KR101926183B1 - Safe starter for the production of soybean fermented food and soybean fermented food therefrom - Google Patents

Abstract

The present invention relates to a fungal strain used in the production of soy sauce, which comprises fungal toxins such as aflatoxin and Aspergillus oryzae which do not produce cyclopiazonic acid (CPA) MJS14 (Accession No .: KCTC18552P) Bacillus strains used for the production of strains and pastes include Bacillus subtilis , which does not produce biogenic amines, D119C (Accession No .: KCTC18551P). In the present invention, the combination of the fungus strain and the bacterium strain selected in the present invention simultaneously exhibits a complex microbiological action and can exhibit a flavor different from that of the soybean fermented by the single inoculation, and the conventional fermentation by the natural inoculation When compared with meju and doenjang, the inoculated strains and the fermentation period can be kept constant, so that uniformity of quality and safety can be secured.

Description

[Background Art] [0002] Soybean fermentation strains which have been proved to be safe and fermented soybean fermented food products produced using the same,

The present invention relates to a safe and excellent seed which can be used for fermentation of soy sauce, and a soy sauce made using the same. More particularly, the present invention relates to a safflower which does not produce fungal toxins ( Aspergillus Bacillus subtilis D119C (accession number: KCTC18551P) which does not produce oryzae MJS14 (accession number: KCTC18552P) and biogenic amines, and a soup made using the same.

In Korea, traditional soy sauce is made by molding soya bean as a main meju after immersion and thickening, and then hanging it with straw to make fermentation by natural inoculation. Although the meju and soy sauce fermented in this manner has a simple merit, it can not maintain a constant quality because naturally inoculated microorganisms can not be controlled, and there is a high possibility that toxins are produced due to contamination of harmful microorganisms.

In case of natural inoculation, Meju may be contaminated with Aspergillus flavus strain, which is very similar in appearance to Aspergillus oryzae , a Meju fermentation fungus. This strain is a strong carcinogen To produce aflatoxin. In addition, the naturally inoculated meju fermenting bacteria are highly likely to produce biogenic amines, which have recently become an issue of harmful substances.

Therefore, it is very difficult to secure the safety of meju and soy sauce when natural fermentation is carried out by inoculation. Therefore, it is necessary to newly develop a seed-bacterium which has been proved to be stable and to use it actively in the production of the soybean.

Korean Patent Publication No. 10-2014-0057436 (published on Apr. 2014, 2005) discloses a method for preparing Korean Meju doenjang using a novel strain Bacillus amyloliquefaciens CJ3-27 and Aspergillus oryzae CJ KG Is described. Korean Patent Laid-open Publication No. 10-2014-0012411 (published on Apr. 201, 02.03) discloses a strain of Bacillus licheniformis SCKB11 having an antibacterial activity against a harmful microorganism and a biogenic amine-decomposing activity, Contents are described.

Currently, in the industry, for the fermentation of soybean paste, A. oryzae , The strain is mainly used. However, these strains already used in the industry are mainly strains that focus only on the functions such as enzymatic activity, and there is little test for safety. In addition, there is no additional validation of the safety of fermented soybeans after fermentation.

Although the fermentation of soy sauce was reported using seeds, it is reported that the production method of producing soy sauce by simultaneous inoculation of the combined strains is mainly performed by using a single seed culture or a method of mixing after fermentation with each seed culture Almost never.

DISCLOSURE OF THE INVENTION The present invention has been made in order to solve such problems, and it is intended to provide a seed candidate which is stable from industrial koji, traditional meju and pasta, and can impart excellent sensory characteristics.

In addition, the present invention aims to provide a soy sauce having excellent stability and a superior sensory characteristics by using a strain with safety.

The invention Aspergillus duck material (Aspergillus oryzae ) MJS14 (Accession No .: KCTC18552P).

The Aspergillus of the present invention's duck material (Aspergillus oryzae ) MJS14 (Accession No .: KCTC18552P) is a strain that does not produce aflatoxin and cyclopiazonic acid (CPA), which are preferably mycotoxins.

Bacillus subtilis , D119C (Accession No .: KCTC18551P).

The Bacillus subtilis of the present invention, D119C (Accession No .: KCTC18551P) is preferably a strain that does not produce a biogenic amine.

