WO2024144322A1 - Korean fermented soybean lump and soybean paste having reduced aflatoxin and uniform quality using seed fermented soybean lump, and method for producing same - Google Patents

Abstract

The present invention relates to a method for producing a Korean fermented soybean lump and soybean paste having reduced aflatoxin and uniform quality using a seed fermented soybean lump. The concept and conditions of a seed fermented soybean lump and a method for producing a fermented soybean lump and soybean paste using same have been established, and a Korean fermented soybean lump and soybean paste not only having significantly reduced aflatoxin production but also having consistent quality and excellent flavor can be produced. Thus, the present invention can be helpfully used as a novel method for producing a Korean fermented soybean lump and soybean paste that addresses the issues of the existing method for producing a Korean fermented soybean lump and soybean paste, and as a fermented soybean lump and soybean paste produced by the method.

Description

Korean meju and soybean paste with reduced aflatoxin and uniform quality using sea meju, and method for producing the same

The present invention relates to Korean meju and soybean paste with reduced aflatoxin and uniform quality using sea meju, and a method for producing the same.

This application claims priority based on Korean Patent Application No. 10-2022-0187250 filed on December 28, 2022, and all contents disclosed in the specification and drawings of the application are incorporated in this application.

Soybean paste, one of the representative traditional fermented foods, is a frequently consumed food that is very important in the Korean diet as a seasoning with harmonious taste and flavor and a source of protein. In addition, functional properties such as anticancer activity, angiotensin converting enzyme (ACE) inhibitory activity, tyrosinase inhibitory activity, and immune-boosting effect have been reported and are attracting attention as a health food (Cha et al., 2014; Jung, Park, & Park, 2006; Kim et al., 2014).

Meju is not only nutritionally excellent as a raw material for various sauces such as soy sauce, soybean paste, and red pepper paste, but also contains a large amount of high-quality vegetable protein, which reduces the risk of arteriosclerosis and heart disease, and has functional properties such as smooth blood flow. This is excellent. Meju is classified into “Korean Meju” and “Improved Meju” according to the fermentation method according to the Food Code. “Korean Meju” refers to a product made by steaming or boiling soybeans, molding them, and fermenting them naturally using the traditional Korean method. “Improved Meju” refers to products made by fermenting steamed or boiled soybeans using selected starter bacteria.

Soybean paste is divided into “Korean soybean paste” and “(improved) soybean paste” depending on the fermentation method of meju. “Soybean paste” is made by cultivating yeast, etc., using soybeans as the main ingredient, then mixing it with table salt, fermenting and maturing it, or soaking meju in saline solution, fermenting it, separating the filtrate, and processing it. “Korean-style soybean paste” is “Korean-style meju.” After fermenting by adding saline solution, the filtrate is separated and it is called traditional soybean paste.

Korean soybean paste made using Korean meju has a variety of flavors and is preferred over improved soybean paste, which has a stronger sweet taste. In addition, the active ingredients of soybeans, such as isoflavones, remain intact during the process of fermenting meju into soybean paste, soy sauce, etc., and in addition, the proteins are decomposed by the action of various microorganisms such as Aspergillus. oryzae. It contains various functional substances such as various amino acids and is characterized by excellent nutritional content.

However, when manufacturing Korean meju, it is contaminated by various bacteria floating in the air during the molding and floating stage in a certain shape, which leads to the growth of not only beneficial microorganisms involved in fermentation but also harmful microorganisms that produce toxins such as aflatoxin. This causes problems in controlling the soybean paste fermentation environment and is a major factor in maintaining uniform quality. As a result of an inspection of 517 Korean soybean paste and Korean meju products conducted by the Ministry of Food and Drug Safety, aflatoxin was detected in 33 products above the standard level, leading to suspension of sales and recall. In addition, since the quality and flavor of soybean paste varies depending on the manufacturing method, manufacturing location, manufacturing period, and manufacturing region of meju, not only aflatoxin production but also problems of uneven quality may occur.

In order to solve this problem, various bacteria such as Aspergillus and Bacillus are inoculated in the production of Koji to control artificial fermentation and block the incorporation of miscellaneous bacteria. Not only is the unique flavor reduced, but it also has the disadvantage of not being able to sell the product by labeling it as Korean soybean paste because it does not use Korean meju manufactured using traditional methods.

Therefore, while maintaining the unique taste and flavor of traditional soybean paste, the taste and flavor are maintained consistently for each product produced, and the development of a method that does not produce carcinogenic substances such as aflatoxin and an industrialization process using the same is necessary for the industrialization, modernization and development of traditional soybean paste. It is desperately needed for internationalization.

However, despite the diversity of research on strains or combinations thereof optimized for soybean paste, soy sauce, cheonggukjang, etc., or manufacturing methods for the same, the concept of seed meju was invented and a uniform meju with excellent flavor and functionality without artificial strain inoculation was produced. No research has been attempted on the manufacturing method of soybean paste and the meju and soybean paste produced by the above method.

One object of the present invention is to provide a method for preparing Korean soybean paste comprising the following steps:

(S1) manufacturing cimeju;

(S2) preparing Korean-style meju by adding the sea meju; and

(S3) Step of producing Korean soybean paste from the Korean meju.

Another object of the present invention is to provide soybean paste prepared by the above method.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art to which the present invention belongs from the description below. There will be.

The present invention provides a method for preparing Korean soybean paste comprising the following steps:

(S1) manufacturing cimeju;

(S2) preparing Korean-style meju by adding the sea meju; and

(S3) Step of producing Korean soybean paste from the Korean meju.

In one embodiment of the present invention, the sea meju may satisfy any one or more of the following conditions, but is not limited thereto:

a1) Aflatoxin was not detected;

a2) Aflatoxin biosynthetic gene was deleted from the C. meeju isolate; and

a3) Aflatoxin is not produced from the C. meeju isolate.

In one embodiment of the present invention, the isolate is Aspergillus sp.; Alternatively, one or more of the Penicillium sp. or Mucor sp. may be the dominant strain, but are not limited thereto.

In one embodiment of the present invention, the aflatoxin biosynthetic gene may be one or more selected from the group consisting of aflO, aflP, and aflR, but is not limited thereto.

In one embodiment of the present invention, the deletion may be a loss of the function of the gene, but is not limited thereto.

In one embodiment of the present invention, the step (S2) may include, but is not limited to, the following steps:

(b1) soaking, steaming, and cooling soybeans;

(b2) manufacturing unripened meju by molding the ground soybeans after cooling;

(b3) adding sea meju to step (b2); and

(b4) Fermenting the unripe meju.

In one embodiment of the present invention, the amount of seed meju added in step (b3) is based on the total weight of the ground soybeans, and the dominant strain of the seed meju is respectively

In the case of Aspergillus sp., it is more than 0.5% by weight,

In the case of Penicillium sp. or Mucor sp. , it may be 2.5% by weight or more, but is not limited thereto.

In one embodiment of the present invention, the fermentation may proceed at room temperature for 5 to 10 days, but is not limited thereto.

In one embodiment of the present invention, the step (S3) may include, but is not limited to, the following steps:

(c1) immersing the meju prepared in step (S2) in brine;

(c2) primary fermentation of the soaked meju; and

(c3) Secondary fermentation of solids separated from the primary fermented meju.

In one embodiment of the present invention, the brine is 15 to 25% (w.v) and may be three times the volume of the weight of the meju, but is not limited thereto.

In one embodiment of the present invention, the primary fermentation may be carried out at room temperature for 30 to 60 days, but is not limited thereto.

In one embodiment of the present invention, the secondary fermentation may proceed for more than 3 months, but is not limited thereto.

The present invention provides soybean paste produced by the above method.

In one embodiment of the present invention, the soybean paste may be characterized in that aflatoxin is not detected, but is not limited thereto.

According to the manufacturing method of Korean meju and soybean paste with uniform quality and reduced aflatoxin using sea meju, the concept and conditions of sea meju and the meju and soybean paste manufacturing method applying the same have been established, which not only significantly reduces the production of aflatoxin but also improves the quality. In that it is possible to produce Korean meju and soybean paste that are uniform and have excellent flavor, it is useful as a new Korean meju and soybean paste production method that solves the problems of the existing Korean meju and soybean paste production method and the meju and soybean paste produced by the above method. It can be.

Figure 1 is a diagram showing the manufacturing process of meju and soybean paste according to an embodiment of the present invention.

Figure 2 is a graph showing the ability to inhibit aflatoxin production of meju produced using meju dominated by fungi of the genus Penicillium or Mucor as seed meju.

Figure 3 is a graph showing the ability to inhibit aflatoxin production of meju produced using meju dominated by Aspergillus genus mold as seed meju.

