JP2013252069A - Method for producing fermented seasoning - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a fermented seasoning obtained by fermentation using Bacillus subtilis which facilitates hygiene control regarding microorganisms during a production process because of the absence of spores in the fermented seasoning, and enables sterilizing treatment under a mild condition.SOLUTION: A method for producing a fermented seasoning includes: a fermentation process of inoculating spore-forming ability-defective Bacillus subtilis strains obtained by a method by natural mutation into protein-containing raw material to aerobically ferment the raw material; and a sterilization process of sterilizing the fermented material after the fermentation.

Description

  The present invention relates to a method for producing a fermented seasoning using a spore-forming ability-deficient strain of Bacillus subtilis.

Bacillus subtilis is used in various fermented foods.
The main fermented foods produced by Bacillus subtilis include natto and tofu in Japan (Okinawa region), odor tofu and tofu in China and Taiwan, Chong-guk-chan in Korea, and kinema in Nepal. Fermented foods from Bacillus subtilis have a characteristic flavor derived from lower fatty acids and pyrazine compounds, have high nutritional value and contain various functional ingredients, but Bacillus subtilis has spore-forming ability. There are vegetative cells and spores, and there are problems such as quality deterioration and contamination of production facilities or other products due to Bacillus subtilis.

  In order to kill Bacillus subtilis spores, for example, sterilization treatment under severe conditions such as 120 ° C for 20 minutes or more under pressure heat sterilization or 180 ° C for 30 minutes or more is required. When sterilized under various conditions, the flavor and color of the fermented food after sterilization are greatly impaired. For this reason, the range of use of fermented foods by Bacillus subtilis in the food industry is limited, and there is no example in particular used as a seasoning.

  For natto, one of the fermented foods of Bacillus subtilis, in order to enhance its preservability, for example, dry natto is sterilized by sterilization at 105 ° C under reduced pressure moist heat sterilization or gas moist heat sterilization so that no spores remain. Although a method for producing natto (Patent Document 1) has been disclosed, a drying process and a heating process at a temperature higher than 100 ° C. are necessary, and thus maintaining the original flavor of fermented foods by Bacillus subtilis Have difficulty. Further, since Bacillus subtilis having a conventional spore-forming ability is used, many spores exist in natto before sterilization, at least dry natto, and thus there is still a problem of bacterial contamination in the production facility.

  As for a spore-forming deficient mutant, for example, a vitamin K high-producing natto mutant with a reduced or reduced spore-forming ability obtained by treating natto bacteria with a mutagen such as ultraviolet rays is cultured. A method for producing vitamin K (Patent Document 2) is disclosed. Regarding this method, it has been pointed out in the literature that when a culture is carried out using a medium generally used for cell culture, it is not suitable for wide industrial use. Moreover, there is no description regarding that the culture obtained by cultivating the spore-forming ability-deficient mutant can be sterilized by heat sterilization at 100 ° C. or lower. Furthermore, in general, mutants obtained by treatment with mutagens such as ultraviolet rays, radiation, or nitrosoguanidine have unknown mutation sites, and at the same time, multiple genes may be mutated. There is a problem that the properties possessed are greatly changed, and there is a problem that the ability to spore is restored by reversion.

  In addition, Bacillus natto mutant strains in which a specific gene was destroyed by genetic recombination and the germination rate of spores decreased or the spore formation ability decreased, and natto with few spores difficult to disinfect and sterilize using the mutant strain ( Patent Documents 3 and 4) are disclosed. However, these mutant strains have a problem that their traits are easily changed, and a problem that germination ability and spore formation ability are restored. In addition, the genetic recombination method is inefficient and has technical problems because it is necessary to selectively destroy specific genes of Bacillus subtilis. Furthermore, the use in the food industry is greatly limited because of the use of genetically modified microorganisms and the need for selective pressure with antibiotics such as kanamycin to maintain the gene disrupted state.

JP 2006-6117 A JP 2000-83653 A JP 2010-115139 A JP 2011-10634 A

  As described above, for fermented foods by Bacillus subtilis, there is a demand for fermented foods, particularly fermented seasonings that are sterilized under sterilization conditions that do not have spores in the fermented foods and do not impair the flavor and nutritional components. However, there is no report about such fermented seasonings.

