KR20160097423A - Salt-tolerant Yeast (Zygosaccharomyces mellis TK-01) Isolated from Soy Paste and Characterization Thereof - Google Patents

Abstract

The present invention relates to a Zygosaccharomyces mellis TK-01 strain, which is salt-tolerant yeast isolated from soybean paste, and an applications thereof and, more specifically, to the evaluation of glutaminase and glutamyl transferase activities using a culture of the Zygosaccharomyces mellis TK-01 strain, and to the hydrolysis of plant protein using the Zygosaccharomyces mellis TK-01 strain. The Zygosaccharomyces mellis TK-01 strain is obtained by the following steps: isolating salt-tolerant yeast from traditional soybean paste containing high concentration table salt; and isolating the Zygosaccharomyces mellis TK-01 strain from the yeast and identifying the same.

Description

{Salt-tolerant Yeast (Zygosaccharomyces mellis TK-01) Isolated from Soybean Paste and Characterization Thereof} The present invention relates to a salt-tolerant Yeast (Zygosaccharomyces mellis TK-01)

The present invention relates to the isolation of the salt tolerant yeast Zygosaccharomyces mellis TK-01 strain in a salt-rich traditional soybean paste. The present invention also relates to the production of a hydrolyzate of vegetable protein using the yeast strain Zygosaccharomyces mellis TK-01 or a culture thereof.

The taste of food is composed of 5 kinds of sweet taste, salty taste, sour taste, bitter taste plus flavor based on four flavors. The richness is a major factor that determines the harmony of the taste of food. It is known that monosodium glutamate (MSG), nucleic acid type sodium inosinate (IMP) and sodium guanylate (GMP) give a typical flavor. MSG is a common substance used to improve the taste of food. It is prepared by extracting from early kelp. However, since mass production of wheat gluten was started in the 1920s by acid hydrolysis, it became possible to produce glutamic acid by microbial fermentation in the 1950s, and sodium glutamate was added to make glutamic acid by fermentation method to improve water solubility.

In order to use Hydrolyzed Vegetable Protein (HVP) as a seasoning ingredient, Hydrolyzed Protein (aHVP) is added to glycerin and hydrochloric acid (chlorine (3 - chloro - 1, 2 - propanediol, MCPD) and 1, 3 - dichloro - 2 - propanal (DCP) which are generated by the reaction of chlorohydrin . In addition, as the consumer's preference for natural has increased, the use of enzyme hydrolysis protein (eHVP) using vegetable proteins such as soy protein, corn protein and wheat gluten has been increasing. However, the production of eHVP using a commercially available proteolytic enzyme has a limitation in that the production of bitter tastes due to incomplete degradation of the protein and the economical efficiency are inferior.

In addition, the commercially available protease has a low activity of the endo / exo-protease, resulting in a low content of glutamic acid, and the effect of short-medium peptides on flavor characteristics (eg, bitter peptides) quality degradation, microorganisms having proteolytic and amino group conversion ability are isolated and identified. Thus, excellent strains with typical hyper-protease and glutaminase activity have been developed and used for protein degradation. However, most of the strains used Due to the fungi of Aspergillus genus, it is difficult to store the strain and to develop efficient fermentation production process (eg, Sporulation & Degeneration).

Hydrolyzate (HVP) from plant protein is the main flavor source of traditional fermented foods such as soy sauce and doenjang, which is based on free amino acids and short / medium peptide hydrolysates of proteins. Various methods have been tried to produce such amino acid and peptide solutions through fermentation. However, there is a problem in that the quality and yield of the hydrolyzate deteriorate due to frequent bacterial growth in the process of producing protein hydrolyzate from plant protein raw materials. To solve this problem, it is common to add a large amount of a bacterium such as a salt or an alcohol, and these additives inhibit the action of the enzyme, thereby increasing the decomposition time.

