KR102240109B1 - A Novel Bacillus subtilis TSD and methods for preparing Doenjang using the same - Google Patents

Abstract

The present invention relates to a novel Bacillus subtilis TSD strain and a fermented soybean paste manufacturing method using the same. The present invention provides a novel Bacillus subtilis TSD strain and uses thereof. According to the present invention, when the novel Bacillus subtilis TSD strain is fermented with steamed soybeans, the content of amino acid nitrogen can be increased to produce fermented soybean paste with further improved flavor. The Bacillus subtilis TSD strain can be used in various ways in preparation of intestinal preparations, probiotics, food additives, and fermented products, and in particular, can be usefully used as a seed starter for soybean paste fermentation. In addition, shiitake mushroom fermented soybean paste manufactured by using shiitake mushroom and the Bacillus subtilis TSD strain according to the method of the present invention maximizes functionality of the shiitake mushroom and increases palatability of the fermented soybean paste. The method comprises a separation step, a strain preparation step, a soybean paste manufacturing step, and a fermentation step.

Description

A Novel Bacillus subtilis TSD and methods for preparing Doenjang using the same}

The present invention relates to a novel Bacillus subtilis TSD strain and a method for preparing soybean paste using the same.

In Korea, along with the increase in national income and the resulting changes in social and cultural lifestyles, the consumption pattern of soybean paste is gradually changing from a form that is manufactured and consumed by each household directly to a form of consumption that is consumed by purchasing commercialized soybean paste. . As a result, soybean paste has secured a strong domestic market, and as its unique flavor and excellent anticancer effect are known to the international community, soybean paste is gradually taking its place in the international market.

Doenjang is excellent nutritionally because it contains essential amino acids, fatty acids, organic acids, minerals, and vitamins, which are easy to lack in grain protein. Useful substances related to physiological activity contained in soybean paste include saponin, phytic acid, lectin, oligosaccharide, and isoflavone derived from soybeans. In particular, it is known that isoflavone, one of the useful components of soybeans, is converted into aglycone/glycoside by microorganisms during the fermentation process of soybean paste, thereby increasing the absorption rate in vivo. In addition, physiological activities such as anti-obesity, anti-hypertension, anti-cancer, antioxidant and anti-thrombosis, liver function enhancement, dementia prevention effect, and osteoporosis are known. Is being carried out in various ways.

On the other hand, according to the recently reported research results, some of the many European countries had significantly lower cancer incidence levels, and in common, these countries generally consume a lot of fermented foods, especially Finland. , In the case of Bulgaria, there have been reports that the incidence of cancer is significantly lowered because of the large intake of yogurt and other foods. In addition, it has been reported that miso, a representative fermented food in Korea, is consumed by most of the people and has a lower cancer incidence rate than other countries.

On the other hand, the present inventors have tried to develop a new strain that can improve the quality and sensory properties of doenjang. As a result, the Bacillus subtilis TSD strain was newly isolated and identified from doenjang, and the present invention was completed by experimentally confirming the applicability of the strain to a fermented product. In particular, in the case of preparing doenjang using the above strain, it was specifically confirmed that the flavor can be improved by increasing the amino acid nitrogen content, and thus doenjang having high preference can be prepared.

KR 10-2010-0023695 A

It is an object of the present invention to provide a Bacillus subtilis TSD strain [accession number: KCCM12595P] suitable for fermentation of doenjang.

Another object of the present invention is to provide a method for preparing doenjang using Bacillus subtilis TSD strain [accession number: KCCM12595P] or a culture solution thereof.

Another object of the present invention is to provide a method for preparing shiitake mushroom miso using shiitake powder, and Bacillus subtilis TSD strain [accession number: KCCM12595P] or a culture solution thereof.

In order to achieve the above object, the present invention provides a Bacillus subtilis TSD strain [accession number: KCCM12595P].

In addition, the present invention provides a method for producing miso comprising a step of inoculating and fermenting the steamed beans with Bacillus subtilis TSD strain [accession number: KCCM12595P] or a culture solution thereof.

In addition, the present invention is a mixture of shiitake mushroom powder and steamed beans Bacillus subtilis (Bacillus subtilis) TSD strain [accession number: KCCM12595P] or the step of fermenting after inoculating a culture solution thereof; Provides a manufacturing method.

The present invention provides Bacillus subtilis TSD strains and uses thereof. In addition, when the Bacillus subtilis TSD strain of the present invention is fermented with steamed soybeans, the content of amino acid nitrogen may be increased to prepare a doenjang having more excellent flavor.

The Bacillus subtilis TSD strain according to the present invention can be variously used in the manufacture of intestinal agents, probiotics, food additives, and fermented products, and can be particularly useful as a seed starter for fermentation of soybean paste.

In addition, shiitake mushroom miso prepared using shiitake mushroom powder and Bacillus subtilis TSD strain according to the method of the present invention further increases the palatability of soybean paste while maximizing the functionality of shiitake mushrooms.

1 is a photograph showing the morphological characteristics of the cells of Bacillus subtilis TSD strain (KCCM12595P).
Figure 2 shows the sequence listing of Bacillus subtilis TSD strain.
3 is a graph showing the number of viable cells according to the growth time of Bacillus subtilis TSD strain.
Figure 4 is a graph showing the protease activity of Bacillus subtilis TSD strain according to growth time.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention relates to a Bacillus subtilis TSD strain [accession number: KCCM12595P].

Bacillus subtilis according to the present invention (Bacillus subtilis) TSD strain [accession number: KCCM12595P] is a new strain of Bacillus subtilis derived from traditional fermented food. Although the Bacillus subtilis TSD strain in the present invention was isolated and identified from soybean paste, the acquisition route is not limited thereto.

