KR20170018222A - Composition comprising microoraganism having protease activity - Google Patents

Abstract

The present invention relates to a composition containing a strain having proteolytic activities, and to a method for producing protein hydrolysates using the composition. According to the present invention, owing to proteolytic activities and glutaminase activities of the strain, the strain can be useful for producing seasoning components close to natural flavors.

Description

[0001] The present invention relates to a composition comprising a microorganism having proteolytic activity,

The present invention relates to a composition comprising a strain having a protein hydrolyzing activity.

In Korea, there are many fermented foods. Among them, soybean paste and fermented fish are a type of protein which is hydrolyzed by enzymes produced by microorganisms to produce a variety of amino acids or peptides, It is a hydrolyzate.

Known methods for producing protein hydrolyzate for seasoning include acid decomposition, enzyme digestion, and fermentation. In the past, acid decomposition methods for hydrolyzing proteins by using acids such as hydrochloric acid have been widely used, but they have been rarely used recently due to the risk. In recent years, enzymatic degradation techniques using various protein hydrolytic enzymes with different substrate specificity have been attracting attention as consumers' demand for seasonings close to natural flavor has increased. As the protein hydrolyzing enzyme, a bacterial-derived protease containing no peptidase and a fungal-derived protease containing a peptidase are used. In the fermentation method in which microorganisms are inoculated directly into the protein material and fermented, a variety of protease activity, such as Aspergillus oryzae and Aspergillus sojae) and Aspergillus (koji mold) is mainly used as, a long time has been used in the manufacture of Jiang, miso. Generally Aspergillus When a strain of the genus Escherichia coli is used as an enzyme source in solid fermentation or liquid fermentation, various types of amino acids or peptides are produced by various kinds of enzymes.

Glutamic acid was found to be the main ingredient of umami (umami, savory or jimi) classified as basic five flavors in addition to sweet, sour, bitter and salty taste. . Therefore, it is advantageous to produce a protein hydrolyzate containing a large amount of glutamic acid when preparing a protein hydrolyzate for seasoning.

Vegetable Protein In order to develop a natural seasoning material, it is common to mix wheat gluten hydrolyzate because soy protein hydrolyzate alone, which is most widely used, lacks a rich flavor. However, wheat gluten has difficulties in solubilization and hydrolysis compared to soybean protein, and commercial enzymatic hydrolysis has limitations in obtaining high amination rate and free amino acid ratio.

Therefore, in order to produce a hydrolyzate which can be used as a natural seasoning material and a salty enhancer, it is important to obtain an excellent fermentation microorganism that can be applied to practical industrial applications in addition to commercial protease. In particular, in order to produce glutamic acid in a large amount, glutaminase activity This excellent strain is required. Glutaminase is an enzyme that acts on L-glutamine to hydrolyze an amide group to produce glutamic acid and ammonia.

It is an object of the present invention to provide a composition comprising Bacillus subtilis TP6 (Accession No. KFCC 11343P), a strain having a protein hydrolyzing activity.

The present invention relates to a method for fermenting a composition comprising a protein source at a normal pressure with a glutaminase activating strain or glutaminase activating strain and a protein hydrolyzing enzyme and further subjecting the fermented composition to a fermentation under a pressure higher than normal pressure Wherein the protein hydrolyzate is a protein hydrolyzate. In one embodiment of the invention, the protein source is an insoluble vegetable protein, and in another embodiment of the invention the protein source is selected from the group consisting of wheat gluten, corn gluten, defatted soybean and soy protein, in another embodiment, the glue Tommy better activity strain is Bacillus subtilis TP6's tiller (Bacillus subtilis TP6). The Bacillus subtilis TP6 strain is known from Korean Patent No. 10-0753002.