  In order to solve the above-mentioned problems, the present invention is not a Bacillus subtilis spore-forming deficient mutant constructed by genetic recombination or artificial mutation, but a Bacillus subtilis spore-forming ability obtained by a method using natural mutation. It aims at providing the manufacturing method of the fermented seasoning characterized by inoculating and fermenting a defect | deletion strain | stump | stock to the foodstuff containing protein.

  As a result of diligent research focusing on fermentation by microorganisms belonging to Bacillus subtilis, the present inventors have found that they are not mutants lacking spore-forming ability of Bacillus subtilis constructed by genetic recombination or artificial mutation, but by a method using natural mutation. By inoculating the resulting Bacillus subtilis spore-forming ability-deficient strain into a protein-containing food and fermenting it, fermentation that can be sterilized under sterilization conditions that do not cause spores and do not impair the flavor and nutritional components. It discovered that a seasoning could be manufactured efficiently and completed this invention.

The present invention includes the following aspects.
Item (1)
In the method for producing a fermented seasoning, a fermentation process of aerobically fermenting a spore-forming deficient strain of Bacillus subtilis obtained by a method by natural mutation after inoculating a food containing protein,
A sterilization step of sterilizing the fermented material after the fermentation;
The manufacturing method of the fermented seasoning characterized by including.
Item (2)
The method for producing a fermented seasoning according to item (1), wherein the food containing protein is a food containing 30% or more protein per solid matter.
Item (3)
The method for producing a fermented seasoning according to Item (1) or Item (2), wherein the method using natural mutation is a method utilizing a catabolite repression-like phenomenon.
Item (4)
The method for producing a fermented seasoning according to any one of Items (1) to (3), comprising a step of treating a food containing protein with a protease before fermentation or in parallel with the fermentation step.
Item (5)
The method for producing a fermented seasoning according to any one of Items (1) to (4), wherein the sterilization step is performed at 100 ° C or lower.

  According to the present invention, since the spore is not present in the obtained fermented seasoning, hygiene management of the production equipment and the product in terms of microorganisms becomes easy in the processing of the fermented seasoning or the processing using the fermented seasoning. Furthermore, the range of use of the fermented seasoning in the food industry can be expanded. Moreover, according to this invention, since a spore does not exist in this fermentation seasoning, it can sterilize on moderate conditions and can provide a fermentation seasoning with favorable flavor.

  The present invention provides a method for producing a fermented seasoning obtained by inoculating a spore-forming ability-deficient strain of Bacillus subtilis obtained by a method by natural mutation into a food containing protein and fermenting it, and further sterilizing it. Is.

  The present invention provides a fermentation process for fermenting aerobically after inoculating a spore-forming deficient strain of Bacillus subtilis obtained by a method by natural mutation in a method for producing a fermented seasoning into a food containing protein, And a sterilization step of sterilizing the fermented product after the fermentation.

  In the present invention, the step of inoculating a spore-forming ability-deficient strain of Bacillus subtilis obtained by a method using spontaneous mutation into a food containing protein will be described in detail below.

  Bacillus subtilis used as a parent strain in the present invention is an aerobic Gram stain-positive rod and has a spore-forming ability.

  Bacillus subtilis used as a parent strain in the present invention can be obtained from the National Institute of Technology and Evaluation, etc. Specific examples include Bacillus subtilis NBRC3009, Bacillus subtilis NBRC3013, Bacillus subtilis NBRC13169, and the like.

  Methods for obtaining spore-forming ability-deficient strains include genetic recombination methods that create spore-forming ability-deficient strains by knocking out spore formation-related genes using genetic recombination techniques, and various methods such as radiation, ultraviolet rays, nitroso compounds, and alkylating agents. A method by artificial mutation that induces a spore-forming ability-deficient strain by treating with a mutagen, a method by natural mutation to screen for a strain that has lost its spore-forming ability naturally during culture, etc. In the present invention, a spore-forming ability-deficient strain is obtained by a method using spontaneous mutation. Since the spore-forming ability-deficient strain obtained in the present invention is obtained by a method using spontaneous mutation, its use in the food industry is not limited.

