JP2006055151A - Method for producing natto - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide natto (fermented soybeans) enhancing nutritive value of proteins originated from cereals such as rice, wheat and the like, being constituent amino acids of body-constituting enzymes and containing L-lysine, L-threonine, branched amino acids expected to express physiological actions as energy source of muscles in higher concentration than that of conventional natto. <P>SOLUTION: Natto is produced by inducing and selecting AEC (S-(β-aminoethyl)-cysteine) resistant strain, AHV (α-amino-β-hydroxyvaleric acid) resistant strain or HL (β-hydroxyleucin) resistant strain through artificial mutation or spontaneous mutation of Bacillus natto, enhancing productivity of L-lysine, L-threonine or branched amino acids, respectively, and using the amino acid-producing strains. The natto containing L-lysine, L-threonine or branched amino acids expected to have effects for health and nutrition is produced. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

The present invention relates to the breeding of microorganisms and the use thereof, more specifically, the present invention relates to the breeding of Bacillus natto with improved productivity of essential amino acids, particularly L-lysine, L-threonine and branched chain amino acids, The present invention relates to breeding natto bacteria, a method for producing natto containing many of these amino acids using the breeding natto bacteria, and natto produced using the breeding natto bacteria.

L-lysine, L-threonine, and branched chain amino acids (L-valine, L-leucine, L-isoleucine) are all known to be important amino acids as essential amino acids. As the industrial production method of these amino acids, the direct fermentation method using sugar as the main raw material using mutants or recombinants such as Brevibacterium lactofermentum, Corynebacterium glutamicum, Escherichia coli, etc. has become the mainstream. ing.

L-lysine is present in proteins, and is liberated in shellfish such as clams and crows and many germinating plants. It is a physiologically important amino acid and a typical essential amino acid. Since humans and livestock cannot biosynthesize L-lysine in the body, it is necessary to take L-lysine from food. Rice and flour are staple foods at the global level and are an important source of proteins composed of amino acids. However, rice and wheat flour are most deficient in L-lysine compared to the ideal breast milk amino acid pattern.

L-threonine is known as an important protein constituent amino acid. It is a typical essential amino acid, which is the amino acid that is deficient next to L-lysine in wheat flour.

L-leucine, L-valine, and L-isoleucine are known as branched chain amino acids. L-leucine is present in proteins and is present in a free state in the liver, spleen, germinating plants and the like. L-valine is present in proteins such as flaxseed protein and is present in a free state in germinating plants. Furthermore, L-isoleucine is present in proteins, is present in proteins such as elastin and casein, and is present in a free state in sugar beet molasses and germinating plants. All of these branched chain amino acids are typical essential amino acids. In wheat flour, L-leucine is the second missing amino acid after L-lysine and L-threonine, and L-valine and L-isoleucine are also deficient. Is an amino acid. These three branched-chain amino acids play various important roles such as increasing protein in the body, muscular energy source when exercising, improving endurance during exercise, and recovery from fatigue. I am doing.

Natto, a fermented soy bean fermented with natto bacteria, is a traditional nutritional food from Japan. Recently, the number of enthusiasts has also increased in Europe and America.

When fermenting soybeans with Bacillus natto, the soybean protein is decomposed by the protease of Bacillus natto to produce peptides and amino acids, thereby improving the digestion and absorption of the protein.

Natto fermented with natto bacteria can be said to be a highly preferred food as an amino acid source.

However, when rice or wheat flour is used as a staple food, L-lysine, L-threonine, branching, which are essential amino acids derived from the current natto as a side meal, are sufficient to bring out the physiological effects of the contained protein. Since it is considered that the chain amino acid content is not sufficient, it is necessary to develop natto containing L-lysine, L-threonine, and branched chain amino acids at higher concentrations. However, there is no inventive idea to increase the contents of L-lysine, L-threonine and branched chain amino acids in natto.

Although it is conceivable to separately add L-lysine, L-threonine, and branched chain amino acids to natto, this is not a preferable method because it leads to an increase in the cost of inexpensive natto. Therefore, it is required to increase the contents of L-lysine, L-threonine and branched chain amino acids in a natural manner by artificially improving the ability of Bacillus natto.

Problems to be solved by the invention

The present invention increases L-lysine, L-threonine, and branched chain amino acids that increase the nutritional value of cereal-derived proteins such as rice and wheat flour, become constituent amino acids of living body constituent enzymes, and can be expected to have physiological effects as muscle energy sources The purpose is to provide natto containing natto at a higher concentration than commercial natto.

