JP5944585B2 - Method for producing Korean-style miso tama miso using Bacillus amyloliquefaciens CJ3-27 and Aspergillus oryzae CJKG which are novel strains - Google Patents

Description

The present invention uses Bacillus amyloliquefaciens CJ 3-27 and Aspergillus oryzae CJ KG, which are strains excellent in proteolytic activity isolated from traditional miso balls. The present invention relates to a method for producing a Korean-style miso ball miso.

For Koreans who traditionally have a vegetarian diet, soy has been used as a source of protein and essential fatty acids because it contains high protein and fat. In particular, miso is a fermented food produced using miso balls fermented with soybeans as the main ingredient, and various anti-cancer, hypoglycemic, suppression of mutations by various physiologically active substances produced through the fermentation process, Nutritionally superior indigenous Korea that has various physiological activities such as thrombolytic ability and contains antioxidant components such as tocopherol, isoflavones and phenolic acids transferred from soybean It is a traditional fermented food.

Traditional Korean traditional miso is made with soybeans, made into miso balls, shaped into bricks or rectangles, naturally fermented, soaked in saline and aged for 6-12 months, and then soy sauce is taken out. It is produced by aging solids.

Although traditional Korean traditional miso has excellent nutrition and physiological activity in terms of functionality as described above, its production method varies slightly from region to region. Since fermentation is carried out by microorganisms, not only the quality variation is severe, but also there is a problem that the quality of taste is not constant because various harmful microorganisms affect the fermentation.

As examples of the harmful microorganisms, Aspergillus flavus produces aflatoxin that is harmful to human body (mycotoxin), and Bacillus cereus produces emetic toxin. Or produces diarrheic toxins. In addition, histamine, which is a representative biogenic amine that can be generated by microorganisms during fermentation in protein-containing foods, is difficult to breathe, fever flushing, sweating, headache when ingested at a concentration above a predetermined level. Causes diarrhea, convulsions, increased and decreased blood pressure, urticaria.

In order to grow traditional Korean traditional miso as a global food, establish a standardized manufacturing method to ensure food safety, minimizing taste quality variation and sensory superior quality products It can be said that the development of the process for this is essential.

In recent years, in order to publicize the excellence of miso for the globalization of Korean food, research on the standardization of the manufacturing method of miso and the nutritional components and functional effects of miso has been actively conducted.

As part of the research, we screened and developed excellent strains in traditional soy sauce, and developed a manufacturing method that overcomes the problems that appear in traditional soy sauce by inoculating such excellent strains as starters. . However, most of the research is related to a method for producing an improved miso bean-shaped miso-tamago, rather than research on the method of making miso from a traditional Korean-style miso-tamago in bulk. Although improved miso has advantages in terms of shortening the fermentation and ripening period and quality control, it is technically impossible to realize the rich taste of traditional miso expressed in traditional miso onion miso There are serious limitations.

The present invention solves the quality problems of traditional Korean traditional miso and has a sensory superior taste quality that improved miso does not have. The purpose is to develop and provide the technology to manufacture.

Therefore, the object of the present invention is to identify Bacillus amyloliquefaciens CJ 3-27 and Aspergillus oryzae (Aspergillus oryzae), which are isolates identified from traditional miso balls and are excellent in proteolytic enzyme activity. Aspergillus oryzae) CJ KG.

Another object of the present invention is to provide a method for producing a Korean-style miso tama miso using the novel strain.

In order to achieve the above-mentioned object, the present invention identifies Bacillus amyloliquefaciens CJ 3-27, which is an isolated strain identified from traditional miso tamago and has excellent proteolytic enzyme activity. Aspergillus oryzae CJ KG is provided.

The present invention also includes a step of selecting, washing and immersing soybeans, a step of cooling the soybeans after steaming, and a step of culturing Bacillus amyloliquefaciens CJ3-27 to produce a culture solution. Chopping after inoculating the chilled soybean with the culture solution of Bacillus amyloliquefaciens CJ 3-27, molding the chopped cooled soybean, and the surface of the molded miso ball A step of culturing the Aspergillus oryzae CJ KG to produce a seed meal, spraying the seed miso on the formed miso egg and inoculating it, and fermenting the fermented miso A step of salting the balls for salt water separation, and a step of aging the salt water-separated miso To provide a process for the production of miso ball miso of Korean-style to be.