  The method for obtaining a spore-forming ability-deficient strain of Bacillus subtilis used in the present invention is not limited as long as it is a method using natural mutation to screen for a strain that is deficient due to a change in spore-forming ability during culture. It is preferable to obtain a spore-forming-deficient strain by culturing Bacillus subtilis repeatedly under certain culture conditions. For example, a high-temperature culture method in which culture is performed at a high temperature of 48 ° C. to 50 ° C., a random method that distinguishes between wild-type and defective-type colonies by coloring melanin pigments, and a method that uses a catabolite repression-like phenomenon (J. F. Michel, B. Cami, P. Schaeffer: Ann. Inst. Pasteur, 114, 11; 21 (1968)). Among these, a method using a catabolic metabolite suppression-like phenomenon is more preferable. A spore-forming ability-deficient strain obtained by a method utilizing a catabolic metabolite suppression-like phenomenon is deficient in lysozyme activity and transformation ability as well as spore-forming ability, and can be passaged without any problem due to lysis. It is possible to maintain the trait of deficiency in forming ability.

Methods that utilize catabolic metabolite suppression-like phenomena include, for example, methods of cultivating Bacillus subtilis repeatedly diluted in a medium that uses nitrate as the only nitrogen source, or plants that use citric acid or histidine as a single carbon source. The method of succession etc. are mentioned. Preferably, Bacillus subtilis is repeatedly diluted and cultured in a medium containing nitrate as the only nitrogen source. Specifically, logarithmic growth is carried out in a medium containing nitrate such as NaNO ₃ and KNO ₃ as the only nitrogen source. In this method, Bacillus subtilis cells that have reached the end of the term are diluted and cultured repeatedly to obtain a spore-forming deficient strain of Bacillus subtilis. In the case of using a medium containing nitrate as the only nitrogen source, any medium can be used as long as it can acquire the spore-forming ability-deficient strain of Bacillus subtilis in the present invention. For example, the following [Medium A] Is mentioned.

[Composition of Medium A]
Glucose: 10 g / L, NaNO ₃ : 3 g / L, K ₂ HPO ₄ : 2.5 g / L, KH ₂ PO ₄ : 1 g / L, MgSO ₄ · 7H ₂ O: 0.5 g / L, CaCl ₂ · 2H ₂ O: 0.2 g / L, FeCl ₃ · 6H ₂ O: 3 mg / L, H ₃ BO ₃ : 68 mg / L, MnCl ₂ · 4H ₂ O: 7 mg / L, ZnCl ₂ : 0.6 mg / L, CoCl _{2 · 6H 2 O: 3mg /} L, NiSO 4 · H 2 O: 0.5mg / L, CuSO 4 · 5H 2 O: 20μg / L, Na 2 MoO 4 · 2H 2 O: 50μg / L, L- ascorbic Acid: 320 mg / L, Nicotinamide: 44 mg / L, Calcium pantothenate: 24 mg / L, Thiamine hydrochloride: 17 mg / L, Pyridoxine hydrochloride: 5 mg / L, Riboflavin: 4 mg / L, Folic acid: 0 8 mg / L, biotin: 0.3 mg / L, cyanocobalamin: 8 μg / L

In the present invention, a method for obtaining a spore-forming ability-deficient strain of Bacillus subtilis is not particularly limited, but for example, a method in which Bacillus subtilis is repeatedly diluted and cultured in a medium containing nitrate as the sole nitrogen source. If there are, the following methods are specifically mentioned.
Specifically, Bacillus subtilis is repeatedly diluted at 30 ° C. for 5 to 7 days and cultured in a medium containing nitrate as the sole nitrogen source, thereby accumulating spore-forming ability-deficient strains. The accumulated culture broth was appropriately diluted and cultured in a standard agar medium, and colonies that appeared were picked up and further cultured in an appropriate medium such as the Schaffer medium described below at 37 ° C. for 72 hours to obtain The culture solution is heat-treated (for example, 80 ° C., 10 minutes) and then cultured on a standard agar medium, and a strain in which no colonies appear is defined as a spore-forming ability-deficient strain.

[Composition of Schaeffer medium]
Nutrient broth (Difco): 8 g / L, KCl: 1 g / L, MgSO ₄ .7H ₂ O: 0.12 g / L, CaCl ₂ : 1 mM, MnCl ₂ : 10 μM, FeSO ₄ : 1 μM, pH 7.0

  In the present invention, the food containing the raw material protein only needs to contain protein in the food, but food containing 30% or more of protein per solid is preferable. Examples of such foods include beans, milk, livestock meat, seafood, yeast, and sake lees. Among them, soybeans and broad beans are preferable for beans, chicken, pork and beef are preferable for livestock meat, and skipjack, mackerel and sardines are preferable for seafood. In the case of seafood, nodules (coarse, naked, dead, etc.) obtained from these seafood may be used. The food containing the raw material protein may be either unheated or heat-treated.