The TSD strain isolated from doenjang through the following examples was found to have the nucleic acid sequence of SEQ ID NO: 1 as a result of 16S rRNA sequence analysis for microbial identification and classification. As a result of confirming the homology of the nucleic acid sequence through a blast search program provided by NCBI GenBank (http://www.ncbi.nin.gov), Bacillus subtilis NCIB 3610 strain and It could be confirmed that it was 99% identical.

Therefore, the microorganism of the present invention having the 16S rRNA nucleotide sequence of SEQ ID NO: 1 was named Bacillus subtilis TSD strain, and was deposited with the Korea Seed Association (KCCM) on October 2, 2019 (consignment). No. KCCM12595P).

The Bacillus subtilis TSD strain of the present invention is a Gram-positive bacterium, is a facultive anaerobe capable of growing in both aerobic and anaerobic conditions, and takes the form of a bacillus (see Fig. 1).

When the Bacillus subtilis TSD strain of the present invention is fermented with steamed soybeans, the content of amino acid nitrogen can be increased to produce soybean paste having more excellent flavor, so it is very useful for the production of soybean paste.

In one embodiment, the present invention provides a starter composition for fermentation comprising the Bacillus subtilis TSD strain (Bacillus subtilis TSD) strain.

In the present invention, the term "starter" refers to a preparation containing microorganisms involved in the fermentation of food, and refers to microorganisms that grow predominantly in mature foods by adding them during fermented food production. In the present invention, the starter refers to a preparation essentially containing a microorganism, that is, Bacillus subtilis TSD strain, which constantly adjusts the taste of fermented food and provides excellent sensory taste.

In one embodiment, the starter composition may be used for the manufacture of fermented food. The fermented foods may include fermented foods such as miso, kimchi, and cheese, but are not limited thereto, and may be prepared by various methods known in the art.

In one embodiment of the present invention, the Bacillus subtilis TSD strain may be included in a starter composition for fermentation during the manufacture of doenjang, and when using this, the taste of well-aged soybean paste can be maintained for a long period of time while helping the maturation of soybean paste. Helps to maintain.

In the present invention, the fermentation starter composition may be formulated as a liquid formulation, a powder formulation, or the like, and may be used, for example, as a powder formulation. The powder formulation can be obtained by collecting the cultured strain by centrifugation or the like and freeze drying. In the case of adding the starter strain to fermented food, it is common to add the wet-type cells isolated from the bacterial culture, but the liquid formulation has a disadvantage that it is difficult to store and maintain quality during distribution, making it difficult to distribute it as a product. On the other hand, powder formulations have the advantage of being easy to supply, maintain quality, and use. The starter composition may further include ingredients commonly added to the starter, such as Bacillus subtilis TSD strain, as well as soy flour, skim milk, amino acids, sugars, sugar alcohols, and starch.

Another aspect of the present invention relates to a method for preparing doenjang using Bacillus subtilis TSD strain [accession number: KCCM12595P] or a culture solution thereof.

In one embodiment, the present invention relates to a method for producing miso comprising a step of inoculating and fermenting a Bacillus subtilis TSD strain [accession number: KCCM12595P] or a culture solution thereof to steamed beans.

In the above, the present invention may further include the step of mixing the fermented soybeans in a mixture of meju and brine mixed with the fermented soybean. The meju mixture may be a mixture of meju and 13 to 16% brine. The meju is not particularly limited as long as it is prepared by a conventional method.

In another embodiment, the present invention comprises the steps of: (a) separating the cured meju from the brine after immersing the meju in brine; (b) inoculating steamed beans with Bacillus subtilis TSD strain [accession number: KCCM12595P] or a culture solution thereof and fermenting to prepare a seed fermentation product; (c) preparing miso by mixing the seed fermented product and the cured meju; (d) fermenting the miso prepared from step (c).

In the method for preparing soybean paste, the Bacillus subtilis TSD strain may be added as a strain itself or as a culture solution. The culture solution may include a culture solution obtained by culturing the strain in a liquid medium, a concentrated solution obtained by concentrating the culture solution, and the like.

^{The Bacillus subtilis TSD strain is inoculated at a concentration of 4X10 9} to 5X10 ¹⁰ CFU based on 1 kg of soybean paste, and if the concentration of the strain is less than the lower limit, miso with improved flavor can be obtained even if a strain isolated from soybean paste is used. Not possible, and if it exceeds the upper limit, antioxidant activity and functionality may be deteriorated.

When the Bacillus subtilis TSD strain is used for the production of doenjang, the miso contains a higher content of amino acid nitrogen, as well as improved taste and aroma such as umami, bitterness, and sweetness compared to doenjang without the strain added. Can be manufactured.

In the present invention, the Bacillus subtilis TSD strain may be added as a powder formulation or a liquid formulation when making doenjang in order to exhibit excellent sensory taste and an effect of increasing the amino acid nitrogen content.

Another aspect of the present invention is the step of fermenting after inoculating a mixture of shiitake mushroom powder and steamed beans with Bacillus subtilis TSD strain [Accession Number: KCCM12595P] or a culture solution thereof; It relates to a method of making mushroom miso.

In another embodiment, the present invention comprises the steps of: (a) separating the cured meju from the brine after immersing the meju in brine; (b) inoculating a mixture of shiitake mushroom powder and steamed soybeans with Bacillus subtilis TSD strain [accession number: KCCM12595P] or a culture solution thereof and fermenting to prepare a seed fermentation product; (c) preparing miso by mixing the seed fermented product and the cured meju; (d) fermenting the miso prepared from step (c) above; may be a method for producing shiitake mushroom miso.

In the method for preparing shiitake mushroom miso of the present invention, in the step (a), the meju is immersed in brine, and then the cured meju is separated from the brine. At this time, the salted meju may have a salinity of 11 to 14%, preferably a salinity of 11.5 to 14.5%, more preferably 12 to 13%.

In step (b), a mixture of shiitake mushroom powder and steamed soybeans is inoculated with Bacillus subtilis TSD strain or a culture solution thereof, followed by fermentation.

