KR101241288B1 - Method for preparing composite microorganism producing glutamic acid and flavoring material - Google Patents

Abstract

The present invention relates to a novel high glutamic acid producing bacterium Bacillus subtilis (KFCC 11492P) and a method for producing a natural seasoning material using the same. In addition, the present invention relates to a natural seasoning material prepared using the strain of the present invention.

Description

Glutamic acid producing strain and manufacturing method of natural seasoning material {METHOD FOR PREPARING COMPOSITE MICROORGANISM PRODUCING GLUTAMIC ACID AND FLAVORING MATERIAL}

The present invention relates to a glutamic acid producing strain and a method for producing natural seasoning material using the same. The present invention also relates to a natural seasoning material prepared using the strain of the present invention.

Recently, due to the controversy over the harmfulness of chemical seasonings (MSG), major processed food companies are launching chemical additive-free products, including MSG, but the development of alternative natural materials is insufficient.

As natural seasonings, amino acids, animal and plant extracts, brewed products, etc., which have broken down proteins are used.

Peptides have been noted as ingredients that enrich the taste of food, and several components have recently been separated. That is, glutathione in beef extract, garlic's allin, (+)-S-methyl-L-cysteine sulfoxide, γ-L-glutamyl-S-allyl-L-cysteine and onion's trans (+)-S -Propenyl-L-cysteine sulfoxide and γ-glutamyl-peptide were isolated and confirmed.

In addition, it is reported that di- and tri-peptides containing a lot of glutamic acid not only have a rich taste but also have a bitter taste blocking effect. In addition, it is known that the peptide fraction of the molecular weight 500-1000 in an acid hydrolysis peptide shows sweetness and umami.

Recently, natural seasonings were developed using cultivated domestic bacteria from skim soybeans and decomposing proteins using various enzymes obtained therefrom, but the sensory effects were insufficient.

Accordingly, an object of the present invention is to obtain natural seasoning materials by decomposing various natural protein materials by proteases produced by bacteria using Korean bacteria and converting them into peptides having glutamic acid and a molecular weight of 500 to 1000 using selected enzymes.

The object of the present invention is achieved by isolating and identifying bacteria from rice straw and then providing Bacillus subtilis (KFCC 11492P), a selected glutamic acid high producing strain.

The object of the present invention is also achieved by a seasoning material containing amino acids and peptides obtained by inoculating and culturing the glutamic acid-producing strain Bacillus subtilis (KFCC 11492P) in a medium containing a natural protein.

The high glutamic acid-producing strain of the present invention can obtain a natural seasoning material containing abundant glutamic acid and a peptide, and can provide a new material to supplement and replace a chemical seasoning such as MSG or nucleic acid.

1 is a graph showing changes in glutamic acid content according to protein sources.
2 is a diagram of a mass culture production process using the strain of the present invention.

Hereinafter, preferred embodiments of the present invention and physical properties of the respective components will be described in detail with reference to the accompanying drawings. However, the present invention is not limited thereto, And this does not mean that the technical idea and scope of the present invention are limited.

The method for producing glutamic acid producing strain and natural seasoning material according to the present invention is achieved by providing glutamic acid high producing strain Bacillus subtilis (KFCC 11492P).

Glutamic acid can be obtained by inoculating the above-mentioned glutamic acid-producing strain Bacillus subtilis (KFCC 11492P) in a medium containing a natural protein and culturing, and the above-mentioned Bacillus subtilis (KFCC 11492P) containing a natural protein. By inoculating and incubating the medium, a seasoning material containing amino acids and peptides can be obtained.

At this time, it is preferable to use the above-mentioned medium soybean meal or wheat gluten or a mixture thereof, and more preferably, a mixture of 1 to 3 parts by weight of skim soybean meal and 1 to 5 parts by weight of wheat gluten is used.

Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Isolation and Identification of Bacteria

The rice straw was cut at 1 cm intervals and placed in a test tube containing 1 ml of sterilized physiological saline, and left at room temperature for 2 hours. The test tubes were shaken every 30 minutes. After heat treatment at 70 ° C. for 2 minutes after 2 hours, the supernatant was plated on LB agar medium and incubated at 37 ° C. for 1-2 days. Cultured colonies were used for bacterial identification.

A total of 1200 isolates were isolated from rice straws in Gangwon-do, Chungcheong-do, Gyeongsang-do, Jeju-do, China (Namkyung, Hangzhou), Japan (Osaka, Okayama, and Hakone), and 266 Bacillus subtilis were selected through identification.

Example 2 Glutamic Acid High Production Strain Selection

266 Bacillus subtilis bacteria identified in Example 1 were inoculated in 5 ml of 5% skim soybean meal liquid medium and shake-cultured at 37 ° C. The culture was centrifuged (1,2000 rpm, 5 minutes, 4 ℃) to determine the glutamic acid content of the supernatant. The glutamic acid content of the sample was diluted to 50 ~ 100mg / L and used by color development method of L-glutam liquid embryo kit (Yam S Shoyu, Japan). The standard solution was L-glutam liquid embryo (100 mg). / L) was used. More than 50% of the 266 total bacteria produced less than 50 mg / L glutamic acid and a total of two strains were found to produce more than 300 mg / L glutamic acid. In particular strain 172 produced 346.9 mg / L of the highest glutamic acid.