The present invention provides a Korean-style miso tama miso that has a uniform taste quality while shortening the fermentation period and aging period of the miso tama during the production of traditional Korean-style miso, and ensuring safety against pathogenic microorganisms. It has an excellent effect in manufacturing.

It is a diagram according to the process for manufacture of the Korean-style miso ball miso of this invention. It is a figure which shows the degree of the crack outside the miso ball by the presence or absence of cooling of steamed soybeans. It is a figure which shows the reduction | decrease amount of the water | moisture content by the degree of the external crack of the miso ball in a fermentation process. It is a figure which shows the description analysis result with respect to the detailed taste attribute and intensity | strength of each miso.

Best Mode for Carrying Out the Invention

Hereinafter, the present invention will be described in detail.

The present invention identifies Bacillus amyloliquefaciens CJ 3-27 and Aspergillus oryzae CJ, which are isolates identified from traditional miso balls and are excellent in proteolytic enzyme activity. KG is provided.

Specifically, in the present invention, various strains were isolated from traditional Korean miso balls, and then, among others, Bacillus spp. Strains having excellent protease activity were isolated. The Bacillus strains isolated by 16s rDNA base sequence analysis (16srDNA sequencing) were identified. The results are as follows.

The isolated Bacillus strain was identified as Bacillus amyloliquefaciens by the phylogenetic tree based on the above 16srDNA sequence analysis (SEQ ID NO: 1). This strain having excellent antibacterial activity was named Bacillus amyloliquefaciens CJ 3-27, and was deposited with the Korean Preservation Center on October 22, 2012 (Accession No. KCCM11317P).

In addition, the present invention isolates various fungi from traditional Korean miso balls, and then isolates an Aspergillus sp. Strain that excels in proteolytic enzyme production efficiency. Identification of Aspergillus strains isolated by 18s rDNA sequence analysis (18s rDNA sequencing) was performed. The results are as follows.

The isolated Aspergillus genus strain was identified as Aspergillus oryzae by the phylogenetic tree based on 18srDNA base sequence analysis as described above (SEQ ID NO: 2). This strain having excellent proteolytic enzyme production efficiency was named Aspergillus oryzae CJ KG and deposited on September 27, 2012 at the Korean Microbiology Preservation Center (Accession No. KCCM11301P).

The present invention also relates to a strain excellent in the production efficiency of Bacillus amyloliquefaciens CJ 3-27 and a proteolytic enzyme, which are excellent in the activity of proteolytic enzymes isolated from the traditional miso balls. A method for producing a Korean-style miso tama miso using Aspergillus oryzae CJ KG is provided.

More specifically, the present invention includes a step of selecting, washing and immersing soybeans, a step of cooling the soybeans after steaming, and culturing Bacillus amyloliquefaciens CJ 3-27 to obtain a culture solution. Chopping after inoculating the chilled soybean with the culture medium of Bacillus amyloliquefaciens CJ 3-27, molding the chopped cooled soybean, and the molded Drying the surface of the miso ball, cultivating Aspergillus oryzae CJ KG to produce the seed cake, spraying the seed cake on the molded miso egg and inoculating the seed cake, and the fermentation And a step of salting the prepared miso balls to separate the salt water, and aging the salt water separated miso To provide a method of manufacturing miso ball miso Korean with excellent top quality.

The steamed soybean is preferably cooled to a predetermined temperature or higher, and more preferably is cooled to a temperature of 50 to 65 ° C.

The cooling step not only blocks the growth potential of Bacillus cereus, which is a pathogenic microorganism that cannot grow above 50 ° C., and is also a spore-like Bacillus amyloliquefaciens strain CJ 3-27 It suppresses the growth of Bacillus cereus by promoting germination and predominating with early bacteria. In addition, the cooling step induces cracks outside the miso balls during the surface drying process. The cracks on the outside of the miso ball allow the aerobic bacteria and mold to grow sequentially from the surface of the miso ball to the inside of the miso ball by inducing the inflow of air from the surface of the miso ball to the inside of the miso ball. The enzyme titer of miso tama is increased by the enzyme secretory action due to the growth of the strain to shorten the fermentation period and ripening period of the miso tama.