Figures 4 to 9 show the physicochemical quality and enzyme activity evaluation results of meju produced using meju dominated by fungi of the genus Penicillium or Mucor as seed meju, respectively, showing moisture content, salinity, pH, and color according to the amount of seed meju added. , amino acid nitrogen content, and protease activity.

Figures 10 to 15 show the physicochemical quality and enzyme activity evaluation results of meju produced using meju dominated by Aspergillus genus mold as seed meju, respectively, moisture content, salinity, pH, color, and amino acid nitrogen according to the amount of seed meju added. This is a graph showing content and protease activity.

The present invention provides a method for producing Korean soybean paste comprising the following steps and a soybean paste prepared by the method:

(S1) manufacturing cimeju;

(S2) preparing Korean-style meju by adding the sea meju; and

(S3) Step of producing Korean soybean paste from the Korean meju.

Soybean paste produced by the method of the present invention is Korean soybean paste, and Korean soybean paste refers to soybean paste manufactured using Korean meju. It may have the same meaning as traditional soybean paste or traditional soybean paste, and can be made from improved meju produced by inoculating specific bacteria. It can be distinguished from manufactured (improved) soybean paste.

In addition, in the present invention, Korean meju and Korean soybean paste can only be labeled as meju and soybean paste manufactured in a traditional way, and cannot be labeled as Korean meju and soybean paste manufactured including the step of inoculating specific bacteria.

However, the soybean paste produced using the sea meju of the present invention is not only a Korean soybean paste because it does not use specific bacteria, but is also a Korean soybean paste that has excellent taste and nutritional content and uniform quality because no aflatoxin toxin is produced. You can.

In the present invention, seed soybean paste is a certain amount of meju added during the meju manufacturing process to produce soybean paste. It may be the same concept as seed soy sauce traditionally used in soy sauce production and seed soybean paste used in soybean paste production, but in the conventional soybean paste production process. There may be materials and terms that have never been used in.

Seed soy sauce and seed soybean paste are used in households that manufacture and consume the seed soy sauce and seed soybean paste to maintain their taste and increase their quantity. The seed meju of the present invention can also be used for the above purposes, but in addition, By adding meju, the beneficial bacteria, nutritional components, sensory properties, and/or functionality of sea meju can be applied as is to meju and soybean paste or used for the purpose of increasing the function, but are not limited thereto.

As described above, the seed meju of the present invention is the same concept as seed soy sauce and seed soybean paste, so when producing seed meju, Korean-style meju and/or Korean-style soybean paste can be produced using a portion of the seed meju. Therefore, it is not possible to manufacture one Korean meju and/or Korean soybean paste by manufacturing one meju of the present invention, and a plurality of Korean meju and/or Korean soybean paste can be manufactured through one meju. Accordingly, it is possible to produce Korean meju and/or Korean soybean paste by repeating the step of producing Korean meju and Korean soybean paste by performing a single cimeju manufacturing step several times. In the present invention, when step (S1) is performed once, steps (S2) and (S3) can be performed repeatedly, and thus several Korean meju and/or Korean soybean paste can be produced.

The meju of the present invention may have the characteristic of not producing aflatoxin, and the meju and soybean paste produced accordingly may also have the above characteristic, but are not limited thereto.

In addition, the sea meju of the present invention can not only be used in meju and soybean paste as disclosed in an embodiment of the present invention, but can also be applied to the use of other sauces that can be manufactured using meju, such as soy sauce. It is not limited to this.

In one embodiment of the present invention, the sea meju may satisfy any one or more of the following conditions, but is not limited thereto:

a1) Aflatoxin was not detected;

a2) Aflatoxin biosynthetic gene was deleted from the C. meeju isolate; and

a3) Aflatoxin is not produced from the C. meeju isolate.

In the present invention, the sea meju is characterized in that aflatoxin is not detected, and this feature may include both that no contamination occurs through external factors and that no contamination occurs through internal factors. At this time, the internal factor may include the aflatoxin biosynthetic gene not functioning properly and aflatoxin not being produced. External factors may include factors other than the inherent characteristics of the seedlings, regardless of the aflatoxin biosynthetic gene.

Cmeju of the present invention may satisfy all of the above conditions, but is not limited thereto.

Aflatoxin is a secondary metabolite produced by certain fungi ( Aspergillus flavus and Aspergillus parasiticus ) that grows on soil, rotting plants, hay and grain under conditions of 24 - 35℃ and moisture content of 7% or more. It is classified as B1, B2. , G1, G2, M1, M2, etc. In addition, mycotoxins can be largely divided into Aspergillus genus, Penicillium genus, and Fusarium genus mold toxins depending on the producing bacteria.

Pure aflatoxin B1 is a white to yellow crystal and has no odor. The molecular weight of aflatoxin B1, B2, G1, G2, M1, and M2 is 310 to 330, and is soluble in chloroform, acetone, acetonitrile, and methanol. B1 and B2 have blue fluorescence, G1 and G2 have Blue-Green fluorescence, and M1 and M2 have Blue-Violet fluorescence. Aflatoxin is very stable in a dry state and is decomposed only when heated to 280-300℃. However, in the presence of moisture and at high temperatures, it is destroyed after a certain period of time and the lactone ring portion is hydrolyzed in an alkaline solution. Oxidizing agents such as sodium hypochlorite, potassium permanganate, and hydrogen peroxide react with aflatoxin and cause aflatoxin to lose its fluorescence, and when hydrogen is added to aflatoxin B1 and G1, it changes into aflatoxin B2 and G2.

Aflatoxin is known to occur mainly in nuts such as rancid walnuts, peanuts, cashews, and pistachios, and is a toxic substance that causes mutations, carcinogenesis, and deformities. Exposure to aflatoxin causes growth failure, developmental delay, liver damage, and liver cancer, and is known to be one of the most carcinogenic substances. Although adults have a high tolerance for exposure, it is not safe, and animals are known to have no immunity. In particular, IARC (International Agency for Research on Cancer) defines it as a Group 1 carcinogen, and aflatoxin B1, B2, G1, G2, and M1 are the main toxins. Among them, aflatoxin B1 is the most commonly found and has the strongest toxicity. have Aflatoxin B1 is activated by cytochrome p450 in the liver, and in the kidney, it is converted to aflatoxin B1 8,9-oxide by peroxidase and then binds to DNA, showing a strong carcinogenic effect. In fact, aflatoxin is contaminated in soybean fermented foods, including Korean soybean paste, as well as douchi (Qiu, Shi, Wang, Ma, & Wang, 2019), kanjiang (Lee, Lee, & Lee, 2012), and tempeh (Yudiono, Ayu). It is reported that this happens. In particular , Aspergillus is involved in fermentation in the process of producing Korean meju or soybean paste. oryzae and Aspergillus flavus and Aspergillus parasticus , which produce aflatoxin, are impossible to distinguish with the naked eye.

If a person is infected with mold toxins, the disease is called mycotoxicosis. This disease is not contagious between humans and animals and is poisonous only when ingested orally. It is closely related to the season, and the causative bacteria has been detected in the causative food. It has been confirmed that it cannot be treated with antibiotics or other drugs and that it causes cancerous tumors in major organs such as the liver and kidneys. In addition, there are chitoriopyridine and patulin, which cause nervous system disorders, chitorinin, which causes kidney failure, and fusarium toxins (red mold poison), which have effects similar to hematopoietic dysfunction and follicular hormones. Aflatoxin inhibits messenger-RNA synthesis and also affects DNA synthesis. The site of action on RNA is mainly the nucleus, which appears to prevent the precursor from binding to RNA. Additionally, cytoplasmic RNA is also thought to change as a result of inhibiting the activity of DNA-dependent RNA polymerase. Inhibition of messenger-RNA synthesis inhibits protein synthesis, and the associated decrease in fat transport ability is an early lesion mainly seen in the liver of animals infected with this toxin. Aflatoxin is widely known as a substance that causes liver cancer in humans. Liver cancer is caused by the occurrence of deletion mutations in the P53 tumor-suppressor gene and the active activity of oncogenes.

In the body, aflatoxin can be metabolized in the liver to a reactive epoxide intermediate or hydroxylated to the less harmful aflatoxin M1. Aflatoxin is commonly ingested orally, and the most toxic, aflatoxin B1, can penetrate through the skin. The U.S. Food and Drug Administration (FDA) action level for aflatoxin contained in food or feed is 20 to 300 ppb.

