KR920001204B1 - Process for making soy sauce using zygosaccharo myceces rouxii and torulopsis versatilis - Google Patents

Abstract

New hybridoma ST723-F31 (I) is prepd. by protoplast fusion of zygosaccharomyces rouxii and torulopsis versatilis in the presence of 40 % PEG 4000. The obtd. (I) is stabilized by several succeed culture at 30 deg.C in the complete medium contg. 2.0 % glucose, 0.2 % yeast extract, 0.2 % tryptone, 0.2 % (NH4)SO4, 0.1 % polypeptone, 0.1 % KH2PO4, 0.05 % MgSO47H2O, 0.05 % KCl and 0.001 % FeSO47H2O. The obtd. (I) is useful for the production of soysauce having a good flavor and taste.

Description

Novel hybridomas and their preparation, which produce the unique scent of soy sauce obtained from Zygosaccharomyces rouxii and Torulopsis versatilis, and methods of preparing soy sauce using the same

1 is a gas chromatogram of volatile components in a culture of hybridoma ST723-F31 (KCTC 8472 P) cultured in aroma production medium;

2 is a gas chromatogram of aroma components produced by hybridoma ST723-F31 (KCTC 8472 P) analyzed by GC-mass;

3 is the mass spectrum of 4-hydroxy-2- (or 5) -ethyl-5 (or 2) -methyl-3- (2H) -furanone;

4 is the mass spectrum of 4-ethylguaiacol;

5 is a gas chromatogram of volatile components in a culture of hybridoma ST723-F31 (KCTC 8472 P) cultured in amino acid soy sauce.

The present invention provides a novel hybridoma and a method for preparing the same, and a method for preparing soy sauce using the same, which produces a unique aroma of soy sauce obtained from Zygosaccharomyces rouxii and Torulopsis versatilis. It is about a method.

Korean traditional soy sauce is made by fermenting and ripening meju with brine. Microorganisms that exist in nature are involved in this manufacturing process, and the related yeasts are known as Zaigo Saccharomyces luxi and tolulopsis versatiles [Ref. Characteristics of Fragrances Generated by Microorganisms during Fermentation, Journal of Resource Problem Research, Yeungnam University, 5 (1), 91-96 (1986)].

Japanese fermented soy sauce is produced by using natural bacteria, by artificially using bacteria, and by fermentation with a bioreactor. First, the method using natural bacteria is a process in which Koji made from skim soybean and wheat is soaked in brine and fermented for 6 months to 1 year. At this time, fungi and bacteria of Bacillus in Aspergillus , Lactic acid bacteria, etc. multiply to break down proteins to produce amino acids and peptides, and also produces lactic acid and various acids. In particular, the yeast Zaigo Saccharomyces luxi and Tolulopsis spepis breeds to produce alcohol and esters to enhance the flavor of soy sauce.

Next, the method of artificially using bacteria is a method of artificially fermenting the soy sauce during the soaking process by adding artificial Zagosaccharomyces luxi and tolulopsis spp.

Lastly, the fermentation method using a bioreactor is fermented and matured in a bioreactor for soybean fermentation using Pediococcus halophilus, Zaygo Saccharomyces luxyi and Tolulopsis versatileis. As a production method, it is used to shorten the fermentation period of soy sauce [see K.OSAKI, Y.OKAMOTO, T.AKAO, S.MAGATA & H. TAKAMATSU: Fermentation of Soy Savce with Immobilized Whole Cells, J. of Food Science, 50m 1289-1292 (1985).

However, the conventional conventional soy sauce manufacturing method as described above is involved in the fermentation and maturation of various microorganisms in nature, so it is difficult to control the taste and flavor because the production of aromatic components or the balance of components is difficult, and also shows excellent taste and aroma. In order to take a long ripening period has a disadvantage. In addition, the main yeasts of Korean traditional soy sauce and Japanese brewed soy sauce fermentation, Zaigosaccharomyces luxi and tolulopsis versatiles, 4-hydroxy-2- (or 5) -ethyl-5 (or 2) ) -Methyl-3- (2H) -furanone (HEMF) and 4-ethylguaiacol (4-EG) are respectively produced and alcoholic fermentation is also determined to determine the flavor of soy sauce. It grows and is involved in the fermentation of liver, and when it reaches late maturation, Z. luxi is extinguished and T. versatiles proliferates and is involved in ripening.

Therefore, fermentation and maturation takes a lot of time, and two bacteria must be used, respectively, so that the management of bacteria is difficult and cumbersome.

In order to solve the above-mentioned problems, the present invention provides a novel hybridoma having characteristics of Z. luxi and T. versatiles, which are fermentation aging yeasts. According to the present invention can shorten the fermentation period and can solve the hassle of using two bacteria separately in the manufacturing process.

The novel hybridomas of the present invention can be prepared by the following method. That is, the amino acid demand mutant strain was mutated with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) (1.5 mg / ml) of Zaigosaccharomyces luxi WFS4 and tolulobis versatiles IAM 4993. Arginine demand SMn7 and leucine demand TMn23 were selected to produce soy fragrance of the mutant strains with aroma odor of cultures better or similar to that of wild strains.

Hybridomas ST723-F31 were stabilized by multiple passages of hybridomas obtained by protoplast fusion (using methods such as Fournier (1977)) in the presence of 40% PEG 4000. Screened. At this time, the composition of the used medium is shown in Table 1, the culture temperature and the treatment temperature was 30 ℃.