Since the enzymes produced by salt-tolerant or hawthornic microorganisms are relatively salt-tolerant, these salt-tolerant enzymes have the advantage of reducing contamination of germs in the enzymatic production process and the enzymatic degradation process. However, only few bacterial strains, including Zygosacchromyces rouxii and some Candida species, have been known as salt tolerant microorganisms.

Doenjang is a traditional fermented food that is unique to Korea and produces unique flavor and aroma in the process of decomposing soybean protein into peptides and amino acids by enzymes produced by fungi, bacteria and yeast at a high salt concentration. Has been used as a vegetable seasoning. In particular, yeasts participating in the fermentation of doenjang are known to be involved in the formation of deep flavor of miso in the salt tolerant environment, thereby determining the quality of doenjang.

Yeast is a safe microorganism that is generally accepted as safe (GRAS, generally recognized as safe). It is not only used for baking, brewing, and other foods, but also contains proteins, nucleic acids, vitamins and minerals . Yeast extracts produced from yeast are relatively inexpensive compared with other materials having the same strength and are widely used as natural flavor materials. In addition, the yeast extract contains not only amino acids but also nucleic acid and peptide components, which have complexity and rich taste. Therefore, it already has a function that can not be seen in other seasonings such as masking odor.

Yeast has a variety of proteolytic enzymes (eg, protease A, protease B, amino-peptidase and carboxy-peptidase) meal, etc.), which can contribute to the development of natural seasoning materials by forming new peptides profiles. Also, since the difference in the flavor and composition of the product is very different according to the production method of the yeast extract, the variation in the price is also very diverse, and the yeast extract having excellent flavor can be used as a high value added seasoning material.

When producing natural seasonings containing miso, soy sauce and other protein hydrolysates, it is important to increase the amount of glutamic acid among the various flavoring substances to produce a stronger umami. Glutamic acid is produced in two ways: first, when glutamine is liberated from a protein by a protease, then the liberated glutamine is hydrolyzed by glutaminase to produce glutamic acid. Glutaminase is an enzyme widely distributed in the biological system and acts on L-glutamine to hydrolyze an amide group to form L-glutamic acid and ammonia. If the free ratio of glutamic acid to the total glutamic acid content during protein hydrolysis is not very high, this is considered to be due to insufficient glutaminase activity. Therefore, an attempt has been made to improve a strain having high activity of protease and glutaminase by cell fusion of a strain having a high protease activity and a strain having a high glutaminase activity (Ushijima S. et al. Biochem. 1987).

Glutamyltransferase reacts with γ-Glutamyl compound to form an intermediate, which is known to have reaction selectivity depending on pH. When this intermediate reacts with water, hydrolysis occurs and glutamic acid is produced. When it reacts with other amino acids or peptides, transpeptidation occurs and a new γ-glutamyl compound is produced (FIG. 1). γ-Glutamyl compounds are generally known to have high solubility, resistance to peptidase, and good taste. For example, theanine (γ-L-glutamylethylamide) is well known as a umami key-compound for determining the grade of Japanese green-tea, and γ-glutamylation of bitter amino acid not only effectively reduces the bitter taste, lemons-like sour taste.

Gamma-Glutamyltransferase (GGT) is a hetero-dimeric enzyme and is involved in the metabolism of glutathione. As far as it is known, it is derived from animals and microorganisms. The electrons are calcium-dependent enzymes and are distributed in the organs, skin, blood and the like of animals (K. Ikura et al., Biochemistry 1988, MAPhilips et al., Proc. Natl. Acad. Sci. ), And the latter has been found to be calcium independent from bacteria of the genus Streptoverticilium (EP 0 481 504 A1). However, since microorganism-derived glutamyltransferase is produced by purifying the microorganism from a culture of fungi, there is a problem such as a feed amount and efficiency, and production of glutamyltransferase by genetic engineering methods is attempted. Therefore, proteins and genes related to glutamyltransferase have been reported, and a method of expressing these proteins in E. coli by expressing them has been reported. However, there is a problem that the amount of expression is very small in such secretion expression.