Means for solving the problem

The inventors have studied various means for improving the content of free amino acids such as L-lysine, L-threonine and branched chain amino acids in natto, and artificially improved the ability of natto itself to produce these amino acids. The present invention was promoted because it was possible to do so.

Natto bacteria adhere to rice straw and are widely present in nature. Although taxonomically classified as Bacillus subtilis, there is also a literature that classifies as Bacillus natto or classifies as a variant of Bacillus subtilis because it produces a sticky material specific to natto and requires biotin.

The biosynthesis regulation mechanism of L-lysine, L-threonine and branched chain amino acids of Bacillus natto has not been completely elucidated, and no attempt has been made to improve the biosynthesis ability of these amino acids. there were.

Accordingly, as a result of various studies, the present inventors obtained a mutant Bacillus natto obtained by artificial mutation treatment as S- (β-Aminoethyl) -cysteine (an analog of L-lysine). When the L-lysine secreting ability of the resistant strain obtained by culturing in the medium containing AEC) was examined, new findings were obtained that this resistant strain secreted L-lysine. Furthermore, it discovered that the resistant strain with respect to 4-Azalysine and Oxalysine which are other analogs of L-lysine also had the above-mentioned characteristic.

In addition, the present inventors conducted various tests, deleted L-threonine resolution from mutant Bacillus natto obtained by artificial mutation, and then mutated the mutant again to give L-threonine. Of a resistant strain obtained by culturing in a medium containing α-Amino-β-hydroxyvaleric acid (AHV), which is one of the analogs of L-threonine, the L-threonine secreting ability was examined. I found out.

Furthermore, the present inventors have conducted various studies and obtained by culturing mutant Bacillus natto obtained by artificial mutation treatment in a medium containing β-hydroxyleucin (HL), which is one of the branched chain amino acid analogs. The obtained resistant strain was examined for the ability to secrete branched chain amino acids, and it was found that L-valine, L-leucine and L-isoleucine were secreted alone or simultaneously. Furthermore, it discovered that the resistant strain with respect to (alpha) -Aminobutyric acid and Norvaline which are other analogs of branched chain amino acid has the same characteristic.

That is, the present inventors succeeded in breeding these amino acid highly secreting natto using a medium containing an analog such as L-lysine, L-threonine, and branched chain amino acid as a selective medium. By confirming that it is possible to produce natto containing a large amount of L-lysine, L-threonine, and / or branched chain amino acids by producing natto using a conventional natto production method using breeding natto bacteria The invention has been completed.

As the parent strain of Bacillus natto used in the present invention, an isolated strain from the natural world, an industrially used strain, a strain imparted with auxotrophy or drug resistance, and the like can be used.

Any mutation method may be used as a method for mutation of Bacillus natto used in the present invention. That is, natural mutation, physical mutation such as ultraviolet irradiation or X-ray irradiation, and chemical mutation such as ethylmethylsulfonic acid or N-methyl-N-nitro-N-nitrosoguanidine (NTG) can be used.

In this invention, it has the characteristics in the selective culture medium of the target breeding natto. In order to obtain Bacillus natto secreting L-lysine, it is desirable to add an analog of L-lysine to the selective medium. As an analog of L-lysine, AEC, 4-Azalysine, Oxalosine, etc. can be used. The concentration of the L-lysine analog added should be determined by examining the degree of analog resistance of Bacillus natto performing the mutation operation, but it may be added to the selective medium at a concentration of about 50 to 5000 μg / ml.

In order to obtain Bacillus natto secreting L-threonine, it is desirable to add an analog of L-threonine to the selective medium. In the case of Bacillus natto having the resolution of L-threonine, the L-threonine resolution may be deleted in advance by mutation. As an analog of L-threonine, AHV or the like can be used. The concentration of the L-threonine analog should be determined by examining the degree of analog resistance of the natto bacteria to be used, but it may be added to the selective medium at a concentration of about 100 to 8000 μg / ml.

Furthermore, in order to obtain Bacillus natto secreting branched chain amino acids, it is desirable to add an analog of the branched chain amino acid to the selective medium. As an analog of a branched chain amino acid, HL, α-Aminobutyric acid, Norvaline, or the like can be used. The addition concentration of the branched chain amino acid analog may be added to the selective medium at a concentration of about 100 to 8000 μg / ml, although it depends on the resistance of Bacillus natto performing the mutation operation.

The Bacillus natto to be improved is mutated and then inoculated on a solid selective medium supplemented with an analog such as AEC, AHV or HL and cultured at 37 ° C. for 5 days to purify the formed colonies. Natto is made using this purified Bacillus natto, and Bacillus natto containing a large amount of L-lysine, L-threonine or branched chain amino acids can be obtained and selected.