It is preferable to use the Bacillus amyloliquefaciens CJ 3-27 strain as a culture solution cultured in a spore state. The culture solution of the strain is preferably inoculated with 0.1% to 5% of the cooled steamed soybeans based on the weight of the raw soybean grains.

The culture medium for culturing the bacteria is preferably a soy sauce medium. The soy sauce medium is selected from the group consisting of 1-10% soy sauce selected from the group consisting of Korean-style soy sauce, forged soy sauce and mixed soy sauce, and glucose, sucrose, galactose, and maltose. It is produced by mixing 0.1 to 10% of a sugar source.

In a preferred embodiment of the present invention, a purely isolated and stored Bacillus amyloliquefaciens CJ 3-27 strain is inoculated into a soy sauce medium (manufactured soy sauce + glucose) under a temperature condition of 30 ° C. to 42 ° C. The culture solution of Bacillus amyloliquefaciens CJ 3-27 strain was produced by culturing for 20 to 42 hours until spores were produced.

In the present invention, chopping means a step of finely grinding soybean, and the size is not particularly limited.

The shape of the miso ball covers a solid shape such as a rectangular parallelepiped shape, a rectangular parallelepiped shape, a columnar shape, a donut shape, an oval shape, and a spherical shape. Preferably, the miso ball is a rectangular parallelepiped having a size of 2 × 2 cm ³ to 30 × 30 cm ³ and a weight of 100 g to 5 kg.

In the present application, the term surface drying means drying the molded miso balls.

The surface drying is preferably performed by hot air drying under a temperature condition of 50 ° C. to 65 ° C. for 5 to 24 hours. The surface drying step has an effect of suppressing external contamination and growth of harmful microorganisms in the air during the fermentation period by drying the moisture outside the miso balls within a short time.

The Aspergillus oryzae CJKG seed meal is preferably inoculated in an amount of 0.01 to 1% based on the weight of raw soybean grains.

Fermentation is preferably performed for 7 to 30 days under a temperature condition of 10 ° C to 45 ° C.

After the fermentation, 18-25% salt water was added to the fermented miso balls 1.8-2.5 times the weight of the miso balls and salted for 10-50 days to separate the salt water.

Miso, which has been subjected to salt water separation, is aged at room temperature for 4 to 6 months, maximizing the surface in direct contact with air.

Hereinafter, the contents of the present invention will be described in more detail with reference to examples. However, these examples are presented only to help understanding of the contents of the present invention, and the scope of rights of the present invention should not be construed to be limited by these examples.

[Example]
Example 1: Isolation and identification of strains from traditional miso tamales Traditional miso recovered from traditional food manufacturers located in Gyeonggi, Gangwon, Chungcheongbuk, Gyeongsangbuk, and Jeollanam-do to isolate strains A ball was used as a sample.

After diluting traditional miso balls in sterilized water, the bacteria are spread on a plate counting agar (PCA) medium and cultured at 37 ° C., and a plate on which about 30-50 colonies appear is selected. Pure separation was performed by performing three-stage separation using a platinum loop. Molds were applied to potato dextrose agar (PDA) and aseptically separated from the colonies that appeared while culturing at 28 ° C. A total of 893 strains were isolated, including 773 strains of bacteria and 120 strains of mold.

In order to select a strain having a high proteolytic enzyme activity from the isolates, after inoculating on a soy bean slurry agar medium containing 1% skim milk, the bacterium was 37 ° C. and the mold was 28 Culturing was carried out at 3 ° C. for 3 to 7 days. The size of a clear zone that appears thereafter was measured, and one kind of bacteria and one kind of bi were selected from bacteria having high enzyme activity. The identification of the strain was performed on a purely isolated strain. Bacteria were identified by 16s rDNA sequencing (16s rDNA sequencing), and molds were identified by 18s rDNA sequencing (16s rDNA sequencing).

