JP6269149B2 - Method for inhibiting the growth of thermostable spore-forming thermophilic bacteria - Google Patents

Description

  The present invention relates to a method for suppressing the growth of thermostable spore-forming high-temperature bacteria, which are flat caustic rot-causing bacteria, which is often a problem in container-packed beverages that are sold by heating.

  Conventionally, addition of sucrose fatty acid esters to beverages and the like is generally known for preventing flat sour deterioration in heated beverages such as milk coffee and potage soup.

  However, it is generally known that such a conventional technique significantly hinders its antibacterial effect by ingredients contained in beverages and the like, particularly starch. For example, in a high starch content beverage such as potage soup, it is necessary to add a high concentration of sucrose fatty acid ester of several thousand ppm, and this high concentration of sucrose fatty acid ester has an effect on the flavor of beverages and the like. (Non-patent document 1).

  In addition to sucrose fatty acid esters, di, tri or polyglycerin fatty acid esters can be mentioned as typical additives in preventing flat sour deterioration (Patent Document 1). There have been some reports of technologies that make it difficult for the antibacterial action of starch to be inhibited by these additives, but none of them has left the field of using fatty acid esters, and new technologies such as biosurfactants There is no report on the prevention of corruption using substances.

JP 2009-118772 A

Toyo Food Research Lab. 71-76 (2009)

  The present invention has been made against the background of such prior art problems. That is, an object of the present invention is to provide a method for suppressing the growth of thermostable spore-forming thermophilic bacteria at a low concentration and without being particularly affected by the antibacterial action of starch, among ingredients contained in beverages and the like. There is.

As a result of intensive studies, the present inventors have found that the above problems can be solved by the following means, and have reached the present invention.
That is, this invention consists of the following structures.
(1) A method for suppressing the growth of heat-resistant spore-forming thermophilic bacteria, characterized in that, in a food or drink containing starch, mannosyl erythritol lipid (MEL) is contained in an amount of 0.0001 to 0.5% by weight.
(2) The method according to (1), wherein the MEL is at least one selected from MEL-A, MEL-B, and MEL-C.
(3) The method according to (1) or (2), wherein the thermostable spore-forming thermophilic bacterium is a flat sour deterioration causative bacterium.
(4) The thermostable spore-forming thermophilic bacterium is selected from the group consisting of Geobacillus stearothermophilus, Bacillus coagulans, Moorella thermoacetica, Thermoanaerobacter (Themoanaerobacter mathranii, T. thermohydrosulfricus, etc.), Clostridium (Clostridium sporogenes etc.), The method as described in any one of (1)-(3) which is at least 1 sort (s) chosen from Alicyclobacillus acidocaldarius (etc.).
(5) The method according to any one of (1) to (4), wherein the starch concentration is 0.01 to 5% by weight.
(6) A heat-resistant spore-forming high-temperature bacterial antibacterial agent for use in the method according to any one of 1 to 5.
(7) Food-drinks containing 0.01 to 5 weight% of starch and 0.0001 to 0.5 weight% of mannosyl erythritol lipid (MEL).
(8) A container-packed coffee beverage containing 0.01 to 5% by weight of starch and 0.0001 to 0.5% by weight of mannosyl erythritol lipid (MEL).
(9) Container-packed soup containing 0.01 to 5% by weight of starch and 0.0001 to 0.5% by weight of mannosyl erythritol lipid (MEL).
(10) A container containing 0.01 to 5% by weight of starch and 0.0001 to 0.5% by weight of mannosyl erythritol lipid (MEL).

  According to the present invention, it is possible to select biosurfactants such as MEL as an additive for the purpose of suppressing the growth of heat-resistant spore-forming high-temperature bacteria in a container-packed beverage or the like sold by heating. Compared with the prior art, the present invention is not subject to inhibition of the antibacterial action by components contained in beverages and the like, particularly starch, and can exhibit its antibacterial action at a low concentration.

  Hereinafter, the present invention will be described in detail. Examples of the thermostable spore-forming high-temperature bacterium showing the effect of the present invention include Geobacillus stearothermophilus, Bacillus (Bacillus coagulans, etc.), Moorella thermoacetica, Thermoanaerobacter genus (Themoanaerobacter mathranii, T. thermohydrosulfricus, etc.) Genus Clostridium (Clostridium sporogenes etc.) and Alicyclobacillus acidocaldarius (eg Alicyclobacillus acidocaldarius etc.). Among them, Geobacillus genus and Morella genus are preferable.