The present invention relates to a process for producing a fermented soybean fermented by fermenting soybeans with boiled soybeans, wherein the fermenting bacteria are mainly Aspergillus oryzae , MJS14 (Accession No .: KCTC18552P) strain and Bacillus subtilis And a strain of D119C (accession number: KCTC18551P). The present invention also provides a method for producing a filament.

In the method of the present invention, the aspergillus oryzae ( Aspergillus oryzae ) MJS14 (Accession No .: KCTC18552P) strain and Bacillus subtilis The D119C (Accession No .: KCTC18551P) strain is preferably inoculated with two strains simultaneously.

In the method of the present invention, the above-mentioned field (s) may be, for example, miso or soy sauce.

The present invention relates to a fungal strain for use in the production of soy sauce, asbestos does not produce mycotoxins and aflatoxins the CPA (cyclopiazonic acid) Aspergillus's duck material (Aspergillus oryzae ) MJS14 (Accession No .: KCTC18552P).

In addition, the present invention relates to a bacterium strain used for the production of soy sauce, which comprises Bacillus subtilis which does not produce a biogenic amine, D119C (Accession No .: KCTC18551P).

In the present invention, by simultaneously inoculating the fungus strain and the bacterium strain selected in the present invention, a complex microbiological action is exhibited, and the flavor may be different from that of soybean fermented by single inoculation.

In addition, when compared with conventional meju and doenjang in which mixed fermentation due to natural inoculation occurs, the present invention can keep the inoculated strains and the fermentation period constant, thereby ensuring uniformity of quality and securing safety.

FIG. 1 is a cross-Aspergillus oryzae) ≪ / RTI > MJS14 (Accession No .: KCTC18552P) strain.
2 is a cross-SmaⅠ Restriction enzyme cleavage method Aspergillus oryzae (Aspergillus oryzae) MJS14 (Accession No .: KCTC18552P).
FIG. 3 is a graph showing the results obtained by the PCR using the Aspergillus oryzae of the present inventionAspergillus oryzae) MJS14 (Accession No .: KCTC18552P) clusters of aflatoxin biosynthetic genes.
FIG. 4 is a graph showing the results obtained by the PCR using the Aspergillus oryzae of the present inventionAspergillus oryzae) MJS14 (Accession No .: KCTC18552P).
FIG. 5 is a graph showing the distribution ofAspergillus oryzae) MJS14 (Accession No .: KCTC18552P), Bacillus subtilis (Bacillus subtilis) D119C (Accession No .: KCTC18551P), which is a mixed fermented soybean meal.
FIG. 6 is a graph showing the distribution of the fungus strain Aspergillus oryzae of the present inventionAspergillus oryzae) MJS14 (Accession No .: KCTC18552P), the bacterial strain Bacillus subtilis of the present inventionBacillus subtilis) D119C (Accession No .: KCTC18551P) and the mixed proteolytic activity and starcholytic enzyme activity of the mixed fermented soybean meju.
FIG. 7 is a PCA graph showing flavor components of doenjang prepared by simultaneous inoculation of two strains of the present invention. FIG.

The invention Aspergillus duck material (Aspergillus oryzae ) MJS14 (Accession No .: KCTC18552P). The fungal strain A. oryzae MJS14 (Accession No: KCTC18552P) was tested for quantitative production and molecular genetic assays that do not produce aflatoxin and CPA, the mycotoxins that can be produced by Aspergillus fungi.

On the other hand, the present invention relates to a method for producing Bacillus subtilis , D119C (Accession No .: KCTC18551P). B bacteria developed in the present invention. subtilis D119C (Accession No .: KCTC18551P) was found not to produce biogenic amines, toxic substances that bacteria can produce, and was found to have high protease, amylase and cellulase activities.

The present invention also relates to a process for producing a fermented soybean fermented by fermenting soybeans with boiled soybeans, wherein the fermenting bacteria are mainly Aspergillus oryzae , MJS14 (Accession No .: KCTC18552P) strain and Bacillus subtilis And a strain of D119C (accession number: KCTC18551P). The present invention also provides a method for producing a filament.