In general, during the manufacturing process, the molded soybeans go through a floating stage. At this stage, contamination occurs by various bacteria floating in the air, which leads to the growth of harmful microorganisms that produce toxins such as aflatoxin in addition to beneficial bacteria. In addition to the problem of maintaining uniform quality, it is difficult to produce a uniform product due to the relatively long manufacturing period and diverse quality of meju, and it is not easy to control the natural fermentation environment, which is harmful to microorganisms that produce fungal toxins such as aflatoxin. Despite the preference for Korean soybean paste due to the difficulty in suppressing the growth of soybean paste, improved soybean paste can be produced in most factories, so the main task is to prevent aflatoxins from being produced when manufacturing soybean paste containing soybean paste, and the present invention According to the method, soybean paste with uniform quality and excellent sensory properties can be produced using meju that does not produce aflatoxin.

In order to check whether or not aflatoxin is detected in the meju used in an embodiment of the present invention, a method for determining whether or not it is below the detection limit can be applied. In one embodiment of the present invention, as a result of analyzing aflatoxin, if the amount is quantified below the detection limit, it can be judged as non-detection, and the limit of detection (LOD) is a method based on the standard deviation of the reaction and the slope of the calibration curve. It can be calculated by applying the reflected formula below, but is not limited to this.

Detection limit = 3.3 * σ/S (σ: standard deviation of calibration curve, S: slope of calibration curve)

Here, the slope S was obtained from a calibration curve prepared using aflatoxin concentration, and the standard deviation σ can be used as the area standard deviation of the lowest concentration in the calibration curve.

The detection limit calculated in this example was found to be 0.011 μg/kg for AFB1, 0.003 μg/kg for AFB2, 0.028 μg/kg for AFG1, and 0.010 μg/kg for AFG2 in the meju medium, and 0.234 μg/kg for AFB1 in the soybean paste medium. , AFB2 0.038 μg/kg, AFG1 0.142 μg/kg, and AFG2 0.027 μg/kg, but are not limited thereto.

In one embodiment of the present invention, the isolate is a dominant strain of one or more of the genus Aspergillus ( Aspergillus sp. ), Penicillium sp., or Mucor sp. However, it is not limited to this.

In the present invention, the isolate may refer to a strain isolated from a candidate seed strain in an embodiment of the present invention, but is not limited thereto.

Microorganisms belonging to the genus Aspergillus of the present invention are a type of fungus and may be used in the present invention as the same term as mold.

For example, Aspergillus acidus ( A. acidus ), Aspergillus awamori ( A. awamori ), Aspergillus clavatus ( A. clavatus ), Aspergillus nidulans ( A. nidulans ), Aspergillus niger ( A. niger ), Aspergillus versicolor ( A. versicolor Vuill. ), but is not limited thereto. As another example, the Aspergillus microorganisms include Aspergillus acidus Kozak ( A. acidus Kozak 41731), Aspergillus Awamori Nakaz 41844 ( A. awamori Nakaz 41844), Aspergillus clavatus demagier 40071 ( A clavatus Desmazieres 40071), Aspergillus nidulans 44342 ( A. nidulans (Ediam ) G. Winter 44342), Aspergillus niger van Tieghem 41018), Aspergillus niger van Tieghem 44333 ( It may include, but is not limited to, Aspergillus niger van Tieghem 44333) and Aspergillus versicolor 42602 (A. ve rsicolor (Vuill.) Tirab. 42602).

Penicillium or the microorganism belonging to the genus Penicillium of the present invention is a type of blue mold fungus and may be used in the present invention as the same term as mold. For example, Penicillium Citrinum , Penicillium verruculosum , Penicillium commune, Penicillium oxalicum , Penicillium echinul It may include, but is not limited to , Penicillium echinulatum and Penicillium chrysogenum .

Microorganisms belonging to the genus Mucor of the present invention are a genus of about 40 types of fungi in the Mucoraceae family, and may be used in the present invention as the same term as mold. For example, Mucor amphibiorum , Mucor circinelloides , Mucor ellipsoideus , Mucor fragilis , Mucor hiemalis hiemalis ), Mucor hiemalis f. Silvaticus ( Mucor hiemalis f. silvaticus ), Mucor indicus ( Mucor indicus ), Mucor mucedo, Mucor paronychius ( Mucor paronychius ), Mucor piriformis , radish Mucor plumbeus , Mucor pseudolusitanicus , Mucor racemosus , Mucor ramosissimus , Mucor varicolumellatus ( Mucor ) variicolumellatus ) and Mucor velutinosus, but are not limited thereto.

In one embodiment of the present invention, the aflatoxin biosynthetic gene may be one or more selected from the group consisting of aflO, aflP, and aflR, but is not limited thereto.

In one embodiment of the present invention, the deletion may be a loss of the function of the gene, but is not limited thereto.

In the present invention, “deletion” may mean a state in which the function of the gene is lost due to qualitative or quantitative changes in the aflatoxin biosynthetic gene.

In the present invention, “state in which gene function is lost” may include all cases in which the aflatoxin gene is not biosynthesized normally, and may mean a gene that is not wild type, and “wild” means the state in which no mutation has occurred. , even if a mutation occurs at one or more points or positions in the base sequence or amino acid sequence of the domain, it may include, but is not limited to, cases where the phenotype (function) of the domain is not affected.

In the present invention, “mutation” refers to a phenomenon that causes a qualitative or quantitative change in a gene due to an abnormality in a gene or chromosome, causing a change in genetic traits. For example, the mutation may be a mutation at the gene level or amino acid level, but is not limited thereto.

Additionally, the mutation may be a point mutation or multiple mutations, preferably selected from substitutions, insertions and deletions. Additionally, the mutation may be a silent mutation, a neutral mutation, a missense mutation, a nonsense mutation, a frame shift mutation, etc., but is not limited thereto.

In one embodiment of the present invention, the step (S2) may include, but is not limited to, the following steps:

(b1) soaking, steaming, and cooling soybeans;

(b2) manufacturing unripened meju by molding the ground soybeans after cooling;

(b3) adding sea meju to step (b2); and

(b4) Fermenting the unripe meju.

In step (b3) of the present invention, the seed meju may be added to ground soybeans and then molded together to be used to produce unripened meju, but is not limited thereto.

In the present invention, soybean is a type of bean whose origin is Glycine soja . In Korea, it is called white soybean, soybean soybean, soybean sprout, etc., and in English, it is called soybean. White and hard. Since it has the most uses and is the most cultivated bean worldwide, it is in fact the most common bean. Of course, it has many uses, so it is used to make various sauces, soybean oil, soy milk, tofu, bean sprouts, and other food ingredients such as soy protein.

In the present invention, soybeans may refer to soybeans in a clean state after being washed with tap water 2 L/circuit a total of 5 times, but are not limited thereto.

In the present invention, “immersion” means soaking in a liquid. In the present invention, the immersion process can be carried out for 24 hours by preparing soybeans and tap water in a ratio of 1:3 (w/v), but is not limited thereto.

In the present invention, the ratio of soybeans to tap water is (soybeans:tap water), 1:1, 1:2, 1:3, 1:4, 1:5, 2:5, 3:5, 4:5, 2:4 , or 2:3, but is not limited thereto.

In addition, in the present invention, the soaking process is 16 to 32 hours, 16 to 28 hours, 16 to 24 hours, 16 to 20 hours, 16 to 18 hours, 18 to 32 hours, 18 to 28 hours, 18 to 24 hours, 18 to 24 hours. It can be 20 hours, 20 to 32 hours, 20 to 28 hours, 20 to 24 hours, 22 to 32 hours, 22 to 28 hours, 22 to 24 hours, 24 to 32 hours, 24 to 28 hours, or 24 to 26 hours. However, it is not limited to this.

In the present invention, “steaming” means boiling. In the present invention, the steaming process may be carried out at 121° C. for 60 minutes or at atmospheric pressure for 4 to 6 hours, but is not limited thereto.

In the present invention, the steaming temperature is 100 to 135 ℃, 100 to 130 ℃, 100 to 125 ℃, 100 to 121 ℃, 105 to 135 ℃, 105 to 130 ℃, 105 to 125 ℃, 105 to 121 ℃, 110 to 135 ℃ , 110 to 130 °C, 110 to 125 °C, 110 to 121 °C, 115 to 135 °C, 115 to 130 °C, 115 to 125 °C, or 115 to 121 °C, but is not limited thereto.

In the present invention, the steaming time is 45 to 75 minutes, 45 to 70 minutes, 45 to 65 minutes, 45 to 60 minutes, 50 to 75 minutes, 50 to 70 minutes, 50 to 65 minutes, 50 to 60 minutes, 55 to 75 minutes. , 55 to 70 minutes, 55 to 65 minutes, or 55 to 60 minutes, but is not limited thereto.