Furthermore, in the present invention, food containing protein can be used after being subjected to shredding treatment, grinding treatment, extraction treatment and the like. These processing methods may be carried out by known means alone or in combination.
For example, in the method of chopping or pulverizing, various cutting machines and pulverizers that use cutting, crushing, friction, air pressure, water pressure, etc., can be mentioned. Specifically, cutters, slicers, dicers, etc. A chopper, a grinder, a mixer, a mill or the like can be used. Examples of the chopped or crushed material include kinako, kure, minced meat, and fish meal.
Further, for example, extraction methods include room temperature extraction, heat extraction, pressure extraction, stirring extraction, ultrasonic extraction, and the like, and preferably, extraction processing is performed by heat extraction. Usually, the extraction treatment is solvent extraction with water or the like that can be used for food processing, and preferably extraction with water, alcohol, or a water-alcohol mixed solution. The extraction time and the extraction temperature are appropriately determined depending on the physical properties of the substance to be extracted and the extraction method. The extraction temperature is usually 20 to 150 ° C., and the extraction time is usually 1 minute to 24 hours. .
In the present invention, the extract of the food containing protein can be a liquid part obtained as it is after extraction or by solid-liquid separation, but preferably a liquid part obtained by solid-liquid separation after extraction is used. . In any case, the concentrate can be used. Examples of those subjected to extraction treatment include soy milk, livestock meat extracts, seafood extracts, yeast extracts and the like.

  In the present invention, the food containing the raw material protein is used as a paste-like, slurry-like or liquid fluid containing water and having fluidity. The method of turning the food containing the raw material protein into a paste, slurry, or liquid fluid is not particularly limited, and is performed by a method generally used for processing foods. A paste, slurry, or liquid may be used, and the pulverized raw material may be dispersed in water to obtain a paste, slurry, or liquid.

  In the present invention, the food containing the raw protein is preferably sterilized before the fermentation process. The sterilization conditions may be a temperature and a time at which microorganisms can be sterilized. Examples of the sterilization conditions include a normal pressure of 80 ° C. or higher for 5 to 120 minutes, and a pressurized pressure of 100 to 140 ° C. for 0.1 to 40 minutes. Moreover, it can sterilize more effectively if it sterilizes in an acidic liquid.

  In the present invention, the food containing the raw material protein is fermented with a spore-forming ability-deficient strain of Bacillus subtilis under aerobic conditions. That is, the food is inoculated with a spore-forming ability-deficient strain of Bacillus subtilis and fermented by shaking, aeration or stirring, or a combination thereof. The temperature and time of fermentation should just be the conditions by which fermentation is performed appropriately, temperature is 20-45 degreeC normally, Preferably it is 30-40 degreeC, and time is 1 hour-48 hours normally, Preferably it is 1 Time to 36 hours, more preferably 2 to 24 hours.

  In the present invention, in order to further improve the flavor of the obtained fermented seasoning, it is preferable to treat the food containing protein with a protease before fermentation or in parallel with the fermentation process. By treating with a protease, an amino acid such as glutamic acid or a proteolysate such as a peptide is produced in the fermented seasoning, and the umami and richness are increased and the flavor is improved. The usage-amount of protease can be determined according to the titer of enzyme activity, and although it does not specifically limit, Usually, it is about 0.01-500 U / g with respect to the weight of the foodstuff containing a protein.

  The protease used in the present invention may be any protease as long as it can be used in foods, and animal-derived, plant-derived or microorganism-derived proteases can be used alone or in combination. Examples of animal-derived proteases include pepsin and trypsin, plant-derived proteases such as papain, and microorganism-derived proteases such as those derived from filamentous fungi and Bacillus subtilis.

  Commercially available proteases used in the present invention include, for example, equinezyme G (manufactured by Amano Enzyme Co., Ltd., derived from Aspergillus genus), Sumiteam (registered trademark) FP (manufactured by Shin Nippon Chemical Industry Co., Ltd., derived from Aspergillus genus), and nucleicin (registered trademark). (Manufactured by HBI Corporation, derived from the genus Bacillus), purified papain FL-3 (manufactured by Asahi Food and Healthcare Co., Ltd., derived from papaya) and the like.

