carbon and desalted with H-type and OH-type ion resins according to a conventional method. Purified and further concentrated to obtain a branched α-glucan solution having a concentration of 50% by mass. This branched α-glucan has a ratio of 2,3,6-trimethylated to 2,3,4-trimethylated in methylation analysis of 1 to 1.3, and 2,3,6-trimethylated and 2 , 3,4-trimethylated product accounted for 70.3% of the partially methylated product. Further, 2,4,6-trimethylated product and 2,4-dimethylated product were 3.0% and 4.8% of partially methylated product, respectively. The branched α-glucan had a weight average molecular weight of 6,220 daltons and a value (Mw / Mn) obtained by dividing the weight average molecular weight by the number average molecular weight was 2.2. Further, this branched α-glucan produced 35.1% by mass of isomaltose per solid of digested product by digestion with isomalt dextranase, and the water-soluble dietary fiber content determined by the enzyme-HPLC method was 75.8. It was mass%. This product can be used as a lipid metabolism improving agent as it is, partially purified, fractionated, and further in the form of a composition containing these. In addition, this product is used as it is, partially purified, fractionated, and further in the form of a composition containing these, an inhibitor or preventive agent for blood insulin and neutral fat, and a visceral fat accumulation inhibitor. It can also be used as an agent or preventive agent.

  After adding calcium carbonate to 30% tapioca starch milk to a final concentration of 0.1% by mass, the pH was adjusted to 6.5, and α-amylase (manufactured by Novo, trade name Termamir 60 L) was added per gram starch. It added so that it might become 0.2 mass%, and it was made to react at 95 degreeC for 15 minutes. The reaction solution was autoclaved (120 ° C.) for 10 minutes, then cooled to 40 ° C., and the concentrated crude enzyme solution of α-glucosyltransferase prepared by the method of Example 2 was added to the α-glucosyltransferase. 10 units are added per gram of substrate solid as activity, and 1 unit of CGTase derived from Bacillus stearothermophilus (manufactured by Hayashibara Biochemical Laboratories Co., Ltd.) is added at 40 ° C., pH 6.0. For 72 hours. The reaction solution is kept at 95 ° C. for 10 minutes, then cooled and filtered, and the filtrate obtained is decolorized with activated carbon, purified by desalting with H-type and OH-type ion resins, and concentrated. Spray-dried to obtain branched α-glucan powder. This branched α-glucan showed a ratio of 2,3,6-trimethylated to 2,3,4-trimethylated 1: 1.6 in methylation analysis, The total of 2,3,4-trimethylated product accounted for 80.0% of the partially methylated product. In addition, 2,4,6-trimethylated product and 2,4-dimethylated product showed 1.4 and 1.7% of partially methylated product, respectively. The branched α-glucan had a weight average molecular weight of 10,330 daltons and a value (Mw / Mn) obtained by dividing the weight average molecular weight by the number average molecular weight was 2.9. Furthermore, this branched α-glucan produced 40.7% by mass of isomaltose per digested solid in isomalt-dextranase digestion, and the water-soluble dietary fiber content determined by the enzyme-HPLC method was 68.6. It was mass%. This branched α-glucan can be used as a lipid metabolism improving agent as it is, partially purified, fractionated, and further in the form of a composition containing these. In addition, this product is used as it is, partially purified, fractionated, and further in the form of a composition containing these, an inhibitor or preventive agent for blood insulin and neutral fat, and a visceral fat accumulation inhibitor. It can also be used as an agent or preventive agent.