Example 2: Production of Bacillus amyloliquefaciens CJ 3-27 strain culture solution Soy sauce (1% to 10%) and glucose (0.1% to 5%) were mixed, and then the pH was adjusted with edible sodium hydroxide. The soy sauce medium was manufactured by correcting to neutrality and sterilizing at 120 ° C. for 15 minutes. One sterilized Bacillus amyloliquefaciens CJ 3-27 colony, which had been stored purely in Nutrient agar, was inoculated into the sterilized soy sauce medium. This was cultured for 20 to 42 hours under the temperature condition of 30 ° C. to 42 ° C. until spores were produced to produce a Bacillus amyloliquefaciens CJ 3-27 culture solution. Table 1 shows the results of changes in the number of bacteria and the number of spores according to the culture time of the Bacillus amyloliquefaciens CJ 3-27 strain.

Example 3: Production of Aspergillus oryzae CJ KG strain seed meal Purified water is added to wheat bran, water is added so that the water content is in the range of 40% to 60%, and sterilized at 121 ° C for 15 to 30 minutes. A platinum loop of Aspergillus oryzae CJ KG strain, which has been cooled to 40 ° C. or lower and purely cultured in potato dextrose agar (PDA) medium, is then inoculated at a temperature of 25 ° C. to 40 ° C. for about 5 days to After stationary culture for 10 days to produce spores, the seeds were pulverized under aseptic conditions.

Example 4: Manufacture of Korean-style miso tama miso (1) Selection and washing of soybeans Soybeans were washed through a selection step for removing foreign substances from soybeans, and then soaked with water three times the weight of soybeans.

(2) Steaming soybeans and removing the soybean water soaked in cooling water and steaming, the mixture was cooled to a temperature of 50 to 65 ° C. within 30 minutes using a blower.

(3) Inoculation of Bacillus amyloliquefaciens CJ 3-27 strain and shaping of miso ball The strain culture solution produced according to Example 2 was added to the cooled steamed soybeans in an amount of 0. After inoculating to 1 to 5% and mixing, chopping was performed to form miso balls in a rectangular parallelepiped shape with a weight of 1.2 to 2 kg.

(4) Surface drying of molded miso balls and inoculation with Aspergillus oryzae CJ KG strain The surface of the molded miso balls is dried at 50 to 65 ° C. for 10 to 24 hours using a hot air dryer. It was. The surface-dried miso balls were taken out from the dryer, and the strain culture solution produced according to Example 3 was inoculated so as to be 0.01 to 1% based on the weight of the raw soybean grains.

(5) Fermentation, salting and aging of miso balls After the produced miso balls are fermented at a temperature of 10 to 45 ° C. for about 7 to 30 days, the weight of the miso balls is 1.8 to 2. Salt water having a salt concentration of 5 times 18% to 25% was added, and salted for 10 to 50 days, followed by salt water separation.

The miso separated after the salt water separation was placed in an aging container, and further salt was sprinkled on the upper side of the miso, and the surface in direct contact with air was minimized using a pressing plate and aged at room temperature. The salt used at this time may be any one selected from the group consisting of purified salt, grilled salt, bamboo salt, rock salt, regenerated salt, and earth plate salt.

While aging at room temperature, the maturity of the miso (amino nitrogen content, mg%) was measured, tasted to confirm the quality, and the aging was completed. Table 2 below shows changes in the amino nitrogen content (mg%) of the Korean-style miso tama miso with the aging period.

Comparative Example 1: Production of traditional Korean traditional miso The method described in Example 4 with the exception of the step of cooling the steamed soybean and the step of inoculating the strain produced according to Example 2 and Example 3. Similarly, a traditional Korean traditional miso was produced and used as a control.

Example 5: Comparison of degree of external cracking and moisture reduction of miso balls with and without cooling of steamed soybeans Miso balls and steamed soybeans formed without performing the step of cooling steamed soybeans using a blower at 50 to 65 ° C Miso balls that were cooled and molded were produced. Each of the produced miso balls was dried at 50 to 65 ° C. for 10 to 24 hours using a hot air dryer.