In the present invention, the fermented product obtained by the fermentation process is sterilized.
In the present invention, since there is no spore in the fermented seasoning, it can be sterilized under mild conditions, and the flavor, color and texture of the fermented seasoning after the sterilization treatment are not impaired, and the fermentation has a good flavor. Seasoning can be provided.
In the present invention, the sterilization conditions by heat sterilization may be set as long as the physical flavor after sterilization is acceptable, and can be set as appropriate. The heating temperature is at least 100 ° C., preferably 70 The heating time is determined depending on the temperature, and is usually 1 to 60 minutes.

Since there is no spore in the fermented seasoning according to the production method of the present invention, when processing the fermented seasoning, a strong sterilization treatment that kills the spore is not required for the production equipment and microorganisms of the product, Hygiene management is easy.
In addition, the fermented seasoning after sterilization according to the present invention has a remarkably small number of bacteria as a fermented food by Bacillus subtilis and can be widely used in the food industry if sterilization is appropriately performed.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited by the following examples. In addition, in this invention, all the mixture% or density | concentration of each component or raw material used for a culture medium are a weight part basis.

[Acquisition Example 1] (Acquisition 1 of a spore-forming ability deficient strain)
A spore-forming strain of Bacillus subtilis was obtained by a method using catabolic repression-like phenomenon, which is a method based on natural mutation. Specifically, the following method is used.
Bacillus subtilis NBRC3013 strain (hereinafter referred to as “NBRC3013 strain”) was cultured three times with shaking culture medium (described in paragraph No. 0021 of the present specification) using nitrate as the sole nitrogen source. (30 ° C., 120 hours), the culture solution was appropriately diluted and smeared on a standard agar medium, and cultured at 30 ° C. for 2 days. Sixteen strains were picked up from the colonies generated after the culture, and each was cultured with shaking at 37 ° C. for 72 hours using a Schaeffer medium (described in paragraph 0023 of the present specification). The obtained culture broth was heated at 80 ° C. for 10 minutes, smeared on a standard agar medium, and cultured at 30 ° C. for 48 hours. Colonies of 9 out of 16 strains appeared. Therefore, 7 strains in which no colony appeared among 16 strains were determined to be spore-forming ability-deficient strains, and one strain was arbitrarily selected from the 7 strains. 3013ΔSpoA strain.

[Test Example 1] (Confirmation test for spore-forming ability-deficient strain)
B. In order to confirm that the 3013ΔSpoA strain is a spore-forming ability-deficient strain of Bacillus subtilis, tests were conducted on the measurement of the growth temperature and the number of heat-resistant bacteria.

  Tests for growth temperature were performed using NBRC3013 strain and B. cerevisiae. The 3013ΔSpoA strain was cultured for 20 hours at 30, 37, 40, 45, and 50 ° C. using a standard agar medium, respectively, and the degree of growth was tested. The results are shown in Table 1.

  The test for measuring the number of heat-resistant bacteria was carried out using NBRC3013 strain and B.I. 3013ΔSpoA strains were each subjected to shaking culture at 37 ° C. for 72 hours using a Schaeffer medium (described in paragraph No. 0023 of this application), and then heat-treated at 80 ° C. for 10 minutes. Numbers were checked by routine methods. The results are shown in Table 2.

  For Table 1, NBRC3013 strain and B. The 3013AΔSpo strain showed the same characteristics at 37, 40, and 45 ° C. with large growth and optimum temperature.

About Table 2, about 10 < ⁶ > piece / g live bacteria were detected even after NBRC3013 stock | strain heat processing for 10 minutes at 80 degreeC, and it was confirmed that the spore is formed. On the other hand, B. Since no viable bacteria were detected after heat treatment at 80 ° C. for 10 minutes, the 3013ΔSpoA strain was confirmed to have no spore formation, that is, a spore-forming ability-deficient strain.