  Sodium bisulfite was added to a 27.1 mass% corn starch liquor (hydrolysis rate 3.6%) to a final concentration of 0.3 mass%, and calcium chloride was added to a final concentration of 1 mM. Thereafter, the mixture was cooled to 50 ° C., and 11.1 units of the concentrated crude enzyme solution prepared by the method of Example 1 was added as α-glucosyltransferase activity per gram of substrate solid, and further, 50 ° C., pH 6. It was allowed to act at 0 for 68 hours. The reaction solution is kept at 80 ° C. for 60 minutes, then cooled and filtered, and the filtrate obtained is decolorized with activated carbon, purified by desalting with H-type and OH-type ion resins, and concentrated. Spray-dried to obtain branched α-glucan powder. In this branched α-glucan, in the methylation analysis, the ratio of 2,3,6-trimethylated product to 2,3,4-trimethylated product, which is a partially methylated product, was 1: 2.5, and 2,3,6 -The sum of trimethylated and 2,3,4-trimethylated accounted for 68.4% of partially methylated. Further, 2,4,6-trimethylated product and 2,4-dimethylated product showed 2.6 and 6.8% of partially methylated product, respectively. The branched α-glucan had a weight average molecular weight of 4,097 daltons and a value (Mw / Mn) obtained by dividing the weight average molecular weight by the number average molecular weight was 2.1. Furthermore, this branched α-glucan produced 35.6% by mass of isomaltose per solid of the digest in digestion with isomaltodextranase, and the water-soluble dietary fiber content determined by the enzyme-HPLC method was 79.4. It was mass%. This branched α-glucan can be used as a lipid metabolism improving agent as it is, partially purified, fractionated, and further in the form of a composition containing these. In addition, this product is used as it is, partially purified, fractionated, and further in the form of a composition containing these, an inhibitor or preventive agent for blood insulin and neutral fat, and a visceral fat accumulation inhibitor. It can also be used as an agent or preventive agent.

  Using purified α-glucosyltransferase derived from Bacillus circulans PP710 (FERM BP-10771) prepared by the method of Experiment 6 in place of the concentrated crude enzyme solution, an isoamylase derived from Pseudomonas amylodelamosa (Hayashibara Biochemical Research Co., Ltd.) A branched α-glucan powder was obtained in the same manner as in Example 5 except that 1,000 units were added per gram of substrate solid. This branched α-glucan showed a ratio of 2,3,6-trimethylated and 2,3,4-trimethylated, which is a partially methylated product, to 1: 4 in methylation analysis. The sum of the chloride and 2,3,4-trimethylated accounted for 67.9% of the partially methylated. In addition, 2,4,6-trimethylated product and 2,4-dimethylated product showed 2.3% and 5.3% of partially methylated product, respectively. The branched α-glucan had a weight average molecular weight of 2,979 daltons and a value (Mw / Mn) obtained by dividing the weight average molecular weight by the number average molecular weight was 2.0. Furthermore, this branched α-glucan produced 40.6% by mass of isomaltose per digested solid in digestion with isomaltodextranase, and the water-soluble dietary fiber content determined by the enzyme-HPLC method was 77% by mass. there were. This branched α-glucan can be used as a lipid metabolism improving agent as it is, partially purified, fractionated, and further in the form of a composition containing these. In addition, this product is used as it is, partially purified, fractionated, and further in the form of a composition containing these, an inhibitor or preventive agent for blood insulin and neutral fat, and a visceral fat accumulation inhibitor. It can also be used as an agent or preventive agent.

  The branched α-glucan prepared in Example 5 was examined for physicochemical properties according to a conventional method, and is summarized in Table 43 as an example of the properties of the branched α-glucan used in the present invention.