In the present invention, the time when the steaming process is carried out at atmospheric pressure is 2 to 10 hours, 2 to 8 hours, 2 to 6 hours, 3 to 10 hours, 3 to 8 hours, 3 to 6 hours, 3 to 4 hours, 4 hours. It may be from 10 hours to 10 hours, 4 to 8 hours, or 4 to 6 hours, but is not limited thereto.

In the present invention, “leaving out to cool” may refer to the process of cooling by lowering the high temperature maintained during the steaming process to a lower specific temperature, but is not limited thereto.

In the present invention, the standing cooling process may mean cooling by lowering the temperature of the steaming process to 40°C, but is not limited thereto. For example, from the boiling temperature, 50℃, 49℃, 48℃, 47℃, 46℃, 45℃, 44℃, 43℃, 42℃, 41℃, 40℃, 39℃, 38℃, 37℃, The temperature may be lowered to 36°C or 35°C for cooling, but is not limited thereto.

In the present invention, “molding” means making a certain shape. The forming process may include, but is not limited to, forming soybeans that have undergone washing, soaking, steaming, and cooling into a certain shape. For example, in the present invention, soybeans steamed after the cooling step are ground, and seed meju is added to the ground soybeans, mixed uniformly, and then molded to form meju.

In the present invention, the unripened meju may mean “meju before the fermentation stage,” and the meju may mean “meju after fermentation,” but are not limited thereto.

In the present invention, seed meju may be added at least 0.5% by weight based on the total weight of ground soybeans, but is not limited thereto, and may be added differently depending on the type of dominant strain separated from the seed meju. At this time, a different addition amount may be determined based on the effect of reducing apollotoxin production by the isolated dominant strain, but is not limited thereto.

In one embodiment of the present invention, the amount of seed meju added in step (b3) is based on the total weight of the ground soybeans, and the dominant strain of the seed meju is respectively

In the case of Aspergillus sp., it is more than 0.5% by weight,

In the case of Penicillium sp. or Mucor sp. , it may be 2.5% by weight or more, but is not limited thereto.

In the present invention, meju produced by adding sea meju, which is a dominant strain of the Aspergillus genus, can inhibit the production of more than 99% of aflatoxin even if only 0.5% by weight of sea meju is added. In addition, meju produced by adding sea meju, which is a dominant strain of the genus Penicillium or Mucor genus, can inhibit the production of more than 90% of aflatoxin when more than 2.5% by weight of sea meju is added.

In the present invention, the amount of sea meju added is 0.5 to 12.5% by weight, 0.5 to 10% by weight, 0.5 to 8% by weight, 0.5 to 5% by weight, 1 to 12.5% by weight, and 1 to 10% by weight, based on ground soybeans. , 1 to 8% by weight, 1 to 5% by weight, 2 to 12.5% by weight, 2 to 10% by weight, 2 to 8% by weight, 2 to 5% by weight, 3 to 12.5% by weight, 3 to 10% by weight, 3 to 8 wt.%, 3 to 5 wt.%, 4 to 12.5 wt.%, 4 to 10 wt.%, 4 to 8 wt.%, 4 to 5 wt.%, 5 to 12.5 wt.%, 5 to 10 wt.%, or 5 to 5 wt.% It may be 8% by weight, but is not limited thereto.

In one embodiment of the present invention, the weight percentage is the amount added based on 100 of the weight of the steamed and ground soybeans to which the seameju is added, so it can be replaced with parts by weight.

Meju formed in the present invention may undergo a fermentation process after the outer drying step, and may be under conditions of 40° C. for 1 day (24 hours), but is not limited thereto.

In the present invention, the drying period is 16 to 32 hours, 16 to 28 hours, 16 to 24 hours, 16 to 20 hours, 16 to 18 hours, 18 to 32 hours, 18 to 28 hours, 18 to 24 hours, 18 to 24 hours. It can be 20 hours, 20 to 32 hours, 20 to 28 hours, 20 to 24 hours, 22 to 32 hours, 22 to 28 hours, 22 to 24 hours, 24 to 32 hours, 24 to 28 hours, or 24 to 26 hours. However, it is not limited to this.

In the present invention, the rolling temperature may be 25 to 55 ℃, 25 to 50 ℃, 25 to 45 ℃, 25 to 40 ℃, 30 to 55 ℃, 30 to 50 ℃, 30 to 45 ℃, or 30 to 40 ℃. , but is not limited to this.

In one embodiment of the present invention, the fermentation may proceed at room temperature for 5 to 10 days, but is not limited thereto.

In the present invention, the fermentation period is 4 to 10 days, 4 to 9 days, 4 to 8 days, 4 to 7 days, 5 to 10 days, 5 to 9 days, 5 to 8 days, 5 to 7 days, and 6 to 10 days. , 6 to 9 days, 6 to 7 days, 6 to 10 days, 6 to 9 days, 6 to 8 days, 6 to 7 days, or 7 days, but is not limited thereto.

In the present invention, the fermented meju may be subjected to a washing process to remove foreign substances on the surface, and at this time, it may be washed three times per side with tap water using a food brush, but is not limited to this.

In one embodiment of the present invention, the step (S3) may include, but is not limited to, the following steps:

(c1) immersing the meju prepared in step (S2) in brine;

(c2) primary fermentation of the soaked meju; and

(c3) Secondary fermentation of solids separated from the primary fermented meju.

In the present invention, “brine water” may mean water with salt content, and may include any type and condition of brine generally used in the art to produce soybean paste.

In the present invention, brine can be used by measuring salinity (concentration) and volume based on washed meju, but is not limited thereto.

In one embodiment of the present invention, the brine is 15 to 25% (w.v) and may be three times the volume of the weight of the meju, but is not limited thereto.

The salinity of the brine in the present invention is 8 to 35% (w.v), 8 to 32% (w.v), 8 to 30% (w.v), 8 to 28% (w.v), 8 to 25% (w.v), 10 to 35% (w.v). % (w.v), 10 to 32% (w.v), 10 to 30% (w.v), 10 to 28% (w.v), 10 to 25% (w.v), 12 to 35% (w.v), 12 to 32% ( w.v), 12 to 30% (w.v), 12 to 28% (w.v), 12 to 25% (w.v), 15 to 32% (w.v), 15 to 30% (w.v), 15 to 28% (w.v) , or 15 to 25% (w.v), but is not limited thereto.

In the present invention, the volume of brine is 5 times more than 1, 4.5 times more than 1, 4 times more than 1, 3.5 times more than 1, 3 times more than 1, 2 to 5 times, 2 to 4.5 times, and 2 to 4 times more than 1. It may be 2 to 3 times, 3 to 5 times, 3 to 4 times, or 3 times, but is not limited thereto.

In one embodiment of the present invention, the primary fermentation may be carried out at room temperature for 30 to 60 days, but is not limited thereto.

In the present invention, the primary fermentation period may be 30 to 60 days, 30 to 50 days, 30 to 40 days, 30 to 38 days, 30 to 36 days, 30 to 34 days, 30 to 32 days, or 30 days. It is not limited. In addition, as the primary fermentation period increases, the sensory effects of soybean paste or meju may decrease, especially in terms of aroma, sweetness, and salty taste, but are not limited thereto.

After the primary fermentation, the solid and liquid parts are separated through a fermentation step, for example, using a cloth wrapper or food net, and the solid part can be made into soybean paste and the liquid into soy sauce through secondary fermentation. It is not limited to this.

In one embodiment of the present invention, the secondary fermentation may proceed for more than 3 months, but is not limited thereto.

In the present invention, the secondary fermentation period is 3 to 12 months, 3 to 11 months, 3 to 10 months, 3 to 9 months, 3 to 8 months, 3 to 7 months, 3 to 6 months, 3 to 5 months, 3 to 5 months. It may be 4 months, but is not limited thereto. If the secondary fermentation period is less than 3 months, the sensory effect in color or overall satisfaction of soybean paste or meju may be reduced, but is not limited to this.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Establishment of Cmeju conditions

In order to use fermented meju as seed meju, criteria for selecting seed meju were established. Specifically, ① aflatoxin was not detected in meju, ② the Aspergillus genus isolate isolated from meju that satisfied the conditions of ① had a deletion of at least one of the major aflatoxin biosynthetic genes (aflO, aflP, aflR), and ③ Three conditions were established, including confirmation that the Aspergillus genus isolate isolated from meju that satisfied the conditions of ① does not produce aflatoxin. The specific details for each condition are as follows.

