JP2010178728A - Concentrated culture medium with bacteriostat - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid culture medium transportable and/or storable at room temperature and easily proliferatable necessary bacteria on site: and to provide a method for easily culturing them, in view of the fact that, on site of using useful bacteria where there is no equipment for producing a liquid medium, methods for enabling necessary bacteria to be proliferated when they are needed have been demanded. <P>SOLUTION: This concentrated liquid culture medium is such that: a nutrient suitable for bacteria to be used is included at higher concentration than normal levels and also a bacteriostat is added. Besides, such a bacteriostat is also included in a starter-containing concentrated liquid culture medium and the starter. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a concentrated medium that can be distributed and stored at room temperature by adding a bacteriostatic agent, a method for producing the same, and a method for growing microorganisms.

In recent years, attempts have been made to propagate various bacteria as fermented foods, environmental improvements, bio-pesticides, and the like to utilize the ability of microorganisms. There are various types of microorganisms. Lactic acid bacteria and yeast are used for alcoholic beverages, miso, soy sauce, pickles and fermented milk of traditional fermented foods, and Bacillus bacteria are used for natto, and a very wide variety of bacterial species and strains are used. ing. In addition to foods that have been used for a long time, various microbial species and strains are used for mass production of chemical acrylamide, fermented fats and oils, microbial insecticides, fungicides, herbicides, removal of environmental pollutants, etc. It is expected to expand in the future.

Various bacteria are also used in special environments such as outdoors. For example, aerobic bacteria such as oil-degradable yeast and bacteria are used to reduce oil content (BOD) in wastewater treatment for environmental improvement, and anaerobic bacteria such as methane bacteria to obtain fuel gas along with degradation of livestock manure Bacillus bacteria, Aerobacteria bacteria, etc. are widely used as biopesticides. Although it is semi-outdoor, Bacillus bacteria, lactic acid bacteria, and yeast are used to counter odors in cattle houses, pig houses, and poultry houses, and in recent years, the scope of use has become increasingly widespread.

In wastewater treatment plants that use fungi outdoors, deodorization sites in livestock barns, and plantation farms that use biopesticides, such fungi are added to facilities in large quantities or sprayed. A method of increasing the number of bacteria in stages, such as making a mother starter from a seed culture, then producing a bulk starter and carrying out the main culture in order to produce and use the target product in large quantities by performing the main culture of these useful bacteria Is generally done. Alternatively, there are freeze-dried microbial cells and deep frozen starters that use and apply this process.

In the production of traditional fermented milk such as yogurt, first, a skim milk medium is prepared with a liter-sized Erlenmeyer flask containing skim milk, heated at 65 to 95 ° C. for 30 minutes, and then cultured at a temperature of 35 to 45. Cooled to about 0 ° C, inoculated with 1 to 3% seed culture, and incubated for 16 to 24 hours is a mother starter, prepared in large numbers and cooled to 3 to 10 ° C and stored until used for bulk starter. Similarly, 30 to 100 times the amount of skim milk is sterilized and cooled, and 1 to 3% of mother starter is inoculated to produce a bulk starter. It is described that 1 to 3% of this is added to a yogurt mix and fermented to produce yogurt (Non-patent Document 1).

As described above, in order to produce fermented milk from a mother starter, various facilities such as measurement, dissolution, heating, cooling, sterilization, and culture are required, and the skilled workers who have a hygienic concept are involved in the handling. It was essential to do. In addition, storage of each starter is basically cold storage, and it is very important to store it at a low temperature in order to prevent deterioration of the sterilized bulk liquid, and to incubate after warming to the culture temperature at the time of use. Met. Storage and transportation of tank liquids for bulk starters at room temperature is not carried out because troubles such as lactic acid bacteria do not grow as intended due to the growth of residual bacteria at the time of sterilization and the growth of environmental pollutants.

A device technology (Patent Document 1) for continuously administering a freeze-dried starter solution into a liquid fermentation medium is disclosed. Although a method using a concentrated culture solution is described therein, it is not described that this technology can be stored and transported at room temperature. Rather, it requires complicated equipment such as a tank for thawing the frozen frozen concentrated culture.

In addition, spore bacteria used to treat waste water containing BOD components are supplied to aeration tanks, etc. in a state just before the growth and germination of the spores. A technique for adding glucose, skim milk, starch and the like (Patent Document 2) is disclosed, and it is said that odorless wastewater treatment has been established with this bacterium. However, this technique is a method in which a sludge mass of various spores and pellets thereof are directly put into a wastewater treatment tank, and a technique related to a concentrated medium is not shown.

Yogurt Business Books Co., Ltd. 156-179 pages Special table 2008-506410 JP2004-344886

In order to produce a fermented product, the seed culture is hygienically inoculated to produce a mother starter, which is then planted to produce a bulk starter for main culture. Production of a liquid medium has complicated processes such as measurement, mixing, dissolution, sterilization, and cooling of culture components, and hygiene management in various processes has been essential. In addition, when there was a time interval between planting, refrigerated storage was necessary.