<Lipid metabolism improving agent>
Dissolve 2 parts by weight of the branched α-glucan solution obtained by the method of Example 3 and 3 parts by weight of sucrose in 100 parts by weight of raw milk, sterilize by heating with a plate heater, then concentrate to a concentration of 70%, and canned under aseptic conditions. Thus, a lipid metabolism improving agent in the form of sweetened condensed milk was obtained. If you eat sweets and dishes that use this product, you can effectively improve lipid metabolism. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
175 parts by weight of skim milk powder, 50 parts by weight of a branched α-glucan powder obtained by the method of Example 4 and 50 parts by weight of a lactosucrose-rich powder (Hayashibara Shoji Co., Ltd., registered trademark “milk fruit oligo”) Dissolved in 500 parts by mass, sterilized at 65 ° C. for 30 minutes, cooled to 40 ° C., inoculated with 30 parts by mass of a starter of lactic acid bacteria, and cultured at 37 ° C. for 8 hours according to a conventional method. A lipid metabolism improving agent was obtained. Since this product is liquid, if it is ingested, it can improve lipid metabolism more effectively than ingesting the same amount of branched α-glucan in the form of a solid composition. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
10 parts by mass of branched α-glucan powder obtained by the method of Example 4, 20 parts by mass of hydrous crystal trehalose, 20 parts by mass of anhydrous crystalline maltitol, anhydrous citric acid with respect to 33 parts by mass of orange juice powder produced by spray drying. 0.65 parts by mass, 0.1 parts by mass of malic acid, 0.2 parts by mass of 2-O-α-glucosyl-L-ascorbic acid, 0.1 parts by mass of sodium citrate, and appropriate amounts of powdered fragrance are mixed and stirred well. Then, it is pulverized into a fine powder, which is charged into a fluidized bed granulator, the exhaust temperature is 40 ° C., and an appropriate amount of the branched α-glucan solution obtained by the method of Example 3 is sprayed as a binder for 30 minutes. Granulated, weighed and packaged to obtain a lipid metabolism improving agent in the form of powdered juice. If this product is dissolved in water and ingested as a juice, lipid metabolism can be improved more effectively than ingesting the same amount of branched α-glucan in the form of a solid composition. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
100 parts by mass of corn starch, 30 parts by mass of the branched α-glucan solution obtained by the method of Example 3, 70 parts by mass of water-containing trehalose crystal, 40 parts by mass of sucrose, and 1 part by mass of sodium chloride were mixed thoroughly, and 280 parts by mass of chicken egg In addition, gradually add 1,000 parts by weight of boiled milk to this, and continue to stir over fire. When the corn starch is completely gelatinized and the whole becomes translucent, stop the fire and cool it. An appropriate amount of vanilla flavor was added, and weighed, filled and packaged to obtain a lipid metabolism improving agent in the form of custard cream. Lipid metabolism can be improved by ingesting this product by packing it in a shoe. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
In accordance with a conventional method, water was added to 10 parts by mass of the raw red beans, boiled, astringent and extracted, and water-soluble impurities were removed to obtain about 21 parts by mass of red bean granule cake. Add 14 parts by weight of sucrose, 5 parts by weight of the branched α-glucan solution obtained by the method of Example 3 and 4 parts by weight of water to this boiled oil, and boil it. As a result, about 35 parts by mass of a lipid metabolism improving agent in the form of cocoon was obtained. Lipid metabolism can be improved if this product is used to make and ingest strawberry bread, manju, dumpling, mid-career, ice confectionery, etc. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
100 parts by weight of wheat flour, 2 parts by weight of yeast, 5 parts by weight of sucrose, 1 part by weight of branched α-glucan powder obtained by the method of Example 4 and 0.1 part by weight of inorganic food are kneaded with water according to a conventional method. The seeds were fermented at 26 ° C. for 2 hours, then ripened and baked for 30 minutes to obtain a bread-form lipid metabolism improving agent. Ingestion of this product can improve lipid metabolism. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
40 parts by weight of a soy peptide solution for food (Fuji Oil Co., Ltd., trade name “Hynewt S”) is mixed with 2 parts by weight of the branched α-glucan powder obtained by the method of Example 4 to give a plastic. The product was placed in a vat, dried under reduced pressure at 50 ° C., and pulverized to obtain a lipid metabolism improving agent in powder form. If this product is taken in water and taken, water metabolism can be improved more effectively than the same amount of branched α-glucan in the form of a solid composition. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
100 parts by mass of branched α-glucan powder obtained by the method of Example 4, 200 parts by mass of hydrous crystals of trehalose, 200 parts by mass of powder containing high maltotetraose, 270 parts by mass of powdered egg yolk, 209 parts by mass of skim milk powder, sodium chloride 4 parts by mass, 1.8 parts by mass of potassium chloride, 4 parts by mass of magnesium sulfate, 0.01 parts by mass of thiamine, 0.1 parts by mass of sodium L-ascorbate, 0.6 parts by mass of vitamin E acetate and 0. A formulation consisting of 04 parts by mass was prepared, and this formulation was filled into a moisture-proof laminated sachet by 25 grams and heat sealed to obtain a lipid metabolism improving agent in a liquid food form. Ingesting this product can improve lipid metabolism more effectively than ingesting the same amount of branched α-glucan in the form of a solid composition. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
30 parts by mass of branched α-glucan powder obtained by the method of Example 4, 0.1 part by mass of tea extract, 0.01 part by mass of guava leaf extract, 0.02 part by mass of L-carnitine tartrate, sucrose 1 Add water to 2 parts by mass, 2 parts by mass of α, α-trehalose and 0.001 part by mass of thiamine to make a total of 500 parts by mass and fill 350 ml each with a plastic bottle to prepare a lipid metabolism improving agent in the form of a soft drink. did. Ingesting this product can improve lipid metabolism more effectively than ingesting the same amount of branched α-glucan in the form of a solid composition. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
The roasted and ground regular coffee beans were subjected to noodle drip using boiling tap water at 90 ° C. for 5 minutes by a conventional method to obtain a coffee extract. 50 parts by mass of this coffee extract, 4 parts by mass of branched α-glucan prepared by the method of Example 6, 3.5 parts by mass of milk, and 6 parts by mass of granulated sugar are mixed and boiled to 100 parts by mass as a whole. Tap water was added. Next, the pH is adjusted to 6.5 using a 5% by mass aqueous sodium hydrogen carbonate solution, 250 ml each is filled into a steel can, preheated to 93 ° C. in a hot water bath, sealed, Sterilized at 123 ° C. for 15 minutes and then quenched in running water for 1 hour to prepare a canned coffee form lipid metabolism improving agent. Ingesting this product can improve lipid metabolism more effectively than ingesting the same amount of branched α-glucan in the form of a solid composition. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