[Example 1] (Preparation of sterilized soybean fermented seasoning)
204 g of kinako was mixed with 996 g of water in a 2 L jar fermenter and sterilized at 121 ° C. for 15 minutes using an autoclave. After the sterilization treatment, it is cooled to 40 ° C. 6 g of a preculture of 3013ΔSpoA strain (viable bacteria number of about 10 ⁸ cells / g) was inoculated, 0.6 g of protease (horsezyme G) was added, and fermentation was performed with aeration and stirring at 40 ° C. for 15 hours. In addition, about a preculture liquid, B.I. A culture solution obtained by inoculating a colony of 3013ΔSpoA strain in a medium containing 2% by weight of yeast extract and 2% by weight of glucose in water and shaking culture at 40 ° C. for 7 hours was used as a preculture solution. .
A fermented product obtained by aeration and agitation fermentation (general viable cell count: 6.7 × 10 ⁷ cells / g) is sterilized at 80 ° C. for 10 minutes, thereby sterilizing soybean fermented seasoning (Example 1) 1100 g (Brix 13.6 °) was obtained. About this sterilized soybean fermented seasoning, the number of general viable bacteria was examined by a conventional method. Moreover, L-glutamic acid content was measured using F-kit L-glutamic acid (manufactured by Roche Diagnostics), which is a test combination for food analysis. Each result is shown in Table 3.
Moreover, when the obtained sterilized soybean fermented seasoning was sensory-evaluated, it sufficiently had a preferable flavor and umami unique to Bacillus subtilis fermented product, and had a good flavor as a seasoning material.

[Comparative Example 1]
Aerated and stirred fermentation was carried out in the same manner as in Example 1 except that the NBRC3013 strain having spore-forming ability was used as the fermentative bacterium. A fermented product (general viable count: 4.8 × 10 ⁸ cells / g) obtained by aeration and agitation fermentation is sterilized at 80 ° C. for 10 minutes, so that the sterilized soybean fermented seasoning (Comparative Example 1) 1100 g (Brix 13.5 °) was obtained.
About this sterilized soybean fermented seasoning, the number of viable bacteria and the L-glutamic acid content were measured in the same manner as in Example 1. Each result is shown in Table 3.

About Table 3, the sterilized soybean fermented seasoning (Example 1) obtained by the present invention was sterilized soybean fermented seasoning of Comparative Example 1 obtained by fermentation using the parent strain NBRC3013. In addition, it contained the same amount of glutamic acid and no viable bacteria were detected. Moreover, since the sensory evaluation result of the sterilized soybean fermented seasoning (Example 1) obtained by the present invention is a mild sterilization condition of 80 ° C. for 10 minutes, there is almost no influence by heating, and the flavor is good. there were.
On the other hand, since the sterilized soybean fermented seasoning of Comparative Example 1 contains spores, 3.0 × 10 ⁶ cells / g of general viable bacteria were detected.

[Acquisition Example 2] (Acquisition of strains deficient in spore formation 2)
B. subtilis-deficient strain B. subtilis B. subtilis B. subtilis NBRC13169 strain was used as the parent strain in the same manner as in Acquisition Example 1. 13169ΔSpoB strain was obtained.

[Example 2] (Production of sterilized fermented pork extract)
10 g of pork extract (Brix 60 °) was mixed with 39 g of water and 1 g of glucose in a 200 ml Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes using an autoclave. After the sterilization treatment, the mixture was cooled to 37 ° C., and B. subtilis spore forming ability-deficient strain B. 1 g of the preculture of 13169ΔSpoB strain (viable cell count of about 10 ⁸ cells / g) was inoculated, and 0.05 g of protease (Sumiteam FP) was added, followed by shaking fermentation at 37 ° C. for 20 hours. In addition, about a preculture liquid, B.I. A colony of 13169ΔSpoB strain was inoculated into a medium containing 2% by weight of yeast extract and 2% by weight of glucose in water, and a culture solution obtained by shaking culture at 37 ° C. for 7 hours was used as a preculture solution. .
Fermented pork extract (Example 2) 40 g after being sterilized by heat-sterilizing a fermented product (general viable count: 1.2 × 10 ⁸ cells / g) obtained by shaking fermentation at 70 ° C. for 30 minutes. (Brix 13.8 °) was obtained.
About this sterilized fermented pork extract, the number of general viable bacteria and the L-glutamic acid content were measured in the same manner as in Example 1. Each result is shown in Table 4.
Moreover, when the obtained sterilized fermented pork extract was subjected to sensory evaluation, it had a favorable flavor and umami unique to Bacillus subtilis fermented products, and had a good flavor as a seasoning material.

[Comparative Example 2]
Shaking fermentation was carried out in the same manner as in Example 2 except that the NBRC13169 strain having spore-forming ability was used as the fermentative bacterium. Fermented pork extract (Comparative Example 2) 40 g that has been sterilized by heat sterilizing a fermented product (general viable count: 5.6 × 10 ⁸ cells / g) obtained by shaking fermentation at 70 ° C. for 30 minutes. (Brix 13.8 °) was obtained.
About this sterilized fermented pork extract, the number of general viable bacteria and the L-glutamic acid content were measured in the same manner as in Example 1. Each result is shown in Table 4.