Example 1-1. Selection of meju with no aflatoxin detected

In order to select meju in which aflatoxin was not detected, aflatoxin in meju was analyzed according to the following method. To 10 g of meju sample, 50 mL of 80% methanol (v/v), 1 g of sodium chloride, and 25 mL of hexane were added, homogenized (6,200 rpm, 3 minutes), and then mixed with Whatman No. Filtered through 4. 5 mL of the filtrate was diluted with 35 mL of 0.1% Tween 20 solution (v/v) and filtered through glass fiber filter paper (GF/B). At this time, 20 mL of the filtrate was injected into the immunoaffinity column and passed through at a rate of one drop per second, and then the column was washed with 15 mL of distilled water and air was passed through to remove any water remaining in the column. Mycotoxins present in the column were eluted using 2 mL of methanol containing 0.1% (v/v) acetic acid. After all 2 mL of elution solvent had passed through the column, it was allowed to stand for 5 minutes, and then 2 mL of elution solvent was added to elute the toxin once more. A total of 4 mL of the elution solution was dried under nitrogen at 45°C and re-dissolved in 0.5 mL of 50% methanol. The re-dissolved solution was filtered through a 0.2 μm PVDF syringe filter and was used as a test solution. 50 μL of the test solution was injected into a liquid chromatograph equipped with a fluorescence detector and analyzed. Derivatization was performed using PHRED (photochemical reactor for enhanced detection), and aflatoxin was separated on a C18 column (4.6 x 150 mm, 3.5 μm). It was performed at 40°C at a flow rate of 1 mL/min. Mobile phase A was distilled water containing 0.5% acetic acid, and mobile phase B was a mixture of methanol/acetonitrile (85:15, v/v) containing 0.5% acetic acid. The concentration gradient was 40% for mobile phase B for the first 8 minutes. %, then increased to 70% within 2 minutes and maintained for 5 minutes, then decreased to 40% within 2 minutes and maintained for 3 minutes. The wavelengths of the fluorescence detector were set at 360 nm for excitation and 450 nm for fluorescence.

As a result of analyzing aflatoxin under the analysis conditions described above, if the quantification was below the detection limit, it was judged as non-detection, and the limit of detection (LOD) was calculated according to the formula below, based on the standard deviation of the reaction and the slope of the calibration curve. did.

Detection limit = 3.3 * σ／S

(σ: standard deviation of calibration curve, S: slope of calibration curve)

At this time, the slope S was obtained from a calibration curve prepared using aflatoxin concentration, and the standard deviation σ was the area standard deviation of the lowest concentration in the calibration curve.

As a result, the detection limit calculated in this example was found to be at the level of AFB1 0.011 μg/kg, AFB2 0.003 μg/kg, AFG1 0.028 μg/kg, and AFG2 0.010 μg/kg in the meju medium, and in the soybean paste medium, AFB1 The levels were 0.234 μg/kg, AFB2 0.038 μg/kg, AFG1 0.142 μg/kg, and AFG2 0.027 μg/kg.

Example 1-2. Meju with no aflatoxin detected Aspergillus Deletion of at least one of the aflatoxin biosynthetic genes (aflO, aflP, aflR) of the genus isolate was confirmed.

In order to isolate Aspergillus genus mold from meju selected according to Example 1-1 in which aflatoxin was not detected, 10 g of meju sample was diluted with sterilized saline to prepare dilutions of the 101 to 105 series. 1 mL of the dilution solution was dispensed into a fungal selection medium (DRBC medium (dichloran rose bengal chloramphenicol agar)) according to the dilution factor, and cultured at 25°C for 5 to 7 days. A plate medium that produced 15 to 300 colonies on the cultured DRBC medium was selected, and pure culture was performed on PDA medium (potato dextrose agar) using a sterilized loop until colonies of a single strain were generated (25°C). , 5 to 7 days). This process was repeated several times until one type of colony was finally created in one medium, and the pure isolated strain was identified by sequencing the ITS region.

To confirm the possibility of aflatoxin production by Aspergillus fungi among the isolates, the presence or absence of major aflatoxin biosynthesis genes (structural genes (aflO-1,333 pb, aflP-797 bp), regulatory genes (aflR-1,032 bp)) were confirmed by multiplex PCR. . The base sequences of the PCR primers used are shown in Table 1.

The PCR reaction conditions were initial denaturation at 94°C for 4 minutes, followed by denaturation at 95°C for 1 minute, binding at 64°C for 2 minutes, and elongation at 72°C for 2 minutes. After performing a total of 30 amplification reactions, a final extension reaction was performed at 72°C for 10 minutes.

Example 1-3. Isolated from meju with no aflatoxin detected Aspergillus Confirmation of aflatoxin non-productivity of isolates from the genus

In order to confirm whether Aspergillus fungi among the isolates isolated according to Example 1-2 actually produce aflatoxin, the aflatoxin produced in the medium was quantified after culturing in PDA medium at 25°C for 7 days. Specifically, 0.5 g of medium cultured with Aspergillus isolates was placed in an EP tube, 1 mL of methanol was added, and sonication extraction was performed for 1 hour. The extract was transferred to a test tube, then 1 mL of methanol was added to the medium, and sonication extraction was performed for 1 hour (repeated a total of 3 times). The extract extracted repeatedly three times was dried by adding nitrogen in a heating block at 45°C, and then re-dissolved by adding 1 mL of 50% methanol (v/v). After re-dissolution, it was centrifuged (12,000 × g, 10 min), and the supernatant was filtered through a 0.2 μm PVDF syringe filter and used as the test solution. The test solution was confirmed for the presence or absence of aflatoxin according to the liquid chromatograph-fluorescence analyzer conditions in the same manner as in Example 1-1.

Example 2. Cmeju selection

In order to select meju that can be used as seed meju to be used in the present invention, suitable seed meju was selected from 55 pieces of commercially available Korean meju according to the seed meju selection conditions and method of Example 1. Specifically, as a result of investigating the status of aflatoxin contamination among 55 pieces of commercially available Korean meju, it was confirmed that it was not detected in 45 pieces. Among them, we isolated molds distributed among brick-shaped meju made only from soybeans, and obtained 190 isolates belonging to 12 genera and 44 species. The presence or absence of aflatoxin biosynthetic gene deletion and aflatoxin production were confirmed for the above isolates, and the meju to be used as seed meju were selected as five mejus dominated by fungi of the genus Penicillium or Mucor and five mejus dominated by molds of the genus Aspergillus ( Table 2).

Example 3. Preparation of meju and soybean paste with added sea meju

According to Example 2, a method for producing meju and soybean paste using the selected meju as seed meju was established (Figure 1). Specifically, the sea meju was pulverized and added to the forming step of the ground and steamed soybeans during the meju manufacturing process. To make meju, selected soybeans were washed and soaked for 12 to 24 hours, and the soaked soybeans were steamed under pressure (110 to 121°C, 40 to 60 minutes) or steamed for 4 to 6 hours at atmospheric pressure. After steaming was completed, the beans were left to cool to about 40°C. The steamed soybeans were ground by hand, and at this time, crushed seed meju was added to the ground steamed soybeans and mixed uniformly to form square meju. The formed meju was dried indoors (30 ~ 40℃) and then fermented at room temperature. After fermentation was completed, the meju was washed three times per side with tap water using a food brush to remove foreign substances on the surface. The washed meju was immersed in 15 to 25% (w/v) brine equivalent to three times the weight of meju and fermented at room temperature for 30 to 60 days. Afterwards, the solid and liquid parts were separated using a linen wrapping cloth or food net, and the solid part was aged into soybean paste for more than 3 months to complete meju and soybean paste using sea meju.

Example 4. Confirmation of excellent aflatoxin production inhibition ability of meju manufactured from sea meju

The ability of meju produced by the method of Example 3 using the sea meju selected according to Examples 1 and 2 and the sea meju was confirmed to inhibit aflatoxin production. Specifically, to evaluate the ability to inhibit aflatoxin production by adding meju, A. flavus (KCCM 60330), which produces aflatoxin, was inoculated during the meju forming step. In addition, in order to confirm the optimal amount of sea meju added to suppress aflatoxin production, each experiment was performed by adding 0.5%, 1.0%, 2.5%, 5.0%, 7.5%, 10.0%, and 12.0% of sea meju based on the weight of the steamed soybeans. carried out.

First, as a result of confirming the ability to inhibit aflatoxin production in meju made from seed meju, which is dominated by fungi of the genus Penicillium or Mucor genus, it was observed that the amount of aflatoxin production decreased as the amount of seed meju added relative to the weight of meju increased. In particular, an aflatoxin production inhibition rate of more than 90% was confirmed when the seed meju ratio was 2.5% or more (Figure 2).