In the present invention, a strain resistant to inorganic salts isolated from soybean paste was identified, and the selected yeast strain was designated as Zygosaccharomyces mellis TK-01 strain, and salt resistance, glue Tamarinase, glutamyltransferase, and protease activity. Thus, the present invention has been completed. The object of the present invention is to provide a salt tolerant yeast Zygosaccharomyces mellis TK-01 strain isolated from miso. The present invention also relates to a method for hydrolyzing not only the proteins of the yeast itself but also the added non-yeast proteins (for example, wheat gluten, bran, soya bean meal, etc.) using the autoclaving enzymes of the screening strains and the cultures thereof.

In order to achieve the above object,

The present invention provides a Zygosaccharomyces mellis TK-01 strain (Accession No .: KCCM-11605P) having salt tolerance, glutaminase activity and glutamyltransferase activity.

Also provided is a culture of Zygosaccharomyces mellis TK-01 strain (Accession No .: KCCM11605P) having salt tolerance, glutaminase activity and glutamyltransferase activity.

In order to achieve the object, a method for hydrolyzing vegetable proteins using an active ingredient of Zygosaccharomyces mellis TK-01 strain is provided.

Hereinafter, the present invention will be described in detail as follows

The present invention relates to the use of Zygosaccharomyces sp.) strain. Specifically, Zygosaccharomyces mellis TK-01 strain isolated from five commercially available soybean soybean isolates, preferably Zygosaccharomyces mellis TK-01, isolated from commercially available soybean is provided.

The Zygosaccharomyces sp. Strain of the present invention was named Zygosaccharomyces mellis TK-01 and the strain Zygosaccharomyces mellis TK-01 according to the present invention was named Zygosaccharomyces mellis TK- 01 (Zygosaccharomyces mellis TK-01) was deposited with the Korea Culture Center of Microorganisms, an international depository organization, on November 20, 2014, and received the accession number KCCM11605P.

The present invention also provides a method for isolating a strain of Zygosaccharomyces sp. From miso. The inventors of the present invention isolated Zygosaccharomyces melis TK-01 from doenjang and cultured the strain to obtain a culture.

In addition, it provides a function of decomposing vegetable proteins due to salt resistance, glutaminase activity, and glutamyltransferase activity of Zygosaccharomyces mellis TK-01 strain of the present invention. The present invention provides plant protein hydrolysis at the same time as the production of Zygosaccharomyces mellis TK-01 or an extract of this strain.

Figure 1 shows the reaction mechanism of gamma glutamyl transpeptidase.
Figure 2 shows the colonization of yeast isolated from miso.
3 shows a phase contrast microscope photograph (x400 times) of a yeast strain.
4 is a photograph showing evaluation of the salt tolerance of the yeast strain.
Figure 5 shows the identification of yeast strains by 18s RNA analysis
FIG. 6 is a graph showing the distribution of Zygosaccharomyces mellis Lt; RTI ID = 0.0 > TK-01. ≪ / RTI >
7 shows a calibration curve of gamma glutamyl transpeptidase.
Figure 8 shows the 18S rRNA nucleotide sequence.

Hereinafter, the present invention will be described in more detail by way of examples

[Example 1] Screening of salt tolerant yeast

Considering that the intended product is a food product, the present invention has been selected from microorganisms conventionally used in food or brewing fields. The yeasts used were yeast strains of six commercially available yeast strains (Saf-Instant Red, Saf-Instant Gold, Bakels Instant Active Drye Yeast, Vega Instant Yeast, Mauripan Instant Dry Yeast and E-mart Instant Dry Yeast) Two strains (Saccharomyces cerevisiae KCTC 7951, Zygosaccharomyces rouxii KCCM 50054) and three yeast isolates from commercial doenjang (SJ 07, SJ 09, SJ 21) were used (Table 1 and 2).