The method of the present invention for producing a bacterium of the present invention is a method for producing a bacterium comprising Aspergillus oryzae , MJS14 (Accession No .: KCTC18552P) strain and Bacillus subtilis D119C (Accession No .: KCTC18551P)). Therefore, in addition to the use of these strains, other technical ideas necessary for the production of soy sauce, such as boiling meju, fermentation and aging in soybean paste or soy sauce The techniques may use known techniques. Therefore, a detailed description thereof will be replaced with a known technique.

On the other hand, sheet production method of the present invention is Aspergillus duck material (Aspergillus oryzae ) MJS14 (Accession No .: KCTC18552P) strain and Bacillus subtilis D119C (accession number: KCTC18551P) may be used alone, or both of them may be used. However, it is preferable that two strains are simultaneously inoculated and used. When the two species selected in the present invention are mixed and fermented in the form of a seed meal, it can be confirmed that the enzyme activity is higher than that of the single fermentation of the strain .

From these results, it was found that the mixed fermentation was more effective in fermentation than the fermentation in the form of the soybean meju using the fungus strain and the bacterium strain selected in the present invention. It was confirmed that the microbial interaction showed a higher level of enzyme activity without affecting the activity.

When fermented soybean paste was fermented using mixed fermented soybean meal, fermented soybean paste showed higher fermentation rate than traditional soybean paste fermented by natural inoculation. From these results, it was found that more efficient fermentation was performed in a relatively short time in the soybean paste using the mixed seed of the present invention.

In addition, the taste component analysis by the sensory evaluation revealed that the unpleasantness was lower than the traditional doenjang, and the relatively high flavor and sweetness were shown to be relatively high, there was.

From the standpoint of safety, it was confirmed that a very low level of biogenic amine was detected when the biogenic amine of the present invention was analyzed, and it was confirmed that the safety was very high when compared with the conventional soybean paste It means.

As a result, it was found that when the fermented meju was fermented and the soy sauce was produced by the mixed fermentation of the two germs of the present invention which secured the safety, the quality was improved as well as the safety was improved compared to the traditional soy sauce. Accordingly, the two species derived from the present invention and the mixed fermentation can be applied to the industrial production of safflower which is safe and has excellent flavor.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

[ Example  One: Aspergillus  Duck Aspergillus oryzae ) MJS14  (Accession No .: KCTC18552P) strain and Bacillus Subtilis  ( Bacillus subtilis ) D119C  (Accession No .: KCTC18551P) Isolation and Identification of Strain]

Fungal strains were isolated from Korean traditional meju. Morphological identification was carried out for the identification of fungal strains. MJS14 (accession number: KCTC18552P) has a colony shape similar to that of A. oryzae / A. flavus when aerial hyphae are observed in the form of a large colony and spore commonly used for the identification of fungi. Spore color was greenish to purple. In addition, the spores were smooth rounded, which was also consistent with the spore characteristics of A. oryzae / A. flavus (Fig. 1). However, A. oryzae and A. flavus were highly homologous to their morphological characteristics, which made it difficult to distinguish species. Therefore, MJS14 (Accession No .: KCTC18552P) was identified by additional molecular genetic PCR methods.

For the molecular identification of Aspergillus sp. Microorganisms, we use a method to amplify aflatoxin gene cluster gene. In the molecular genetic identification of fungi, species with common characteristics among species belonging to the same genus are grouped together and identified one more time in the section. A. oryzae and A. flavus belong to the section flavi in the above classification system, and the PCR product of the aflT gene, which is a gene of the aflatoxin gene cluster, showed amplified product, and the MJS14 strain was identified as Aspergillus section flavi . Real-time PCR amplification of the afaflT gene in the aflT gene revealed that the MJS14 strain was classified as A. oryzae / A. flavus group among Aspergillus section flavi .

However, the Sma I restriction enzyme digestion method was further used to identify MJS14 isolates from A. flavus . A. flavus usually has a band of 3.8kb in addition to the 1.7kb band, while A. oryzae typically has a band of 1.7kb, 1.0kb, and 2.7kb. The MJS14 strain of the present invention isolated from meju was faint, but showed band characteristics of A. oryzae including up to a band of 1.0 kb. Therefore, the strain was identified as A. oryzae strain.