<Lipid metabolism improving agent>
Branched α-glucan powder prepared by the method of Example 6 3 parts by mass, ferrous fumarate 0.06 parts by mass, inositol 0.1 parts by mass, carnitine chloride 0.2 parts by mass, thiamine nitrate 0.01 parts by mass , 0.01 parts by weight of riboflavin sodium phosphate, 0.01 parts by weight of pyridoxine hydrochloride, 1 part by weight of 2-O-α-glucosyl-L-ascorbic acid, 2 parts by weight of aminoethylsulfonic acid, 4 parts by weight of xylitol, α, After adding an appropriate amount of sodium citrate to 5 parts by weight of α-trehalose, 5 parts by weight of erythritol, 0.8 parts by weight of citric acid, 0.06 parts by weight of sodium benzoate and 0.1 parts by weight of mixed fruit flavor, and then dissolving in purified water. The pH was adjusted to 3.0, and purified water was added to make the total amount 100 parts by mass. This solution was filtered with filter paper, and the filtrate was sterilized by heating at 80 ° C. for 25 minutes using a sterilizer, and then 100 ml each was filled into a glass bottle and capped to prepare a lipid metabolism improving agent in the form of an internal solution. Ingesting this product can improve lipid metabolism more effectively than ingesting the same amount of branched α-glucan in the form of a solid composition. The product can also be used as an inhibitor or preventive agent for blood insulin or neutral fat, and as an inhibitor or preventive agent for visceral fat accumulation.

  The lipid metabolism-improving agent of the present invention has high safety of branched α-glucan as an active ingredient, is not inferior to commercially available indigestible dextrin, and has an excellent lipid metabolism-improving action. In addition, the lipid metabolism improving agent of the present invention has no side effects even when taken for a long time, and can be used with confidence. The present invention is an invention that exhibits such remarkable effects, and is a truly significant invention that contributes greatly to the world.

Starch part which is a substrate of the gel filtration HPLC chromatograms of glucan A and B prepared from partially decomposed starches using α-glucosyltransferase preparations derived from Bacillus circulans PP710 and Arthrobacter globiformis PP349, respectively. It is the figure compared with that of the decomposition product. It is a figure which shows typically the structure of the starch partial decomposition product and the branched alpha-glucan used by this invention. It is a figure which shows the optimal temperature of a Bacillus circulans PP710 origin alpha-glucosyltransferase. It is a figure which shows the optimal pH of a Bacillus circulans PP710 origin alpha-glucosyltransferase. It is a figure which shows the temperature stability of a Bacillus circulans PP710 origin alpha-glucosyltransferase. It is a figure which shows the pH stability of a Bacillus circulans PP710 origin alpha-glucosyltransferase. It is a figure which shows the optimal temperature of the alpha-glucosyltransferase derived from Arthrobacter globiformis PP349. It is a figure which shows the optimal pH of the alpha-glucosyltransferase derived from Arthrobacter globiformis PP349. It is a figure which shows the temperature stability of alpha-glucosyltransferase derived from Arthrobacter globiformis PP349. It is a figure which shows the pH stability of alpha-glucosyltransferase derived from Arthrobacter globiformis PP349. It is a figure which shows the optimal temperature of a Bacillus circulans PP710 origin amylase. It is a figure which shows the optimal pH of a Bacillus circulans PP710 origin amylase. It is a figure which shows the temperature stability of Bacillus circulans PP710 origin amylase. It is a figure which shows the pH stability of Bacillus circulans PP710 origin amylase. It is the figure which compared the chromatogram of the gel filtration HPLC of the branched alpha-glucan prepared using the alpha-glucosyltransferase purified product and the amylase purified product derived from Bacillus circulans PP710 together with that of the partial starch degradation product as a substrate. . It is the figure which compared the chromatogram of the gel filtration HPLC of the branched alpha-glucan prepared using Bacillus circulans PP710 origin alpha-glucosyltransferase purified product and isoamylase together with that of the starch partial decomposition product which is a substrate. It is the figure which compared the chromatogram of the gel filtration HPLC of the branched alpha-glucan prepared combining the alpha-glucosyltransferase purified product derived from Bacillus circus PP710, and alpha-amylase with that of the starch partial decomposition product which is a substrate. . It is the figure which compared the chromatogram of the gel filtration HPLC of the branched alpha-glucan prepared using Bacillus circulans PP710 origin alpha-glucosyltransferase purified product and CGTase together with that of the starch partial decomposition product which is a substrate. It is the figure which compared the chromatogram of gel filtration HPLC of the α-glucosyltransferase purified product derived from Bacillus circulans PP710 and the branched α-glucan prepared by using isoamylase and CGTase together with that of the partial starch degradation product as a substrate. is there.