Shiitake (Lentinus edodes) is an edible mushroom belonging to the basidiomycete oyster and pine mushroom, and is grown in old trees or farms. In addition, shiitake mushrooms remove poison in the human body, help prevent hunger from feeling hunger, and have a curative effect by allowing blood to pass smoothly, and help blood circulation by suppressing the accumulation of cholesterol in the blood, and help blood circulation smoothly, such as arteriosclerosis and high blood pressure. It has a preventive effect and is known to be effective in treating anemia. In particular, it prevents rickets in pregnant women and has recently been in the spotlight as a low-calorie food, and among other ingredients of shiitake mushrooms, lentinan, an anticancer and antitumor polysaccharide substance, has been reported to increase immunity and suppress tumor growth.

An object of the present invention is to provide a method for preparing shiitake mushroom miso that can further enrich the taste and aroma of soybean paste while easily ingesting various physiologically active ingredients of shiitake mushrooms by adding shiitake mushroom powder in the process of preparing doenjang.

To this end, the shiitake powder is 1.0 to 5.0% by weight, preferably 1.25 to 3.25% by weight, more preferably 1.5 to 3.0% by weight, more preferably 1.6 to 2.9% by weight, more preferably based on the total weight of the miso paste. Preferably, it may be added in an amount of 1.75 to 2.75% by weight, more preferably 1.75 to 2.25% by weight, and most preferably 2% by weight.

If shiitake mushroom powder is added in excess of the upper limit when making soybean paste, the unique taste of shiitake mushrooms becomes stronger, and the thick taste and tasteful aftertaste are strengthened, which reduces the flavor of the miso. There is a problem that the content is lowered. In addition, as the volume occupied by shiitake mushrooms increased, the spawn did not grow well.

In addition, when shiitake mushrooms are added below the lower limit, the effect due to the addition of shiitake mushrooms becomes insignificant.

The shiitake powder and steamed beans of step (b) may be mixed in a weight ratio of 1: 8 to 10, preferably 1: 8.5 to 9.5.

In addition, the fermentation of step (b) may be a solid-state fermentation of the shiitake mushroom powder and the steamed soybean mixture at 28 to 35° C. for 35 to 55 hours.

And, in step (c), miso is prepared by mixing the seed fermented product obtained in step (b) and the cured meju obtained in step (a).

The seed fermented product for preparing shiitake mushroom miso is 10 to 25% by weight, preferably 15 to 25% by weight, more preferably 17.5 to 22.5% by weight, more preferably 19 to 21% by weight of the total weight of the doenjang. It can be mixed to make miso.

Specifically, the seed fermented product is a weight ratio of 0.5 to 3: 7 to 9.5 with the cured meju, preferably 0.5 to 2: a weight ratio of 8 to 9.5, more preferably 1 to 2: a weight ratio of 8 to 9, further Preferably, it may be mixed in a weight ratio of 1.5 to 2: 8 to 8.5.

The cured soybean paste may be mixed in an amount of 70 to 80% by weight based on the total weight of soybean paste, and the salinity may be adjusted by appropriately adding steamed soybeans.

The soybean paste prepared in step (c) is used to adjust the salinity of the soybean paste to 8 to 12%, preferably 9 to 11%, and more preferably 9 to 10% by using steamed soybeans or cured meju.

In the method for producing shiitake mushroom miso according to the present invention, when shiitake mushrooms are added in the form of a seed fermented product fermented in advance with Bacillus subtilis TSD strain or a culture solution thereof, the shiitake mushroom powder and Bacillus sub Compared with the doenjang prepared by adding the Tilis TSD strains, the flavor and umami are further enhanced, and the taste of shiitake mushrooms does not appear at all, so it is possible to manufacture soybean paste with higher preference. In addition, when the shiitake mushroom miso is prepared by the production method according to the present invention, miso having a higher amino acid nitrogen content and beta glucan content can be prepared.

Specifically, in the following examples, as a result of comparing the soybean paste inoculated with the Bacillus subtilis TSD strain and soybean paste in the production of soybean paste, the strain was a dominant species in the soybean paste inoculated with the strain while leading the fermentation and amino acid It was confirmed to increase the nitrogen content and beta glucan content. In addition, although not described in the following examples, a specific experiment was conducted to show that when the strain was added during the manufacture of soybean paste, the strain was present as a fermentation-initiating bacterium from the beginning of maturation of soybean paste, thereby increasing the antioxidant effect, antibacterial effect and anticancer effect in the doenjang It was confirmed through.

In addition, the present invention is prepared according to the above method, characterized in that it comprises the Bacillus subtilis (Bacillus subtilis) TSD strain [accession number: KCCM12595P] or a culture solution thereof, it provides a shiitake mushroom miso.

The shiitake mushroom miso contains 10 to 25% by weight of a seed fermentation product fermented with shiitake mushroom powder and steamed beans with Bacillus subtilis TSD strain, 70 to 80% by weight of cured meju, and 2.5 to 15% by weight of steamed beans I can. At this time, the shiitake mushroom powder may be included in an amount of 1.5 to 3.0% by weight based on the total weight of the shiitake mushrooms.

Hereinafter, the present invention will be described in detail by examples, but the present invention is not limited by the following examples.

<Example>

Example 1: Soybean paste fermentation seed selection

1-1. Preparation of the strain

Various types of soybean paste prepared by a conventional method for producing soybean paste were collected and used as strain-separated soybean paste samples. The soybean paste sample was collected, diluted 10-fold with 0.85% saline, inoculated with 0.1 ml of LB agar medium, and then plated with a glass rod. Thereafter, the plate was incubated for 1 day in an incubator at 37°C. Each of the resulting colonies was inoculated by stroke on an LB agar plate, and cultured at 37° C. for 1 day to separate strain colonies. The following various experiments were performed using the strain colonies.