On the other hand, bacterial strain D119C was isolated from Korean traditional soybean paste and physiological identification using API 50E kit and molecular genetic identification using 16S rRNA gene were carried out. The isolate D119C (accession number: KCTC18551P) was identified as B. subtilis Respectively.

The results of identification of the MJS14 and D119C strains are summarized in Table 1 below.

MJS14
Morphological identification Aspergillus oryzae / flavus ITS sequence analysis Aspergillus oryzae / flavus SmaI restriction enzyme digestion Aspergillus oryzae AVM test Aspergillus oryzae D119C
API50E kit Bacillus subtilis 16S rRNA sequencing Bacillus subtilis

[ Example  2: Evaluation of safety and seed compatibility of selected strains]

 One. MJS14  (Accession number: KCTC18552P ) Evaluation of strains

PCR was performed to confirm the mycotoxin biosynthesis ability of the fungal candidate strain selected in Example 1 above at the molecular genetic level.

Aspergillus to which A. oryzae belongs The biosynthetic genes for aflatoxin toxins, which are likely to be produced by section Flavi species, are known as in FIG. Based on this, the aflatoxin biosynthesis gene of A. oryzae MJS14 (accession number: KCTC18552P) isolated and identified was confirmed by PCR.

In FIG. 3, it was confirmed that part of the norB / cypA gene of the aflatoxin biosynthesis gene of MJS14 (Accession No .: KCTC18552P) was deleted (same as the analysis result of A. oryzae ). In other words, the A. oryzae MJS14 strain was confirmed to have deleted one of the clusters of aflatoxin biosynthesis gene cluster. Based on this, it could be expected that aflatoxin was not biosynthesized.

On the other hand, A. oryzae may also produce CPA (cyclopiazonic acid). CPA biosynthetic clusters of CPA-producing strains and non-producing strains are shown in Fig. In Fig. 4, the black arrow indicates the CPA biosynthetic gene. As a result, as shown in FIG. 4, the MJS14 strain of the present invention (accession number: KCTC18552P) showed band matching with a strain that biosynthesizes CPA and has all CPA biosynthesis genes. However, as a result of quantitative analysis by HPLC, CPA was not detected at all, and thus MJS14 strain had a CPA biosynthesis gene, but it was confirmed that CPA was not actually produced.

Table 2 summarizes the above experimental results.

Strain Aflatoxin gene cluster CPA gene cluster Biosynthesis
of Aflatoxin Biosynthesis of CPA norB / cypA ^a nor-1 aflR give -One omtA maoA dmaT pks /
nrps AVM TLC HPLC TLC HPLC A. flavus
NRRL3357 Ⅱ + + + + + + + + + + + + A. oryzae
SRRC266 Ⅰ + + + + + + + - - - - - A. oryzae
NBRC4177 ND ND ND ND ND + + + ND ND ND + + A. oryzae
RIB40 ND ND ND ND ND + + - ND ND ND - - MJS14 Ⅰ + + + + + + + - - - - -

ND, No data.

^a I, 400 bp; II, 800 bp

 2. D119C  (Accession number: KCTC18551P ) Evaluation of strains

[0060] D119C (Accession No .: KCTC18551P) In order to confirm the bacterium suitability of the bacterium strains, the antioxidant activity against the Bacillus cereus strain, which is a food poisoning bacterium, was confirmed on the medium in terms of safety.

first, D119C (Accession No .: KCTC18551P) had an amino acid decarboxylase activity, which is an enzyme that produces a biogenic amine from an amino acid. Amino acid was added as a nutrient source on the medium, and then bromocresol purple was added as a pH indicator to confirm the formation of biogenic amines. If the strain produces biorenic amines, the pH on the medium is increased and can be observed as a color change. By this method, strains having low amino acid decarboxylase activity can be selected. As a result, the B. subtilis D119C (accession number: KCTC18551P) of the present invention showed no amino acid decarboxylase activity (DC activity) (Table 3)

On the other hand, from another safety standpoint, the antagonistic action against B. cereus , a food poisoning bacterium, was confirmed by the presence or absence of clear zone on the medium. The topical agar was used to inoculate the B. cereus strain, and the paper disk was placed thereon to inoculate the bacterial strain of invention D119C (Accession No .: KCTC18551P), and then the size of the transparent ring was confirmed. As a result of the experiment, it was confirmed that the most transparent circle appeared in the B. subtilis D119C strain of the present invention (accession number: KCTC18551P) (Table 3 below) and B. subtilis D119C (accession number: KCTC18551P) It could be judged to be a strain.