As a result, it was found that there was a difference in the degree of cracking on the outside of the miso ball depending on whether or not the steamed soybean was cooled. In the case of miso balls formed after cooling the steamed soybeans, it was found that there were significantly more cracks and a slightly lighter shade than the miso balls formed without the cooling step. This is shown in FIG.

In addition, it was found that the amount of water loss of miso balls formed by cooling the steamed soybeans during the fermentation process to induce external cracks was large, and the results are shown in FIG. During the fermentation process, the weight loss rate of the early Korean miso balls was measured, and the water loss rate of the Korean miso balls formed after cooling the steamed soybeans was molded without the cooling step. It was found that the amount of weight reduction was greater than that of the control group. This is presumably because the amount of evaporation of internal moisture was increased by the formation of cracks on the outside of the miso balls. The phenomenon of cracking on the outside of the miso ball helps to propagate bacteria and mold, which are aerobic microorganisms, by allowing air to flow inside the miso ball. In addition, this reduces the moisture to form a moisture gradient inside the miso ball and grows mold that requires a low moisture content on the outside, and bacteria and lactic acid bacteria inside the miso ball that have a relatively high moisture content It has the effect of promoting the growth of various microorganisms and enabling the combined fermentation of various microorganisms in a single miso ball.

Example 6: Comparison of post-fermentation quality with or without inoculation of Bacillus amyloliquefaciens CJ3-27 and Aspergillus oryzae CJ KG strains Bacillus amyloliquefaciens -Component of microorganisms and pH, proteolytic enzyme titer (protease activity), a component of biogenic amine, which are quality indicator items of Korean miso tama miso after fermentation with or without inoculation of Aspergillus oryzae CJ KG strain The content of histamine is analyzed, and the results are shown in Table 3 below.

When the quality of the Korean miso tamago after fermentation was analyzed, the Korean miso tamago produced according to Example 4 had a pH higher than that of the Korean miso tamago produced according to Comparative Example 1 which is a control zone. Measured low, which correlates with the number of lactic acid bacteria. That is, it is analyzed that the growth of lactic acid bacteria is induced as the molding temperature of the initial miso-dama is lowered, and the titer of proteolytic enzyme is also analyzed 2.9 times higher than that of Comparative Example 1 which is a control group. It was. It was observed that mold was actively proliferating inside the miso balls, but this is considered to be due to the effect.

In the case of Bacillus cereus, both the control and Example 4 Korean miso balls start to ferment under high temperature conditions of 50 ° C. or higher at the beginning and do not grow due to the influence of temperature control. When histamine, which is a kind of amine, was measured, the Korean miso tamago produced according to Example 4 was analyzed to be lower than the control group. The Bacillus amyloliquefaciens CJ 3-27 strain applied in the production of the Korean-style miso candy has a very low activity of decarboxylase involved in the generation of histamine among biogenic amines. Is recognized.

After the ripening of the Korean-style miso tama miso was completed, the pH and amino nitrogen content (mg%), which are quality indicators, were measured, and the results are shown in Table 4 below.

After the ripening was completed, the Korean miso tama miso was analyzed, and the Korean miso tama miso of the example was fermented without inoculating the strain through the surface drying process without cooling the miso tamago. Compared to Korean miso tama miso, the pH is relatively low as in the case of miso tama at the completion of fermentation, and consistently small amounts of Bacillus cereus are detected. When the amino nitrogen content was analyzed, it was found that the Korean miso tama miso of Example 4, which had a higher proteolytic enzyme titer, was about 1.6 times higher than the control group.

Experimental Example 1: Sensory evaluation of Korean miso tama miso according to the present invention A sensory evaluation was performed on the control zone prepared according to Comparative Example 1 and the Korean miso tama miso manufactured according to Example 4. This sensory evaluation was conducted on 70 married women who are the main purchasers of food items in a 25- to 39-year-old household living in Seoul. The results are shown in Table 5 below.