1-2. Evaluation of protease activity

In order to select a fermentation seed suitable for doenjang fermentation, a strain capable of improving soybean protein degradation, that is, a strain having excellent protease activity, was selected.

Accordingly, among the various strain colonies, 25 strains were selected to compare protease activity.

Proteolytic enzyme activity of the strains was measured by inoculating each strain in TSB (tryptic soy broth) medium, incubating at 37° C. for 24 hours, collecting the culture supernatant, and then using Azocasein assay. The protease activity of each strain measured in this way is shown in Table 1 below.

Strain name Protease activity (unit/mL) Strain name Protease activity (unit/mL) SYD 523 BYD 633 HJD1 1,647 WSJD 711 SND 126 HJD2 796 YKD 588 YSD 1,176 SKD 1,664 GP6 238 KMD 1,622 GP8 462 GDD 571 J2 318 SJD 101 J3 432 EJD 715 J4 1,240 HMD 437 GG1 39 MHD 385 GG2 1,546 IGD 508 GG3 689 TSD 1,471

In Table 1, HJD1, SKD, KMD, TSD, YSD, J4 and GG2 strains, which are strains having a protease activity of 1,000 unit/mL or more, were selected for a total of 7 strains.

1-3. Fermentation flavor evaluation

In order to confirm the fermentation flavor when the soybean paste was prepared using the selected seven strains, it was attempted to evaluate the flavor of the fermented soybeans after fermenting soybeans with each strain.

To this end, the soybeans were sufficiently immersed in water, increased, and then ground. Each strain (HJD1, SKD, KMD, TSD, YSD, J4 and GG2) was inoculated with 1% (0.2 mL) culture solution to 20 g of the ground soybeans and fermented at 30° C. for 3 days. Thereafter, the number of viable cells and protease activity of the fermented steamed soybeans were measured, and the fermentation flavor was evaluated, and the results are shown in Table 2 below.

Strain name Protease activity
(unit/g) Viable cell count (cfu/g) Flavor HJD1 2,493 2.8×10 ⁹ The smell of stale meju and ammonia scent SKD 3,240 2.4×10 ⁹ There is a odor such as ammonia fragrance KMD 3,756 4.5×10 ⁹ Strong ammonia and scent TSD 3,612 3.6×10 ⁹ Soybean fermentation flavor YSD 3,635 1.2×10 ⁹ Meju flavor J4 1,236 1.3×10 ⁷ Bean fishy scent (nearly no fermentation scent) GG2 4,416 4.8×10 ⁸ Weak ammonia scent and fragrant scent

Looking at Table 2, the flavor of the TSD strain was the best, and the proteolytic activity and the number of viable bacteria were also stable, so the TSD strain was selected as a doenjang fermentation strain.

1-4. Morphological characteristics

In the analysis of the morphological characteristics of the TSD strain, the shape and color of the colonies of the strain were observed, and the strain was observed with an optical microscope after Gram stain kit (BD Co., USA). Is shown in FIG. 1.

As shown in FIG. 1, the isolated strain, the TSD strain, was a Gram-positive strain, and as a result of observation with an optical microscope, it was confirmed that it was in the form of a bacillus, and the colony color was light ivory.

1-5. Molecular biological identification of isolated strains

In order to investigate the molecular biological characteristics of the TSD strain selected in Example 1-3, chromosomal DNA was isolated from the strain culture solution, and then the chromosomal DNA was analyzed for the entire nucleotide sequence of the 16s rRNA gene to identify microorganisms. The identified strain was investigated to be a strain belonging to Bacillus subtilis , and the nucleotide sequence (16s rRNA sequence) of the identified strain is shown in SEQ ID NO: 1 (see FIG. 2).

As a result of comparing the nucleotide sequence of the strain with other strains registered in GenBank, it exhibited 99% homology with the standard strain Bacillus subtilis NCIB 3610 strain, and a strain having 100% homology with SEQ ID NO: 1 already exists. It was confirmed that it did not. Therefore, the present inventors deposited these strains with the Korea Seed Association (KCCM) on October 2, 2019, and received the following accession numbers.

Bacillus subtilis -accession number KCCM12595P (aka "TSD")

1-4. Optimization of culture conditions for isolated strains

It was intended to select the optimal culture conditions of the Bacillus subtilis TSD strain of the present invention. TSB (tryptic soy broth) medium was used as a medium for the TSD strain.

Culture temperature of TSD strain

In order to select the culture temperature condition of Bacillus subtilis TSD strain, the TSD strain was cultured at 30°C, 33°C, 35°C, 37°C, 39°C, 41°C and 50°C, respectively, and the number of viable cells and proteolysis of the TSD strain. The enzyme activity was measured and shown in Table 3 below. At this time, the protease activity was measured by the Azocasein assay method.

Growth temperature (℃) Viable bacteria count (Log cfu/mL) Protease activity (Unit/mL) 30 8.22 1,394 33 8.49 1,425 35 8.82 1,559 37 9.76 1,615 39 8.59 1,497 41 7.03 567 50 6.82 293

Looking at Table 3, the Bacillus subtilis TSD strain had relatively stable viable cell count and protease activity at 30 to 39°C, stable at 35 to 39°C, and showed the best results at 37°C.

Incubation time of TSD strain

In order to select the culture time condition of Bacillus subtilis TSD strain, while incubating the TSD strain at 37° C. for 120 hours, the number of viable cells and protease activity of the TSD strain according to the cultivation time were measured for each culture time, and FIG. 3 and It is shown in Figure 4.

3 is a graph showing the number of viable cells according to the growth time of the Bacillus subtilis TSD strain, and FIG. 4 is a graph showing the protease activity according to the growth time of the Bacillus subtilis TSD strain to be.

3 and 4, Bacillus subtilis TSD strain was excellent in viable cell count and protease activity when cultured at 37° C. for 4 to 24 hours, and even better when cultured for 8 to 20 hours, 16 In the case of culturing for a period of time, the best results were shown.