  “Biosurfactant” is a general term for surface-active substances produced by living organisms. It has not only excellent surface activity and high biodegradability, but also various physiological functions. There is a possibility of developing behaviors and functions different from those of drugs. A typical example of a biosurfactant is a surfactant produced by a microorganism. Currently, the above-mentioned surfactants produced by microorganisms are roughly classified into five types: sugar type, acyl peptide type, phospholipid type, fatty acid type, and polymer compound type.

  As biosurfactant used in the present invention, mannosyl erythritol lipid (MEL), and mannosyl alditol lipid (MAL) other than MEL include mannosyl mannitol lipid (MML), mannosyl sorbitol lipid (MSL), mannosyl arabitol lipid (MAraL). ), Mannosyl ribitol lipid (MRL), etc., among which MEL is preferable.

(MEL)
MEL is a substance discovered from Ustilago nuda and Shizonella melanogramma. Later, it was reported that it was also produced by yeasts such as Candida antarctica (currently Pseudozyma antarctica) and Kurtzmanomyces, which are mutant strains of itaconic acid production. Has been.

  The structure of MEL is shown in general formula (1). In the general formula (1), the substituent R1 is an aliphatic acyl group having 4 to 24 carbon atoms which may be the same or different. MEL is classified into four types, MEL-A, MEL-B, MEL-C, and MEL-D, based on the presence or absence of acetyl groups at the 4th and 6th positions of mannose.

  Specifically, in MEL-A, in the general formula (1), the substituents R2 and R3 are both acetyl groups. In MEL-B, in the general formula (1), the substituent R2 is an acetyl group, and the substituent R3 is hydrogen. In MEL-C, in general formula (1), substituent R2 is hydrogen and substituent R3 is an acetyl group. In MEL-D, in the general formula (1), the substituents R2 and R3 are both hydrogen.

  The carbon number of the substituent R1 in the above MEL-A to MEL-D depends on the carbon number of the fatty acid constituting the triglyceride, which is an oil and fat to be contained in the MEL production medium, and the degree of utilization of the fatty acid of the MEL-producing bacterium to be used. Change. In addition, when the triglyceride has an unsaturated fatty acid residue, it is possible to include an unsaturated fatty acid residue as the substituent R1 unless the MEL-producing bacterium assimilates up to the double bond portion of the unsaturated fatty acid. is there. As apparent from the above description, the obtained MEL is usually in the form of a mixture of compounds having different fatty acid residue portions of the substituent R1.

  The composition of the present invention contains mannosyl erythritol lipid having the structure represented by the general formula (2) or the general formula (3).

  The substituent R1 in the general formula (2) and the general formula (3) is an aliphatic acyl group having 4 to 24 carbon atoms which may be the same or different. The number of carbon atoms of the substituent R1 is not particularly limited as long as it is within the above range, but it is more preferably 8 to 14. Further, the substituent R1 in the general formulas (2) and (3) may be a saturated aliphatic acyl group or an unsaturated aliphatic acyl group, and is not particularly limited. When it has an unsaturated bond, you may have a some double bond, for example. The carbon chain may be linear or branched. In the case of an oxygen atom-containing hydrocarbon group, the number and position of oxygen atoms contained are not particularly limited.

  In the general formula (2), the substituent R2 is the same or different hydrogen or acetyl group. In the general formula (3), the substituent R2 is the same or different hydrogen or acetyl group.

  The structure of MAL (mannosyl alditol lipid) other than MEL is shown in general formula (4). Mannitol, arabitol, ribitol, and sorbitol are added as sugar alcohols (alditol) other than erythritol (n = 4: mannitol, sorbitol, n = 2: arabitol, ribitol). According to the general formula (4), MAL is a saturated or unsaturated straight chain having 2 to 20 carbon atoms, preferably 4 to 18 carbon atoms, more preferably 6 to 14 carbon atoms at the 2nd and 3rd positions of mannose. It has an alkanoyl group having a chain or a branch.

  (In the formula, the substituents R1 may be the same or different and each has a saturated or unsaturated straight or branched chain having 2 to 20 carbon atoms, preferably 4 to 18 carbon atoms, more preferably 6 to 14 carbon atoms. (In the formula, the substituent R2 is a hydrogen atom or an acetyl group, which may be the same or different, and is preferably a compound in which both of the substituents R2 are acetyl groups.)

