KR102110995B1 - Composition for culturing Bacillus genus microorganisms for promoting gamma-glutamyltransferase activity - Google Patents

Abstract

The present specification relates to a composition for promoting gamma-glutamyltransferase activity of Bacillus genus microorganisms and a culturing method using the same. The composition for culturing Bacillus genus microorganisms comprising sugar as a carbon source and soybean peptone as a nitrogen source have gamma-glutamyltransferase activity when the Bacillus genus microorganisms are cultured in a medium comprising the composition, thereby improving flavor and appealability of a fermented food produced using the same.

Description

Composition for culturing Bacillus genus microorganisms for promoting gamma-glutamyltransferase activity to promote gamma-glutamyl transferase activity}

Disclosed herein is a composition for culturing microorganisms of the genus Bacillus, and in one aspect, a composition for enhancing gamma-glutamyl transferase activity of the microorganisms of the genus Bacillus and a fermentation method using the same.

Fermented food refers to food using the fermentation of microorganisms, and fermentation refers to a process in which flavor components of various characteristics are produced by enzymes produced by microorganisms. Therefore, microorganisms as well as raw materials used in manufacturing fermented foods are very important. Even if the same substrate (bean, rice, barley, etc.) is used, the taste and aroma characteristics of the fermented food may vary depending on the type of fermenting microorganism, the degree of growth, and enzyme characteristics.

To make soy and miso, fermented foods of soybeans, meju is needed. Fermentation microorganisms grow in Meju and produce enzymes. Traditional meju recipes naturally inoculate various microorganisms present in the air. On the other hand, the commercial meju manufacturing method produces an improved meju by inoculating specific microorganisms in order to mass-produce stable quality products.

In order to obtain a taste component from soy protein, most commercially improved meju uses Bacillus spp., A microorganism having excellent proteolytic and converting enzyme activity, as a main fermentation microorganism. The quality of the fermented foods of soybeans is determined by the enzyme properties and production of the microorganisms used. On the other hand, in terms of decomposition and conversion of soy protein, it is possible to increase enzyme production or increase activity through genetic mutation of microorganisms. However, genetically modified microorganisms in food are not used commercially for safety reasons. In light of this commercial use reality, it is necessary to have optimal conditions for enhancing the enzyme activity and production of soybean fermentation microorganisms.

It is important to control the temperature, moisture, oxygen content, and nutrients appropriate for the growth of microorganisms. Temperature determines the growth rate and amount of microorganisms, and is also related to the enzyme activity of microorganisms. And most of the microorganisms are absolutely aerobic, so the growth rate may vary depending on the amount of oxygen supplied. In particular, the nutrient source is the most important factor in the growth of microorganisms, the carbon source and nitrogen source require a large amount, and minerals (Ca, K, Na, Mg, P, S, etc.) require a small amount. The carbon source is used as the cell's composition and energy source for the cell, and the nitrogen source is needed to synthesize intracellular components such as proteins and nucleic acids. Among minerals, sulfur (S) constitutes substances such as cysteine, cystine, and methionine, which play an important role in the growth and differentiation of microorganisms, and phosphorus (P) constitutes DNA, RNA, phospholipids, vitamins, and NAD. Mineral components in minerals play various roles such as growth and spore formation of microorganisms, structural components of metabolites, and structural formation and activation of enzymes. However, if the minerals (minerals and inorganic components) are excessive, the growth of microorganisms can be suppressed.

In the manufacture of fermented soybean food, fermented microorganisms play an important role in determining the properties of the product. Therefore, if it satisfies the nutritional needs of microorganisms, it will help to enhance the enzyme activity required for soybean fermentation. In addition, fermented foods having excellent umami and gokumi (persistence, weight) characteristics may be manufactured.

KR 10-1749991 B1 KR 10-1814024 B1 CN 108588138 A CN 101363014 B

In one aspect, an object of the present invention is to provide a composition for culturing microorganisms including a carbon source and a nitrogen source that allow microorganisms in Bacillus to have gamma-glutamyltransferase activity.

In another aspect, an object of the present invention is to provide a fermentation method using the composition.

The activity of enzymes produced by microorganisms is important in producing flavor components from soybean, the main raw material of soybean fermented food. At this time, the enzyme activity of the microorganism is greatly influenced by the medium component, and the type of the enzyme whose activity is increased depends on the medium component. Therefore, in order to mass-produce specific enzymes, it is necessary to supply nutrients that can increase the production of specific enzymes.

In one aspect, the present invention is a composition for culturing microorganisms of the genus Bacillus containing sugar as a carbon source and soypeptone as a nitrogen source, wherein the microorganism of the genus Bacillus cultured in a medium containing the composition is Provided is a composition for culturing microorganisms of the genus Bacillus, which has gamma-glutamyltransferase activity.

In another aspect, the present invention provides a fermentation method comprising culturing a microorganism in the genus Bacillus in a medium containing the composition.

In another aspect, the present invention is a microorganism in the genus Bacillus ( Bacillus ) cultured in a medium containing the composition, at least one of the lysate and the culture; Or it provides a fermented food comprising a gamma-glutamyltransferase (gamma-glutamyltransferase) enzyme solution isolated from any one or more of the microorganisms, the crushed product and the culture.

In another aspect, the present invention provides a method for producing gamma-glutamyltransferase comprising culturing a microorganism in Bacillus in a medium containing the composition.

The composition for culturing microorganisms of Bacillus genus, which includes sugar as a carbon source of the present invention and soybean peptone as a nitrogen source, has a gamma-glutamyltransferase activity when culturing Bacillus microorganisms in a medium containing the composition. It has an excellent effect of improving the flavor and organoleptic properties of the fermented food prepared by using it.

