KR0138703B1 - Culturing method with enzyme treated soybean - Google Patents

Abstract

The present invention provides a use of soybean meal enzyme digest obtained by treating soybean meal with proteolytic enzyme as an organic nitrogen source or carbon source of a culture medium for microorganisms. Soybean meal enzyme digests added to the microbial culture medium according to the present invention have a degree of hydrolysis in the range of 10-60% and a protein content in the range of 40-70% by weight based on total nitrogen.

The culture medium containing soybean meal enzyme degradation product according to the present invention can improve the productivity of fermentation, and is easily removed even in the recovery and purification process after fermentation, and thus has little effect on the quality of the final product. In addition, the enzyme decomposition products can be prepared to have a suitable degree of hydrolysis in accordance with the characteristics of the fermentation.

Description

Use of soybean meal enzyme degradant as a medium for culturing microorganisms

The present invention relates to a new use of soybean meal enzyme digests, and more particularly to a new use of soybean meal enzyme digests obtained by digesting soybean meal with proteolytic enzymes as an organic nitrogen source or carbon source in a culture medium for microbial culture.

Soybean meal, a by-product of oil obtained from soybeans, is abundantly used as an animal feed additive as a rich carbon source and protein source. Soybean meal is also used as a raw material for preparing acid-decomposed soy sauce by decomposing it with hydrochloric acid and preparing it properly. It is rarely used as an additive added to a culture medium for microbial culture, but it is used for fermentation for microbial culture in the form of soybean roasted or ground soybean meal or in the form of decomposition of soybean meal with hydrochloric acid or sulfuric acid. However, soy flour contains carbohydrates, proteins, fats, etc., but it is difficult to use microorganisms, so it takes a long time for fermentation and is insoluble, and thus has a bad effect on recovery and purification of useful materials after fermentation. In addition, soybean meal acid decomposition products are prepared by adding acid to soybean meal and heating them to react for a long time (4 to 24 hours) at a high temperature (100 to 125 ° C) to decompose proteins contained in soybean meal to most free amino acids. Amino acid content is 75 ~ 95% and is used as nitrogen source in fermentation. However, in the process of acid degradation, some amino acids such as asparagine, glutamine and tryptophan may be degraded and thermally denatured to other substances (for example, glutamic acid to pyroglutamic acid). It is also known that various materials contained in soybean meal react with each other under high temperature and strong acid treatment conditions to produce many impurities. Therefore, when acid decomposing products are used for fermentation, there are problems such as lack of some nutrients and incorporation of toxic substances.

Soy protein is known to contain many biologically active substances (Japanese Food and Development, Vol. 26, No. 11, pages 28-36, 1991). These are mainly oligopeptides with 2 to 6 amino acids. However, in order to obtain such a bioactive peptide, soy protein must be digested with enzyme. In the case of acid decomposing products obtained by treating soybean meal with acid, soybean protein is almost hydrolyzed to amino acids, so there is little bioactive substance. Therefore, it can be said that it is more advantageous to use an enzyme degradation product than to use soybean meal acid degradation product for fermentation.

The present invention relates to a novel use of such soybean meal enzyme digestion in the medium for microbial culture.

That is, it is an object of the present invention to provide a new use of soybean meal enzyme digestion by adding it to a culture medium for microbial culture.

Another object of the present invention is to provide a method of using soybean meal enzyme digest obtained by treating soybean meal with proteolytic enzyme as an organic nitrogen source or organic carbon source of the microbial culture medium.

Another object of the present invention is a method for producing a microbial culture medium or soybean meal enzyme digestion, characterized in that the addition of soybean meal enzyme digestion obtained by treating soybean meal with proteolytic enzymes, containing an organic nitrogen source or an organic carbon source. It is to provide a medium composition for.

Other objects and applications of the present invention will become apparent to those skilled in the art from the following detailed description of the invention.

Hereinafter, the present invention will be described in more detail.

There are many known methods of preparing soy products by treating soybean meal with proteolytic enzymes (European Patent 408,063A, European Patent 148,600A, European Patent 141,615A), and soybean meal using the soybean meal enzyme degradant used in the present invention. It is obtained by treating with an enzyme, and there is no limitation in the preparation method thereof. For example, a method for preparing a soybean meal enzyme degradation product that can be used in the present invention is as follows: Soybean meal is properly pretreated and then treated with proteolytic enzymes. Enzymes that can be used include exo-type and endo-type, which can be used as appropriately mixed. The enzymatic reaction can be completed in one step depending on the degree of hydrolysis, or can go through a multi-step reaction.