  The biosurfactants preferably used in the present invention are MEL-A, MEL-B and MEL-C shown above.

  In addition, although a biosurfactant may be used independently, 2 or more types of biosurfactants can also be used together.

(Biosurfactant production method)
The method for producing the biosurfactant is not particularly limited, and a fermentation method using microorganisms may be arbitrarily selected. For example, culture production of MEL (MEL-A, MEL-B, MEL-C) can be produced by Pseudozyma antarctica (NBRC 1073) according to a conventional method, and Pseudozyma antarctica, Pseudozyma sp. it can. It is a well-known fact that an MEL mixture can be easily obtained with any microorganism. The MEL mixture can be purified using silica gel column chromatography to isolate MEL-A, MEL-B and MEL-C. Moreover, as bacteria which produce MEL-B, Pseudozyma antarctica and Pseudozyma tsukubaensis are known, and these bacteria may be used. Pseudozyma hubeiensis, Pseudozyma graminicola, etc. are known as bacteria that produce MEL-C, and these bacteria may be used. The microorganism having the ability to produce MEL is not particularly limited, and can be appropriately used depending on the purpose.

  Fermentation medium for producing biosurfactant includes N sources such as yeast extract and peptone, C sources such as glucose, glycerol and fructose, and inorganic salts such as sodium nitrate, dipotassium hydrogen phosphate and magnesium sulfate heptahydrate. A medium having a general composition can be used, and oil and fats such as olive oil, soybean oil, sunflower oil, corn oil, canola oil and coconut oil, and water-insoluble such as hydrocarbons such as liquid paraffin and tetradecane can be used. Sexual substrates can be used alone or added with two or more.

  Fermentation conditions such as pH and temperature, culture time, and the like can be arbitrarily set, and the culture solution after fermentation can be used as it is as the biosurfactant of the present invention. In addition, it is possible to appropriately add any operation such as filtration, centrifugation, extraction, purification, sterilization and the like to the culture solution after fermentation, and the obtained extract can be diluted, concentrated and dried. .

  As oils and fats used as raw materials, vegetable oils and fats are preferable. Vegetable fats and oils are not particularly limited, and can be appropriately selected according to the purpose. For example, soybean oil, rapeseed oil, corn oil, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, palm oil, etc. Among them, soybean oil and olive oil are biosurfactants (especially MEL) production efficiency , Production rate, and yield) are particularly preferable. These may be used alone or in combination of two or more.

  There is no restriction | limiting in particular as an inorganic nitrogen source, Although it can select suitably according to the objective, For example, ammonium nitrate, urea, sodium nitrate, ammonium chloride, ammonium sulfate, etc. are mentioned.

  There are no particular limitations on the method for recovering and purifying the biosurfactant, and it can be appropriately selected according to the purpose. For example, it can be recovered by centrifuging the culture solution to recover the oil, and extracting and concentrating with an organic solvent such as ethyl acetate.

  Examples of the extraction solvent include water, alcohols (for example, lower alcohols such as methanol, absolute ethanol, and ethanol, or polyhydric alcohols such as propylene glycol and 1,3-butylene glycol), ketones such as acetone, diethyl ether, and dioxane. , Esters such as acetonitrile and ethyl acetate, and organic solvents such as xylene, benzene, and chloroform can be used alone or in any combination of two or more kinds, and each solvent extract is combined. Can also be used.

  There are no particular limitations on the extraction method, but usually it may be in the range of the boiling point of the solvent from room temperature to normal pressure. After extraction, it is filtered or adsorbed, decolored and purified using an ion exchange resin to form a solution. , Paste, gel, and powder. In many cases, it can be used as it is, but if necessary, further purification treatment such as deodorization and decolorization may be added as long as the effect is not affected. As a purification treatment means such as deodorization and decolorization, an activated carbon column or the like may be used, and a normal means generally applied depending on the extracted substance may be arbitrarily selected. If necessary, a high-purity biosurfactant can be obtained by purification using a silica gel column.

  The biosurfactant thus obtained can be used by being added to a container-packed beverage or the like that is sold by heating. For example, coffee beverages such as milk coffee, soups such as corn potage soup, and shirako can be mentioned, but any of them can be applied, but is not limited thereto.

  The biosurfactant may be added to foods as long as it is a commonly used method. That is, it may be added directly to the food, or may be added by being added to other food ingredients, and is not limited thereto.