Figure 1 shows the comparison of gamma-glutamyl transferase enzyme activity according to the type of nitrogen source.
Figure 2 is a comparison of the gamma-glutamyl transferase enzyme activity according to the content of soybean peptone. The content (%) of FIG. 2 refers to the weight% based on the total weight of the medium, and relative activity (%) is 100% of the highest gamma-glutamyltransferase enzyme activity in the experimental group. It means the relative activity of time.
Figure 3 shows a comparison of gamma-glutamyl transferase enzyme activity according to the type of carbon source.
Figure 4 is a comparison of the gamma-glutamyl transferase enzyme activity according to the content of sugar. The content (%) of FIG. 4 refers to the weight% based on the total weight of the medium, and the relative activity (%) is 100% of the highest gamma-glutamyltransferase enzyme activity in the experimental group. It means the relative activity of time.
Figure 5 shows the effect of the components in the medium composition on the enzyme activity as a result of sub-factor experiments for selecting a factor showing a major effect on gamma-glutamyl transferase enzyme activity.
6 is a response surface analysis method for optimizing the composition of the medium, showing the optimized composition of the medium composition.
Figure 7 shows the comparison of the gamma-glutamyl transferase enzyme activity according to the medium composition.
Figure 8 is a comparison of the amino acid content in the liver according to whether or not the addition of the gamma-glutamyl transferase enzyme solution obtained by culturing Bacillus amyloliquiparations using the medium composition.
Figure 9 is a comparison of the taste characteristics strength of the liver according to whether or not the addition of the gamma-glutamyl transferase enzyme solution obtained by culturing Bacillus amyloliquiparations using the medium composition.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention, as a composition for culturing microorganisms of the genus Bacillus containing a carbon source and a nitrogen source, the carbon source is sugar (sucrose), the nitrogen source is soypeptone (soypeptone), in a medium containing the composition The cultured microorganism of the genus Bacillus is Bacillus genus, having at least one enzyme activity selected from the group consisting of gamma-glutamyltransferase, gamma-glutamylpeptidase and glutaminase. Provided is a composition for culturing microorganisms.

The microorganisms of the genus Bacillus are Bacillus amyloliquefaciens , Bacillus subtilis , Bacillus methylotrophicus , Bacillus licheniformis , Bacillus licheniformis , Bacillus velezensis Bacillus sonorensis and Bacillus valismortis may be one or more selected from the group consisting of Bacillus amyloliquefaciens , specifically fermented foods. If it is a microorganism of the genus Bacillus used in manufacturing, the type is not limited.

The composition may include a carbon source and a nitrogen source.

The carbon source may be one or more selected from the group consisting of sugar, mannitol, fructose, xylose, and maltose, and specifically, may be sugar. The composition may include 1 to 5% by weight of sugar based on the total weight of the medium containing the composition, specifically 1 to 3% by weight of sugar, and more specifically 1% by weight or more , 1.1% or more, 1.2% or more, 1.3% or more, 1.4% or more, 1.5% or more, 1.6% or more, 1.7% or more, 1.8% or more, 1.9% or more, 1.91% or more , 1.92 wt% or more, 1.93 wt% or more, 1.94 wt% or more, 1.95 wt% or more, 1.96 wt% or more, 1.97 wt% or more, 1.98 wt% or more, 1.99 wt% or more, 2 wt% or more, 2.01 wt% or more , 2.02 wt% or more, 2.03 wt% or more, 2.04 wt% or more, 2.05 wt% or more, 2.06 wt% or more, 2.07 wt% or more, 2.08 wt% or more, 2.09 wt% or more, 2.1 wt% or more, 2.11 wt% or more , 2.12 wt% or more, 2.13 wt% or more, 2.14 wt% or more, 2.15 wt% or more, 2.16 wt% or more, 2.17 wt% or more, 2.18 wt% or more, 2.19 wt% or more, 2.2 wt% or more, 2.21 wt% or more, 2.22 wt% or more, 2.23 wt% or more, 2.24 wt% or more, 2.25 wt% or more, 2.26 wt% or more, 2.27 wt% or more, 2.28 wt% or more, 2.29 wt% or more, 2.3 wt% or more, 2.31 wt% or more, 2.32 wt% or more, 2.33 wt% or more, 2.34 wt% or more, 2.35 wt% or more, 2.36 wt% or more, 2.37 wt% or more, 2.38 wt% or more, 2.39 wt% or more, 2.4 wt% or more, 2.41 wt% or more, 2.42 wt% or more, 2.43 wt% or more, 2.44 wt% or more, 2.45 wt% or more, 2.46 wt% or more, 2.47 wt% or more, 2.48% or more, 2.49% or more, 2.5% or more, 2.51% or more, 2.52% or more, 2.53% or more, 2.54% or more, 2.55% or more, 2.56% or more, 2.57% or more, 2.58 wt% or more, 2.59 wt% or more, 2.6 wt% or more, 2.7 wt% or more, 2.8 wt% or more, 2.9 wt% or more, 3 wt% or more, or 4 wt% or more Can contain sugar, 5% or less, 4.9% or less, 4.8% or less, 4.7% or less, 4.6% or less, 4.5% or less, 4.4% or less, 4.3% or less, 4.2% or less Less than, 4.1% by weight, 4% by weight or less, 3.9% by weight or less, 3.8% by weight or less, 3.7% by weight or less, 3.6% by weight or less, 3.5% by weight or less, 3.4% by weight or less, 3.3% by weight or less, 3.2% by weight Or less, 3.3% or less, 3.2% or less, 3.1% or less, 3% or less, 2.9% or less, 2.8% or less, 2.7% or less, 2.6% or less, 2.59% or less, 2.58% or less Or less, 2.57% or less, 2.56% or less, 2.55% or less, 2.54% or less, 2.53% or less, 2.52% or less, 2.51% or less, 2.5% or less, 2.49% or less, 2.48% or less Or less, 2.47% or less, 2.46% or less, 2.45% or less, 2.44% or less, 2.43% or less, 2.42% or less, 2.41% or less, 2.4% or less, 2.39% or less Or less, 2.38 wt% or less, 2.37 wt% or less, 2.36 wt% or less, 2.35 wt% or less, 2.34 wt% or less, 2.33 wt% or less, 2.32 wt% or less, 2.31 wt% or less, 2.3 wt% or less, 2.29 wt% Or less, 2.28 wt% or less, 2.27 wt% or less, 2.26 wt% or less, 2.25 wt% or less, 2.24 wt% or less, 2.23 wt% or less, 2.22 wt% or less, 2.21 wt% or less, 2.2 wt% or less, 2.1 wt% Or less, 2.09 wt% or less, 2.08 wt% or less, 2.07 wt% or less, 2.06 wt% or less, 2.05 wt% or less, 2.04 wt% or less, 2.03 wt% or less, 2.02 wt% or less, 2.01 wt% or less, 2 wt% Or less, 1.9% or less, 1.8% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less, 1.1% or less or 1% The following sugar may be included, but is not limited thereto, and microorganisms in Bacillus cultured in a medium containing the composition are sensed. If the content of the sugar having ma-glutamyl transferase activity, the content is not limited.