TABLE 1

This hybridoma ST723-F31 (KCTC 8472 P) was inoculated into the scent generating medium (Example 1) and amino acid soy sauce, and cultured at 30 ° C. for 26 days. As a result, it showed excellent soy flavor and the volatile aroma component of the culture was extracted with ethyl ether using a steam distillation-extraction (SDE) apparatus [See Thomas H. Schultz, Robert A. Flath, T. Richard Mon, Sve. B. Eggling & Roy Teranishi; Isolation of Volatile Components from a Model system, J. Agric. Food Chem., 25 (8) 446-454 (1977).

The concentrate was subjected to preparative GC to obtain gas chromatogram, and at the same time, the sensory test of each peak component was performed.

In addition, the mass spectrum was obtained by GC-mass to identify the fragrance components.

In this way, it was possible to confirm 4-EG, which is a special ingredient of HEMF and T. versatiles, which is a special ingredient of soy sauce produced by Z. luxi, and this hybridoma was excellent in soy sauce as in the following example. It was confirmed to produce incense. In addition, the hybridoma has strong salt resistance and grows in a wide pH range, and has characteristics that make it easy to use for soybean fermentation.

Example 1

Hybridoma ST723-F31 (KCTC 8472 P) was incubated for 30 days at 30 ° C in a fragrance-forming medium (FM) and then used as a trained panel agent. Sensory evaluation [Ref .: Korea Industrial Standards; Sensory flavor test, Industrial Advancement Administration, KSA 7002 (1977)], and its growth state were observed.

As a result, the fragrance of fragrance was excellent in the short liver, and the growth state showed the growth characteristics of S. luxi (top yeast) and T. versatiles (bottom yeast) at the same time.

The composition of the fragrance producing medium is glucose 2.0%, yeast extract 0.5%, polypeptone 0.5%, KH ₂ PO ₄ 0.1%, MgSO ₄ 0.1%, soy extract 10.0%, NaCl 10.0%, ferulic acid 0.05%, pH 5.5 Soy extract is prepared by the following method.

Example 2

After extracting the volatile scent component in the culture of Example 1 with the SDE apparatus, the peak of the scent component was confirmed by preliminary GC, and the result of sensory test of each peak component is shown in FIG.

As shown in FIG. 1, the HEMF peak at retention time (RT) 6.308 and the 4-EG peak at R.T 8.963 are shown, indicating that the hybridoma ST723-F31 produces both specific components.

Example 3

The fragrance extract sample of Example 2 was identified by obtaining a mass spectrum using a GC-mass, and library searching with a computer. The gas chromatogram of the GC-mass is shown in FIG. 2, the mass spectrum of the identified HEMF is shown in FIG. 3, and the mass spectrum of 4-EG is shown in FIG. The conditions of GC and GC-mass are as follows.

Example 4

Hybridoma ST723-F31 (KCTC 8472 P) was added to amino acid soy sauce (14% salt, pH 4.5) with 1% soy extract and 3% glucose, followed by stationary incubation at 30 ° C for 26 days. Was investigated.

The fragrance's fragrance showed salty conventional soy sauce. The aroma was extracted from the culture by the SDE apparatus, and the results of sensory examination of the components of each peak by preliminary GC are shown in FIG.

Example 5

The resistance to salts of hybridoma ST723-F31 (KCTC 8472 P) was investigated.

The medium used was inoculated into solid medium and liquid medium in which salts were added at different concentrations (15% to 27%) to the complete medium of Table 1, followed by incubation at 30 ° C. for 8 days. The hybridomas grow very weakly at 25% salt concentration and were very resistant to salts.

TABLE 2

Example 6

PH growth of hybridoma ST723-F31 (KCTC 8472 P) was investigated. After culturing for 8 days at 30 ℃ in a complete medium from pH 3 to pH 10, the results are shown in Table 3. This hybridoma was grown at pH from pH 3 to pH 10 and had broad growth pH.

As described in the above embodiments, the novel hybridoma ST723-F31 (KCTC 8472 P) of the present invention simultaneously generates the characteristic flavor components of soy sauce produced by Zaygo Saccharomyces luxi and Tolulopsis versatileis, respectively. It can solve the problem of difficulty in producing and harmonizing fragrance components when fermenting soy sauce, and the difficulty of mixing culture of Z. luxi and T. versatiles. In addition, when the soybean fermentation is carried out with these two bacteria, T. versatiles grow after Z. luxi grows, but it takes a long time. Therefore, when the liver is fermented in a short time as a bioreactor, one hybridoma may be used instead of two bacteria, Z. luxi and T. versatiles, which simplifies the process and shortens the time. have.

Claims (3)

Zaigo Saccharomyces luxiii (4-hydroxy-2- (or 5) -ethyl-5 (or 2) -methyl-3- (2H) -furanone (HEMF)), a unique flavor component of soy sauce Z.Luxii produced HEMF and T. Versatileis produced by fusing Zygosaccharomyces rouxii) and Torulopsis versatilis, which produces 4-ethylguaiacol (4EG). Novel hybridoma ST723-F31 that produces 4-EG and other characteristic orientations of the soy produced by these Z.Luxii and T.Versatiles. KCTC 8472 P). Zygosaccharomyces rouxii and Torulopsis versatilis IAM 4993 were mutated to N-methyl-N'-nitro-N-nitrosoguanidine to obtain amino acid requirement mutants; Among the obtained mutants, the arginine- and leucine-ureotypic mutants, which have a odor of culture fragrance which produces better or similar to wild strains, are selected; Protoplast fusion of the two mutants in the presence of 40% PEG 4000 to obtain hybridomas; Method for producing a hybridoma as defined in claim 1, characterized in that the hybridoma is passaged several times in a complete medium to stabilize. A method for producing soy sauce with improved flavor, characterized in that it is fermented directly using soybean fermentation using the hybridoma as defined in claim 1, or (ii) fermentation with a bioreactor.

Images