The strain used was inoculated into YMPD (yeast extract 3 g / L, malt extract 3 g / L, peptone 5 g / L, dextrose 10 g / L, pH 5.5) broth and incubated at 30 ° C. for 24 hours. Streaking was performed to obtain a single colony. The isolated strain was inoculated on YMPD broth and cultured at 30 ° C. for 48 hours at 180 rpm. The strain was then frozen in a deep freezer at -80 ° C. with 20% glycerin in the culture medium. .

In order to measure the salt tolerance of the isolated strain, solid medium was prepared by adding 0, 0.5, 1.0, 1.5, 2.0, 2.5 M potassium chloride (KCl) to the YMPD medium and thawing the frozen strain, YMPD agar plate at 30 ° C for 72 hours.

Specifically, as shown in FIG. 4, all of the 11 yeasts formed an elongation up to a concentration of 1.5 M KCl, but the number of colonies formed from 1.0 M KCl concentration drastically decreased in Saccharomyces cerevisiae KTCT 7915 and Bakels Instant Active Drye Yeast (Austria). At 2.0 M KCl concentration, only three isolates and Zygosaccharomyces rouxii KCCM 50054 formed colonies. In 2.5 M KCl, colonies of SJ 07 and Zygosaccharomyces rouxii KCCM 50054 were observed. As a result, the salt tolerance of newly isolated isolates of doenjang SJ 07 was confirmed.

In addition, the 18S rRNA sequencing of the salt tolerant strain SJ 07 revealed that it was 99% homologous to the Zygosaccharomyces cerevisiae strain (Fig. 5). SJ 07 was identified as Zygosaccharomyces melis TK -01 (Zygosaccharomyce mellis TK-01) and used for subsequent experiments.

From the above results, it was confirmed that Zygosaccharomyces mellis TK-01 according to the present invention TK-01) was deposited with the Korean Culture Center of Microorganisms, an international depository organization, on November 20, 2014, and received the deposit number KCCM11605P.

1. Morphological characteristics

Morphological characteristics of Zygosaccharomyces sp. It is similar to the general characteristics of yeast. In other words, when a white cream colony was formed on a YMPD agar plate and observed under a microscope, it showed a subglobe shape with no pseudohyphae and an ovoid shape.

2. Cultural characteristics

The isolated microorganisms can grow in the range of 20 ℃ to 30 ℃ and can grow in the range of pH 3.5 - pH 6.5, but the optimal pH range is pH 4.5 - pH 6.0.

3. Physiological characteristics

Substrate utilization characteristics such as sugars of microorganism Zygosaccharomyces mellis are as follows.

Sequencing comparative data showing 99% homology with 18S rRNA sequencing comparison is also shown in FIG.

[Example 2] Characterization of Zygosaccharomyces mellis TK-01

The morphological characteristics of Zygosaccharomyces mellis TK-01 of the present invention include Zygosaccharomyces sp. It is similar to the general characteristics of yeast. In other words, it forms a white colony on a YMPD agar plate, and when observed with a microscope it does not have a pseudohyphae and shows a subglobe shape close to an ovoid.

The isolated microorganisms can be grown in the range of 20 ℃ to 30 ℃ and growth is possible in the range of pH 3.5 - pH 6.5, but the optimal pH range is judged as pH 4.5 - pH 6.0.

[Example 3] Salt resistance evaluation of Zygosaccharomyces mellis TK-01

0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 M potassium chloride (KCl) were added to the YMPD medium to determine the relationship between the salt concentration and the cell mass of the strain Zygosaccharomyces mellis TK-01 of the present invention. And incubated at 30 ° C for 24 hours. The cells were centrifuged at 8,000 x g for 10 minutes and washed twice with distilled water. The recovered cells were dried at 80 ° C for 24 hours to measure dry cell weight (DCW).

As a result, Zygosaccharomyces mellis TK-01 isolated from doenjang decreased to 92, 84, 76, 68, 65, 64% when the salt concentration was increased by 0.5 M when 100% , And growth was possible in the presence of 3 M (22%) KCl. (Fig. 6).