The nitrogen source may be one or more selected from the group consisting of soybean, soybean powder, defatted soybean, defatted soybean powder and soypeptone, specifically, soybean peptone (soypeptone). The composition may include 2 to 8% by weight of soybean peptone based on the total weight of the medium containing the composition, specifically 2.5 to 6.5% by weight of soybean peptone, and more specifically 2% by weight % Or more, 2.1% or more, 2.2% or more, 2.3% or more, 2.4% or more, 2.5% or more, 2.6% or more, 2.7% or more, 2.8% or more, 2.9% or more, 3% or more % Or more, 3.1% or more, 3.2% or more, 3.3% or more, 3.4% or more, 3.5% or more, 3.6% or more, 3.7% or more, 3.8% or more, 3.9% or more, 4% or more % Or more, 4.1% or more, 4.2% or more, 4.3% or more, 4.4% or more, 4.5% or more, 4.6% or more, 4.7% or more, 4.8% or more, 4.9% or more, 5% or more % Or more, 5.1% or more, 5.11% or more, 5.12% or more, 5.13% or more, 5.14% or more, 5.15% or more, 5.16 % By weight or more, 5.17% by weight or more, 5.18% by weight or more, 5.19% by weight or more, 5.2% by weight or more, 5.21% by weight or more, 5.22% by weight or more, 5.23% by weight or more, 5.24% by weight or more, 5.25% by weight or more, 5.26 Above weight%, above 5.27% by weight, above 5.28% by weight, above 5.29% by weight, above 5.3% by weight, above 5.31% by weight, above 5.32% by weight, above 5.33% by weight, above 5.34% by weight, above 5.35% by weight, above 5.36 Above 5% by weight, above 5.37% by weight, above 5.38% by weight, above 5.39% by weight, above 5.4% by weight, above 5.41% by weight, above 5.42% by weight, above 5.43% by weight, above 5.44% by weight, above 5.45% by weight, above 5.46 Above weight%, above 5.47% by weight, above 5.48% by weight, above 5.49% by weight, above 5.5% by weight, above 5.6% by weight, above 5.7% by weight, above 5.8% by weight, above 5.9% by weight, above 6% by weight, 6.5 It may contain at least 7% by weight, at least 7% by weight or at least 7.5% by weight soybean peptone, 8% by weight or less, 7.5% by weight or less, 7% by weight or less, 6.9% by weight or less , 6.8% or less, 6.7% or less, 6.6% or less, 6.5% or less, 6.4% or less, 6.3% or less, 6.2% or less, 6.1% or less, 6% or less, 5.9% or less , 5.8% or less, 5.79% or less, 5.78% or less, 5.77% or less, 5.76% or less, 5.75% or less, 5.74% or less, 5.73% or less, 5.72% or less, 5.71% or less , 5.7% or less, 5.69% or less, 5.68% or less, 5.67% or less, 5.66% or less, 5.65% or less, 5.64% or less, 5.63% or less, 5.62% or less, 5.61% or less , 5.6% or less, 5.59% or less, 5.58% or less, 5.57% or less, 5.56% or less, 5.55% or less, 5.54% or less, 5.53% or less, 5.52% or less, 5.51% or less , 5.5% or less, 5.49% or less, 5.48% or less, 5.47% or less, 5.46% or less, 5.45% or less, 5.44% or less, 5.43% or less, 5.42 % By weight or less, 5.41% by weight or less, 5.4% by weight or less, 5.3% by weight or less, 5.2% by weight or less, 5.1% by weight or less, 5% by weight or less, 4.9% by weight or less, 4.8% by weight or less, 4.7% by weight or less, 4.6 Weight% or less, 4.5% or less, 4.4% or less, 4.3% or less, 4.2% or less, 4.1% or less, 4% or less, 3.9% or less, 3.8% or less, 3.7% or less, 3.6 Weight% or less, 3.5% or less, 3.4% or less, 3.3% or less, 3.2% or less, 3.1% or less, 3% or less, 2.9% or less, 2.8% or less, 2.7% or less, 2.6 It may include, but not limited to, 2.5% by weight or less, 2.5% or less, 2.4% or less, 2.3% or less, 2.2% or less, or 2.1% or less soybean peptone, in a medium containing the composition. If the microorganism in the cultured Bacillus is a content of soybean peptone having gamma-glutamyl transferase activity, the content is not limited. Does not.