Also, in one embodiment of the present invention, the fermentation at the above-mentioned atmospheric pressure is performed until the strain reaches spore forming section, and in another embodiment of the present invention, the above-atmospheric pressure is 50 MPa to 200 MPa, In another embodiment of the present invention, the protein source is a protein that has been pretreated and solubilized by at least one method selected from the group consisting of heat treatment, acid hydrolysis, and enzymatic hydrolysis. In another embodiment of the present invention, And is contained in an amount of 0.01 wt% to 10 wt% based on the weight of the whole composition.

In one embodiment of the present invention, the enzymatic hydrolysis in the solubilization method of the protein source is a hydrolysis method using a proteolytic enzyme. Endo-type protein hydrolytic enzymes are widely used for solubilization of protein sources, and endo-type enzymes act inside the polymer polypeptide to randomly cleave to produce a large amount of water-soluble relatively low-molecular peptides. Representative commercial enzymes that are widely used include, but are not limited to, for example, Alaclase, Protamex and Neutrase. Enzymes used for solubilization of proteins and methods of solubilization are not limited to endo-type protein hydrolytic enzymes and exo-protein hydrolytic enzymes, but only if they are capable of solubilizing plant proteins.

More preferably, when using alcalase, 0.1-2% of the enzyme is dissolved in water in a weight ratio to the substrate, and then, while stirring, at least one protein selected from the group consisting of wheat gluten, corn gluten, defatted soybean meal and soy protein The roundness is gradually added so as not to be entangled, and the solubilized solution is made to have a final concentration of 10-20%. At this time, the reaction temperature is maintained at 50 ° C and the pH is adjusted to maintain the pH of 5-8.

In one embodiment of the invention the Bacillus sub-blocks scan TP6 (Bacillus subtilis TP6), or Bacillus subtilis TP6 ( Bacillus < RTI ID = 0.0 > subtilis TP6) and a protein hydrolyzing enzyme, wherein in one embodiment of the invention the protein is an insoluble vegetable protein, and in another embodiment of the present invention the protein hydrolyzing enzyme is But are not limited to, chymotrypsis, thermolysin, streptococcal fibrinolysin, streptolysin, flavourzyme, trypsin, papain and proanase (pronase).

In one embodiment of the invention the Bacillus sub-blocks scan TP6 (Bacillus subtilis TP6), or Bacillus subtilis TP6 ( Bacillus < RTI ID = 0.0 > subtilis TP6) and a protein hydrolyzing enzyme.

Another embodiment of the invention the Bacillus subtilis TP6 (Bacillus subtilis TP6) (KFCC 11343P) strains and commercial protein added hydrolase, and at the same time the fermentation or complex, after degrading enzyme hydrolysis Bacillus subtilis TP6 (Bacillus to enter into force as subtilis TP6), or Bacillus subtilis TP6 (Bacillus Subtilis TP6) can be hydrolyzed by adding enzymes intermittently during fermentation, or by adding enzyme after Bacillus subtilis TP6 fermentation. Since the type of protein hydrolytic enzymes produced according to the species are different from the substrate specificity, it is possible to use a variety of protein hydrolytic enzymes of various origins in order to increase the yield of amino acids as well as to produce hydrolysates having a rich taste.

Thus, Bacillus subtilis TP6 with glutaminase activity ( Bacillus < RTI ID = 0.0 > To complement the protein hydrolytic enzyme produced by the subtilis TP6 strain (KFCC 11343P), a commercial enzyme may be added. Further still another object of the fermentation complex protease and Bacillus subtilis TP6 (Bacillus subtilis TP6) (KFCC 11343P) strain is Bacillus subtilis TP6 (Bacillus Since the lag time of 12 hours to 96 hours is elongated until the enzyme is fully produced by the proliferation of subtilis TP6 (KFCC 11343P), proteolysis proceeds preferentially at the beginning of fermentation by the added enzyme, after that, the Bacillus subtilis TP6 (Bacillus It lies in that by the enzyme which is produced subtilis TP6) (KFCC 11343P) further protein to be efficiently proceeding the proteolytic by such hydrolysis.