From these results, it can be confirmed that the Bacillus subtilis TSD strain of the present invention is cultured at 37° C. for 16 hours is the optimal culture condition for the strain.

Example 2: Preparation of Doenjang using TSD strain

2-1. Preparation of meju

After immersing conventional meju in brine for about 40 days, the meju was separated from the brine to prepare meju having a salinity of about 12%.

2-2. Preparation of the strain

The Bacillus subtilis TSD strain isolated and identified in Example 1 was plated on a plate and cultured in a 37°C incubator for 48 hours to isolate a single colony. The separated colonies were collected, added to a liquid MRS medium, and cultured at 37° C. and 200 rpm for 16 hours to activate the TSD strain.

2-3. Preparation of steamed beans

Soybeans (soybeans) were washed twice, and water was added to 1.4 times the volume of the beans, followed by soaking for 20 to 24 hours. Thereafter, the soaked beans were harvested and steamed for 2 to 3 hours in a steam pot at 100 to 150°C, then dried and cooled to 35°C, followed by grinding.

2-4. Manufacture of seed fermentation products

To 100 g of the steamed beans prepared in 2-3, ^{1 mL of the TSD strain culture solution (2×10 9} cfu/ml) prepared in 2-2 was inoculated and fermented at 30° C. for 48 hours to prepare a seed fermentation product.

2-5. Manufacturing of miso

Soybean paste was prepared by mixing the seed fermented product prepared in 2-4 at a weight ratio of 10% of the total weight of soybean paste. At this time, the salinity was adjusted to 10% using the steamed beans prepared in 2-3 and meju prepared in 2-1.

2-6. Fermentation of miso

The prepared soybean paste was fermented for 4 weeks under conditions of 30°C and 75% humidity.

Control: Preparation of TSD strain-free soybean paste

It was carried out in the same manner as in Example 2, but the doenjang was prepared without adding the TSD strain during the doenjang manufacturing process.

Test Example 1: Evaluation of soybean paste prepared using TSD strain

1-1. Analysis of quality characteristics of doenjang prepared using TSD strain

In order to compare the quality characteristics of the control group and the soybean paste prepared in Example 2, the pH, amino acid nitrogen content (mg%), total number of bacteria, and chromaticity of each doenjang sample were measured, respectively, and are shown in Table 4 below.

Specifically, the pH of the doenjang was measured with a pH meter (Model 410, thermo orion, USA) after adding 20 mL of distilled water to 10 g of the doenjang sample, stirring well.

To determine the amino acid nitrogen content of doenjang, add 100 mL of distilled water to 2 g of doenjang sample by Formal titration, stir for 1 hour, sufficiently mix, titrate to a pH of 0.4 with 0.1N NaOH solution, add 20 mL of neutral formalin solution, and then 0.1 Titration was performed with N NaOH solution to pH 8.4 to measure the amino acid nitrogen content (NH2-N).

For the total number of bacteria in the doenjang, take 10g of the doenjang sample, dilute 10 times, take 100 ul of the diluted sample, spread on a plate agar (Difco co.), and incubate at 37°C for 24 hours to obtain the number of colonies per mL. It was expressed as a forming unit (Colony forming unit, CFU).

The chromaticity of doenjang is corrected with a standard color plate (L:96.85, a:-0.28, b: -0.30) using a color difference meter (Chroma meter, Cm-3500d, Monolta, Japan), and then L value (brightness), a Values (redness) and b-values (yellowness) were measured five or more times, respectively, and expressed as averages and deviations.

division pH Amino acid nitrogen (mg%) Total bacteria
(cfu/mL) Chromaticity Brightness (L value) Redness (a value) Yellowness (b value) Control 4.40 217 1×10 ¹¹ 52.83 10.02 21.75 Example 2 4.43 278 9×10 ¹⁰ 50.60 10.70 22.80

Looking at Table 4, it can be seen that the content of amino acid nitrogen is significantly higher in the case of the miso according to Example 2 of the present invention compared to the control (general miso).

1-2. Sensory evaluation of doenjang prepared using TSD strain

For the control group and the doenjang prepared in Example 2, sensory evaluation was performed and shown in Table 5 below.

For the sensory evaluation, 10 experts in taste evaluation of doenjang were composed of a panel, and evaluation was performed according to a 9-point scale method (strength: 1. very bad; 5. moderate; 9. very good). At this time, the SPSS 12.0 statistical program was used for statistical processing, and the significance was verified at the significance level p<0.05 by the DMR-test (Duncan multiple range test).

division color flavor incense Salty Umami bitter sweetness Comprehensive symbol map Control 6.6 5.6 5.8 6.4 4.8 4.8 5.6 5.0 Example 2 6.8 6.4 6.6 6.8 6.2 6.0 6.2 6.7

Looking at Table 5, in the case of the miso according to Example 2 of the present invention, overall preference was increased compared to the control (general miso), especially the umami was remarkably improved, the flavor was further improved, and the taste such as bitter taste and sweet taste also You can see that it has improved.

Preliminary Test Example 1: Fermentation Characteristics of Shiitake Mushrooms

1-1. Preparation of Doenjang Prepared by Adding Shiitake Mushroom Powder

Shiitake mushrooms contain 50% of carbohydrates, pentosan, and 20% of protein. They contain a high content of amino acids such as glutamine, allicin, and leucine in addition to vitamin B2, and the content of vitamin C is known to be the highest among mushrooms. In addition, shiitake mushrooms not only contain a lot of physiologically active ingredients useful for health promotion, but also contain a large amount of savory ingredients, so if you add shiitake mushrooms in the process of making miso, you can further improve the flavor of the miso. Expected.