The composition may further include at least one mineral selected from the group consisting of KH ₂ PO ₄ , Na ₂ HPO ₄ , MgSO ₄ and CaCl ₂ . Specifically, the composition may include 0.1 to 1% by weight of KH ₂ PO ₄ based on the total weight of the medium containing the composition, more specifically 0.1% by weight or more, 0.2% by weight or more, 0.3% by weight Or more, 0.4 wt% or more, 0.5 wt% or more, 0.6 wt% or more, 0.7 wt% or more, 0.8 wt% or more, or 0.9 wt% or more may include KH ₂ PO ₄ , 1 wt% or less, 0.9 wt% or less , 0.8 wt% or less, 0.7 wt% or less, 0.6 wt% or less, 0.5 wt% or less, 0.4 wt% or less, 0.3 wt% or less, or 0.2 wt% or less of KH ₂ PO ₄ , but is not limited thereto. no. Alternatively, the composition may include 1 to 2% by weight of Na ₂ HPO ₄ based on the total weight of the medium containing the composition, more specifically 1% by weight or more, 1.1% by weight or more, 1.2% by weight or more , 1.3 wt% or more, 1.4 wt% or more, 1.5 wt% or more, 1.6 wt% or more, 1.7 wt% or more, 1.75 wt% or more, 1.8 wt% or more, or 1.9 wt% or more of Na ₂ HPO ₄ , 2% or less, 1.9% or less, 1.8% or less, 1.75% or less, 1.7% or less, 1.6% or less, 1.5% or less, 1.4% or less, 1.3% or less, 1.2% or less or It may include Na ₂ HPO _{4 up} to 1.1% by weight, but is not limited thereto. Alternatively, the composition may include 1 to 3 mM MgSO ₄ based on the total weight of the medium containing the composition, more specifically 1 mM or more, 1.2 mM or more, 1.4 mM or more, 1.6 mM or more, 1.8 MgSO ₄ may include at least mM, at least 2 mM, at least 2.2 mM, at least 2.4 mM, at least 2.6 mM, or at least 2.8 mM, 3 mM or less, 2.8 mM or less, 2.6 mM or less, 2.4 mM or less, 2.2 mM or less, MgSO ₄ of 2 mM or less, 1.8 mM or less, 1.6 mM or less, 1.4 mM or less, or 1.2 mM or less, but is not limited thereto. Alternatively, the composition may include 0.01 to 0.2 mM CaCl ₂ based on the total weight of the medium containing the composition, and more specifically, 0.01 mM or more, 0.02 mM or more, 0.04 mM or more, 0.06 mM or more, 0.08 CaCl ₂ may be included in mM or more, 0.1 mM or more, 0.12 mM or more, 0.14 mM or more, 0.16 mM or more, or 0.18 mM or more, 0.2 mM or less, 0.18 mM or less, 0.16 mM or less, 0.14 mM or less, 0.12 mM or less, 0.1 mM or less, 0.08 mM or less, 0.06 mM or less, 0.04 mM or less, or 0.02 mM or less of CaCl ₂ may be included, but is not limited thereto.

The composition may be that the microorganism of the genus Bacillus cultured in a medium containing the composition has enzyme activity. The enzyme may be at least one selected from the group consisting of gamma-glutamyltransferase, gamma-glutamyltransferase, gamma-glutamylpeptidase, and glutaminase, specifically gamma-glutamyltransferase. It may be a raise, but the type is not limited as long as it is an enzyme related to enhancing flavor and organoleptic properties of the fermented food.

Having the enzyme activity may have one or more activities selected from the group consisting of gamma-glutamyltransferase, gamma-glutamylpeptidase, and glutaminase. And may specifically have gamma-glutamyltransferase activity, which reacts the substrate by at least one catalytic action selected from the group consisting of gamma-glutamyltransferase, gamma-glutamylpeptidase and glutaminase. By doing so, it means that the chemical reactions necessary for life phenomena can be carried out inside living things. More specifically, as a result of measuring the activity using the gamma-glutamyl transferase analysis kit (Biovision, cat.K-784-100), the value is 8000 mU / mL or more, 8500 mU / mL or more, 9000 mU / mL or more, 9500 mU / mL or more, or 10000 mU / mL or more. Or, having the enzyme activity, when the microorganism of the genus Bacillus is cultured for 20 to 170 hours at a temperature of 0 to 45 ° C in a medium containing the composition, 8000 mU / mL or more, 8500 mU / mL or more, 9000 It may have a gamma-glutamyl transferase activity of mU / mL or more, 9500 mU / mL or more, or 10000 mU / mL or more.

The microorganism culture in the Bacillus may be cultured for 20 to 170 hours, specifically 20 hours or more, 25 hours or more, 30 hours or more, 35 hours or more, 40 hours or more, 45 hours or more, 48 hours or more, 50 hours or more , Over 52 hours, Over 54 hours, Over 56 hours, Over 58 hours, Over 60 hours, Over 62 hours, Over 64 hours, Over 66 hours, Over 68 hours, Over 70 hours, Over 80 hours, Over 90 hours, 100 More than hour, more than 110 hours, more than 120 hours, more than 130 hours, more than 140 hours, more than 150 hours or more than 160 hours can be cultured, 170 hours or less, 160 hours or less, 150 hours or less, 140 hours or less, 130 hours Or less, 120 hours or less, 110 hours or less, 100 hours or less, 90 hours or less, 80 hours or less, 72 hours or less, 70 hours or less, 68 hours or less, 66 hours or less, 64 hours or less, 62 hours or less, 60 hours or less, 58 hours or less, 56 hours or less, 54 hours or less, 52 hours or less, 50 hours or less, 48 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, 30 hours or less, or 25 hours or less may be cultured, but is not limited thereto. .

The microorganism culture in the Bacillus may be cultured at a temperature of 0 to 45 ° C, specifically, 0 ° C or higher, 5 ° C or higher, 10 ° C or higher, 15 ° C or higher, 20 ° C or higher, 25 ° C or higher, 30 ° C or higher, 32 C. C., 34 C or more, 36 C or more, 38 C or more, 40 C or more, 42 C or more, or 44 C or more, may be cultured at a temperature of 45 C or less, 44 C or less, 42 C or less, 40 C or less, 38 ° C or less, 36 ° C or less, 34 ° C or less, 32 ° C or less, 30 ° C or less, 25 ° C or less, 20 ° C or less, 15 ° C or less, 10 ° C or less, or may be cultured at a temperature of 5 ° C or less It does not work.