In addition, as a result of confirming the ability of meju to inhibit aflatoxin production using meju dominated by Aspergillus genus fungi as seed meju, it was found that the seed meju added relative to the weight of meju was similar to the use of meju dominated by Penicillium or Mucor genus molds as seed meju. A tendency for aflatoxin production to decrease as the amount increased was observed. In particular, when using meju dominated by the Aspergillus genus as seed meju, it was confirmed that the production of more than 99% of aflatoxin was significantly suppressed even if only 0.5% of the seed meju based on the weight of the steamed soybeans was added (Figure 3).

Example 5. Confirmation of excellent aflatoxin production inhibition ability of soybean paste prepared from Cmeju

The aflatoxin production inhibition ability of the sea meju and the soybean paste prepared by the method of Example 3 using the sea meju selected according to Examples 1 and 2 was confirmed. Specifically, DMP2.5, DMP5.0, which is a soybean paste made using meju made by adding 2.5% or 5.0% of dominant seed meju of Penicillium or Mucor genus, respectively, and 2.5% or 5.0% of dominant seed meju of Aspergillus genus, respectively. DAsp2.5 and DAsp5.0, soybean paste made using meju, were prepared and analyzed for their ability to inhibit aflatoxin production.

As a result, it was confirmed that aflatoxin was not produced in the soybean paste prepared using sea meju selected according to the standards of the present invention, regardless of the amount of sea meju added (2.5% and 5.0%) (ND: not detected). In addition, given that the production ability has not been confirmed for all types of aflatoxin B1, B2, G1, and G2, when using sea meju according to the present invention, aflatoxin toxin production does not occur even when Korean soybean paste is manufactured without inoculating specific bacteria. It was confirmed that uniform and excellent soybean paste could be manufactured.

Example 6. Evaluation of physicochemical quality and enzyme activity of meju produced from sea meju

The physicochemical quality (moisture content, pH, salinity, color, and amino acid nitrogen content) and protease activity of meju prepared by adding sea meju selected according to Example 2 were evaluated.

Specifically, the moisture content was measured according to the standard normal pressure heating and drying method for food products. The process of taking 5 g of sample, drying it in a dryer at 105°C for about 4 hours, cooling it in a desiccator for about 30 minutes, and measuring the weight was repeated. The weight of the sample when it reached a constant weight was compared with the difference in weight of the first sample. The moisture content was calculated.

The pH was measured by diluting 5 g of the sample 4-fold in distilled water using a pH meter (S220 Seven™ pH/ION), and the salinity was measured by dispensing 0.5 mL of this dilution into the recognition part of the salinity meter (PAL-03S, ATAGO). It was measured. Color was measured using a colorimeter (Konica minolta, CR-400) after preparing 5 g of meju in a plastic dish and flattening it.

The content of amino acid nitrogen was measured according to the whole molar titration method of the Food Code. Neutralized formol solution was prepared by adding 1 ml of 0.5% phenolphthalein solution to 5 g of sample to 50 ml of 30 - 40% formaldehyde, and then adding 0.2N sodium hydroxide solution until it turned light red. Neutralized formol solution was added to 20 ml of test solution. 10 ml of molar solution and 1 ml of 0.5% phenolphthalein solution were added, and 0.2N sodium hydroxide solution (NaOH) was added until the color was slightly darker than the control solution, and then the color was colored slightly darker than the control solution with 0.2N hydrochloric acid. After that, 0.2N sodium hydroxide solution was added dropwise to make it the same color as the control solution. Two drops of sodium hydroxide solution were added to the control solution to make it dark red. The test solution was titrated with 0.2N sodium hydroxide solution until it became the same color. .

Amino nitrogen was calculated using the following formula.

Amino nitrogen (mg%) = 2.8 × [VT-(VH+Ve)] × F × D × 100/S

(VT: Total amount of 0.2N sodium hydroxide solution (ml),

VH: amount (ml) of 0.2 N sodium hydroxide solution equivalent to 0.2 N hydrochloric acid solution,

Ve: Total amount of 0.2 N sodium hydroxide solution added to the control solution (ml),

F: Total amount of 0.2 N sodium hydroxide solution used in titration (ml),

D: dilution factor, S: sample amount (g)).

Protease activity was measured according to the Food Code. Specifically, 5 g of the sample was shaken in 100 mL of distilled water and filtered to obtain a sample solution, and 1 mL of the sample solution and 1 mL of a 0.6% casein solution were mixed well in a constant temperature water bath at 37°C and reacted for 10 minutes. After that, 2 mL of 0.4M trichloroacetic acid solution was added and left in a constant temperature water bath at 37°C for 25 minutes. Then, 1 mL of the filtered filtrate, 5 mL of 0.4M sodium carbonate solution, and 1 mL of 3-fold diluted porin reagent were mixed well and incubated at 37°C. After reacting for 20 minutes, the colored solution was prepared as a test solution. The blank test solution was made by reacting 1 mL of the test solution at 37°C for 10 minutes, adding 2 mL of 0.4M trichloroacetic acid solution and mixing, adding 1 mL of 0.6% casein solution, leaving it at 37°C for 25 minutes, and then reacting in the same manner as the test solution below. carried out. The calibration curve was prepared by diluting the L-tyrosine solution with 2N HCl to 10 ~ 200 μg/mL, and 1 mL of L-tyrosine solution and 2N HCl at each concentration were mixed with 5 mL of 0.4M sodium carbonate solution and 1 mL of porin reagent diluted three times. was mixed well and reacted at 37°C for 20 minutes, and the absorbance was measured at 660 nm. The amount of L-tyrosine produced by protease was calculated by subtracting the absorbance value of the blank test solution from the absorbance value of the test solution and substituting the value into the calibration curve.

The amount of L-tyrosine in the test solution was calculated by converting it to the titer (Unit/g) of protease contained in 1g of sample according to the following formula.

Protease titer (Unit/g) = Amount of L-tyrosine in test solution (μg/mL) × 100 (enzyme reaction of 1 mL in 100 mL) × 4 (color reaction of 1 mL in 4 mL) × dilution of test solution Multiple / [10 (enzyme reaction time, minutes) × 5 (sample weight, g)]

As a result, the physicochemical quality of meju produced by using meju dominated by fungi of the genus Penicillium or Mucor and meju dominated by molds of the Aspergillus genus as seed meju was confirmed as follows.

First, meju produced by using meju dominated by fungi of the genus Penicillium or Mucor as seed meju has a moisture content of 27.9 to 34.1% (Figure 4), a salinity of 12.3 to 16.5% (Figure 5), and a pH of 5.7 to 5.7. 6.6 (Figure 6), amino acid nitrogen was found to be 759.4 to 1124.3 mg% (Figure 8), and protease activity was found to be 710.5 to 896.2 unit/g (Figure 9).

In addition, meju produced by using meju dominated by Aspergillus fungi as seed meju has a moisture content of 28.0 to 33.4% (Figure 10), a pH of 6.5 to 6.7 (Figure 12), and a protease activity of 763.1 to 882.6 units/ g (Figure 15), salinity was found to be 12.3 to 22.5% (Figure 11), and amino acid nitrogen was found to be 883.8 to 1690.8 mg% (Figure 14), confirming a tendency to increase as the sea meju content increases.

Example 7. Evaluation of physicochemical quality and enzyme activity of soybean paste prepared from Cmeju

The physicochemical quality (moisture content, pH, salinity, color, and amino acid nitrogen content) and protease activity of soybean paste prepared by adding selected sea meju according to Example 2 were evaluated. Specifically, the pH of soybean paste was measured using and applying the AACC method (2000). First, after mixing 5 g of soybean paste sample and 45 mL of distilled water, the supernatant was taken and measured with a pH meter (CP-411, Sechang Instruments., Ltd., Seoul, Korea), repeated 5 times to obtain the average value. To determine the sweetness of soybean paste, add 9 mL of distilled water to 1g of soybean paste sample, stir, let stand for more than 1 hour, and then centrifuge with a centrifuge (HA-12 centrifuge, Hanil Science Industrial Co., Inchun, Korea). The supernatant obtained is measured using a sugar content meter (PAL-12 centrifuge). 1, Atago Co. Ltd., Tokyo, Japan), and the measurements were repeated five times. Salinity was measured at room temperature using a salinity meter (EB-158P, EISHIN, Japan) by mixing 1 g of soybean paste sample with 9 mL of distilled water and taking the supernatant. The test was repeated 5 times.

As a result, the pH, sugar content, and salinity of soybean paste produced using the seed meju of the present invention were confirmed as shown in Table 4 below (control group; basic soybean paste, DMP2.5, DMP5.0; dominant seed meju of the genus Penicillium or Mucor Soybean paste prepared by adding 2.5% or 5.0% of DAsp2.5, DAsp5.0; soybean paste prepared by adding 2.5% or 5.0% of Aspergillus genus, respectively) (***: p<0.001 ) (Mean±SD, values indicated with the same letter in the horizontal column are not significantly different at the p<0.05 level by Duncan's multiple range test).