As a result, the Korean-style miso ball miso according to the present invention shows a higher degree of preference in general taste. In particular, the most important taste attributes of miso are high aroma preference and umami preference (p <0.05) and tend to have a weak bitterness intensity (p <0.1). It is recognized that it had a positive effect on the quality of typical taste. More specifically, the Korean-style miso tama miso according to Example 4 has a better “paste taste / savoryness / salt taste / sweet taste / umami taste preference” (p <0.05) compared to the control group. The degree of liking for this was very high, about 3.9. In the case of the control product, there was a response that it did not smell sour.

Also in the open response, it is mentioned that the Korean-style miso tama miso of Example 4 has good fragrance and umami, and many respondents say that it is like a homemade miso made at home (more than 20%). It was analyzed that the peculiar taste of miso was realized well.

In addition, in order to derive detailed taste attributes and strength of each miso, a description analysis was conducted on 20 trained professional panelists, and the results are shown in FIG.

Measuring the intensity of the sensory properties of both products, it was found that there was a statistical difference between the products in the remaining properties excluding the “burnt scent” property (p <0.05).

Analyzing the products in the control ward, the characteristics of Japanese miso and sweet scents, sweetness, bitterness, darkness, MSG taste, flavor when stewed radish dry leaves, Japanese miso flavor, astringent, not refreshing The powdery characteristics and the appearance brownness, sediment and turbidity were strong. When the product according to Example 4 was analyzed, the Korean traditional miso scent, soy sauce scent, miso ball scent, salty scent, sour scent, bitter scent characteristics, salty taste, sourness, umami, soy sauce flavor, miso The flavor of jade and Korean traditional miso were strong.

From the above results, the new strains Bacillus amyloliquefaciens CJ 3-27 and Aspergillus oryzae CJ KG (Bacillus amyloliquefaciens) CJ 3-27 and Aspergillus oryzae CJ KG were inoculated. By improving the physical structure and process of miso candy that can be successfully grown and optimizing the fermentation temperature, the titer of the proteolytic enzyme is improved and the content of amino nitrogen is increased during the ripening period. It was found that the most important attributes of miso were fragrance and umami, and the overall taste of miso was improved compared to the ward.

Claims (9)

Sorting, washing and soaking soybeans;
Cooling the soybeans after cooking;
Culturing Bacillus amyloliquefaciens to produce a culture solution;
A step of chopping the following inoculation with the strain culture solution into the cooled soybean,
Molding the chopped cooled soybeans;
Drying the surface of the molded miso ball;
Culturing Aspergillus oryzae to produce seeds of said strain;
Spraying the seeded miso balls on the molded miso balls and inoculating them,
Salting the fermented miso balls to separate the salt water;
Aging the miso that has undergone salt water separation;
A method for producing a Korean-style miso tama miso characterized by The method for producing a Korean miso tama miso according to claim 1 , wherein the cooling step is performed under a temperature condition of 50C to 65C. The Korean-style miso according to claim 1 , wherein the culture solution of Bacillus amyloliquefaciens is inoculated in an amount of 0.1 to 5% based on the weight of raw soybean grains. A production method of onion miso. The method for producing a Korean-style miso tama miso according to claim 1 , wherein the step of drying the surface is performed under a temperature condition of 50C to 65C for 5 to 24 hours. The Korean-style miso according to claim 1 , wherein the Aspergillus oryzae seed meal is sprayed and inoculated so as to be 0.01 to 5% of the weight of raw soybean grains. A production method of onion miso. The method for producing a Korean-style miso ball miso according to claim 1 , wherein the fermentation is performed at a temperature of 10 ° C. to 45 ° C. for 3 to 30 days. The salting is performed by adding salt water having a salt concentration of 18% to 25% to the fermented miso balls by adding 1.8 to 2.5 times the weight of the miso balls and immersing them for 10 to 50 days. method of manufacturing miso ball miso Korean according to claim 1, characterized in that. 2. The taste of claim 1, wherein the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens CJ 3-27 (deposit number KCCM 11317P). A production method of onion miso. 2. The method for producing a Korean-style miso tamamiso according to claim 1, wherein the Aspergillus oryzae is Aspergillus oryzae CJ KG (deposit number KCCM 11301P).