According to an embodiment of the present invention, Bacillus amyloliquefaciens was used for 72 hours at 37 ° C in a medium containing a medium composition comprising sugar as a carbon source and soypeptone as a nitrogen source. When cultured, it was confirmed that gamma-glutamyl transferase activity was improved by 3.3 or 39 times or more than when cultured in a medium containing other carbon sources or other nitrogen sources (Examples 1-2 and 1-3), particularly optimized When cultured in the culture medium, it was confirmed that the activity was enhanced by about 50 times or more than when cultured in the control medium M9 medium (Example 2-3).

Another aspect of the present invention provides a fermentation method comprising culturing a microorganism in the genus Bacillus ( Bacillus ) in a medium containing the composition. The composition, the description of the microorganism of the genus Bacillus is as described above.

The microorganism culture in the Bacillus may be cultured for 20 to 170 hours, specifically 20 hours or more, 25 hours or more, 30 hours or more, 35 hours or more, 40 hours or more, 45 hours or more, 48 hours or more, 50 hours or more , Over 52 hours, Over 54 hours, Over 56 hours, Over 58 hours, Over 60 hours, Over 62 hours, Over 64 hours, Over 66 hours, Over 68 hours, Over 70 hours, Over 80 hours, Over 90 hours, 100 More than hour, more than 110 hours, more than 120 hours, more than 130 hours, more than 140 hours, more than 150 hours or more than 160 hours can be cultured, 170 hours or less, 160 hours or less, 150 hours or less, 140 hours or less, 130 hours Or less, 120 hours or less, 110 hours or less, 100 hours or less, 90 hours or less, 80 hours or less, 72 hours or less, 70 hours or less, 68 hours or less, 66 hours or less, 64 hours or less, 62 hours or less, 60 hours or less, 58 hours or less, 56 hours or less, 54 hours or less, 52 hours or less, 50 hours or less, 48 hours or less, 45 hours or less, 40 hours or less, 35 hours or less, 30 hours or less, 30 hours or less, or 25 hours or less may be cultured, but is not limited thereto. .

The microorganism culture in the Bacillus may be cultured at a temperature of 0 to 45 ° C, specifically, 0 ° C or higher, 5 ° C or higher, 10 ° C or higher, 15 ° C or higher, 20 ° C or higher, 25 ° C or higher, 30 ° C or higher, 32 C. C., 34 C or more, 36 C or more, 38 C or more, 40 C or more, 42 C or more, or 44 C or more, may be cultured at a temperature of 45 C or less, 44 C or less, 42 C or less, 40 C or less, 38 ° C or less, 36 ° C or less, 34 ° C or less, 32 ° C or less, 30 ° C or less, 25 ° C or less, 20 ° C or less, 15 ° C or less, 10 ° C or less, or may be cultured at a temperature of 5 ° C or less It does not work.

The fermentation method may further include preparing a medium containing the composition, and the medium manufacturing step may further include sterilizing the prepared medium. The medium may include various types of medium, and may be specifically sterilized medium, but is not limited thereto.

The fermentation method may further include culturing the microorganism in the Bacillus to obtain a culture.

Another aspect of the invention, Bacillus ( Bacillus ) cultured in a culture medium containing the composition, any one or more of the lysate and its culture; Or a group consisting of gamma-glutamyltransferase, gamma-glutamylpeptidase, and glutaminase isolated from any one or more of the microorganisms, their lysate, and its culture. It provides a fermented food comprising one or more enzyme solutions selected from. The composition, the microorganism of the genus Bacillus, and the description of the culture thereof are as described above.

The enzyme solution may be a gamma-glutamyl transferase enzyme solution, but is not limited thereto.

The fermented food is any one or more of the microorganism, its crushed product and its culture; Alternatively, it may further contain 15% or more of glutamic acid compared to the fermented food that does not contain the enzyme solution, specifically, 15% or more, 20% or more, 25% or more, or 30% or more, but is not limited thereto. no. The fermented food is any one or more of the microorganism, its crushed product and its culture; Alternatively, the glutamine content may be reduced by 85% or more compared to the fermented food that does not contain the enzyme solution, and specifically, may be reduced by 85% or more, 90% or more, or 95% or more, but is not limited thereto.

The fermented foods are meju, Korean soy sauce, brewed soy sauce, mixed soy sauce, Korean miso, miso, seasoned miso, red pepper paste, seasoned red pepper paste, chunjang, cheonggukjang and a mixture containing one or more selected from the group consisting of; Food seasoning material; And it may be one or more selected from the group consisting of food enzymes, but is not limited thereto.

At least one of the microorganisms of the Bacillus genus cultured in the medium containing the composition for culturing the microorganisms of the Bacillus, its debris and its culture is very useful as a food, food additive, food seasoning material, food enzyme preparation, feed additive or pharmaceutical Can be used. Specifically, any one or more of the microorganisms of Bacillus genus, Bacillus , crushed products and cultures cultured in the medium containing the composition for culturing microorganisms of the genus Bacillus can be used as a starter strain. Refers to an agent or composition containing microorganisms involved in fermentation for the manufacture of food and pharmaceutical products. It is used to provide microorganisms that can grow as a dominant species by growing in the fermented product by adding the product at the time of manufacture. When manufacturing the jangjang using the starter, when the product is manufactured by the microorganisms contained in the strain, the quality is constantly controlled, or the odor is not generated or reduced from the jangjang for a specific purpose. It can achieve the purpose of enhancing the taste or soft taste.