Proteolytic enzymes are produced by plants, animals and microorganisms, and are mainly of microbial origin. The enzyme-producing microorganisms are the genus Aspergillus, Bacillus, Staphylococcus and Rizo. Microorganisms such as genus Rhizopus and Serratia can be exemplified and can be selected and used as necessary. After the reaction is completed, the enzyme is inactivated by a method commonly used in the art, and then unreacted solids are separated as necessary. The reaction solution or separation solution is concentrated and dried to obtain a powdered soybean meal enzyme decomposition product. If necessary, it can be concentrated and used as a paste without drying. Soybean meal enzymes prepared according to the conventional method has a protein content of 40 to 70% based on the total nitrogen and the free amino acid content varies depending on the degree of hydrolysis, but is within the range of 10 to 30% of the protein content. In addition, since an excipient is added for efficient drying, the carbohydrate content is 20 to 50%. Therefore, the protein and carbohydrate content may vary depending on the preparation method.

The production method of soybean meal enzyme degradant used in the microbial culture medium according to the present invention is not particularly limited. If soy protein is enzymatically decomposed and used as fermentation medium, there is no commercialized product, but there is soytone (product of Difco) which enzymatically decomposes soy flour. However, this product is enzymatically decomposed soybean powder, and the hydrolysis degree is constant, so there is a restriction in use that it can not be widely used in all fermentation. In addition, there are peptone, tryptone, and polypeptone which are enzyme-degraded casein, but they are different from those hydrolyzed soybean meal, and the degree of hydrolysis is constant. there is a problem.

Among the properties of soybean meal enzyme degradants, the degree of hydrolysis and average molecular weight of proteins are very important factors in the use of fermentation medium. The degree of hydrolysis of the protein is defined as follows and the average molecular weight is measured by gel permeation chromatography method.

The degree of hydrolysis of the enzymatic degradation products can be varied by controlling the use ratio and mixing ratio of the endo-type and exo-type of proteolytic enzymes used for the preparation of soybean meal enzyme degradation products. In addition, it is possible to change the degree of hydrolysis by controlling the enzyme reaction time. In general, the higher the degree of hydrolysis, the smaller the average molecular weight, so it can be used as a medium for various fermentations depending on the degree of hydrolysis.

For example, in the case of antibiotic fermentation which requires a long time for fermentation, it is preferable to use a degradation product having a low degree of hydrolysis. Antibiotic fermentation is usually to obtain the second to third metabolites of microorganisms, so it is common to produce useful substances after the growth of the bacteria is usually added to the medium such as soy flour. Therefore, it is possible to cause abnormal fermentation by using a hydrolyzed (high hydrolyzable) decomposition product readily available to microorganisms. If the hydrolysis degree is low, there is no fear of abnormal fermentation, and the growth of bacteria is faster than the use of soy flour or soybean meal acid decay, which shortens the overall fermentation time and increases the productivity under the influence of the bioactive substance contained in the enzyme decomposition product. . Generally, it is preferable to use about 10 to 60% of hydrolysis. The average molecular weight at this time is about 800-3000. In addition, in the fermentation that produces rapid growth like bacteria and produces amino acids, nucleic acids, enzymes, etc., it is preferable to use a high hydrolysis, 30 ~ 60% of the hydrolysis is effective. The average molecular weight at this time is about 200 to 600.

There is no limitation on the amount of soybean meal enzyme digestion added to the microorganism culture medium. However, generally considering the balance between economical aspects and other media components, 0.1 to 10% by volume is appropriate, preferably in the range of 0.1 to 5% by volume.

Soybean meal enzyme digestion can be used in place of soybean meal acid degradation products which have been added to the medium for cultivating microorganisms, as well as CSL (corm steep liquor), soy flour, pharma media, and marinated dong, especially containing insoluble substances. Therefore, when the medium has a physical property that does not dissolve well in water, the replacement effect is greater in the recovery, purification. In particular, when the cells are used after the fermentation is finished, insoluble substances are not mixed, which is more convenient.When using the supernatant from which the cells are removed, impurities and pigments are less easily recovered and purified, and the quality of the product can be improved. .

The advantages of using a fermentation medium composition containing soybean meal enzyme digest according to the present invention are as follows:

(1) Bioactive Peptides Peptides, including their own or active sites, are used in fermentation broth to increase the growth of fermentation and increase fermentation productivity.

(2) In the preparation of soybean meal enzyme degradation products, hydrolysis can be controlled by controlling the reaction conditions, so that it can be used for fermentation using almost all microorganisms.

(3) Soybean meal enzyme digestion is water-soluble and contains little impurities or pigments, so it is easy to recover the product after fermentation and improve the recovery rate.