On the other hand, the activities of protease, amylase and cellulase were measured on the medium to confirm the activity of the most basic enzymes necessary for the fermentation of doenjang. Each of the strains was cultured on a medium supplemented with a substrate, and then confirmed with a transparent ring or a coloring reagent. As a result of the experiment, it was confirmed that the basic level of activity of the enzymes was shown in B. subtilis D119C (accession number: KCTC18551P), which is the most advantageous in terms of safety.

The results of the safety tests and the enzyme activity of the selected strains and other strains are shown in Table 3 below. The quantitative evaluation results of the biogenic amines by HPLC of the strain B. subtilis D119C (accession number: KCTC18551P) Respectively.

strain Strain No. origin DC activity B. cereus
Antagonism protease amylase cellulase Bacillus amyloliquefaciens zip6 rice straw, Korea + + + + ++ Bacillus subtilis KCCM 11316 - - + - - Brevibacillus non reactive D065B doenjang , Korea - - + - + B. subtilis / amyloliquefaciens D077A doenjang , Korea + + + - - B. subtilis / amyloliquefaciens D108C doenjang , Korea + - + - ++ B. subtilis D119C doenjang , Korea - ++ + + ++ B. subtilis / amyloliquefaciens D125D doenjang , Korea - - + - + B. subtilis / amyloliquefaciens D131B doenjang , Korea + - + - - Geobacillus  thermoglucosidasius D135A1 doenjang , Korea - - + - ++ B. licheniformis D136C doenjang , Korea - - - - + B. subtilis / amyloliquefaciens D140D doenjang , Korea + ++ + - + B. subtilis / amyloliquefaciens D162C doenjang , Korea - - - - + B. subtilis / amyloliquefaciens D170C doenjang , Korea - - + - -

Strain Strain No. PUT CAD HIS TYR F B. subtilis D119C ND * ND ND 1.78 + 0.17 ND *; Not detected, PUT; Putrescine, CAD; Cadaverine, HIS; Histamine, TYR; Tyramine

[ Example  3: Using the selected strain Bean of meju  Fermentation]

To investigate the effects of the selective fermentation on the fermentation of soybean meju using the selected strain (MJS14 (accession number: KCTC18552P) and B. subtilis D119C (accession number: KCTC18551P)). For this purpose, each of the selected strains was inoculated into bean curd bean at a level of 10 ⁶ beans (Fig. 5).

As a result of the fermentation, it was confirmed that the proteolytic enzyme activity and the starcholytic enzyme activity were high in the mixed fermented soybean meju compared to the fungus-only fermentation and the bacterium-exclusive fermentation (FIG. 6). The activity of starch hydrolyzing enzyme was higher in fungus - only fermentation sample than that of bacterium - only fermentation sample. However, in the mixed fermentation samples, both enzyme activities were not only high but also had higher enzyme activity than individual fermentation. This suggests that when two microorganisms are cultured in one batch, both strains are growing together successfully, meaning that the two strains interact with one another and provide advantages to each other.

The above experimental results show that the enzyme activity is higher when cultured with bacteria than when the fungi are cultured alone, which means that the sugar and amino acid components affecting taste and flavor are increased. These results indicate that if the two microorganisms of the present invention are inoculated into the finished meju at once to immerse the miso, the two strains not only do not antagonize each other, but also interact with each other, It is possible to produce miso.