According to an embodiment of the present invention, fermented foods can be prepared using microorganisms of Bacillus genus cultured in a medium containing the composition for culturing microorganisms of the genus Bacillus , their lysate and the culture as a starter strain, Or at the time of preparing the fermented food, one or more enzyme liquids selected from the group consisting of gamma glutamyl transferase, gamma glutamylpeptidase and glutamines separated from any one or more of the microorganisms, crushed products, and cultures thereof are used as food seasoning materials It is possible to prepare fermented foods by preparing them as food enzymes and adding them, and the prepared fermented foods have enhanced enzyme activity, specifically, the enzyme activity of gamma-glutamyl transferase, thereby improving the flavor and organoleptic properties of the fermented foods. It was confirmed (Examples 3 and 4). Food seasoning material or food enzyme preparation according to the present invention may be prepared as a solution, powder, suspension, dispersion, emulsion, oily dispersion, paste or granule, but is not limited thereto.

In another aspect of the present invention, gamma-glutamyltransferase (gamma-glutamyltransferase), comprising the step of culturing microorganisms in Bacillus ( Bacillus ) in a medium containing the composition, gamma-glutamylpeptidase (gamma-glutamylpeptidase) ) And glutaminase (glutaminase) provides an enzyme preparation method comprising at least one selected from the group consisting of. The composition, the microorganism of the genus Bacillus, and the description of the culture thereof are as described above.

The enzyme preparation is not limited to its formulation, content, manufacturing method, etc., if it is an enzyme preparation comprising at least one selected from the group consisting of gamma-glutamyl transferase, gamma-glutamylpeptidase and glutamines.

The enzyme preparation may be an enzyme preparation including gamma-glutamyltransferase, but is not limited thereto.

The production method comprises gamma-glutamyltransferase, gamma-glutamylpeptidase and glutaminase from any one or more of the cultured microorganisms, their lysate and its culture. It may further include the step of separating at least one enzyme solution selected from the group consisting of.

Hereinafter, it will be described in more detail through production examples and examples according to the present invention. However, the following preparation examples and examples are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[ Manufacturing example  One] Bacillus  For cultivating genus microorganisms Medium composition  Produce

Sugar 2%, Soypeptone 5%, Na ₂ HPO ₄ 1.75%, KH ₂ PO ₄ 0.8%. The composition of the optimized medium was prepared to be ₂ mM MgSO ₄ and 0.1 mM CaCl ₂ .

[ Manufacturing example  2] Optimization medium  Manufacturing and culture

To confirm the effect of enhancing the gamma-glutamyl transferase activity according to the composition of the medium composition, the medium composition of Preparation Example 1 was prepared and sterilized at 121 ° C for 15 minutes. Then, the pre-cultured Bacillus amyloliquefaciens SMB-469 ( Bacillus amyloliquefaciens SMB-469; accession number KCTC12822BP) culture medium was inoculated to 1% and cultured at 37 ° C for 72 hours. The control group is M9 medium (Glucose 0.4%, NH ₄ Cl 0.1%, Na ₂ HPO ₄₎ which is the minimum medium for cultivating microorganisms in Bacillus. 1.28%, KH ₂ PO ₄ 0.3%, NaCl 0.05%, MgSO ₄ 2mM, CaCl ₂ 0.1mM).

[ Example  One] Medium composition  Main Nutrition Center ( Carbon source , Nitrogen source)

[ Example  1-1] Medium composition  Enzyme activity analysis method

Bacillus genus culture medium was inoculated with a strain of Bacillus genus at about 10 ⁶ CFU / ml, incubated at 37 ° C. for 72 hours, and then the supernatant was taken to obtain a gamma-glutamyltransferase analysis kit (Biovision, cat. K-784-100) was used to measure the activity. Gamma-glutamyl-p-nitroanilide (γ-glutamyl-p-nitroanilide), which is used as a substrate, is decomposed by enzymatic action to produce p-nitroaniline and measure the degree of yellow development For the analysis, it was analyzed at 418 nm using a spectrophotometer.

[ Example  1-2] Selection of nitrogen source

M9 medium prepared by modifying the composition of M9 medium (Glucose 0.4%, NH ₄ Cl 0.1%, Na ₂ HPO ₄ 1.28%, KH ₂ PO ₄ 0.3%, NaCl 0.05%, MgSO ₄ 2mM, CaCl ₂ 0.1mM) (Glucose 0.4%, NH ₄ Cl 0.4%, Na ₂ HPO ₄ 1.28%, KH ₂ PO ₄ 0.3%, NaCl 0.05%, MgSO ₄ 2mM, CaCl ₂ 0.1mM) nitrogen source 0.4% NH ₄ Cl (Ammonium chloride) Was replaced with other nitrogen sources in FIG. 1 to prepare a medium, and the gamma-glutamyl transferase activity was compared by the method of Example 1-1, and the results are shown in FIG. 1.

As shown in FIG. 1, when soypeptone was used as a nitrogen source, the enzyme activity was the highest, and it was confirmed that the enzyme activity was improved by about 39 times or more compared to M9 medium containing NH ₄ Cl as a nitrogen source.

In addition, gamma-glutamyl transferase activity in the medium prepared by varying the content of the selected nitrogen source, soybean peptone, was measured in the same manner as above, and the results are shown in FIG. 2.

As shown in Figure 2, when the soybean peptone content is 5.5% by weight or 6.5% by weight, gamma-glutamyl transferase activity was confirmed to the maximum, and when the soybean peptone content is 2.5 to 6.5% by weight, gamma-glutamyltransfer It was confirmed that the raise activity was greatly enhanced.