In order to use L-lysine, L-threonine, or a highly secreted branched-chain amino acid strain that has been developed for breeding natto as described above, actual natto production equipment can be used as it is. There is no need for new capital investment. In this way, by using a natto bacterium that has been bred and developed, it is possible to produce a nutritious new natto containing a high concentration of L-lysine, L-threonine or branched chain amino acid.

Using the technology described in this patent, the content of L-lysine, L-threonine or branched chain amino acids was improved by using similar breeding strains of various microorganisms of the genus Bacillus, using soybeans and various cereals as raw materials. Fermented foods, feeds, etc. can be produced and are within the scope of this patent.

Examples of the present invention will be described below.

The medium used in this example is an LB medium or a modified Spizzen minimal medium.

The composition of the LB medium is tryptone 10 g, yeast extract 5 g, salt 5 g, water 1 L, pH 6.8. Agar 2% was added to the solid medium.

The modified Spizzen minimal medium composition is 5 g glucose, 1 g Na citrate, 2 g ammonium sulfate, 6 g 1 potassium phosphate, 14 g dipotassium phosphate, 0.2 g magnesium sulfate heptahydrate, 0.1 mg biotin, 1 L water, pH 6.8. Agar 2% was added to the solid medium.

The Bacillus natto NA-1 strain isolated from commercial natto was cultured overnight in LB medium, washed with sterile physiological saline, and then subjected to NTG mutation treatment in the same physiological saline by a conventional method. The washed bacterial solution after the NTG mutation treatment was seeded on a modified Spizzen minimal medium containing 1000 μg / ml of AEC. After culturing at 37 ° C. for 1 week, the appearing colonies were purified with LB medium, and then analog resistance was confirmed with a modified Spizizen minimal medium containing 1000 μg / ml of AEC.

No. which is an AEC resistant strain with confirmed analog resistance. Natto was prepared using 1 strain and its parent NA-1 strain. After soaking the soybeans, it was drained and then steamed. 3000 spores per 1 g of steamed soybeans were inoculated, 45 g each were placed in a PSP tray, covered and put into fermentation, and fermented at 40 ° C. and high humidity.

About two produced natto, free L-lysine content in natto was quantified. As a result, when parent strain NA-1 was used, L-lysine content 60 mg / natto 100 g, AEC resistant strain No. When one strain was used, the L-lysine content was 240 mg / natto 100 g, and an increase in the L-lysine content was observed in natto using the AEC resistant strain. (Figure 1)

Breeding the non-degradable L-threonine strain from the parental strain NA-1 by the usual method,

In the same manner as described in the above, mutant No. showing resistance to AHV 3000 μg / ml was used. Two shares were acquired. NA-1 strain and No. Two strains were used to produce natto by the above method. Next, as a result of quantifying the L-threonine content in the produced natto, when the parent strain NA-1 strain was used, the L-threonine content was 5 mg / natto 100 g. When two strains were used, the L-threonine content was 210 mg / natto 100 g.

From parent strain NA-1

In the same manner as described in the above, mutant no. Acquired 3 shares. NA-1 strain and No. Using 3 strains, natto was prepared by the above method. Next, as a result of measuring the branched chain amino acid content in the produced natto, when the parent strain NA-1 was used, L-valine 35 mg / natto 100 g, L-leucine 80 mg / natto 100 g, L-isoleucine 15 mg / It was 100 g of natto. On the other hand, HL resistant strain No. When three strains were used, L-valine 150 mg / natto 100 g, L-leucine 145 mg / natto 100 g, and L-isoleucine 80 mg / ml.

The mutant Bacillus sp. Obtained in each of the above examples. No. 1 strain, no. 2 strains and No. 2 The three strains are deposited with the National Institute of Advanced Industrial Science and Technology as FERM AP-20135, FERM AP-20136, and FERM AP-20137, respectively.

It is a figure which shows one example of this invention. In the figure, the vertical axis represents the L-lysine content (mg / natto 100 g), and the horizontal axis represents the parent strain NA-1 and the breeding strain No. One strain is shown.

Claims (3)

A method for producing L-lysine-rich natto, characterized by using a natto bacterium having resistance to an analog of L-lysine and secreting L-lysine. A method for producing natto with a high L-threonine content, characterized by using a Bacillus natto having resistance to an analog of L-threonine and secreting L-threonine. A method for producing natto having a high content of branched chain amino acids, comprising using a Bacillus natto having resistance to an analog of a branched chain amino acid and secreting the branched chain amino acid.

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