KR20230158695A - Method of producing B.clausii and alkaline protease by using solid-state fermentation and thereof feed additives - Google Patents

Abstract

The present invention relates to a production method of B.clausii and alkaline proteolytic enzymes using solid-state fermentation and a feed additive using the same. The production method according to the present invention enables simultaneous production of enzymes and live bacteria using a solid-state fermentation method, and enzymes It provides optimal conditions for both activity and production of viable bacteria, and has the effect of producing an economical and nutritionally excellent feed additive by using soybean meal as a substrate.

Description

Method of producing B.clausii and alkaline protease by using solid-state fermentation and feed additives using the same {Method of producing B.clausii and alkaline protease by using solid-state fermentation and its feed additives}

The present invention relates to a method for mass production of B.clausii and alkaline proteolytic enzymes using solid-state fermentation and a feed additive using the same.

Over the past several decades, antibiotics, antibacterial agents, etc. have been used as livestock feed additives to increase livestock productivity. However, the indiscriminate misuse of antibiotics and antibacterial agents is causing serious side effects, such as increased pathogen resistance or residual problems in livestock products. It also increases resistance to diseases in livestock while preventing bioaccumulation of antibiotics or the emergence of resistant bacteria. The development of feed additives is required.

Accordingly, probiotics are attracting attention as substances that can replace antibiotics and antibacterial agents. Probiotics, which have the opposite etymological meaning of antibiotics, are live microbial additives that can induce beneficial effects on host animals by improving the intestinal microbial balance. In addition, probiotics not only have effects such as promoting enzyme secretion in the animal body, improving the digestibility of fluorinated fiber, and reducing anti-nutritional factors, but also have the effect of preventing diseases and regulating immunity.

Microorganisms currently used as probiotics include lactic acid bacteria, Bacillus subtilis, Bacillus, and yeast, but among them, strains of the genus Bacillus, including Bacillus clausii, have many advantages that distinguish them from other microorganisms. For example, because it forms spores, it has acid resistance to strong stomach acid and can maintain its activity until it reaches the intestines, and it produces bacteriocins or BLS (bacteriocin-like substances) with a wide antibacterial spectrum and various structures. In addition, the important products produced by Bacillus strains, such as protease, amylase, cellulase, and glucanase (glucan digestive enzyme), are sources of enzymes needed in industry, and especially when added to livestock feed, they improve feed efficiency and reduce odor. It can exert effects such as suppressing outbreaks.

In particular, Bacillus clausii has many advantages as a probiotic candidate that can be added to livestock feed. It has been proven through various studies that Bacillus claugi is excellent for preventing and treating diarrhea (Sudha MR et al (2013) Beneficial Microbes 4: 211-216; Nista et al (2004) Aliment Pharmacol Ther 20: 1181- 1188; Keya L et al. (2015) IOSR Journal of Dental and Medical Sciences 14(5): 74-76; Jayanthi N et al (2015) J Yoga Phys Ther 5(4): 1.), of IFN-γ It is known to have excellent immunomodulatory activity by increasing production and inducing proliferation of CD4 + T cells (Urdaci et al., J Clin Gastroenterol vol.38, 2004). Therefore, when Bacillus claugi is used as a probiotic as a substitute for antibiotics and antibacterial agents, it will not only increase the productivity of livestock but also have the effect of disease prevention and immune regulation.

Meanwhile, solid state fermentation (SSF) is a method used to manufacture traditional fermented foods and feed additives. It is relatively inexpensive and easy to efficiently mass-produce fermented products without special equipment, so it is widely used in the fermentation industry. It is preferred. In addition, during solid-state fermentation, relatively inexpensive substrates can be used, and substrates hydrolyzed by microorganisms during the fermentation process can be used as high-quality and high-value feed raw materials. In addition, because the amount of moisture used during solid-state fermentation is small, the drying process is simpler compared to liquid fermentation, which is advantageous for preserving the number of viable bacteria, making recovery of finished products easy, and is a fermentation method that is environmentally more advantageous due to less energy usage and wastewater generation.

In addition, the liquid fermentation method may be capable of producing both enzymes and live bacteria, but liquid fermentation may rarely produce live bacteria depending on the fermentation method. Therefore, it is necessary to develop a solid-state fermentation method that can produce both viable bacteria and enzymes, and in particular, a solid-state fermentation method that focuses on increasing the number of viable bacteria in the final product is needed.

