KR920005750B1 - New microorganism bacillus subtilis strain - Google Patents

Abstract

A soybean paste is prepd. by (a) steaming soybean, (b) innoculating the steamed soybean with the starter and fermenting, and (c) mixing the fermented soybean with other additives. The starter is Bacillus subtilis PM3 (KCTC 8486P) (I), Bacillus subtilis SS9 (KCTC 8487P) (II), or the mixt. made by adding ST723-F31 or Bacillus licheniformis SSA3-2M1 (KCTC 8489P) (III) into the (I) or (II), and mixing. The microorganism (I), (II) and (III) are new. The obtd. soybean paste has a good color and taste.

Description

Microorganisms for Korean Traditional Soybean Paste Preparation and Method for Making Soybean Paste Using the Same

FIG. 1 is a gas chromatogram of GC-functionality test of neutral fraction in volatile fraction of culture in which Bacillus subtilis PM3 was cultured in pure soybean medium.

FIG. 2 is a gas chromatogram of GC-functionality test of neutral fraction in volatile fraction of culture in Bacillus subtilis SS9 cultured in pure soybean medium.

3 is a gas chromatogram of neutral fractions of volatile fractions of cultures in which Bacillus subtilis PM3 was cultured in pure soybean medium.

4 is a gas chromatogram of neutral fractions of volatile components of a culture in which Bacillus subtilis SS9 was cultured in pure soybean medium.

The present invention relates to a microorganism capable of producing Korean traditional piglets, a method of culturing the same, and a method of preparing miso using the same.

According to a conventional method of making doenjang, primary fermentation is performed at room temperature for 2-3 months from February by dipping conventional meju or an improved meju made with Aspergillus oryzae mainly made by bacteria in saline. After separating the solid portion and again subjected to secondary fermentation at room temperature for more than 4 months.

This conventional method of making miso has the following problems.

First: prolong the duration of miso production

Second: 1. When traditional meju is used as a raw material, it is difficult to standardize product quality (taste, aroma, color) due to many bacteria in meju and many bacteria from the air during fermentation. In most cases, it is also difficult to internationalize food because of its orientation. 2. When using modified meju mainly made of Aspergillus oryzae as a raw material, doenjang fragrance is not produced unless bacteria are introduced into the air during fermentation. Therefore, it is difficult to mass-produce soybean paste at the plant with improved meju because the quality of the product varies according to the type of bacteria.

Third: The color of the surface of the doenjang is blackened by the bacteria in the meju and the influx of doenjang during fermentation.

Fourth: In terms of quality, it is difficult to produce standardized miso products.

As a result of earnestly researching to solve these problems, the present inventors have found that Bacillus subtilis is a bacterium that has been isolated from traditional miso and Bacillus subtilis. By using this bacterium, the taste and aroma of conventional miso It was found that can be produced in a short time by a simple operation with a few bacteria that can produce and do not produce black pigment, and completed the present invention.

Therefore, it is an object of the present invention to provide bacteria that give off the flavor and taste of conventional doenjang, and to provide a method for producing doenjang that the quality is standardized by using the bacteria, the production period is shorter.

Hereinafter, the present invention will be described in detail.

Strains newly provided according to the present invention are commercially available from soy sauce and soybean paste soaked by conventional methods in a general household and aged for at least one year, for example, C.H.Collins & L.Lyne. Microbial methods 4th, (1976); And W. F. Harris & E. Micane; Laboratory methods in food and diary microbiology (1976)], and then isolated bacteria cultured in boiled soybean medium, and then known methods [Industrial Promotion Agency: Korea Industrial Standard KSA-7001, General sensory test (1976) And KISA: Sensory Flavoring Test, Korean Industrial Standard, KSA-7002 (1977)] can be obtained by selecting the bacteria that smell and taste of miso.

At this time, the boiled soybean containing seed medium and culture medium are provided by the present invention for the first time, thereby culturing and selecting the Bacillus subtilis according to the present invention.