[ Example  4: by mixed fermentation of the selected strain Bean meju  Preparation of used miso]

The soybean miso soybean paste was prepared by mixing and fermenting the selected strain of the present invention. In addition, physicochemical analysis, safety analysis and sensory evaluation were conducted by using doenjang.

sample Formalin nitrogen
(mg%) NaCl
(%) Moisture content
(%) pH Acidity Chromaticity L a b sample 1126 12.0 53.1 6.23 25.3 37.62 10.79 23.55 Reference 890 11.0 56.6 5.04 34.4 41.34 8.52 18.95 sample; Soybean paste prepared by using the mixed fermented soybean meal with the mixed seed of the present invention, Reference; Traditionally-made miso

As a result of comparing the physicochemical properties of the basic soybean paste, as shown in Table 5, the content of formalin nitrogen, which is an index of the degree of fermentation, was higher in the doenjang prepared using the mixed seed of the present invention than that of the conventional soybean paste I could confirm. As a result, it can be concluded that the soybean paste of the present invention is more advanced fermented soybean fermented than the traditional soybean paste fermented at a relatively short period of time, there was.

On the other hand, the pH and acidity affecting the acidity were higher in the traditional miso than the miso in the present invention using the mixed acid, and the acidity of the traditional miso was higher.

On the other hand, using the taste component analysis data of specialty panels, the PCA analysis of the present invention was conducted along with traditional miso (Fig. 7). In the case of traditional miso, the taste components contained a large amount of unpleasant taste components, but the miso did not feel the corresponding flavors of the present invention. In addition, the results of PCA analysis showed that the taste distribution of traditional doenjang and mixed doenjangs of the present invention were very different, and it was confirmed that the mixed bean paste of the present invention showed relatively richness and sweetness compared to traditional doenjang . It was judged that this was due to the high proteolytic enzymes of the mixed yeasts and doenjang of the present invention and the amino acids and sugar components of the starch hydrolyzate. Therefore, the soybean paste prepared using the mixed seeds of the present invention was superior in terms of commerciality, because it was able to reduce unpleasant odor in traditional doenjang while having excellent taste and flavor.

Meanwhile, in order to measure the safety of the doenjang using the mixed seed of the present invention, the biogenic amine was extracted from doenjang and measured (Table 6). Traditional soybean paste was used as a control.

sample Biogenic amines (占 퐂 / g) TRP PHE PUT CAD HIS TYR SPD SPM Total BA Sample ND ND ND ND ND 6.91
± 0.00 ND 5.31
± 0.00 12.22
± 0.00 Reference ND 486.97
± 19.00 ND ND 4.87
± 1.31 916.20
± 41.00 ND 0.06 ± 0.00 1408.10
± 61.31 TRP; Tryptamine, PHE, 2-Phenylethylamine, PUT; Putrescine, CAD; Cadaverine, HIS; Histamine, TYR; Tyramine, SPD; Spermidine, SPM; Spermine

As a result of the experiment, it was found that a very low level of biogenic amine was detected in the soybean paste of the present invention using mixed seeds compared to traditional soybean paste. However, the contents of 2-phenylethylamine and tyramine among 8 kinds of biogenic amines in traditional doenjang were found to be particularly high. As a result, it was found that when the miso was prepared using the mixed seed of the present invention, the safety was very high as compared with the fermented soy sauce fermented by natural inoculation.

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC18551

Checked on: 20170228

Institution name: Korea Biotechnology Research Institute

Accession number: KCTC18552

Checked on: 20170228

Claims (7)

Aspergillus oryzae MJS14 (Accession No .: KCTC18552P) strain and Bacillus subtilis D119C (Accession No .: KCTC18551P) strain.
The method according to claim 1,
The strain Aspergillus oryzae MJS14 (accession number: KCTC18552P)
It does not produce the mycotoxins aflatoxin and CPA (cyclopiazonic acid)
The Bacillus subtilis D119C (accession number: KCTC18551P)
A fermentation strain mixture for fermentation according to claim 1, which does not produce a biogenic amine.
delete delete In the production of a bean jam produced by inoculating fermented soybeans with boiled soybeans and fermenting them,
Aspergillus oryzae , MJS14 (Accession No .: KCTC18552P) strain and Bacillus subtilis D119C (Accession No .: KCTC18551P).
6. The method of claim 5,
The strain Aspergillus oryzae MJS14 (accession number: KCTC18552P)
It does not produce the mycotoxins aflatoxin and CPA (cyclopiazonic acid)
The Bacillus subtilis D119C (accession number: KCTC18551P)
Wherein the biogenic amine is not produced.
6. The method of claim 5,
The above-
Soy sauce, soy sauce, soy sauce, soy sauce, soy sauce or soy sauce.

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