[ Example  1-3] Carbon source  selection

M9-2 medium prepared by modifying the composition of M9 medium (Glucose 0.4%, NH ₄ Cl 0.1%, Na ₂ HPO ₄ 1.28%, KH ₂ PO ₄ 0.3%, NaCl 0.05%, MgSO ₄ 2mM, CaCl ₂ 0.1mM) (Glucose 0.4%, Soypeptone 0.4%, Na ₂ HPO ₄ 1.28%, KH ₂ PO ₄ 0.3%, NaCl 0.05%, MgSO ₄ 2mM, CaCl ₂ 0.1mM). Each was replaced to prepare a medium, and the gamma-glutamyl transferase activity was compared by the method of Example 1-1, and the results are shown in FIG. 3.

As shown in FIG. 3, when sugar (Sucrose) was used as a carbon source, the enzyme activity was highest, and it was confirmed that the enzyme activity was improved by about 3.3 times or more compared to M9 medium containing glucose as a carbon source.

In addition, gamma-glutamyl transferase activity in the medium prepared by varying the content of the selected carbon source sugar was measured in the same manner as above, and the results are shown in FIG. 4.

As shown in Figure 4, when the sugar content is 2% by weight, gamma-glutamyl transferase activity was confirmed to be the maximum, when the sugar content is 1 to 3% by weight, gamma-glutamyltransferase activity is very enhanced Was confirmed.

[ Example  2] Medium composition  Optimization of raw material content

[ Example  2-1] Selection of main factors

An experiment was conducted to select factors that showed a major effect on enzyme activity. The content of sugar (Sucrose), a nitrogen source, soypeptone (soypeptone) and yeast extract (yeast extract) selected from Example 1, and inorganic contents of Na ₂ HPO ₄ , KH ₂ PO ₄ , NaCl, MgSO ₄ and CaCl ₂ As a factor, a partial factor experiment was performed.

Specifically, after setting the content of each raw material as shown in Table 1 below, using the MiniTab 16 (MinTab Inc., State College, Pa, USA), the conditions including 8 individuals, 2 levels, and center point through the partial factor test method were 17. The experimental groups of the dogs were designed (% in Table 1 below refers to the weight percent based on the total weight of the medium). The medium was prepared according to the raw material content of each experimental group, and the process of culturing microorganisms and analyzing gamma-glutamyltransferase enzyme activity in the same manner as in Example 1-1 was repeated three times.

factor factor Level 1 Level 2 Nitrogen source Soypeptone (%) 0.4 3.5 Yeast extract (%) 0.4 3.5 Carbon source Sugar (%) 0.1 1.0 Mineral Na ₂ HPO ₄ (%) 0.1 1.5 KH ₂ PO ₄ (%) 0.1 1.5 NaCl (%) 0.05 5.0 MgSO ₄ (mM) 2.0 20.0 CaCl ₂ (mM) 0.1 5.0

A correlation between the raw material content and the gamma-glutamyltransferase enzyme activity of the experimental group designed using Minitab was derived, and as shown in FIG. 5, sugar (Sucrose), soybean peptone (Soypeptone), and sodium diphosphate (Na) in minerals ₂ HPO ₄ ) was found to have a major effect on enzyme activity.

[ Example  2-2] Optimization of medium composition

In order to set the medium composition for maximizing gamma-glutamyl transferase activity, a response surface methodology was used.

Specifically, the experiment was designed by the Central Composite Design among the reaction surface methods using MiniTab 16 (Minitab Inc., State College, Pa, USA).

At this time, the experiment was designed by setting sugar (Sucrose), soypeptone, and sodium dibasic phosphate (Na ₂ HPO ₄ ) selected in the sub-factor experiment of Example 2-1 as factors, KH ₂ PO ₄ was 0.8%, MgSO ₄ was 2 mM, and CaCl ₂ was 0.1 mM, respectively. After setting the content of the modulator as shown in Table 2, 31 experimental spheres were designed including 7 center points (% in Table 2 below refers to the weight percent based on the total weight of the medium). . The medium was prepared according to the raw material content of each experimental group, and the process of culturing microorganisms and analyzing gamma-glutamyl transferase enzyme activity in the same manner as in Example 1-1 was repeated three times.

division Raw material Level 1 Level 2 Regulator Sugar (%) 0.75 2.00 Soypeptone (%) 2.00 5.00 Na ₂ HPO ₄ (%) 1.00 2.50 Yeast extract (%) 1.00 3.00 Fixed factor KH ₂ PO ₄ (%) 0.80 MgSO ₄ (mM) 2.00 CaCl ₂ (mM) 0.10 NaCl (%) 0

Optimized media composition was derived through Minitab using the measurement values of the gamma-glutamyl transferase enzyme activity of the experimental group. As shown in Figure 6, it was found that the content of sugar (Sucrose) and soypeptone (Soypeptone) showed an effect on gamma-glutamyl transferase activity, sugar (Sucrose) 2%, soybean peptone (Soypeptone) 5% At this time, gamma-glutamyltransferase enzyme activity was best predicted.

[ Example  2-3] Gamma by medium- Glutamyl transferase  Comparison of activities

In order to confirm the effect of enhancing the enzyme activity of the medium composition, the enzyme activity of gamma-glutamyl transferase, an enzyme present in the culture of Preparation Example 2, was analyzed in the same manner as in Example 1-1. Same as 7.

As shown in FIG. 7, the culture medium of Preparation Example 2 obtained by culturing Bacillus amyloliquiparations in the medium composition of Preparation Example 1 showed gamma-glutamyl transferase enzyme activity of about 10,000 mU / mL or more, as a control. It was confirmed that the activity was enhanced by about 50 times or more than the medium M9 medium.

[ Example  3] Confirming the effect of improving flavor in soy sauce

Bacillus amyloliquefaciens SMB-469 (Accession No. KCTC12822BP) containing the medium composition of Preparation Example 1 to confirm that the flavor is improved when applied to the preparation of fermented soybean using the medium composition Gamma-glutamyl transferase enzyme solution obtained by culturing in the medium was added to prepare soy sauce. In addition, amino acid analysis and sensory evaluation were conducted to analyze the content of the flavor components. As for amino acid analysis, water-soluble free amino acids were analyzed using an amino acid analyzer (Amino acid analyzer biochrom 30+ from Biochrom). The results are shown in FIG. 8.