[Example 4] Measurement of protease activity of Zygosaccharomyces mellis TK-01 strain

In the present invention, the enzyme activity of Zygosaccharomyces mellis TK-01 was measured on a YM agar plate supplemented with 2.5 M KCl. The single colony was treated with yeast extract 7 g / L, malt extract 3 g / L, amiga plus 18 15 g / L of potassium phosphate monobasic, 223.62 g / L of potassium chloride (KCl) and 30 g / L of glucose, and incubated at 30 ° C for 48 hours at 180 rpm. The culture broth was frozen at -80 ° C in a deep freezer and added with 20 g of glycerin. The yeast extract (7 g / L, malt extract 3 g / L, amiga plus 18 15 g / L, ammonium sulfate (NH4) L, potassium phosphate monobasic 1 g / L, potassium chloride (KCl) 223.62 g / L and glucose 30 g / L, and incubated at 30 ° C for 48 hours at 180 rpm.

To evaluate the proteolytic activity, the supernatant was centrifuged, and Azo-casein (Megazyme ACS 2/99) was added to the substrate solution (100 mM sodium acetate buffer, pH 4.5 or sodium phospahte buffer, pH 6.0) Respectively. 1 mL of the diluted enzyme solution and 1 mL of the substrate were mixed and reacted at 40 ° C for 10 minutes. Then, 6 mL of 5% trichloroacetic acid (TCA) was added and left at room temperature for 5 minutes to terminate the reaction. After the reaction was completed, the supernatant was centrifuged at 3000 rpm for 10 minutes, and the absorbance was measured at a wavelength of 440 nm. The protease activity was calculated according to the following equation (1).

[Equation 1]

Protease (mUnit / mL) = 146 x Abs. (440 nm) - 4

R = 0.99 (Linear absorbance range = 0.1 to 1.0)

* Fungal protease ( Aspergillus niger from Sigma Chemical Co.)

The results are shown in Table 4.

As shown in Table 4, protease activity was higher than that of acidic condition. Therefore, it is considered desirable to perform self-extinguishing after adjusting the pH to neutral for effective proteolysis.

[Example 5] Measurement of glutaminase activity of Zygosaccharomyces mellis TK-01

 To measure the glutaminase enzyme activity of Zygosaccharomyces mellis TK-01 of the present invention, 0.1 g / L of glutamine was added to the yeast culture prepared by the method of Example 4, and after 24 hours of reaction, the supernatant was separated and analyzed using YSI Biochemistry Analyzer And the enzyme activity was evaluated by measuring the produced glutamic acid. The results are shown in Table 5.

It was confirmed that glutamic acid was produced at a concentration of 0.31 g / L after 24 hours of glutamine addition. The reason why glutamic acid was produced more than the amount of glutamine added was judged to be the conversion of glutamine derived from eHVP used as a nitrogen source in the medium, Lt; RTI ID = 0.0 > glutaminase < / RTI > activity.

[Example 6] Measurement of glutamyltransferase activity of Zygosaccharomyces mellis TK-01

Glutamyl transpeptidase (GGT; EC 2.3.2.2) activity of Zygosaccharomyces mellis TK-01 of the present invention was measured by centrifugation at 10,000 xg for 20 minutes in the same manner as in Example 4, And then measured by the following method using an enzyme solution. 1 mmol / L γ-Glutamyl-p-nitroanilide, 20 mmol / L glycylglycine, enzyme solution (supernatant obtained by centrifuging the microbial culture) and buffer solution were mixed and reacted at 37 ° C. for 30 minutes. The reaction was terminated by the addition of 0.1 mol / L HCl and the absorbance at 410 nm was measured. Using p-nitroaniline as a standard solution, a standard curve of γ-Glutamyltranspeptidase was used and the titer was calculated using the following equation (2) (FIG. 7).

At the same time, the glutaminase activity was measured as in Example 5.