As a conventional technology using solid-state fermentation for the production of live bacteria and enzymes, the Bacillus subtilis strain is described in Korean Patent Publication No. 10-1073986 (New Bacillus subtilis strain and solid-state fermentation method of palm fruit seed meal using the same). A method of producing by solid-state fermentation using palm fruit seed meal is disclosed, and Republic of Korea Patent Publication No. 10-0645284 (solid-state fermentation using novel Bacillus subtilis ＧR-101 and Aspergillus oryzae ＧB-107) (Method for processing enzyme production ability using enhanced solid-state fermentation soy protein and its uses) discloses a processing method with enhanced enzyme production ability using a solid-state fermentation technique using soybean meal, but simultaneously produces large amounts of proteolytic enzymes derived from Bacillus claugi There was no literature describing the production method.

Accordingly, while researching a method for simultaneous mass production of Bacillus claugi and Bacillus claugi-derived proteolytic enzymes, the present inventors discovered the Bacillus claugi probiotic activity and Bacillus claugi-derived proteolytic enzyme activity during solid-state fermentation using soybean meal as a substrate. By discovering the optimal production method for this increase, the present invention was completed.

The purpose of the present invention is to provide a method for mass production of live bacteria and enzymes using solid-state fermentation.

Another object of the present invention is to provide a solid-state fermentation product containing live bacteria and enzymes produced by the above method.

Another object of the present invention is to provide a feed additive containing the solid fermentation product.

Another object of the present invention is to provide an odor reducing agent containing the solid fermentation product.

In order to achieve the above object, one aspect of the present invention is,

a) sterilizing 50 g of soybean meal at 121°C for 15 minutes to obtain sterilized soybean meal;

b) cooling the sterilized soybean meal at room temperature and then adding a sterilized aqueous sodium carbonate solution to obtain a soybean meal suspension with an initial moisture content of 60 to 80% of the weight of the soybean meal;

c) inoculating the soybean meal suspension with 500 μl of Bacillus clausii seed culture cultured in TSB (Tryptic soy broth) for 24 hours to obtain inoculated soybean meal; and

d) solid-state fermentation of the inoculated soybean meal in a shaking incubator at 37°C for 36 to 60 hours.

The present invention provides an optimal manufacturing method that enables simultaneous production of enzymes and live bacteria through a solid-state fermentation method, rather than a liquid fermentation method that only allows enzyme production, and is excellent in both enzyme activity and live bacteria production, and is economical by using soybean meal as a substrate. However, it is effective in producing nutritionally excellent feed additives.

Figure 1 shows the relative enzyme activity (bar graph) and viable cell count (dot graph) of eight types of substrates (soybean meal, sugar coat meal, ethanol meal, confectionery meal, soybean hull, wheat pick, protein hull, and rice hull). .
Figure 2 shows the relative enzyme activity of eight types of substrates (soybean meal, sugar coat meal, ethanol meal, confectionery meal, soybean hull, wheat pick, protein hull, and rice husk) by time (24 hours, 48 hours, and 72 hours). will be.
Figure 3 shows the relative enzyme activity (bar graph) and viable cell count (dot graph) according to the concentration of sodium carbonate.
Figure 4 shows relative enzyme activity (bar graph) and viable cell count (dot graph) according to initial moisture content.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention is,

a) sterilizing 50 g of soybean meal at 121°C for 15 minutes to obtain sterilized soybean meal;

b) cooling the sterilized soybean meal at room temperature and then adding a sterilized aqueous sodium carbonate solution to obtain a soybean meal suspension with an initial moisture content of 60 to 80% of the weight of the soybean meal;

c) inoculating the soybean meal suspension with 500 μl of Bacillus clausii seed culture cultured in TSB (Tryptic soy broth) for 24 hours to obtain inoculated soybean meal; and

d) solid-state fermentation of the inoculated soybean meal in a shaking incubator at 37°C for 36 to 60 hours.

The live bacteria produced by the above production method are Bacillus claugi (Accession number KCTC 10277BP, Republic of Korea Patent Publication No. 10-0465852). The Bacillus claugi is a live bacterium known as a proteolytic enzyme-producing strain isolated from the mudflats of the West Sea.