Example 3 Protein Source Selection of High Production of Glutamic Acid

In order to screen the optimal medium of high glutamate strains, strains isolated from 10 types of rice straw randomly selected on 5% skim soybean meal, 1% skim soybean meal + 5% corn gluten, 1% skim soybean meal + 5% wheat gluten medium After inoculating 20 species, the cells were incubated for 24 hours using 37 ° C orbital-shaker (200rpm). After incubation, the glutamic acid content of the culture supernatant was measured and the results are shown in FIG. 1.

According to the measurement of glutamic acid production according to the medium composition, glutamic acid production was more than 10 times higher in 1% skim soybean meal + 5% wheat gluten medium than other medium.

Therefore, glutamic acid high producing strains were selected using 1% skim soybean meal + 5% wheat gluten medium.

After inoculating 266 Bacillus subtilis bacteria identified in Example 1, it was incubated for 24 hours using 37 ℃ orbital-shaker (200rpm). After incubation, the glutamic acid content of the culture supernatant was measured. As a result, the distribution of Table 1 was confirmed. Of these, four strains with high glutamic acid production capacity were selected first.

Table 1 Measurement of glutamic acid content in 1% skim soybean meal + 5% wheat gluten medium

Example 4 Strain Rescreening

The four strains selected in Example 3 were 1% skim soybean meal + 5% wheat gluten, 1% skim soybean meal + 10% wheat gluten, 2% skim soybean meal + 5% wheat gluten, 2% skim soybean meal + 10% wheat gluten medium After inoculation to each, shaking culture was performed for 24 hours using 37 ℃ orbital-shaker (200rpm). The culture was then sterilized at 121 ° C. for 20 minutes and centrifuged at 1,2000 rpm for 5 minutes to determine the glutamic acid content of the supernatant. The medium with the highest amount of glutamic acid production was selected. As a result, it was confirmed that the most glutamic acid was produced in the 2% skim soybean meal + 10% wheat gluten medium, and the best bacterium with the highest glutamic acid production capacity was selected and deposited in the Korean microbial preservation center with accession number KFCC 11492P.

Example 5 Mass Culture

After confirming the reproducibility of the culture and glutamic acid content through a flask or pilot-scale culture experiment using the strain of Example 4, mass production was performed based on the conditions, and the yield and glutamic acid content of the culture medium according to the media conditions and culture time. I measured the back.

The medium was fermented by varying the mixing ratio of skim soybean meal and wheat gluten. In addition, the characteristics of the culture medium were examined by using a mixture of skim soybean meal and wheat gluten.

As a result, in the culture medium for each medium, the yield and glutamic acid content were the lowest when using the defatted soybean meal alone, 50.7% and 4.23 g / L, respectively. When the fermented eggplants were mixed at a ratio of 1: 5, the yield and glutamic acid content were 76.27% and 13.92 g / L, respectively, which showed high yield and glutamic acid content. In culture time by 12 hours, the yield and glutamic acid content were the lowest at 56.32% and 4.68 g / L, respectively, and the culture yield and glutamic acid content were higher at 48 hours than at 24 hours rather than 12 hours at 24 hours. .

Based on these results, the mass culture process conditions were established and shown in FIG. 2.

Medium culture of wheat gluten and skim soybean meal 5: 1 mixture for 24 hours or more, more preferably 48 hours or more, yield and glutamic acid content of about 70%, 15g / L showed the most suitable culture conditions.

<Table 2> Characteristics of Culture Media by Condition

Example 6 flavor improvement

In order to purify the glutamic acid component in the culture solution and to remove the fermentation odor inevitably generated during fermentation, activated carbon treatment and fractionation by molecular weight of the culture solution were performed. Treatment with different concentrations using activated charcoal resulted in reduced fermentation odor.

As a result of the fractionation by molecular weight, about 70% of the fraction having a molecular weight of 5000 or less is known to affect the flavor.

During the fermentation process, a mailard reaction was carried out using peptides, amino acids and sugars produced from proteins of wheat gluten and skim soybean meal. Starch liquefaction and saccharifying enzymes were treated in the culture medium to produce free sugar in the culture medium itself, and the reaction was performed at 98 ° C. for 90 minutes by adding garlic, onion, and yeast extract to enhance the Jimmy.

Sensory evaluation of the reactants showed better results than commercial Japanese products.

Korea Microbial Conservation Center (Domestic) KFCC11492P 20100707

Claims (5)

Glutamic acid high producing strain Bacillus subtilis (KFCC 11492P).
delete A method for producing glutamic acid, characterized by inoculating Bacillus subtilis (KFCC 11492P) into a medium containing vegetable protein derived from grains.
4. The method of claim 3, wherein the medium is skim soybean meal or wheat gluten or a mixture thereof.
The method of claim 4, wherein the medium is a mixture of skim soybean meal and wheat gluten in a weight ratio of 1: 5.

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