  The blending amount of the biosurfactant in beverages and the like is not particularly limited, but is usually 0.00005 to 2.0% by weight, preferably 0.0001 to 1.0% by weight, particularly preferably 0.0001 to 0.5% by weight. % Blended.

  The heated beverage container-packed beverage thus obtained prevents the deterioration of flat sour caused by these biosurfactants due to the effect of suppressing the germination or growth after germination of heat-resistant spore-forming high-temperature bacteria. And a product with excellent storage stability.

  In particular, the biosurfactant is excellent in that it does not receive an inhibition of the antibacterial action by starch found in sucrose fatty acid esters. In particular, an excellent antibacterial action is exhibited even in the presence of a high concentration of starch having a starch concentration of 0.01 to 5% by weight. Because of its characteristics, even in high-starch beverages such as corn potage soup, it is possible to obtain its antibacterial effect at a very low concentration and to obtain a product with little influence on the flavor of the product by additives It is.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the examples.

Example 1 Production of MEL-A Inoculum culture was performed by inoculating a seed culture (20 ml / 500 ml Sakaguchi flask) with 1 loop of a Pseudozyma antarctica NBRC 10736 colony. Cultured overnight at 30 ° C. The obtained culture broth was used as an inoculum. The composition of the seed medium was 4% Glucose, 0.3% NaNO3, 0.02% MgSO4 · 7H2O, 0.02% KH2PO4, 0.1% yeast extract. For the culture, 75 ml of the above inoculum was inoculated into 1.5 L (5 L-jar) of production medium, and cultured using 5 L-jar under the conditions of 30 ° C., 300 rpm (stirring rotation), 0.5 L / min (Air). The composition of the production medium was 5% olive oil, 0.02% MgSO4 · 7H2O, 0.02% KH2PO4, and 0.1% yeast extract. 250 ml of the culture solution was centrifuged (6500 rpm, 30 min), the supernatant was removed, and the precipitate (bacteria) was collected. 50 ml of ethyl acetate was added to the precipitate, and after sufficient stirring, the mixture was centrifuged (8500 rpm, 30 min), separated into a precipitate and a supernatant, and the supernatant was concentrated with an evaporator. Elution with chloroform: acetone = 1: 0, chloroform: acetone = 9: 1, chloroform: acetone 1: 1, chloroform: acetone = 3: 7, chloroform: acetone = 0: 1 using silica gel Got the minute.

Example 2 Production of MEL-B
0.2 ml of Pseudozyma tsukubaensis frozen stock was inoculated into a 20 ml YM medium / 500 ml Sakaguchi flask and cultured overnight at 26 ° C., 180 rpm, and used as an inoculum. 0.2 ml of the inoculum was again inoculated into a 20 ml YM seed medium / 500 ml Sakaguchi flask and cultured overnight at 26 ° C. and 180 rpm to obtain an inoculum. 20 ml of inoculum was inoculated into 2 L of YM medium / 5 L Jar and cultured at 26 ° C., 300 rpm (1/4 VVM, 0.5 L air / min) for 8 days. The culture solution was centrifuged at 7,900 rpm for 60 minutes at 4 ° C. to separate the cells (including MEL-B) and the supernatant. 80 ml of ethyl acetate was added to each bacterial cell fraction, and the mixture was stirred up and down so that the bacterial cells were sufficiently suspended, and then centrifuged at 4,900 rpm for 30 minutes at 4 ° C. An equal amount of saturated saline was added to the obtained supernatant and stirred to obtain an ethyl acetate layer. An appropriate amount of anhydrous sodium sulfate was added to the ethyl acetate layer, and the mixture was allowed to settle for 30 minutes and then evaporated to obtain a crude MEL-B product. The obtained MEL-B crude purified product was eluted with hexane: acetone = 5: 1 and hexane: acetone = 1: 1 using a silica gel column to obtain a MEL-B fraction purified product.