The enzyme is an alkaline protease derived from Bacillus claugi (accession number KCTC 10277BP). The proteolytic enzyme derived from Bacillus claugi is a stable enzyme that maintains activity even under various physical and chemical conditions such as high pH, surfactants, oxidants, organic solvents, and heavy metals (Korean Patent Registration No. 10-0465852).

Step a) is a step of obtaining sterilized soybean meal by sterilizing 50 g of soybean meal at 121°C for 15 minutes.

The soybean meal is generally a by-product remaining after pulverizing soybeans to extract oil, and unlike SBM (soy bean meal, defatted soybean meal) or SPC (soy protein concentrate), which are commonly used in liquid fermentation, it is a low-cost substrate. , It is nutritionally excellent and is advantageous for use as feed. The specific components of soybean meal are 55-56% protein, 13-14% soluble carbohydrate, 21-22% insoluble carbohydrate, 4-6% ash, and about 1% fat based on dry weight (In-gyu Han, Feed Engineering, Advanced Cultural History, pp 67-107, 1998). However, stachyose and raffinose, which are components of soybean meal, are composed of alpha-galactosyl bonds, so animals without alpha-galactosidase enzyme have no ability to digest them. It may fall (Journal of the Korean Fisheries Society 33, pp 469-474). Therefore, in order to use soybean meal as a feed additive, it is necessary to use microbial fermentation technology.

Step b) is a step of cooling the sterilized soybean meal at room temperature and then adding a sterilized aqueous sodium carbonate solution to obtain a soybean meal suspension with an initial moisture content of 60 to 80% of the weight of the soybean meal.

The content of sodium carbonate in the sodium carbonate aqueous solution in step b) is 0.5 to 2.0% of the weight of soybean meal. Preferably, the content of sodium carbonate is 0.5 to 1.5% of the weight of soybean meal, and more preferably 1.0% of the weight of soybean meal. The pH of a soybean meal suspension containing 1.0% sodium carbonate by weight of soybean meal is 7.3. If the sodium carbonate content is 0.5% or less, fermentation does not proceed, and if the sodium carbonate content is 2% or more, relative enzyme activity or viable cell production may decrease.

The initial moisture content of the soybean meal suspension is 60 to 80%, preferably 65 to 75%, and more preferably 70% of the soybean meal weight. If the initial moisture content is less than 60% of the weight of soybean meal, or more than 80%, the relative enzyme production or viable cell production may decrease.

Step c) is a step of inoculating the soybean meal suspension of step b) with 500 μl of Bacillus clausii spawn cultured in TSB (Tryptic soy broth) for 24 hours.

Step d) is a step of solid-state fermentation of soybean meal inoculated with the Bacillus claugi spawn in a shaking incubator at 37°C for 36 to 60 hours. Preferably, the solid-state fermentation is carried out for 42 to 56 hours, and more preferably, it is carried out in a shaking incubator for 48 hours. If the solid-state fermentation time is less than 36 hours or more than 60 hours, the relative enzyme production or viable cell production may decrease.

Another aspect of the present invention is to provide a solid-state fermentation product containing live bacteria and yeast produced according to the above method.

Another aspect of the present invention is to provide a feed additive containing the above solid-state fermentation product.

Another aspect of the present invention is to provide an odor reducing agent containing the above solid fermentation product.

In a specific example of the present invention, the effect of each substrate was analyzed for the optimal method for mass production of live bacteria and enzymes. As a result of selecting and analyzing a total of eight substrates (soybean meal, sugar coat meal, ethanol meal, confectionery meal, soybean hull, wheat hull, protein hull, and rice hull) among the relatively cheaper substrates than the existing SBM or SPC, soybean meal was found to be When used as a substrate, not only was the relative enzyme production and viable cell production the best (Figure 1 and Table 2), but it was also confirmed that it could be used nutritionally excellently due to the high crude protein content (Table 1).

Additionally, the optimal fermentation time for mass production of live bacteria and enzymes was analyzed. As a result of solid-state fermentation for 24 hours, 48 hours, and 72 hours, it was confirmed that the relative enzyme production and viable cell production were the best when fermented for 48 hours (Figure 2 and Table 3).