Pure boiled soybean medium can be prepared according to the following scheme.

Scheme 1. Preparation of pure boiled soy broth

Glyone max.L

↓

Immersion; 16 hours

↓

Capital increase; 106 ℃, 3 hours

↓

Fracture; Moisture: Soybean = 2.5: 1

↓ NaCl added 10% of moisture

Dispensing; 300g each in 500ml Erlenmeyer flask

↓

Autoclaving; 121 ℃, 15 atmospheres, 40 minutes

In this way, two types of bacteria, PM3 and SS9, were selected, and their culture, morphological and physiological characteristics were observed. NSMair et al, Bergey's manual of systematic Bacteriogy Volumn 2 (1986) and REGord et al. , The Genus Bacillus, Agricultural Hand book No. 427, (1973).

As a result, as shown in Table 1, SS9 showed anaerobic growth, anaerobic growth, VP broth (pH> 7), citrate availability, and the Bergey's manual, as compared to the Bacillus subtilis properties. The remaining properties except egg yolk resitinase and indole forming ability are similar, and PM3 grows in VP broth (pH> 7), citrate utilization, indole forming ability, growth in 55 ℃ -65 ℃, and growth in presence of lysozyme Similar to the rest of the properties except.

In addition, compared with the properties of the Bacillus subtilis of the genus Bacillus (SS9), the growth properties at 5 ℃, 55 ℃, other properties except for egg yolk retinase, PM3 is 5 ℃, The growth at 65 ° C., except for the growth in the presence of lysozyme, is similar in various properties, so it is confirmed that the SS9 and PM3 bacteria are Bacillus subtilis.

The strains Bacillus subtilis PM3 and Bacillus subtilis SS9 of the present invention selected by the present inventors were deposited on May 23, 1990 with the accession numbers KCTC 8486 P and KCTC 8387P, respectively, to the Korea Institute of Science and Technology.

TABLE 1

Salt and sugar resistance of screening bacteria Bacillus subtilis SS9 and Bacillus subtilis PM3 were investigated. At this time, the composition of the medium used is shown in Table 2, and the results of observing the growth of liquid medium and solid medium by salt concentration and sugar concentration are shown in Table 3 and Table 4 respectively. The culture temperature was 37 ℃ and incubated for 7 days.

TABLE 2

TABLE 3

As shown in Table 3, Bacillus subtilis SS9 and Bacillus subtilis PM3 were grown at a salt concentration of 15%, indicating that they were relatively resistant to salts.

TABLE 4

As shown in Table 4, Bacillus subtilis SS9 and PM3 were relatively resistant to sugars and were able to grow even at high sugar concentrations of about 50%.

Identifies the fragrance components produced by culturing Bacillus subtilis PM3 and SS9 isolated and identified in the above manner in pure soybean medium and reveals which directions each fragrance component produces, as follows. Extract the total volatiles and obtain a neutral fraction showing the characteristic traditional Doenjang among the extracted fragrances. Concentrate it and investigate the direction of each peak component by GC-Sensory Test, and make the gas chromatogram of these fragrances constant. I identified it.

(1) Extraction of Volatile Components

For the extraction of volatiles, the method of Nickusson and Rickens [G.B.Nickerson, S.T.Likens, Proc. Am, Soc, Brew, Chem., 5 (1964)], an improved continuous steam distillation-extraction apparatus [T.H. Schults et al., J. Agric. Food Chem., 25 (3), 446-449 (1977).

Pure boiled soybean medium was added with equal amount of distilled water to the culture (300g) incubated for 30 days at 40 ° C for 40 days, and then put into diluent ether (100ml) in a solvent container to maintain a constant temperature of 40 ° C. After circulating the solvent, the temperature of the sample container was raised until the sample boiled and extracted for 2 hours.