Example 3 Production of MEL-C
0.2 ml of Pseudozyma hubeiensis and Pseudozyma graminicola frozen stock were inoculated into a 20 ml YM medium / 500 ml Sakaguchi flask and cultured overnight at 26 ° C. and 180 rpm to obtain seeds. 0.2 ml of the inoculum was again inoculated into a 20 ml YM seed medium / 500 ml Sakaguchi flask and cultured overnight at 26 ° C. and 180 rpm to obtain an inoculum. 20 ml of inoculum was inoculated into 2 L of YM medium / 5 L Jar and cultured at 26 ° C., 300 rpm (1/4 VVM, 0.5 L air / min) for 8 days. The culture solution was centrifuged at 7,900 rpm for 60 min at 4 ° C. to separate the cells (including MEL-C) and the supernatant. 80 ml of ethyl acetate was added to each bacterial cell fraction, and the mixture was stirred up and down so that the bacterial cells were sufficiently suspended, and then centrifuged at 4,900 rpm for 30 minutes at 4 ° C. An equal amount of saturated saline was added to the obtained supernatant and stirred to obtain an ethyl acetate layer. An appropriate amount of anhydrous sodium sulfate was added to the ethyl acetate layer and the mixture was allowed to settle for 30 minutes and then evaporated to obtain a crude MEL-C product. The obtained MEL-C crude purified product was eluted with hexane: ethyl acetate = 1: 1, hexane: ethyl acetate = 1: 3, ethyl acetate using a silica gel column to obtain a MEL-C fraction purified product.

Example 4: Spore purification of Geobacillus stearothermophilus
Geobacillus stearothermophilus NBRC12550 was cultured in 802 medium (1.0% polypeptone, 0.2% yeast extract, 0.1% magnesium sulfate heptahydrate) at 55 ° C. and 220 rpm overnight, and this solution was used as a preculture solution. The preculture was smeared on a plate medium and cultured at 55 ° C. for 3 days. Thereafter, the cells were allowed to stand at 4 ° C. for 3 days, and then the cells were collected with phosphate buffered saline. After washing this with phosphate buffered saline, lysozyme chloride (final concentration 10 mg / mL) was allowed to act at 37 ° C. for 15 hours, followed by sodium dodecyl sulfate (final concentration 10 mg / mL) at 37 ° C. for 2 hours. It was. This bacterial cell suspension was washed with phosphate buffered saline to obtain a purified spore solution. For the culture, this purified spore solution was activated at 100 ° C. for 5 minutes to prepare a test bacterium.

Example 5: Preparation of Moorella thermoacetica spores
Moorella thermoacetica JCM 9319 was cultured in a commercially available modified thioglycolate medium (Nissui Pharmaceutical Co., Ltd.) in the presence of 55 ° C. Aneropack (Mitsubishi Gas Chemical Co., Ltd.) for 7 days, and this solution was used as a preculture solution. The preculture was seeded on a plate medium and cultured at 55 ° C. for 7 days. The cells were collected from the plate medium and washed with phosphate buffered saline. This operation was repeated three times, and this solution was used as a spore solution. The spore fluid was activated at 100 ° C. for 5 minutes to obtain a test bacterium.

Table 1 shows the types of antibacterial agents used and the species or sources of their origin.

Table 2 shows the heat-resistant spore-forming high-temperature bacterial strain, culture temperature, and medium used.

Examples 6 to 9, Comparative Example 1: Antibacterial test in culture medium against Geobacillus stearothermophilus
0.1 mL of Geobacillus stearothermophilus prepared in Example 4 was prepared by adding 0.1 mL of various MELs or sucrose palmitate at a concentration of 0 to 32 ppm and mixing them in an 18 mL test tube containing 802 medium (4.8 ml). The spores were added and cultured overnight at 55 ° C. and 220 rpm, and the presence or absence of bacteria was observed with OD660 to evaluate antibacterial properties. The results are shown in Table 3.

  Table 3 shows the antibacterial activity of each type of antibacterial agent and its concentration in the medium, + indicates that growth is observed, and-indicates that no growth is observed. Performed with N = 2. That is, for example, ++ indicates that growth was observed in both systems at N = 2.

  From Table 3, it was revealed that Examples 6 to 9 and Comparative Example 1 have the same level of antibacterial action. In particular, the antibacterial agents of Examples 6, 7, and 9 were found to have an antibacterial action against heat-resistant spore-forming high-temperature bacteria at extremely low concentrations.

Example 10 and Comparative Example 2: Effect of starch on antibacterial action against Geobacillus stearothermophilus MEL-B or sucrose palmitic acid in 802 medium (4.8 ml) containing corn starch (Nacalai Tesque) at a concentration of 0 to 5,000 ppm Add the acid ester to the medium to 10 ppm, mix, add 0.1 mL of Geobacillus stearothermophilus purified spore solution, incubate at 55 ° C and 220 rpm overnight, observe the presence or absence of fungus growth at OD660, in the presence of starch Antibacterial activity was evaluated.