About Table 4, the sterilized fermented pork extract (Example 2) obtained by this invention is equivalent to the sterilized fermented pork extract of the comparative example 2 obtained by fermenting using NBRC13169 strain which is a parent strain. In addition, no viable bacteria were detected. Moreover, since the sensory evaluation result of the sterilized fermented pork extract obtained in accordance with the present invention (Example 2) is a mild sterilization condition of 70 ° C. and 30 minutes, there is almost no influence by heating, and the flavor is good. there were.
On the other hand, since the sterilized fermented pork extract of Comparative Example 2 had spores, 2.2 × 10 ⁶ general viable bacteria / g were detected.

[Example 3] (Production of sterilized fermented chicken extract)
10 g of chicken extract (Brix 40 °) was mixed with 40 g of water and 0.1 g of protease (Sumiteam FP) in a 200 ml Erlenmeyer flask, enzyme-treated at 50 ° C. for 5 hours, and then autoclaved at 121 ° C. for 15 minutes. Sterilized. After the sterilization treatment, the mixture was cooled to 37 ° C., and B. subtilis spore forming ability-deficient strain B. 1 g of a preculture of 3013ΔSpoA strain (viable cell count of about 10 ⁸ cells / g) was inoculated and subjected to shake fermentation at 37 ° C. for 20 hours. In addition, about a preculture liquid, B.I. A colony of 3013ΔSpoA strain was inoculated into a medium containing 2% by weight of yeast extract and 2% by weight of glucose in water, and a culture solution obtained by shaking culture at 37 ° C. for 7 hours was used as a preculture solution. .
A fermented chicken extract (Example 3) 45 g that has been sterilized by heat-sterilizing a fermented product (general viable count: 6.0 × 10 ⁷ cells / g) obtained by shaking fermentation at 70 ° C. for 30 minutes. (Brix 7.5 °) was obtained.
About this sterilized fermented chicken extract, the number of general viable bacteria and the L-glutamic acid content were measured in the same manner as in Example 1. The results are shown in Table 5.
Moreover, when the sterilized fermented chicken extract obtained was sensory-evaluated, it had a favorable flavor and umami unique to Bacillus subtilis fermented products, and had a good flavor as a seasoning material.

[Comparative Example 3]
Shaking fermentation was carried out in the same manner as in Example 3 except that the fermenting bacteria were NBRC3013 strain having spore-forming ability. Fermented chicken extract (Comparative Example 3) 45 g that has been sterilized by heat-sterilizing a fermented product (general viable cell count: 3.8 × 10 ⁸ cells / g) obtained by shaking fermentation at 70 ° C. for 30 minutes. (Brix 7.6 °) was obtained.
About this sterilized fermented chicken extract, the number of general viable bacteria and the L-glutamic acid content were measured in the same manner as in Example 1. The results are shown in Table 5.

For Table 5, the sterilized fermented chicken extract obtained in accordance with the present invention (Example 3) is equivalent to the sterilized fermented chicken extract of Comparative Example 3 obtained by fermentation using the parent strain NBRC3013. In addition, no viable bacteria were detected. In addition, the sensory test result of the sterilized fermented chicken extract obtained in accordance with the present invention (Example 3) is a mild sterilization condition of 70 ° C. for 30 minutes, so there is almost no influence by heating, and the flavor is good. there were.
On the other hand, since the sterilized fermented chicken extract of Comparative Example 3 contains spores, 3.0 × 10 ⁶ cells / g of general viable bacteria were detected.

Claims (5)

In the method for producing a fermented seasoning, a fermentation process of aerobically fermenting a spore-forming deficient strain of Bacillus subtilis obtained by a method by natural mutation after inoculating a food containing protein,
A sterilization step of sterilizing the fermented material after the fermentation;
The manufacturing method of the fermented seasoning characterized by including.   The method for producing a fermented seasoning according to claim 1, wherein the food containing protein is a food containing 30% or more protein per solid matter.   The method for producing a fermented seasoning according to claim 1 or 2, wherein the method using natural mutation is a method utilizing a catabolite repression-like phenomenon. The method for producing a fermented seasoning according to any one of claims 1 to 3, comprising a step of treating a food containing protein with a protease before fermentation or in parallel with the fermentation step.   The method for producing a fermented seasoning according to any one of claims 1 to 4, wherein the sterilization step is performed at 100 ° C or lower.