Specifically, in the case of pH, the control group was found to be the highest at 5.99, but DAsp5.0, DAsp2.5, DMP5.0, and DMP2.5 were also found to be 5.51, 5.38, 5.05, and 4.97, respectively, showing significant differences in all experimental groups. shown ( p< 0.05). The difference in pH value of soybean paste is due to the difference in the fermentation period of the soybean paste and the difference in organic acids produced by lactic acid bacteria and yeast (Jeon et al. 2016). In a study on the physicochemical properties of traditional soybean paste produced in Sunchang Gochujang Folk Village (Kim et al. 2006), the average pH of soybean paste was 5.41, and the pH range of 4.80 to 6.11 was confirmed, so the pH of the soybean paste subject to this experiment was 4.97 to 5.99. It was found to be within the average range.

In the case of sugar content, DAsp5.0 was 3.67 °Brix, DAsp2.5 was 3.60 °Brix, control group was 3.57 °Brix, DMP5.0 was 3.53 °Brix, and DMP2.5 was 3.40 °Brix, with significant differences and lower values. shown ( p< 0.05). It is known that the sugar content of most soybean paste reaches its maximum at the beginning of fermentation, and then decreases as sugar is used as a substrate for alcohol fermentation and organic acid fermentation by microorganisms (Byun et al. 2014).

The control group showed the highest salinity value at 13.27% (w/v), followed by DAsp2.5 at 12.80% (w/v), DMP2.5 at 12.53% (w/v), and DAsp5.0. This decreased by 11.67% (w/v) and DMP5.0 by 10.83% (w/v), showing a significant difference among all experimental groups ( p< 0.05). According to a study on the quality characteristics of improved soybean paste and traditional soybean paste (Jeon et al. 2016), the average value of improved soybean paste was 10.80 ~ 11.40%, and traditional soybean paste was used more than factory-farmed soybean paste to improve storage, and was 11.77 ~ 14.22%. In that the salinity results of this experiment were confirmed to represent the characteristics of traditional soybean paste.

Example 8. Confirmation of excellent sensory properties of meju and soybean paste manufactured from sea meju

Sensory quality evaluation was conducted to confirm the sensory properties of meju and soybean paste produced using the sea meju selected according to Example 2. Specifically, meju prepared without adding sea meju was divided into a control group, meju with Penicillium or Mucor 2.5% (hereinafter MMP2.5) added, meju with Penicillium or Mucor 5.0% (hereinafter MMP5.0) added, and Aspergillus 2.5% (hereinafter MAsp2. 5) Added meju, and 5 types of meju added with Aspergillus 5.0% (hereinafter MAsp5.0) and 5 types of soybean paste samples each made using them. Control group, Penicillium or Mucor 2.5% (hereinafter referred to as DMP2.5) added soybean paste, Penicillium or Mucor 5.0 % (hereinafter referred to as DMP5.0) soybean paste, Aspergillus 2.5% (hereinafter referred to as DAsp2.5) added soybean paste, and Aspergillus 5.0% (hereinafter referred to as DAsp5.0) added soybean paste) were used as samples for sensory evaluation. The samples were stored in a refrigerator and placed at room temperature for evaluation 1 hour before the start of the experiment before being tested and tested.

Panelists for sensory evaluation are adults living in Seoul and the metropolitan area who enjoy eating soybean paste-based foods, have previous experience working as sensory evaluation agents, and can participate in regular meetings during the activity period. We recruited people who would actively participate without dropping out, who had the personality to work as a member of a group, and who were faithful to maintaining security. Next, seven descriptive analysis panelists were selected. The selected panel learned an overview of basic descriptive analysis evaluation. In addition, in order to objectively evaluate meju and soybean paste, the participants experienced the appearance, aroma, and flavor of soybean paste with various flavors to become more familiar with these samples, and additional training was conducted to derive and evaluate descriptive terms. From the time of selection until the main experiment, training was conducted twice a week for 4 hours each.

To evaluate the appearance, scent/smell, and flavor of meju and soybean paste samples, place 3g each in a personal evaluation container (5 cm diameter It was provided by granting. For flavor evaluation, a small disposable spoon was prepared to minimize the variation in taste caused by tasting a large amount of samples. To prevent the dulling of taste, lukewarm water containing bread and lemon was used as a mouthwash to rinse the mouth so that no flavor remained. There was a break of about 5 minutes between each sample, and if the scent or flavor remained for a long time, the next experiment was conducted after a sufficient break in the hallway outside the laboratory.

In a preliminary experiment of descriptive analysis, descriptive terms for the appearance, aroma, and flavor of soybean paste and soybean paste were developed and defined. In addition, the experiment was repeated until all panelists agreed that the standard sample suggested by the panel leader (researcher) was suitable to represent each characteristic. The preliminary experiment continued until all panelists detected differences between samples and evaluated them with reproducibility. During the preliminary experiment, standards for description terms, definitions, standard samples, and intensity scores of standard samples were developed for meju and soybean paste, and definitions of terms for sensory characteristics for descriptive analysis of meju and terminology for sensory characteristics for descriptive analysis of soybean paste were developed. Definitions are shown in Table 5 and Table 6, respectively.

This experiment was repeated 3 times by 7 panelists, evaluated with 21 observations, and the results were analyzed. Using the descriptive terms established during the preliminary experiment, the intensity of sensory characteristics was evaluated on a 9-point item scale for a total of 10 samples, including 5 types of meju and 5 types of soybean paste, on the completed evaluation sheet. The sensory characteristics of soybean paste samples were divided into appearance characteristics, aroma, and overall satisfaction, and the sensory characteristics of soybean paste samples were divided into appearance characteristics, aroma/smell characteristics, flavor characteristics, and overall satisfaction. The appearance characteristics were evaluated by the degree of brightness and darkness, the aroma/smell characteristics were evaluated by holding the sample close to the nose and smelling the sample, and the flavor characteristics were evaluated by the sensory characteristics that appear when the sample is in the mouth after holding it in the mouth. . When evaluating the intensity of sensory characteristics of each sample, the standard sample established during the preliminary experiment and its intensity were presented together to induce a more accurate evaluation of each sample. The intensity scale of each meju and soybean paste sample used a 9-point item scale, with 1 starting from “very weak” and gradually increasing the intensity, with a maximum score of 9 indicating “very strong.” The overall satisfaction scale is a 9-point Hedonic scale, with satisfaction increasing from 1 point, “I dislike it very much,” to “very good,” with a maximum score of 9 points, with satisfaction increasing as the number increases. This sensory evaluation was conducted after review by the Institutional Bioethics Committee (IRB No.: 2-1040966-AB-N-01-2109-HSR-247-0) and receiving a notice of exemption from review.

In addition, data analysis of the sensory evaluation results was performed using one-way ANOVA using the SPSS (Statistical package for the social sciences, Ver 20.0, SPSS Inc., Chicago IL, USA) program, and the results were Expressed as mean ± standard deviation. The significance between the average values of each measurement was verified at the p< 0.05 level by Duncan's multiple range test.

First, the results of evaluating the sensory characteristics of meju by descriptive analysis are shown in Table 7 (control group; basic meju, DMP2.5, DMP5.0; manufactured by adding 2.5% or 5.0% of dominant meju of the genus Penicillium or Mucor, respectively. Meju produced by adding 2.5% or 5.0% of the dominant meju of the genus Aspergillus ; DAsp2.5, DAsp5.0, respectively) ( ^NS : not significant, ^** : p <0.01, ^*** : p <0.001* **: p<0.001) (Mean±SD, values indicated with the same letter in the horizontal column are not significantly different at the p<0.05 level by Duncan's multiple range test).