(2) fraction of volatile components

The extracted volatiles were fractionated in the same manner as in Scheme 2 [M. Fujimaki, T. Tsugita & T. Kurata; Agric. Biol. Chem., 41 (9), 1721-1725 (1977)]. In other words, the total volatiles extracted in diethyl ether are added to the separating funnel, and 5% HCl (100 ml) is added, shaken vigorously and left for a while to separate the aqueous layer and the ether solution layer, and when NaCl is saturated in the separated aqueous layer. After diluting with diethyl ether (50 ml), the mixture was shaken vigorously and left for a while to separate the aqueous layer and the ether layer. The aqueous layer was discarded and the ether layer was combined with the separated ether layer. This process was repeated with the addition of 5% NaOH (100 ml) to obtain an ether layer, anhydrous Na ₂ SO ₄ was added, and left for one day to remove moisture to obtain a neutral fraction.

Scheme 2

(3) GC-Sensory Test of Neutral Fraction

The neutral fraction obtained by fractionating the total volatiles was concentrated to about 2ml by rotary evaporator at atmospheric pressure, passed through nitrogen gas and finally concentrated to about 150μl, and then extracted from the prepared gas chromatograph while obtaining gas chromatogram with crude gas chromatograph. The direction of each peak component on the resulting gas chromatogram was sensory tested. [A. Yasuhara & K. Fuwa; Bulletin of the chemical society of Japan, 50 (11), 3029-3032 (1977)]

TABLE 5

(4) Identification of neutral fractions

Preparation of GC-Sensory Gas Chromatogram obtained by Gas Chromatograph and GC Pattern of Flavor Compounds. The results of KOVATS's comparison with the Sadtier standard gas chromatography retention index library (1986) and retention index are shown in Table 7 and Table 8.

TABLE 6

GC sensory evaluation of neutral fractions of volatiles in cultured soybean medium with Bacillus subtilis PM3 showed hepatic fragrance at peaks of 2.043 and 4.105 at retention time (see Figure 1). , Which was identified as (z) -3-penten-1-ol and 7-octen-4-ol corresponding to peak numbers 11 and 16 in FIG. 3, and the doenjang fragrance was found at each peak at the 4.528-4.897 site. These were aromatic substances corresponding to peak numbers 17 (2-nonanones), 18 (nitric acid, nonyl ester), 19 (?), 20 (benzaldehyde), and 21 (1-octanol) of FIG.

In addition, hepatic fragrances were observed at the peaks of 4.113 and 6.028 of the retention time in the GC sensory analysis of volatile fractions of the volatile components of Bacillus subtilis SS9 cultured in pure soybean medium. Octen-4-ol), 18 (benzaldehyde), 19 (?).

TABLE 7

TABLE 8

The invention is illustrated in more detail by the following examples.

Example 1

In order to separate the bacteria according to a commercial method, a certain amount of soy sauce and soybean paste soaked in a conventional household in a conventional method and aged for one year or more was diluted three times with sterile water, and then purely separated by a separation medium as shown in Table 9.

TABLE 9

Boiled soybeans prepared in accordance with the above description were incubated in boiled soy broth for 30 days at 40 ° C for 40 days, and the color of the culture medium and the direction of the upper interval and distilled water were added to part of the culture to investigate the miso flavor and taste. Bacillus subtilis PM3 and SS9 were selected according to known methods.

TABLE 10

Example 2

After inoculating Bacillus subtilis SS9 and Bacillus subtilis PM3 in pure boiled soybean medium, they were incubated at 30 ° C for 40 and 55 days, respectively, and then observed the aroma, taste, color and pH after boiling the whole cultured aroma. Is shown in Table 11.

TABLE 11

Example 3

Bacillus subtilis SS9 and PM3 with ST723-F31, a fusion yeast of Zygosaccharomyces rouxii and Torulopsis versatilis, inoculated pure life into soybean medium after 30 ℃, 40 The results of observing the aroma of the final culture, the aroma after boiling, the taste and color, and the pH after daily culture are shown in Table 12.