In addition, the optimal pH conditions for mass production of live bacteria and enzymes were analyzed. In microbial culture, pH is an important factor that determines whether microorganisms will grow. Since the degree of growth and the amount of secondary metabolites produced also vary depending on pH, it is one of the important factors in the production of viable bacteria and enzymes. The pH was adjusted by the concentration of sodium carbonate added, and as a result of analysis according to the sodium carbonate concentration ranging from 0% to 3% of the weight of soybean meal, the relative enzyme production and viable cell production were highest when the sodium carbonate content was 1% of the weight of soybean meal. It was confirmed that it was excellent (Figure 3 and Table 5), and the pH of the soybean meal suspension at the above concentration was 7.30 (Figure 3 and Table 4).

In addition, the optimal moisture content conditions for mass production of live bacteria and enzymes were analyzed. In the production of live bacteria and enzymes, moisture content is one of the important factors as it is related to the growth rate of live bacteria and enzymes and inhibition of fermentation. The initial moisture content of the soybean meal used to analyze the effect of moisture content was 11%, and the initial moisture content was adjusted to a range of 50 to 100% using an aqueous sodium carbonate solution of 1% of the weight of the substrate (soybean meal). As a result, it was confirmed that the relative enzyme production and viable cell production were the best when the initial moisture content was 70% (Figure 4 and Table 6).

Hereinafter, the present invention will be described in detail by examples.

However, the following examples only illustrate the present invention, and the content of the present invention is not limited by the following examples.

<Example 1> Comparison of production of live bacteria and enzymes according to substrate

<1-1> Preparation of B.clausii and alkaline proteolytic enzyme

In order to identify substrates suitable for the production of B.clausii and alkaline proteolytic enzymes using solid-state fermentation, eight types of raw materials that can be used as feed were selected and used. Four types of cucurbits and four types of peels were compared, and the raw materials were purchased from Hanjung SS Co., Ltd. Table 1 below shows the crude protein and moisture content for each substrate.

temperament soybean meal ethanol Cookie cake sugar
coat bag soybean husk protein blood wheat peas chaff crude protein 45% 27% 7% 21% 10% 21% 15% 3% moisture
content 11% 11% 9% 22% 13% 11% 13% 10%

50 g of each substrate shown above was placed in a flask and sterilized at 121°C for 15 minutes. After cooling the sterilized substrate at room temperature, sterilized aqueous sodium carbonate solution was added to adjust the initial moisture content to 70% of the weight of the substrate. At this time, the sodium carbonate content was adjusted to 1% of the substrate weight.

A substrate with an initial moisture content of 70% of the substrate weight was inoculated with 500 μl of Bacillus clausii spawn cultured in TSB (Tryptic soy broth) for 24 hours. Next, the inoculated substrate was subjected to solid-state fermentation in a shaking incubator at 37°C for 48 hours.

<1-2> Method of measuring viable cell count and enzyme production

The number of Bacillus claws produced in <1-1> was measured using the plate count method, and the same experiment was repeated three times and the average value was derived. Add 90 ml of physiological saline to 10 g of solid-state fermentation and homogenize it by shaking for 20 minutes. Then, the suspension was mixed with 10-1, 10-2, 10-3... using physiological saline. Serial dilution was performed, etc. 100 μl of each dilution was plated on TSA (Tryptic soy agar) medium, and the plate was cultured at 37°C for 24 hours. The number of colonies produced after culturing was measured, multiplied by the dilution factor, converted to the number of bacteria per g of test sample, and expressed in CFU (colony-forming unit) units.

In addition, the activity of the enzyme prepared in <1-1> was tested three times and the average value was derived. 90 ml of distilled water was added to 10 g of solid-state fermentation, homogenized by shaking for 20 minutes, and then centrifuged (8000 rpm, 10 minutes) to recover the supernatant. Next, a substrate solution was prepared by dissolving casein in 0.1M Glycine buffer (pH11) to a final concentration of 0.5%. To 0.5 ml of the substrate solution, 0.01 ml of supernatant diluted 10 to 1000 times with 0.1 M Glycine buffer (pH 11) was added. Enzymatic reaction was carried out with the mixture of the substrate solution and the diluted supernatant at 60°C for 10 minutes, and then 0.5 ml of 1.8% trichloroacetic acid, 3% sodium acetate, and 1.9% acetic acid solution was added to stop the reaction and placed on ice. Unreacted proteins were precipitated for 10 minutes. After centrifugation at 14000 rpm for 10 minutes, the supernatant was taken, the absorbance was measured at 275 nm, and the amount of tyrosine produced was calculated. At this time, 1U of proteolytic enzyme was defined as the amount of enzyme required to produce 1 μg of tyrosine in 1 minute, and enzyme activity was expressed as relative enzyme activity (relative enzyme production = measured value / highest value × 100). .