Explanation of symbols

1 and 15-19,
B: Elution position corresponding to a molecular weight of 1,000,000 Daltons B: Elution position corresponding to a molecular weight of 100,000 Daltons C: Elution positions corresponding to a molecular weight of 10,000 Daltons D: Elution positions corresponding to a molecular weight of 1,000 Daltons Elution position corresponding to a molecular weight of 100 Daltons In FIG.
a: Gel filtration HPLC chromatogram of partially decomposed starch as a substrate b: Gel filtration HPLC chromatogram of glucan A c: Gel filtration HPLC chromatogram of glucan B 1: Position corresponding to glucose polymerization degree 499 2: Glucose polymerization degree Position corresponding to 6.3: Position corresponding to the degree of glucose polymerization 384 4: Position corresponding to the degree of glucose polymerization 22.2: Position corresponding to the degree of glucose polymerization 10.9 6: Corresponding to the degree of glucose polymerization 1 Position 7: Position corresponding to glucose polymerization degree 433 8: Position corresponding to glucose polymerization degree 22.8 9: Position corresponding to glucose polymerization degree 10.9 10: Position corresponding to glucose polymerization degree 1 In FIG.
1: Schematic diagram of partial starch degradation product 2: Schematic diagram of branched α-glucan used in the present invention a: Non-reducing terminal glucose residue b: α-1,3-linked glucose residue c: α-1 , 4 glucose residues d: α-1, 6 glucose residues e: α-1, 3, 6 glucose residues f: α-1, 4, 6 bonds Glucose residues diagonally broken line: α-1,3 bond horizontal solid line: α-1,4 bond vertical solid line: α-1,6 bond
●: in the absence of calcium ions ○: in the presence of 1 mM calcium ions in FIG.
a: Gel filtration HPLC chromatogram of partially decomposed starch as a substrate b: Gel filtration HPLC of branched α-glucan obtained by acting 10 units of α-glucosyltransferase and 0.1 unit of amylase per gram of substrate Chromatogram c: Gel filtration HPLC chromatogram of branched α-glucan obtained by acting 10 units of α-glucosyltransferase and 0.2 unit of amylase per gram of substrate d: α-glucosyltransferase per gram of substrate Gel filtration HPLC chromatogram of branched α-glucan obtained by acting 10 units of amylase and 0.5 unit of amylase e: obtained by acting 10 units of α-glucosyltransferase and 1 unit of amylase per gram of substrate Gel filtration HPLC chromatogram of the branched α-glucan in FIG.
a: Gel filtration HPLC chromatogram of partially decomposed starch as a substrate b: Gel filtration HPLC chromatography of branched α-glucan obtained by acting 10 units of α-glucosyltransferase and 50 units of isoamylase per gram of substrate Gram c: Gel filtration HPLC chromatogram of branched α-glucan obtained by reacting 10 units of α-glucosyltransferase and 200 units of isoamylase per gram of substrate d: 10 α-glucosyltransferase per gram of substrate Gel filtration HPLC chromatogram e of branched α-glucan obtained by acting 500 units of unit and isoamylase e: Branch obtained by acting 10 units of α-glucosyltransferase and 1000 units of isoamylase per gram of substrate Gel filtration HPLC chromatogram of α-glucan
a: Gel filtration HPLC chromatogram of partially decomposed starch as a substrate b: Gel of branched α-glucan obtained by acting 10 units of α-glucosyltransferase and 0.1 unit of α-amylase per gram of substrate Filtration HPLC chromatogram c: Gel filtration HPLC chromatogram of branched α-glucan obtained by acting 10 units of α-glucosyltransferase and 0.2 unit of α-amylase per gram of substrate d: α per gram of substrate -Gel filtration HPLC chromatogram of branched α-glucan obtained by acting 10 units of glucosyltransferase and 0.5 unit of α-amylase e: 10 units of α-glucosyltransferase per gram of substrate, α-amylase Gel filtration HPLC chromatogram of branched α-glucan obtained by allowing 1.0 unit to act in FIG.
a: Gel filtration HPLC chromatogram of partially decomposed starch as a substrate b: Gel filtration HPLC of branched α-glucan obtained by acting 10 units of α-glucosyltransferase and 0.1 unit of CGTase per gram of substrate Chromatogram c: Gel filtration HPLC chromatogram of branched α-glucan obtained by acting 10 units of α-glucosyltransferase and 0.2 unit of CGTase per gram of substrate d: α-glucosyltransferase per gram of substrate Gel filtration HPLC chromatogram of branched α-glucan obtained by acting 10 units of CGTase and 0.5 units of CGTase: 10 units of α-glucosyltransferase and 1.0 unit of CGTase per gram of substrate Gel filtration HPLC chromatogram of the obtained branched α-glucan
a: Gel filtration HPLC chromatogram of partially decomposed starch as a substrate b: Branched α-glucan obtained by allowing 10 units of α-glucosyltransferase, 50 units of isoamylase, and 1 unit of CGTase per gram of substrate Gel filtration HPLC chromatogram c: Gel filtration HPLC chromatogram of branched α-glucan obtained by acting 10 units of α-glucosyltransferase, 200 units of isoamylase, and 1 unit of CGTase per gram of substrate d: substrate Gel filtration HPLC chromatogram e of branched α-glucan obtained by acting 10 units of α-glucosyltransferase per gram, 500 units of isoamylase and 1 unit of CGTase: α-glucosyltransferase per gram of substrate 10 units, isoamylase 1000 units, CGTase 1 Gel filtration HPLC chromatogram of the resulting branched α- glucan position to act