Accordingly, in the case of making doenjang using shiitake mushrooms, it was attempted to find out the optimal range of addition amount of shiitake mushrooms in which the taste and aroma of shiitake mushrooms and soybean paste can be most harmonized. For this purpose, in the doenjang prepared by adding shiitake mushrooms, the composition of doenjang according to the amount of shiitake mushrooms added is summarized in Table 6 below.

division Miso raw material (Meju) (%) Increased beans (%) Shiitake Mushroom Powder (%) Sum(%) Control 83.3 16.7 0 100 Shiitake 1.0% 83.3 15.7 1.0 100 1.5% shiitake mushrooms 83.3 15.2 1.5 100 Shiitake Mushroom 2.0% 83.3 14.7 2.0 100 Shiitake 2.5% 83.3 14.2 2.5 100 Shiitake 3.0% 83.3 13.7 3.0 100 3.5% shiitake mushrooms 83.3 13.2 3.5 100

Analysis of quality characteristics of doenjang according to the amount of shiitake mushroom

After preparing shiitake mushroom soybean paste with the above composition, in order to compare the quality characteristics of soybean paste according to the amount of shiitake mushroom added, the pH, amino acid nitrogen content (mg%), total number of bacteria, and chromaticity of each doenjang sample were measured. After that, it is shown in Table 7 below.

division pH Amino acid nitrogen (mg%) Total bacteria
(cfu/mL) Chromaticity brightness Redness Yellowness Control 4.40 217 1×10 ¹¹ 52.83 10.02 21.75 Shiitake 1.0% 4.38 218 7×10 ⁹ 51.76 9.87 21.07 1.5% shiitake mushrooms 4.36 230 9×10 ⁹ 50.01 9.61 20.48 Shiitake Mushroom 2.0% 4.27 235 5×10 ¹⁰ 48.69 9.36 19.80 Shiitake 2.5% 4.22 249 7×10 ¹⁰ 48.27 9.43 19.54 Shiitake 3.0% 4.19 237 6×10 ⁹ 48.21 9.32 18.84 3.5% shiitake mushrooms 4.15 229 1×10 ⁹ 48.12 9.15 18.57

Looking at Table 7 above, it was confirmed that the amino acid nitrogen content of the doenjang sample prepared by adding shiitake mushrooms in an amount of 1.5 to 3.0% by weight was higher than that of other experimental groups (control group, shiitake 1.0% experimental group, shiitake 3.5% experimental group). I can.

Sensory evaluation of doenjang according to the amount of shiitake mushrooms added

For the control group and the doenjang prepared in Example 2, sensory evaluation was performed and shown in Table 8 below.

For the sensory evaluation, 10 experts in taste evaluation of doenjang were composed of a panel, and evaluation was performed according to a 9-point scale method (strength: 1. very bad; 5. moderate; 9. very good). At this time, the SPSS 12.0 statistical program was used for statistical processing, and the significance was verified at the significance level p<0.05 by the DMR-test (Duncan multiple range test).

division color flavor incense Salty Umami bitter sweetness Synthesis
Preference Control 6.6 5.6 5.8 6.4 4.8 4.8 5.6 5.0 Shiitake 1.0% 6.6 5.7 5.9 6.5 5.0 4.8 5.6 5.1 1.5% shiitake mushrooms 6.8 6.4 6.4 6.6 6.0 6.4 6.0 6.1 Shiitake Mushroom 2.0% 6.8 7.4 7.2 7.2 7.2 7.0 6.6 7.2 Shiitake 2.5% 6.8 7.2 6.8 6.4 7.0 6.8 6.6 6.8 Shiitake 3.0% 5.8 6.6 6.6 6.4 7.0 6.8 6.4 6.2 3.5% shiitake mushrooms 5.1 5.4 5.5 6.2 6.5 6.3 5.5 5.2

As shown in Table 8, the doenjang of the control group was slightly bitter, the umami was weak, and the taste and aroma were strong, but the doenjang prepared by adding shiitake mushrooms was confirmed to have a strong umami characteristic of shiitake mushrooms.

The acceptability of doenjang added with 1.5 to 3.0% by weight of shiitake mushrooms was high, and in the case of doenjang with 1.0% by weight of shiitake mushrooms, the effect of adding shiitake mushrooms was insignificant, and the effect of adding shiitake mushrooms at 3.5% by weight was high. In the case of soybean paste, it was confirmed that the taste of shiitake mushrooms became stronger, so that the thick taste was strong, and the aftertaste felt was strengthened, so the preference was markedly lowered.

When shiitake mushrooms were added in an amount of 2.0% by weight when making doenjang, the umami and flavor of the doenjang were found to be the most harmonious while not producing a unique scent of shiitake mushrooms.

Example 3: Preparation of Shiitake Mushroom Doenjang Using Shiitake Mushroom Powder and TSD Strains

It was carried out in the same manner as in Example 2, but in the step of adding the TSD strain culture solution to the steamed beans of 2-4., shiitake mushroom powder was further added to prepare a seed fermented product. At this time, the shiitake mushroom powder was added in an amount of 10% by weight based on the total weight of the steamed beans (2% by weight based on the total weight of soybean paste).

In addition, in the step of mixing the seed fermented product and meju in 2-5., the seed fermented product was mixed with 20% by weight of the total weight of the soybean paste to prepare shiitake mushroom miso.

Comparative Example 1: Preparation of Shiitake Mushroom Soybean Paste Using Shiitake Mushroom Powder

It was carried out in the same manner as in Example 2,

The step of preparing the seed fermentation of 2-4. is not carried out,

2-5. In step 2-4, instead of the seed fermentation product prepared in step 2-4, the steamed beans prepared in step 2-3 were mixed with the meju prepared in step 2-1 to prepare soybean paste, and then 2% by weight of shiitake powder based on the total weight of the soybean paste. By mixing, shiitake mushroom miso was prepared.

Comparative Example 2: Preparation of Shiitake Mushroom Doenjang Using Shiitake Mushroom Powder and TSD Strains

It was carried out in the same manner as in Example 2,

In the step of mixing the seed fermented product and meju in 2-5, the seed fermented product is mixed with 10% by weight of the total weight of soybean paste to prepare soybean paste, and then 2% by weight of shiitake mushroom powder is mixed with respect to the total weight of the doenjang. Mushroom miso was prepared.