Figure 1 and Table 2 below summarize the viable cell production and relative enzyme production measured by the above method according to the substrate.

Relative enzyme production
(Relative enzyme activity) Probiotic production
(CFU/g) soybean meal 100% 4.4E+10 Sugar coat bag 29% - ethanol 13% - Cookie cake 14% - soybean husk 58% 6.3E+09 wheat peas 45% 1.1E+10 protein blood 29% - chaff 36% 6.5E+09

As a result of the experiment, among the eight substrates, soybean meal, soybean hull, and wheat hull had the highest productivity of viable bacteria and enzymes in that order. In particular, soybean meal was selected as the optimal substrate because it has a high crude protein content (Table 1) and is good for use as feed due to its excellent nutritional properties.

<Example 2> Optimization according to fermentation time

The optimization method of live bacteria and enzyme production according to fermentation time was analyzed. Live bacteria and enzymes were produced in the same manner as in <1-1> above, and the solid-state fermentation times were set to 24 hours, 48 hours, and 72 hours and compared. The experiment was repeated three times for each fermentation time and the average value was derived, and viable cells and enzyme production were measured in the same manner as in <1-2> above.

Figure 2 and Table 3 below show the relative enzyme production and viable cell production according to time and substrate.

Relative enzyme production
(Relative enzyme activity) Probiotic production
(CFU/g) temperament 24hrs 48hrs 72hrs 24hrs 48hrs 72hrs soybean meal 16% 100% 95% 1.0E+10 4.4E+10 2.1E+10 Sugar coat bag 29% 29% 28% - - - ethanol 14% 13% 13% - - - Cookie cake 16% 14% 14% - - - soybean husk 20% 58% 60% 1.2E+10 6.3E+09 1.0E+09 wheat peas 27% 45% 41% 2.6E+10 1.1E+10 7.2E+09 protein blood 29% 29% 28% - - - chaff 19% 36% 54% 2.1E+08 6.5E+09 5.0E+09

As a result of the experiment, based on 24-hour solid-state fermentation, sugar coat meal and protein peel showed the highest relative enzyme production of 29%, but when solid-state fermentation was performed for 48 hours, soybean meal was the best with relative enzyme production increasing to 100%. As a result of 72-hour solid-state fermentation, all substrates except rice husk showed the same or lower activity than 48-hour solid-state fermentation. Probiotic production also showed a similar trend to that of enzymes.

Therefore, based on the selected substrate, soybean meal, the optimal fermentation time for enzymes and viable bacteria was determined to be 48 hours.

<Example 3> Optimization method according to pH

Optimization methods for live bacteria and enzyme production according to pH were analyzed. Live bacteria and enzymes were produced using soybean meal in the same manner as in <1-1> above, and the sodium carbonate content of the sodium carbonate aqueous solution used can be 0%, 1%, 2%, or 3% of the weight of soybean meal (substrate). The experiment was adjusted to ensure that The experiment was repeated three times for each pH and the average value was derived, and viable cells and enzyme production were measured in the same manner as in <1-2> above. The pH of the soybean meal suspension according to the concentration of sodium carbonate is shown in Table 4 below.

Sodium carbonate concentration Soybean meal suspension pH 0% 6.25 One% 7.30 2% 8.83 3% 9.45

Figure 3 and Table 5 below show the relative enzyme production and viable cell production according to the concentration of sodium carbonate.

Sodium carbonate concentration Relative enzyme production
(Relative enzyme activity) Probiotic production
(CFU/g) 0% 6% - One% 100% 6.5E+10 2% 65% 5.7E+10 3% 41% 5.4E+10

As a result of the experiment, if sodium carbonate was not added, fermentation did not proceed, and as the concentration of sodium carbonate increased, the relative enzyme production and viable cell production decreased. Therefore, the optimal amount of sodium carbonate added for strain and enzyme production was determined to be 1% of the substrate.

Microorganisms each have an optimal pH value for growth, and are classified into acidophilic, neutrophilic, basophilic, and extreme basophilic groups depending on the optimal pH for growth. The optimal pH for acidophiles is 0 to 5.5, for neutrophils, pH 5.5 to 8.0, for alkalophiles, pH 8.5 to 11.5, and for extreme alkalophpiles, pH is 5.5 to 8.0. It is 10.0 or higher.