As shown in FIG. 8, it was confirmed that the content of glutamic acid, the main component of umami, in the soy sauce prepared by adding the gamma-glutamyl transferase enzyme solution was increased by about 30% or more compared to the soy sauce without the enzyme solution. . On the other hand, it was confirmed that glutamine, a non-microbial substance, decreased by 85% or more. Through this, it was found that the medium composition of Preparation Example 1 has an excellent effect of improving the flavor of fermented foods by enhancing gamma-glutamyl transferase activity when culturing microorganisms in Bacillus.

[ Example  4] Sensory evaluation of taste characteristics of soy sauce

Sensory evaluation was performed to compare the taste characteristics of the soy sauce prepared using the medium composition, and specifically, a characteristic strength evaluation of a 15-point scale was performed and the results are shown in FIG. 9.

As shown in FIG. 9, the gamma-glutamyltransferase enzyme solution obtained by culturing Bacillus amyloliquefaciens SMB-469 ( Bacillus amyloliquefaciens SMB-469; accession number KCTC12822BP) in a medium containing the medium composition of Preparation Example 1 In the soy sauce prepared by adding, it was confirmed that the strength of umami (Umami), deep taste (Kokumi deep taste, Kokumi) and salty taste was higher than that of the non-added soy sauce. Through this, it was found that the medium composition of Preparation Example 1 has an excellent effect of enhancing the sensory properties of fermented foods by enhancing gamma-glutamyl transferase activity when culturing microorganisms in Bacillus.

Through the above Examples 1 to 4, a medium composition containing sugar as a carbon source and soypeptone as a nitrogen source is a microorganism of the genus Bacillus , which is a major microorganism of soybean fermentation, specifically Bacillus amyloliquequiptions. It was found that ( Bacillus amyloliquefaciens ) enhances the gamma-glutamyltransferase activity, thereby improving the flavor characteristics of fermented foods.

Since the specific parts of the present invention have been described in detail above, it is obvious that for those skilled in the art, this specific technique is only a preferred embodiment, whereby the scope of the present invention is not limited. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (18)

As a composition for culturing microorganisms of the genus Bacillus containing a carbon source and a nitrogen source,
The carbon source is sugar (sucrose), the nitrogen source is soypeptone (soypeptone),
The composition comprises Na ₂ HPO ₄ ,
The composition comprises 2.5 to 6.5% by weight of soybean peptone based on the total weight of the medium containing the composition,
The microorganism of the genus Bacillus cultured in the medium containing the composition is at least one selected from the group consisting of gamma-glutamyltransferase, gamma-glutamylpeptidase, and glutaminase. Has enzyme activity,
The microorganism of the genus Bacillus is Bacillus amyloliquefaciens , a composition for culturing a microorganism of the genus Bacillus. delete According to claim 1,
The composition comprises 1 to 5% by weight of sugar based on the total weight of the medium containing the composition, Bacillus genus microbial culture composition. delete According to claim 1,
The composition is to further include one or more minerals selected from the group consisting of KH ₂ PO ₄ , MgSO ₄ and CaCl ₂ , Bacillus genus microbial culture composition. According to claim 1,
The microorganism culture of the genus Bacillus will be cultured for 20 to 170 hours, the composition for culturing the microorganism of the genus Bacillus. According to claim 1,
The microbial culture of the genus Bacillus is cultured at 0 to 45 ° C, a composition for culturing the microorganism of the genus Bacillus. According to claim 1,
The enzyme activity is gamma-glutamyl transferase activity of 8000 mU / mL or more, Bacillus genus microbial culture composition. Fermentation method comprising the step of culturing microorganisms of the genus Bacillus ( Bacillus ) in a medium containing the composition of any one of claims 1, 3 and 5 to 8. The method of claim 9,
The fermentation method is to further include the step of preparing the medium, fermentation method. The method of claim 9,
The medium is a sterilized medium, fermentation method. Claim 1, claim 3 and claim 5 to claim 8, the microorganism of the genus Bacillus ( Bacillus ) cultured in a medium containing the composition of any one of claims, crushed material and any one or more of the culture; or
From the group consisting of gamma-glutamyltransferase, gamma-glutamylpeptidase and glutaminase isolated from any one or more of the microorganism, its lysate and its culture. A fermented food product comprising one or more selected enzyme solutions. The fermented food according to claim 12, wherein the enzyme solution is a gamma-glutamyl transferase enzyme solution. The method of claim 12,
The fermented food is any one or more of the microorganism, its crushed product and its culture; Alternatively, the fermented food further comprises 15% or more of glutamic acid compared to the fermented food that does not contain the enzyme solution. The method of claim 12,
The fermented foods are meju, Korean soy sauce, brewed soy sauce, mixed soy sauce, Korean miso, miso, seasoned miso, red pepper paste, seasoned red pepper paste, chunjang, cheonggukjang and a mixture containing one or more selected from the group consisting of;
Food seasoning material; And
Fermented food, one or more selected from the group consisting of food enzyme preparations. Gamma-glutamyltransferase comprising culturing a microorganism of the genus Bacillus in a medium containing the composition of any one of claims 1, 3 and 5 to 8, Method for producing an enzyme preparation comprising at least one selected from the group consisting of gamma-glutamylpeptidase and glutaminase. 17. The method of claim 16, wherein the enzyme preparation is an enzyme preparation comprising gamma-glutamyltransferase. The method of claim 16,
The production method comprises gamma-glutamyltransferase, gamma-glutamylpeptidase and glutaminase from any one or more of the cultured microorganisms, their lysate and its culture. The method further comprises the step of separating at least one enzyme solution selected from the group consisting of.

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