In another embodiment of the present invention, the strain may be subjected to high pressure treatment at 50 MPa to 200 MPa after spore formation to promote the hydrolysis reaction. Treatment of the TP6 culture medium at a high pressure of 50 MPa to 200 MPa for 12 hours to 48 hours can accelerate the enzyme reaction to obtain a high amino acid yield while remarkably shortening the fermentation time.

The term endo-type protease or endo-type protease used in the present invention refers to an enzyme that degrades peptide bonds other than N-terminal or C-terminal in a protein, and exoproteinase or exoprotease is N - < / RTI > enzyme that degrades peptide bonds at the terminal or C-terminus.

Bacillus subtilis forms endogenous sporozoites when they become depleted in nutrients or become difficult to survive or grow. The formation of endogenous spores from the nutrient cells that actively metabolize and proliferate is called spore formation, and the process of returning endogenous spores to the nutrient cells is called. Endogenous spores are formed in a modified form of the general cell division process. When the spore formation is completed, the large cytoplasm and cell walls of the original cell are degraded. Various kinds of protein hydrolysis enzymes are produced for such degradation.

The present invention relates to a method for producing Bacillus subtilis TP6 ( Bacillus < RTI ID = 0.0 > subtilis TP6) (Accession No. KFCC 11343P), and a method for hydrolyzing a protein using the strain.

1 is a graph showing the content of glutamic acid in fermented products of various strains according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example

Example  1. Screening of strains with excellent glutaminase activity

Glue Tommy better activity the inventors a high proteolytic activity of Bacillus subtilis TP6 (Bacillus subtilis TP6) obtained through the previous studies to starting the superior strain (KFCC11343P), soybean derived from Aspergillus (Aspergillus) It received pre-sale activity and the professional Corsica from Aspergillus spp and Korea Seed Association (koji mold) of material Aspergillus (Aspergillus sojae) KCCM 60354, Aspergillus duck material (Aspergillus oryzae) KCCM 60247 and Aspergillus awamori (Aspergillus awamori ) KCCM 60166 were compared.

Each of the above five strains was inoculated into 100 mL of a 10% (w / w) wheat gluten (Royal Ingredients Group B.B., China) dispersion in a 500 mL Erlenmeyer flask and cultured in a shaking incubator at 37 ° C for 2 days. After completion of the incubation, two 10 mL of each culture were taken and centrifuged at 20,000 x g for 20 minutes. The solubilization ratio was calculated by measuring the solids content with 5 mL of the supernatant, and glutamic acid was quantified using HPLC (high-performance liquid chromatography) with the remaining samples and repeated twice.

Table 1 and five from the candidate strain Bacillus subtilis TP6 (Bacillus as shown in FIG. 1 The whey gluten solubilization rate of subtilis TP6 (hereinafter referred to as TP6) was almost 100% and glutamic acid content was also high. These results suggest that the TP6 strain is a highly effective strain of endo - type and exo - protease activity, and that it has a very strong glutaminase activity, making it the most suitable strain for protein hydrolysis.

Strain name Solubilization rate (%) Aspergillus sojae KCCM 60354 55 Aspergillus oryzae KCCM 60247 15 Aspergillus awamori KCCM 60166 54 Aspergillus soy-derived strain 13 Bacillus subtilis TP6 98

Example 2 Effect of Nutritional Components on the Growth of TP6 Bacteria in Wheat Gluten Medium

Growth of TP6 bacterium was investigated by adding 2% glucose or 2% xylose as a carbon source and 0.5% yeast extract as an organic nutrient to a 10% (w / w) dispersion of wheat gluten in hot water at 90 ° C Respectively. 100 mL of wheat gluten to which monosaccharide glucose, xylose or yeast extract had been added was placed in a 500 mL Erlenmeyer flask, sterilized at 121 ° C for 15 minutes, and cultured for 24 hours in NB (nutrient broth) medium (Difco Laboratories, One TP6 culture was inoculated at 5% (v / v), and cultured at 37 ° C and pH 7.0 for 24 hours.