  Table 4 shows the antibacterial activity of MEL-B and sucrose palmitate in the presence of various starch concentrations. + Indicates growth and-indicates no growth. N = 3.

  From Table 4, in Comparative Example 2, the antibacterial effect was inhibited from the starch concentration of 100 ppm. On the other hand, in Example 10, no inhibition of the antibacterial effect was observed even at 5,000 ppm starch. This indicates that the antibacterial effect of MEL-B is not inhibited by starch, and effectively suppresses the growth of thermotolerant thermophilic bacteria at low concentrations.

Examples 11-13 Comparative Example 3: Antibacterial test in medium against Moorella thermoacetica 0.1 mL of various MELs or sucrose palmitates shown in Table 1 were added to 13 mL test tubes containing modified thioglycolate medium (8.8 ml). Add 0.1mL Moorella thermoacetica spore solution prepared in Example 5 at a concentration of 32ppm, and incubate in the presence of 55 ° C Aneropack for 7 days. did. The results are shown in Table 5.

  Table 5 shows the antibacterial activity in the types of various antibacterial agents and their concentrations in the medium, + indicates that growth is observed, and-indicates that no growth is observed. Performed with N = 2. That is, for example, ++ indicates that growth was observed in both systems at N = 2.

  From Table 5, it was revealed that Examples 11 to 13 and Comparative Example 3 have the same level of antibacterial action. In particular, the antibacterial agents of Examples 11 to 13 have an antibacterial action against anaerobic heat-resistant spore-forming high-temperature bacteria at an extremely low concentration, and have the same effects as sucrose fatty acid esters. It was.

Examples 14 to 16 and Comparative Example 4: Effect of starch on antibacterial action against Moorella thermoacetica 0.1% in modified thioglycolate medium (8.8 ml) containing corn starch (Nacalai Tesque) at a concentration of 0 to 5,000 ppm Add MEL-B or sucrose palmitate to 10 ppm in the medium and mix, add 0.1 mL of Moorella thermoacetica spore solution and incubate at 55 ° C for 7 days. Observed and evaluated for antibacterial properties in the presence of starch. The results are shown in Table 6.

  Table 5 shows the antibacterial activity in the types of various antibacterial agents and their concentrations in the medium, + indicates that growth is observed, and-indicates that no growth is observed. Performed with N = 2. That is, for example, ++ indicates that growth was observed in both systems at N = 2.

  From Table 6, in Comparative Example 3, the antibacterial effect was inhibited from the starch concentration of 100 ppm. On the other hand, in Examples 14 and 15, no inhibition of the antibacterial effect was observed even at starch of 5,000 ppm. This indicates that the antibacterial effect of MEL is not inhibited by starch, and effectively suppresses the growth of anaerobic thermophilic thermophilic bacteria at low concentrations.

  According to the present invention, biosurfactants such as MEL can be selected as an additive for the purpose of suppressing the growth of thermostable spore-forming high-temperature bacteria. The technique can exert its antibacterial action at a low concentration without being inhibited by the antibacterial action by starch as compared with a sucrose fatty acid ester used conventionally. Therefore, it can be used for various products including container-packed beverages that are sold by heating, and is expected to make a significant contribution to the industry.

Claims (4)

In food or beverage containing 0.01 to 5 wt% of starch, characterized in that the inclusion mannosylerythritol lipid to (MEL) with 0.0001 to 0.0010 wt%, inhibited the growth of refractory spore-forming high-temperature bacteria how to. The method according to claim 1, wherein the MEL is at least one selected from the group consisting of MEL-A, MEL-B and MEL-C.   The method according to claim 1 or 2, wherein the heat-resistant spore-forming thermophilic bacterium is a flat sour-causing bacterium. The heat-resistant spore-forming high temperature bacteria, Geobacillus sp (Geobacillus stearothermophilus), Bacillus (Bacillus coagulans, etc.), Morera genus (Moorella thermoacetica), Thermoanaerobacter genus (The r moanaerobacter mathranii, T.thermohydrosulfricus etc.), Clostridium ( The method according to any one of claims 1 to 3, which is at least one selected from the group consisting of Clostridium sporogenes and the like, and Alicyclobacillus acidocaldarius and the like.