Specifically, the brightness of meju was evaluated as the brightest in the control group at 6.7 points, followed by MMP2.5 at 5.5 points, MMP5.0 at 4.6 points, MAsp2.5 at 4.5 points, and MAsp5.0 at 3.4 points, showing a significant difference. It appeared low (p<0.05). In terms of scent, the highest value for Shinnae was 5.7 points for MAsp5.0 ( p< 0.05), while MAsp2.5, MMP5.0, MMP2.5 and the control group tended to decrease from 4.5 to 3.6 points, but MAsp2 Only the 4.5 points of .5 and the 3.6 points of the control group showed a significant difference ( p< 0.05). The sweetness of soybean paste was evaluated to be similar with no significant difference in all experimental groups, ranging from 4.8 to 3.87 points. Salty MAsp5.0 decreased to 5.5 points, MAsp2.5 to 5.0 points, MMP5.0 to 4.5 points, MMP2.5 to 4.1 points, and the control group decreased to 3.5 points, but there was no significant difference in all experimental groups and in some groups. There was a significant difference ( p< 0.05). In the savory flavor, the values of MAsp2.5, MAsp5.0, MMP5.0, and MMP2.5 were found to be excellent, ranging from 5.1 to 4.4 points, which was confirmed to be a significant difference from the control group's score of 3.6 ( p < 0.05). Within Gundeok, similar values were shown among all experimental groups, showing no significant differences. The overall satisfaction level of meju showed a similar trend to that of the savory flavor, showing an above average score of 5.7 to 5.3 points in the order of MAsp5.0, MAsp2.5, MMP5.0, and MMP2.5, and a below average score of 4.2 points. It was confirmed that there was a significant difference from the evaluated control group ( p< 0.05).

In addition, the results of the descriptive analysis of soybean paste are shown in Table 8 (control group; basic soybean paste, DMP2.5, DMP5.0; soybean paste made from meju made by adding 2.5% or 5.0% of dominant meju from Penicillium or Mucor genus, respectively; DAsp2 .5, DAsp5.0,; soybean paste made from meju made by adding 2.5% or 5.0% of Aspergillus genus dominant meju, respectively) ( ^NS : not significant, ^* : p <0.05, ^** : p <0.01, ^{** *} : p <0.001) (Mean±SD, values indicated with the same letter in the horizontal column are not significantly different at the p<0.05 level by Duncan's multiple range test).

Specifically, the brightness of the appearance of soybean paste was highest in the control group at 6.5 points, followed by DMP2.5 and DAsp2.5 at 5.5 and 5.0 points, respectively, and DMP5.0 and DAsp5.0 at 4.0 and 3.4 points, respectively. There was a significant difference ( p< 0.05). It is known that the brightness of soybean paste is due to the change in color of soybean paste components through enzymatic and non-enzymatic browning reactions during storage (Shim et al. 2018).

Both DAsp2.5 and DAsp5.0, which smell exciting, showed a significant difference of 5.4 points from the control group's 4.1 point ( p< 0.05), but the remaining four experimental groups excluding the control group had similar values and did not show a significant difference. The sweetness of soybean paste was evaluated as similar with DMP5.0, DAsp5.0, DAsp2.5, and DMP2.5 values ranging from 5.2 to 4.7 points, but all other experimental groups showed significant differences from the control group's 3.9 points ( p< 0.05 ). The salty Aspergillus group DAsp5.0 and DAsp2.5 were evaluated as saltier than the other experimental groups with 5.7 and 5.5 points, respectively, while DMP5.0, DMP2.5, and the control group were significantly less salty with 4.3 to 3.8 points ( p < 0.05).

The savory flavor of soybean paste showed the highest value of 5.4 points for DAsp5.0, a similar value to 4.7 points for DAsp2.5, and a significant difference from 4.1 to 3.9 points for DMP5.0, DMP2.5, and the control group. There was no significant difference in the remaining experimental groups except for B ( p< 0.05) and DAsp5.0. In Gundeoknae, all experimental groups showed similar values of 4.9 to 3.9 points, showing no significant difference. The sour taste of soybean paste, which is known to be caused by organic acids such as lactic acid and malic acid produced by lactic acid bacteria (Shim et al. 2018), showed a similar trend without significant differences in all experimental groups.

It is known (Shim et al. 2018) that the sweet taste of soybean paste is produced by the carbohydrates of soybeans produced by hydrolysis of saccharification enzymes and the increase of sweet amino acids such as glycine, alanine, serine, lysine, proline, and threonine. , Aspergillus experimental group DAsp5.0 and DAsp2.5 scored 5.3 and 5.1 points, respectively, showing a significant difference from the control group's 3.9 points, showing high results ( p< 0.05), but Penicillium or Mucor experimental group DMP5.0 and DMP2.5 was similar to the control group and showed no significant difference.

There was no significant difference in the salty taste of soybean paste, with similar values ranging from 6.1 to 5.7 points in all experimental groups. Like the salty taste, bitter and savory tastes showed similar trends in all experimental groups, so it was evaluated that there was no difference in quality characteristics from the control group. The umami taste of soybean paste showed a similar trend to the sweetness, with Aspergillus experimental group DAsp2.5 and DAsp5.0 scoring 5.1 and 4.9 points, respectively, showing a significant difference from the control group's 3.6 points ( p< 0.05), but Penicillium or Mucor experimental group DMP5 .0 and DMP2.5 showed similar values to the control group, ranging from 4.2 to 3.6 points. The taste of gundeok, like salty, bitter, and savory, showed similar values in all experimental groups, ranging from 4.6 to 3.9 points, with no significant difference. The overall satisfaction with soybean paste was rated above average for the Aspergillus experimental group DAsp2.5 and DAsp5.0, with 5.5 and 5.4 points, respectively, and was significantly higher than the 4.7 and 4.2 points for DMP2.5 and the control group, and DMP5.0 The score was 5.1 points, which showed a significant difference from the control group and was above the average score ( p< 0.05).

According to these results, meju and soybean paste using sea meju that does not produce aflatoxin were confirmed to be superior in functionality and sensory properties compared to the control group. In particular, in the case of meju and soybean paste using sea meju according to this example, although there was no significant difference from the control group in pH, sugar content, salinity, sweetness, etc., it showed significantly superior sensory properties compared to the control group in terms of overall satisfaction. It was confirmed that the combination of sensory characteristics provides the best taste and flavor.

In particular, not only was it confirmed that Aspergillus genus isolates were dominant, it was superior to other genus, but also a significantly superior effect was confirmed in that aflatoxin production was drastically reduced even at a very small amount (less than 0.5) relative to the weight of steamed soybeans.

Overall, the present invention relates to a method for producing meju and soybean paste using cimeju. The conditions for cimeju that were not used in the prior art were established and applied to the meju and soybean paste production method. The sea meju has the characteristics of no or reduced aflatoxin production, so it has excellent functionality and uniform quality, and since it is naturally fermented, it is possible to produce Korean (traditional or conventional) meju and soybean paste with excellent sensory properties in terms of flavor, etc. It was confirmed that it is possible to provide meju and soybean paste that improve the problems of existing Korean meju and Korean soybean paste or their production methods.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

According to the manufacturing method of Korean meju and soybean paste with uniform quality and reduced aflatoxin using sea meju, the concept and conditions of sea meju and the meju and soybean paste manufacturing method applying the same have been established, which not only significantly reduces the production of aflatoxin but also improves the quality. In that it is possible to produce Korean meju and soybean paste that are uniform and have excellent flavor, it is useful as a new Korean meju and soybean paste production method that solves the problems of the existing Korean meju and soybean paste production method and the meju and soybean paste produced by the above method. As far as possible, industrial applicability is recognized.

Claims (9)

Korean soybean paste manufacturing method comprising the following steps: (S1) manufacturing sea meju; (S2) preparing Korean-style meju by adding the sea meju; and (S3) Step of producing Korean soybean paste from the Korean meju. The method of producing Korean soybean paste according to claim 1, wherein the sea meju satisfies any one or more of the following conditions: a1) Aflatoxin was not detected; a2) Aflatoxin biosynthetic gene was deleted from the C. meeju isolate; and a3) Aflatoxin is not produced from the C. meju isolate. According to paragraph 2, The method of producing Korean soybean paste, wherein the isolate is a dominant strain of at least one of Aspergillus sp. , Penicillium sp. , or Mucor sp. According to paragraph 2, The aflatoxin biosynthetic gene is one or more selected from the group consisting of aflO, aflP, and aflR, a method for producing Korean soybean paste. According to paragraph 2, The deletion is a method of producing Korean soybean paste in which the function of the gene is lost. The method for producing Korean soybean paste according to claim 1, wherein the step (S2) includes the following steps: (b1) soaking, steaming, and cooling soybeans; (b2) manufacturing unripened meju by molding the ground soybeans after cooling; (b3) adding sea meju to step (b2); and (b4) Fermenting the unripe meju. The method of claim 6, wherein the amount of seed meju added in step (b3) is based on the total weight of the ground soybeans, and the dominant strain of the seed meju is respectively In the case of Aspergillus sp., it is more than 0.5% by weight, A method for producing Korean soybean paste, wherein the amount is 2.5% by weight or more in the case of Penicillium sp. or Mucor sp. Soybean paste manufactured by the method of paragraph 1. According to clause 8, The soybean paste is characterized in that no aflatoxin is detected.

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