Comparative Example 3: Preparation of Shiitake Mushroom Soybean Paste Using Sterilized Shiitake Mushroom Ferment

It was carried out in the same manner as in Example 3,

The seed fermented product prepared in 2-4 was sterilized at 121° C. for 15 minutes to prepare shiitake mushroom miso.

The manufacturing method and composition of the shiitake mushroom miso of Examples and Comparative Examples 1 to 3 are summarized and shown in Tables 9 and 10 below.

division Remark Control (high salt) 12% salinity regular miso Control (low salt) Normal miso with 9% salinity Example 2 When making soybean paste, fermented seed fermented from steamed soybeans with TSD strain is added. Example 3 When making soybean paste, shiitake mushroom powder and steamed soybeans are fermented with TSD strains, and shiitake & spawn fermentation products are added. Comparative Example 1 Addition of shiitake mushroom powder when making miso Comparative Example 2 Shiitake mushroom powder and TSD seed fermentation products are added to the bean paste manufacturing process. Comparative Example 3 When making soybean paste, shiitake mushroom powder and steamed soybeans are fermented with TSD strain and added after sterilizing the fermented shiitake & spawn products.

division Meju (%) Steamed Beans (%) Total soybean ingredients (%) Shiitake Mushroom Powder (%) Seed (TSD strain) fermented product (%) Shiitake & spawn fermentation product (%) Sum(%) Control (high salt) 100 (12% salinity) - 100 - - - 100 Control (low salt) 75 (9% salinity) 25 100 - - - 100 Example 2 75 15 90 - 10 - 100 Example 3 75 5 80 - - 20 100 Comparative Example 1 75 23 98 2 - - 100 Comparative Example 2 75 13 88 2 10 - 100 Comparative Example 3 75 5 80 - - 20 100

Comparative Examples 4 to 9: Preparation of Shiitake Mushroom Miso with Different Strains

Conducted in the same manner as in Example 4, but instead of the TSD strain, a Bacillus subtilis strain or Aspergillus duckweed strain, which exhibits excellent effect on fermentation of miso, was pre-sold from the Korea Microbiological Conservation Center or the Institute of Agricultural Biotechnology to prepare shiitake mushroom miso I did. The strains used in Comparative Examples 4 to 9 of the present invention are summarized and shown in Table 11 below.

division Strain Strain name Deposit number Organization name Example 3 Bacillus subtilis TSD KCCM12595P Korea Microorganism Conservation Center Comparative Example 4 Bacillus subtilis J46 KCCM12388P Korea Microorganism Conservation Center Comparative Example 5 Bacillus subtilis CSY388 KACC91564P Institute of Agricultural Biotechnology Comparative Example 6 Bacillus subtilis AFY-2 KACC91988P Institute of Agricultural Biotechnology Comparative Example 7 Aspergillus Duck Ash M385 KACC93210P Institute of Agricultural Biotechnology Comparative Example 8 Aspergillus Duck Ash CJ KY KCCM11302P Korea Microorganism Conservation Center Comparative Example 9 Aspergillus Duck Ash CJ KG KCCM11301P Korea Microorganism Conservation Center

Test Example 2: Characterization of Shiitake Mushroom Doenjang Using Shiitake Mushrooms and TSD Strains

Each of the Shiitake Mushroom Doenjang samples prepared in Control, Example 3 and Comparative Examples 1 to 9 were fermented at 30° C. (75% moisture) for 4 weeks and then analyzed for quality characteristics.

2-1. Analysis of Quality Characteristics of Shiitake Mushroom Doenjang

Using the Shiitake Mushroom Soybean Paste Samples of Control, Example 3 and Comparative Examples 1 to 9, pH, amino acid nitrogen content (mg%), total number of bacteria, number of Bacillus bacteria, seed dominance rate (%) and beta glucan content (mg %) was measured and shown in Table 12 below.

division pH Amino acid nitrogen (mg%) Total bacteria
(cfu/mL) Bacillus bacteria
(cfu/mL) Spawn dominance rate
(%) Beta Glucan
(mg%) Control (high salt) 4.92 163 1×10 ¹¹ 1×10 ⁹ - - Control (low salt) 4.25 254 1×10 ¹¹ 7×10 ⁸ - - Example 3 4.47 419 2×10 ⁹ 2×10 ⁹ 86 939 Comparative Example 1 4.27 235 5×10 ¹⁰ 5×10 ⁸ - 823 Comparative Example 2 4.35 364 9×10 ⁸ 1×10 ⁹ 59 810 Comparative Example 3 4.45 283 2×10 ⁸ 2×10 ⁸ - 848 Comparative Example 4 4.38 376 2×10 ⁹ 1×10 ⁹ - 853 Comparative Example 5 4.32 384 3×10 ⁹ 2×10 ⁹ - 856 Comparative Example 6 4.43 360 1×10 ¹⁰ 3×10 ⁹ - 832 Comparative Example 7 4.55 359 5×10 ⁸ 4×10 ⁸ - 865 Comparative Example 8 5.58 377 1×10 ⁹ 2×10 ⁸ - 883 Comparative Example 9 4.62 345 2×10 ⁹ 8×10 ⁸ - 847

Looking at Table 12, it can be seen that in the case of the shiitake mushroom miso of Example 3, the content of amino acid nitrogen is significantly higher than that of the shiitake mushroom miso of Comparative Examples 1 and 3 prepared by different manufacturing methods. In addition, in the case of Comparative Example 2 in which shiitake mushroom powder and TSD strain were pre-fermented and then mixed with meju, each prepared shiitake mushroom miso without mixing with meju, the seed dominance rate and beta glucan content were significantly lower than those of the example.