TABLE 12

Example 4

Bacillus subtilis SS9 and PM3 were inoculated with soybean medium for pure life, and then cultured at 30 ° C. for 10 days, 20 days, and 30 days, respectively, and then cultured with ST723-F31, a fusion yeast, for each period. Finally, after culturing for 40 days, the analysis results of the culture quality are shown in Table 13.

TABLE 13

Example 5

After inoculation with Bacillus subtilis PM3 and mutant Bacillus licheniformis SSA3-2M1 in pure life soybean medium, the quality of the cultures after incubation at 30 ° C. for 40 days is shown in Table 14.

TABLE 14

Example 6

After culturing Bacillus subtilis PM3 and mutant Bacillus rickenformis SSA3-2M1 in pure soybean medium and inoculating the fused yeast ST723-F31 by period, the result of culture quality after 30 ℃, 40 days incubation Same as 15.

TABLE 15

Example 7

The quality test results of the cultures after incubation at 30 ° C. for 40 days with Bacillus subtilis SS9, PM3 with ST723-F31 in pure soybean degreased medium are shown in Table 16.

TABLE 16

Example 8

The quality results of the culture after incubation at 30 ° C. for 40 days with Bacillus subtilis SS9, PM3 in fusion yeast ST723-F31 on pure boiled cotyled soybean are shown in Table 17.

TABLE 17

Example 9

The quality results of the cultures after incubation at 30 ° C. for 53 days with Bacillus subtilis SS9, PM3 with ST723-F31 in pure boiled defatted soybean medium are shown in Table 18.

TABLE 18

Example 10

The quality results of the culture after incubation for 53 days at 30 ° C. with Bacillus subtilis SS9, PM3 in fusion yeast ST-723-F31 on pure boiled cotyled soybeans are shown in Table 19.

TABLE 19

As described above, the method of preparing doenjang using the strain according to the present invention takes 6 months or longer when preparing the doenjang by the conventional method, compared to the conventional method, but the method of the present invention Since the fermentation is directly inoculated for the production bacteria has a merit that can significantly shorten the fermentation period than the time required for manufacturing by the conventional method. In addition, in the conventional conventional method, the conventional or improved meju must be prepared and immersed in saline to undergo primary fermentation, followed by a complicated process of dividing the liquid and the solid portion into secondary fermentation. Doenjang is prepared by inoculating fermented soybean directly in the temperature range from -37 ℃ to soybeans, which makes it easy to prepare soybean paste.In addition, when preparing doenjang by the conventional method, due to the bacteria introduced into the air during the meju or fermentation process, Not only the standardization of the product is not possible, but also may cause a decrease in quality, but the method of the present invention has an excellent advantage such as standardizing the quality (taste, aroma, color) of the product.

Claims (6)

Bacillus subtilis, which produces the flavor (flavor and aroma) of Korean traditional miso. A method of manufacturing doenjang which is inoculated with boiled crushed soybeans to produce flavors of doenjang and fermented directly with doenjang. The method of claim 2, wherein the bacterium is Bacillus subtilis PM3 (KCTC 8486P) or Bacillus subtilis SS9 (KCTC 8487P). Boiled mixture of Bacillus subtilis PM3 (KCTC 8486P) or Bacillus subtilis SS9 (KCTC 8487P) added with yeast fusion ST723-F31 or Bacillus licheniformis SSA3-2M1 (KCTC 8489P) A method of producing doenjang which is inoculated with crushed soybeans and fermented directly with doenjang. The method of claim 2, wherein the boiled crushed soybean is selected from pure boiled soybean, skim boiled soybean, or pure boiled cotyledon soybean. According to claim 1, Bacillus subtilis PM3 (KCTC 8486P) or Bacillus subtilis SS9 (KCTC 8487P), characterized in that the production of flavor (flavor and flavor) of Korean traditional miso Bacillus subtilis.

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