Claims (8)

A lipid metabolism improving agent comprising a branched α-glucan having glucose as a constituent sugar and having the following characteristics in methylation analysis.
(1) The ratio of 2,3,6-trimethyl-1,4,5-triacetylglucitol to 2,3,4-trimethyl-1,5,6-triacetylglucitol is 1: 0.6 To 1: 4.
(2) The sum of 2,3,6-trimethyl-1,4,5-triacetylglucitol and 2,3,4-trimethyl-1,5,6-triacetylglucitol is a partially methylated group. Occupies more than 60% of the citrate acetate;
(3) 2,4,6-trimethyl-1,3,5-triacetylglucitol is 0.5% or more and less than 10% of partially methylated glucitol acetate; and (4) 2,4- Dimethyl-1,3,5,6-tetraacetylglucitol is 0.5% or more of partially methylated glucitol acetate.   The lipid metabolism improvement according to claim 1, wherein the branched α-glucan has a glucose polymerization degree of 10 or more, and a value (Mw / Mn) obtained by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn) is less than 20. Agent.   The branched α-glucan produces isomaltose in an amount of 25% by mass or more and 50% by mass or less per solid of the digested product by digestion with isomalt dextranase (EC 3.2.1.94). The lipid metabolism improving agent according to 1 or 2.   The lipid metabolism according to any one of claims 1 to 3, wherein the branched α-glucan has a water-soluble dietary fiber content of 40 mass% or more determined by high performance liquid chromatography (enzyme-HPLC method). Improver.   The lipid according to any one of claims 1 to 4, comprising one or more selected from food materials, food additives, pharmaceuticals, pharmaceutical additives, and quasi-drug additives together with branched α-glucan. Metabolic improver.   The lipid metabolism improving agent according to any one of claims 1 to 5, wherein the lipid metabolism improving agent is in a liquid form.   The lipid metabolism improving agent according to any one of claims 1 to 6, which is characterized by having an action of improving lipid metabolism.   The lipid metabolism improving agent according to any one of claims 1 to 7, as a blood insulin increase inhibitor or preventive agent, a blood neutral fat increase inhibitor or preventive agent, or a visceral fat accumulation inhibitor or preventive agent Use of.