In addition, in the case of the shiitake mushroom doenjang of Example 3, compared to the shiitake mushroom doenjang of Comparative Examples 4 to 9 prepared using another strain instead of the TSD strain, the amino acid nitrogen content, the seed dominance rate and the beta glucan content were higher.

2-2. Sensory evaluation of shiitake mushroom miso

Sensory evaluation was performed on each of the shiitake mushroom miso samples prepared in the control group, Example 3, and Comparative Examples 1 to 9, and are shown in Table 13 below.

For the sensory evaluation, 10 experts in taste evaluation of doenjang were composed of a panel, and evaluation was conducted according to the 9-point scale (strength: 1. very bad; 5. moderate; 9. very good) At this time, the SPSS 12.0 statistical program was used for statistical processing, and the significance was verified at the significance level p<0.05 by the DMR-test (Duncan multiple range test).

division color flavor incense Salty Umami bitter sweetness Synthesis
Preference Control (high salt) 6.4 5.4 5.6 4.6 5.0 5.8 5.4 5.2 Control (low salt) 5.0 5.0 5.6 6.0 4.8 5.6 5.4 5.0 Example 3 6.8 7.8 7.7 7.2 8.4 7.1 7.2 7.8 Comparative Example 1 5.6 6.2 6.0 6.0 6.0 5.8 5.4 6.0 Comparative Example 2 7.2 6.8 6.8 6.6 6.8 6.6 6.2 6.8 Comparative Example 3 5.6 6.0 6.2 6.6 6.0 4.8 5.6 5.6 Comparative Example 4 6.4 6.2 6.6 6.8 6.2 5.8 5.8 6.9 Comparative Example 5 6.0 6.8 6.2 6.6 6.0 5.8 5.4 6.8 Comparative Example 6 7.2 7.1 6.8 6.6 6.8 6.0 6.0 7.2 Comparative Example 7 6.8 7.1 7.2 6.8 7.1 6.2 6.0 7.5 Comparative Example 8 6.8 6.8 7.2 6.4 6.8 6.2 5.8 7.1 Comparative Example 9 6.6 6.6 6.8 6.4 6.6 5.6 5.4 6.8

Looking at Table 13, in the case of the shiitake mushroom miso of Example 3, the shiitake mushroom miso of Comparative Examples 1 to 3 prepared by different manufacturing methods, and the shiitake mushrooms of Comparative Examples 4 to 9 prepared using other strains instead of TSD strain It can be seen that the umami and flavor are remarkably superior to that of miso.

Although the present invention has been described as a preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the gist and scope of the invention. In addition, the appended claims include such modifications or variations that fall within the gist of the present invention.

<110> Jeonnam Bioindustry Foundation <120> A Novel Bacillus subtilis TSD and methods for preparing Doenjang using the same <130> HPC-9055 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1509 <212> DNA <213> Bacillus subtilis <400> 1 acgctggcgg cgtgcctaaa tacatgcaag tcgagcggac agatgggagc ttgctccctg 60 atgtttagcg gcggacgggt gagtaacacg tgggtaacct gcctgtaaga ctgggataac 120 tccgggaaac cggggctaat accggatggt tgtttgaacc gcatggttca gacataaaag 180 gtggcttcgg ctaccactta cagatggacc cgcggcgcat tagctagttg gtgaggtaac 240 ggctcaccca aggcgacgat gcgtagccga cctgagaggg tgatcggcca cactgggact 300 gagacacggc ccagactcct acgggaggca gcagtaggga atcttccgca atggacgaaa 360 gtctgacgga gcaacgccgc gtgagtgatg aaggttttcg gatcgtaaag ctctgttgtt 420 agggaagaac aagtgccgtt caaatagggc ggcaccttga cggtacctaa ccagaaagcc 480 acggctaact acgtgccagc agccgcggta aatacgtagg tggcaaagcg ttgtccggaa 540 ttattgggcg taaaagggct cggcaggcgg tttcttaagt ctgatgtgaa agcccccggc 600 tcaaccgggg agggtcattg gaaactgggg aacttgagtg cagaagagga gagtggaatt 660 ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga aggcgactct 720 ctggtctgta actgacgctg aggagcgaaa gcgtggggag cgaacaggat tagataccct 780 ggtagtccac gccgtaaacg atgagtgcta agtgttaggg ggtttccgcc ccttagtgct 840 gcagctaacg cattaagcac tccgcctggg gagtacggtc gcaagactga aactcaaagg 900 aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc aacgcgaaga 960 accttaccag gtcttgacat cctctgacaa tcctagagat aggacgtccc cttcgggggc 1020 agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg atcttagttg ccagcattca gttgggcact ctaaggtgac 1140 tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc 1200 tgggctacac acgtgctaca atggacagaa caaagggcag cgaaaccgcg aggttaagcc 1260 aatcccacaa atctgttctc agttcggatc gcagtctgca actcgactgc gtgaagctgg 1320 aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca 1380 ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg gtgaggtaac cttttaggag 1440 ccagccgccg aaggtgggac agatgattgg ggtgaagtcg taacaaggta gccgtatcgg 1500 aaggtgcgg 1509

Claims (7)

delete delete delete (a) separating the cured meju from the brine after immersing the meju in brine;
(b) Bacillus subtilis TSD strain [Accession No.: KCCM12595P] or a culture solution thereof was inoculated into a mixture in which shiitake mushroom powder and steamed beans were mixed in a weight ratio of 1: 8 to 10, and 28 to 35 ℃ Solid-phase fermentation for 35 to 55 hours to prepare a seed fermented product;
(c) preparing miso by mixing the seed fermented product and the cured meju;
(d) fermenting the miso prepared from step (c). The method of claim 4,
The shiitake mushroom powder is a method for producing shiitake mushroom miso, characterized in that it is added in an amount of 1.5 to 3.0% by weight based on the total weight of the miso. delete delete

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