As a result, as shown in Table 2, in the wheat gluten suspension medium not supplemented with the nutrient source, the viable cell count was about 9.4 x 10 ⁶ CFU / mL, but the viable cell number was about 3 when yeast extract and glucose were added And 2.8 x 10 ⁹ CFU / mL, respectively.

Wheat gluten
(10%) Wheat gluten (10%) +
Yeast extract (0.5%) Wheat gluten (10%) +
Yeast extract (0.5%) +
Glucose (2%) Wheat gluten (10%) +
Yeast extract (0.5%) +
Xylose (2%) Number of bacteria (CFU / mL) 9.4x10 ⁶ 9.7 x ^{10 8} 2.8x10 ⁹ 2.5x10 ⁹

Example 3. Complex hydrolysis of wheat gluten with TP-6 alone and enzyme

The protein was solubilized by adding 1% (w / v) of alkalase to a 10% (w / w) wheat gluten suspension and hydrolyzing at 50 ° C and pH 7.0 for 12 hours. Then, 2% of glucose and 0.5% To prepare a protein culture medium composition. This medium composition was dispensed into four 500 mL Erlenmeyer flasks in 100 mL portions and sterilized at 121 DEG C for 15 minutes. The culture of TP6 cultured 24 hours before in NB (nutrient broth) medium was added at a ratio of 1: 1 (v / v), and 1% w / w-protein (Novozyme, Denmark) ) Were further added and cultured in a 37 ° C shaking incubator for 4 days. The degree of hydrolysis of protein was measured by OPA (O-phthalaldehyde) method and the content of glutamic acid by HPLC.

Table 3 shows the results of complex hydrolysis by adding TP-6 alone and culturing alone and TP-6 and flavonoid enzyme at the same time.

Hydrolysis (%) Glutamic acid content (g / L) TP-6 alone fermentation (4 days) 18.1 2.1 TP-6 + Flavourzyme (1%)
Combined fermentation (4 days) 26.9 3.0

Example 4. High-pressure hydrolysis of soybean protein and wheat gluten TP-6 fermentation broth

1% (w / v) of alkalase was added to a 10% (w / w) suspension of isolated soybean protein and wheat gluten and the proteins were solubilized by hydrolysis at 50 ° C and pH 7.0 for 12 hours. (w / v) and 0.5% (w / v) of yeast extract were added to prepare protein fermentation medium, respectively. Each protein fermentation medium was dispensed into two 1 L Erlenmeyer flasks in 250 mL each and sterilized at 121 ° C for 15 minutes. One test group consisted of 1: 1 (v / v) TP6 preincubation culture in which TP6 was cultured in NB medium at 37 ° C for 24 hours in each protein fermentation medium and cultured in a 37 ° C shaking incubator for 5 days. After incubation for 4 days in the same conditions as above, the flask culture was aseptically injected into a pressure-tight transparent bag of 100 mL, sealed, and hydrolyzed at 100 MPa and 37 ° C for 24 hours to obtain total fermentation time 5 days. At this time, a model TFS-20 manufactured by Ino Wei Co., Ltd. was used as the high-pressure processor. The degree of hydrolysis of the hydrolyzate was determined by the OPA method and the amino acid content was measured by the ninhydrin method.

As a result, as shown in Table 4, when soybean protein suspension was solely fermented with TP6 for 5 days, the degree of hydrolysis was 54.1% and the yield of amino acid was 61.8%. The hydrolysis and amino acid yields of TP6 strains were increased to 66.3% and 62.9%, respectively, when the fermentation broth was incubated for 4 days at atmospheric pressure. Particularly, the degree of hydrolysis was remarkably increased. In the atmospheric pressure fermentation condition, it takes 7-10 days to achieve such a degree of hydrolysis.