[Equation 2]

Glutamyltransferase activity (GTU / Ml) = (As-Ab) x F x D

As: Absorbance of enzyme test solution

Ab: Absorbance of Blank

F: p-nitroaniline concentration (F = 0.011 μmol / mL) when the difference in absorbance obtained from the standard curve was 1.000

D: Dilution of sample

* 1 Glutamyltrasferase unit (GTU): Amount of enzyme liberating 1 μmol of p-nitroaniline for 1 minute

The results are shown in Table 6.

As a result of Table 6, at pH 4.5, there was no glutamyltransferase activity but glutaminase activity was shown to produce glutamic acid. At pH 8.0, glutamic acid was not produced and there was no glutaminase activity but glutamyltransferase Activity. These results are consistent with previous studies showing that one enzyme exhibits glutaminase or glutamyltransferase activity depending on the pH condition. This is because Zygosaccharomyces mellis TK-01, an isolate strain, exhibits glutaminase activity under acidic conditions And it was confirmed that it exhibited glutamyltransferase activity under alkaline conditions.

Example 7 Production of Plant Protein Hydrolyzate Using Enzyme Activity of Zygosaccharomyces mellis TK-01

In order to prepare the hydrolyzate of vegetable protein, Zygosaccharomyces mellis TK-01 strain of the present invention was thawed and frozen to obtain yeast extract 7 g / L, malt extract 3 g / L, amiga plus 18 15 g / L, ammonium sulfate NH4) 2SO4), potassium phosphate monobasic 1 g / L, potassium chloride (KCl) 223.62 g / L, and glucose 30 g / L were inoculated with 1% of the culture medium and incubated in a rotary shaking incubator at 180 rpm After incubation for a time, 1 g / L of wheat gluten powder (Shinsoon Industry Co., Ltd., Korea) was added. Prior to addition of the plant protein, the pH was adjusted to 6.0 using 1M KOH and autogenous digestion was induced at 50 < 0 > C and 180 rpm for 24 hours with shaking agitation. Samples were taken every 6 hours during the disassembly process, and the activity of glutaminase, glutamyltransferase and proteolytic enzyme was measured in the same manner as in Examples 4, 5 and 6. After completion of the reaction, the yeast extract was heated at 80 ° C for 30 minutes to terminate the reaction, and the purified yeast extract was obtained by removing the cells and solids through Celite bed filtration.

The results are shown in Table 7.

As shown in Table 7, as the hydrolysis decomposition time of the vegetable protein increased, the glutamic acid content tended to increase. Further, glutamyltransferase activity appeared and hydrolysis of the vegetable protein could produce γ-Glutamyl compound . These results suggest that Zygosaccharomyces mellis TK-01 may contribute to the development of natural glutamic acid by producing glutamic acid, which is the major component of the flavor, by decomposing the self-digesting protein as well as the added non-yeast protein.

Korea Microorganism Conservation Center (overseas) KCCM11605P 20141120

Claims (5)

Zygosaccharomyces mellis TK-01 strain (accession number: KCCM11605P), which is a salt tolerant yeast isolated from commercially available soybean paste, The method according to claim 1, wherein the salt tolerant yeast Zygosaccharomyces mellis TK-01 TK-01) strain has a glutaminase activity, a glutamyltransferase activity and a protease activity, and is characterized in that a strain of Zygosaccharomyces cerevisiae TK-01 Zygosaccharomyces mellis TK-01 (Zygosaccharomyces mellis TK-01) yeast strain, a hydrolyzate of vegetable protein, and a yeast extract using a yeast strain selected from the group consisting of a yeast strain, Culture method of TK-01 strain The method according to claim 3, wherein Zygosaccharomyces mellis TK-01 (Zygosaccharomyces mellis TK-01), which is characterized in that it produces a fermented natural fermented material from vegetable protein hydrolysates. The fermented yeast, Zygosaccharomyces melisis Culture method of TK-01 strain The method according to claim 3, wherein Zygosaccharomyces mellis TK-01 (Zygosaccharomyces mellis TK-01), which is characterized by the hydrolysis of the yeast protein and non-yeast protein using an autolytic enzyme, Culture method of TK-01 strain

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