Hereinafter, the present invention will be described in more detail with reference to various examples, but the present invention is not limited to these examples.

Comparative Example 1

Penicillium brevicompactum IHEM 1196, a microorganism producing the lactose β-galactosidase, was bran 4%, soy flour 2%, KH ₂ PO ₄ 2%, (NH ₄ ) ₂ SO ₄ Inoculated in 100ml of the medium containing 0.5%, 0.2% KCI and incubated for 4 days at 28 ℃. Enzyme activity of the culture was measured according to a known method using ONPG (o-nitroghenylgalactoside), and one unit of enzyme was defined as the amount of enzyme that produces 1 micromole of o-nitrophenol per minute under reaction conditions. As a result, the dry cell weight was 20g / l and the enzyme activity was 0.2U / ml.

Example 1

Culture using the strain of Comparative Example 1, but was used by replacing soybean meal enzyme digest (31% hydrolysis, 45% protein content) 2% soybean meal contained in the medium used. As a result of the culture, the dry cell weight was 21 g / l, which was similar to that of Comparative Example 1, but the activity of the enzyme was increased more than two times to 0.5 U / ml.

Comparative Example 2

Basic medium of Bacillus cirulans ATCC 31382, a microorganism that produces lactose β-galactosidase (lactose 0.5%, K ₂ HPO ₄ 0.1%, (NH ₄ ) ₂ SO ₄ 0.5%, pH 6.8) 0.1% of soy flour as an organic nitrogen source was added to 100 ml and incubated at 37 ° C. for 2 hours. After incubation, the growth of the bacteria was measured, and the enzyme activity of the supernatant from which the cells were removed was measured in the same manner as in Comparative Example 1, and the results are shown in Table 1.

Comparative Example 3

In the same manner as in Comparative Example 2, but instead of soy flour as a source of organic nitrogen, soytone (soytone, manufactured by Difco) was added and cultured, and the dry cell weight and enzyme activity of the culture were analyzed. The results are shown in Table 1.

[Comparative Example 4]

In the same manner as in Comparative Example 2, but was cultured by adding tryptone (manufactured by Difco) instead of soy flour as an organic nitrogen source, and analyzed the dry cell weight and enzyme activity of the culture. The results are shown in Table 1.

[Comparative Example 5]

It carried out similarly to the comparative example 2, but cultured by adding polypeptone (made by Japan Pharmaceutical Co., Ltd.) instead of soy flour as an organic nitrogen source, and analyzed the dry cell weight and enzyme activity of the culture. The results are shown in Table 1.

Example 2

In the same manner as in Comparative Example 2, but was cultured by adding soybean meal enzyme digestion instead of soy flour as an organic nitrogen source, dry cell weight and enzyme activity of the culture was analyzed. At this time, the degree of hydrolysis of the soybean meal enzyme decomposition product was 51%. The results are shown in Table 1.

Table 1. Growth and Enzyme Activity of Bacteria

From the results in Table 1, it is demonstrated that the fermentation medium containing soybean meal enzyme digest according to the present invention can be used as a highly efficient microbial fermentation medium.

Comparative Example 6

Strains of the genus Brevibacterium, which is a strain producing inosine monophosphate (IMP), were cultured in a conventional manner. The medium used at this time contained 2% CSL and the content of IMP after fermentation was 4.1%. The fermentation broth was recovered and purified by a conventional method using an ion exchange resin to obtain a final product. The recovery rate, product purity and chromaticity at this time are analyzed and shown in Table 2.

Example 3

In the same manner as in Comparative Example 6, soybean meal enzyme digestion (hydrolysis degree 42%) was added to the medium at the concentration of 2% and cultured in the same manner. As a result of culture, the content of IMP was 4.2%. Purification and analysis were performed in the same manner as in Comparative Example 6. The results are shown in Table 2.

[Table 2. IMP recovery results comparison table]

Note) * Transmittance: Measured transmittance at 420nm with 10% solution.

From the results of Table 2, it can be seen that the culture medium containing the soybean meal enzyme degradation product according to the present invention is very efficient for the recovery and purification of the product after fermentation.

Claims (3)

A method of treating soybean meal with proteolytic enzymes so that the soybean meal enzyme digested product having a hydrolysis degree of 10-60% as defined in the following formula is used as an organic nitrogen source or organic carbon source of the microbial culture medium. Degree of hydrolysis (%) = [(number of peptide peptide cleaved / total peptide of protein) × 100] The method of claim 1 wherein the soybean meal enzyme digest is used in an amount of 0.1 to 10% of the total volume of the medium. The method according to claim 1, wherein the protein content of the soybean meal enzyme digest is 40 to 70% by weight based on total nitrogen.