On the other hand, the hydrolysis rate was 32.9% and the amino acid yield was 40.0% after 5 days of culturing in wheat gluten, and the hydrolysis efficiency was slightly lower than that of soybean protein. The hydrolysis and amino acid yields of wheat gluten fermented by TP6 fermentation for 24 hours under high pressure of 100 MPa were increased by 40.1% and 46.1%, respectively.

These results suggest that the endothelium and exo - hydrolytic enzymes secreted by the TP6 strain grow in the atmospheric fermentation broth and the activity of these enzymes is increased under the high pressure condition. As another comparative example, although the protein fermentation medium inoculated with TP6 bacteria was not fermented for 5 days at 100 MPa and 37 ° C under very high pressure conditions, the degree of hydrolysis was very low.

Protein source Fermentation method Hydrolysis (%) Glutamic acid content (g / L) Isolated soy protein Atmospheric pressure fermentation (5 days) 54.1 61.8 Atmospheric pressure fermentation (4 days) +
High pressure hydrolysis (1 day) 66.3 62.9 Wheat gluten Atmospheric pressure fermentation (5 days) 32.9 40.0 Atmospheric pressure fermentation (4 days) +
High pressure hydrolysis (1 day) 40.1 46.1

While the present invention has been described with reference to exemplary embodiments, those skilled in the art will appreciate that various changes and modifications can be made thereto without departing from the scope of the invention, . In addition, many modifications may be made to adapt a particular situation and material to the teachings of the invention without departing from the essential scope thereof. Accordingly, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention be construed as including all embodiments falling within the scope of the appended claims.

Claims (13)

A composition comprising a protein source is fermented at a normal pressure with a glutaminase-activating strain or a glutaminase-activating strain and a protein hydrolyzing enzyme,
Further comprising fermenting the fermented composition under a pressure above atmospheric pressure.
The method according to claim 1,
Wherein the protein source is an insoluble vegetable protein.
3. The method according to claim 1 or 2,
Wherein the protein source is selected from the group consisting of wheat gluten, corn gluten, defatted soybean meal and soy protein.
3. The method according to claim 1 or 2,
Wherein the glutaminase activating strain is Bacillus subtilis TP6. ≪ RTI ID = 0.0 > 11. < / RTI >
3. The method according to claim 1 or 2,
Wherein the fermentation is carried out at the normal pressure until the strain reaches spore forming section.
3. The method according to claim 1 or 2,
Wherein the pressure above the atmospheric pressure is 50 MPa to 200 MPa.
3. The method according to claim 1 or 2,
Wherein the protein source is a protein which is pretreated and solubilized by at least one method selected from the group consisting of heat treatment, acid hydrolysis and enzyme hydrolysis.
3. The method according to claim 1 or 2,
Wherein the protein hydrolyzing enzyme is an exo or endo-type protein hydrolyzing enzyme, and the protein hydrolyzate is contained in an amount of 0.01w% to 10w% based on the weight of the whole composition.
Bacillus subtilus TP6 ( Bacillus subtilis TP6), or Bacillus subtilis TP6 ( Bacillus < RTI ID = 0.0 > subtilis TP6) and a protein hydrolyzing enzyme.
10. The method of claim 9,
Wherein the protein is an insoluble vegetable protein.
11. The method according to claim 9 or 10,
The protein hydrolyzing enzyme may be selected from the group consisting of chymotrypsis, thermolysin, streptococcal fibrinolysin, streptolysin, flavorzyme, trypsin, papain, wherein the protein hydrolyzate is at least one selected from the group consisting of papain and pronase.
Bacillus subtilus TP6 ( Bacillus subtilis TP6), or Bacillus subtilis TP6 ( Bacillus < RTI ID = 0.0 > Subtilis TP6) and a method for hydrolyzing a protein source using a protein hydrolyzing enzyme.
A composition comprising a protein source is fermented at a normal pressure with a glutaminase-activating strain or a glutaminase-activating strain and a protein hydrolyzing enzyme,
Wherein the fermented composition is further fermented under a pressure higher than the atmospheric pressure.

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