In other words, when cultivating microorganisms, pH is an important factor that determines whether microorganisms will grow. The degree of growth and the amount of secondary metabolites produced also vary depending on pH. In the Bacillus claugi strain of the present invention, there was also a significant difference in viable cell and enzyme production depending on pH, as shown in Table 5 above. Therefore, the optimized pH for strain and enzyme production was pH with 1% sodium carbonate added compared to the substrate. It was experimentally confirmed that it was 7.30. In addition, the above results confirmed that it is included in neutrophilic organisms.

<Example 4> Optimization method according to moisture content

Optimization methods for live bacteria and enzyme production according to moisture content were analyzed. Live cells and enzymes were prepared using soybean meal in the same manner as in <1-1> above, and the sodium carbonate aqueous solution used was 50% and 60% of the weight of the substrate, respectively. It was added at 70%, 80%, 90%, or 100%. At this time, the content of sodium carbonate was adjusted to 1% of the weight of the substrate (soybean meal). The experiment was repeated three times for each moisture content and the average value was derived, and viable cells and enzyme production were measured in the same manner as in <1-2> above.

Figure 4 and Table 6 below show the relative enzyme production and viable cell production according to moisture content.

moisture content Relative enzyme production
(Relative enzyme activity) Probiotic production
(CFU/g) 50% 9% - 60% 73% 7.2E+10 70% 100% 7.4E+10 80% 75% 3.6E+10 90% 66% 2.9E+10 100% 21% 1.7E+10

As a result of the experiment, enzyme and viable cell production increased until the initial moisture content was 70%, but decreased when the initial moisture content exceeded 70%. Therefore, it is preferable that the initial moisture content for live bacteria and enzyme production is 60 to 80%, and the optimal initial moisture content was confirmed to be 70%.

The above result is because, in the production of live bacteria and enzymes, if the moisture content is low, the microorganisms do not grow or their growth is delayed and the fermentation process does not occur efficiently. Additionally, if the moisture content is too high, the substrate clumps together and air is not properly injected, which inhibits the growth of microorganisms. In addition, the cost of the process increases as the drying time after fermentation increases, and the quality of the fermented product may decrease due to high temperature, so it is important to find an appropriate moisture content in order to find an optimized method for producing live bacteria and enzymes.

Claims (12)

a) sterilizing 50 g of soybean meal at 121°C for 15 minutes to obtain sterilized soybean meal;
b) cooling the sterilized soybean meal at room temperature and then adding a sterilized aqueous sodium carbonate solution to obtain a soybean meal suspension with an initial moisture content of 60 to 80% of the weight of the soybean meal;
c) inoculating the soybean meal suspension with 500 μl of Bacillus clausii seed culture cultured in TSB (Tryptic soy broth) for 24 hours to obtain inoculated soybean meal; and
d) solid-state fermentation of the inoculated soybean meal in a shaking incubator at 37° C. for 36 to 60 hours.
According to paragraph 1,
A method for mass producing live bacteria and enzymes, wherein the live bacteria are Bacillus claugi (accession number KCTC 10277BP).
According to paragraph 1,
A method for mass producing live bacteria and enzymes, wherein the enzyme is an alkaline protease derived from Bacillus claugi.
According to paragraph 1,
A method for mass producing live bacteria and enzymes, characterized in that the sodium carbonate content of the sodium carbonate aqueous solution in step b) is 0.5 to 2.0% of the weight of soybean meal.
According to clause 4,
A method for mass producing live bacteria and enzymes, characterized in that the sodium carbonate content is 1.0% of the weight of soybean meal.
According to paragraph 1,
A method for mass production of live bacteria and enzymes, characterized in that the initial moisture content of the soybean meal suspension in step b) is 70% of the weight of soybean meal.
According to paragraph 1,
A method for mass production of live bacteria and enzymes, characterized in that the solid-state fermentation in step d) is 48 hours.
Bacillus claugi produced according to the method of paragraph 1 (accession number KCTC 10277BP).
Proteolytic enzyme derived from Bacillus claugi (accession number KCTC 10277BP) produced according to the method of claim 1.
A solid-state fermentation product containing live bacteria and yeast produced according to the method of any one of claims 1 to 9.
A feed additive containing the solid fermentation product of claim 10.
An odor reducer containing the solid fermentation product of claim 10.