Although the use of cultured bacteria seems to be beneficial, it is expensive and cheaper if it is cultured in-house, but it actually uses wastewater treatment plants, livestock barns (cattlehouses, pig houses, poultry houses), garbage disposal facilities, and bio-pesticides. In most farms, facilities for producing liquid media are not in place, and this is not possible. It is difficult to cultivate such useful bacteria at the site of use, and it is difficult to handle because there is no facility for storing the starter in a refrigerator or freezer.

In order to solve this problem, there is a need for a method that allows the growth of necessary bacteria at the site of use of useful bacteria that do not have facilities for producing liquid culture media, and it can be transported and stored at room temperature and can be easily propagated at the site of use. A liquid medium and a simple culture method thereof are provided.

The concentrated medium according to claim 1 is characterized in that a bacteriostatic agent is added.

The concentrated medium according to claim 2 is characterized in that a bacteriostatic agent and a starter bacterium are added.

The starter of claim 3 is characterized in that a bacteriostatic agent is added.

In the concentrated medium and starter according to claim 4, the bacteriostatic agent to be added comprises at least one of alcohols, acids and salts.

The starter according to claim 5 comprises a spore bacterium or / and a spore yeast.

The concentrated medium or starter according to claim 6 comprises a method for determining the type, content and water content of a bacteriostatic agent suitable for a starter bacterium using a flat plate medium containing a bacteriostatic agent.

The manufacturing method of the concentrated culture medium and starter of Claim 7, and the growth method of microorganisms are comprised.

The concentrated medium of the present invention contains a nutrient suitable for the bacteria to be used in a concentration higher than the normal amount, dissolved and sterilized to produce a concentrated medium, and after cooling, alcohols, acids and salts which are bacteriostatic agents One or more of these are added. Moreover, the starter-concentrated concentrated medium is obtained by adding a starter thereto. The concentrated culture medium with the bacteriostatic agent added sufficiently suppresses the contamination of a wide variety of germs such as pollutants from the environment even when transported or stored at room temperature.

The method of selecting the type of bacteriostatic agent used in the present invention and setting the addition amount is to use a high concentration type to suppress the starter bacteria used and to confirm the maximum bacteriostatic agent amount that does not inhibit growth. It consists of law. However, since useful bacteria added as a starter do not grow as they are, the amount of water corresponding to appropriately controlled culture component concentration and bacteriostatic agent concentration can be added to optimize the components of the culture solution and dilute concentration of the bacteriostatic agent Was able to be optimized. By this method, after adding fresh water, the temperature was kept at the growth temperature, and the added starter bacteria could be grown only by stirring under aerobic or anaerobic conditions.

According to the present invention, after the concentrated medium is produced in a facility with production facilities with excellent microorganism management, the appropriate bacteriostatic agent selected is added in a fixed amount, so that it can be transported and stored at room temperature, and the incubator and the heat-retaining With equipment, the required amount of water can be produced very easily at the place where the culture solution is needed, and the required amount can be produced at the required time. Now available. In addition, use of food additives is expected for food.

A concentrated medium that can be transported and stored at normal temperature, or a starter or concentrated medium containing a starter by adding the bacteriostatic agent of the present invention, a method for producing and using the same, and a method for setting the bacteriostatic agent will also be described.

The concentrated medium containing the bacteriostatic agent of the present invention contains nutrients suitable for the bacteria to be used in a higher concentration than the normal amount, dissolved and sterilized, cooled, and alcohols and acids as bacteriostatic agents, One or more salts are added. In addition, the concentrated medium with starter measures nutrients suitable for the bacteria to be cultured at a concentration higher than the normal amount, dissolves and sterilizes, cools it, and then adds one of alcohols, acids and salts as bacteriostatic agents. More than seeds and a starter. In addition, the starter to which a bacteriostatic agent is added is obtained by adding one or more of alcohols, acids and salts, which are bacteriostatic agents, to a starter in which a seed culture is cultured.

The type of bacteriostatic agent suitable for the concentrated medium of the present invention and the amount to be added determine the type and content of the bacteriostatic agent suitable for the starter fungus by creating a plate medium having a different amount from the type contained in the bacteriostatic agent. To do. That is, the type of the bacteriostatic agent having a strong inhibitory action against the bacteriostatic agent of the added starter bacterium is selected, and the amount of water to be diluted is set from the maximum content of the growing bacteriostatic agent. The fungus culture method is easy by adding fresh water in consideration of the concentration of the medium and the content of bacteriostatic agent, then setting the growth conditions for the starter fungus (aerobic, anaerobic, etc.), keeping warm and stirring. The desired useful bacteria can be propagated.

The concentrated medium of the present invention may be a known medium component, for example, MRS medium for Lactobacilli, Sabouraud medium for yeast, and standard agar (SPC) medium agar that can be used for other common fungi. Ingredients are measured from 5 to 500 times the normal concentration, and the medium dissolved in water is sterilized by heating and then cooled, and alcohols (for example, ethyl alcohol), acids (for example, acetic acid), salts that are bacteriostatic agents An appropriate amount of one or more of (for example, salt) is added, and this provides a concentrated medium that can be distributed and stored at room temperature. The concentrated medium of the present invention is preferably a liquid, but may be a gel or sol.

The concentrated medium containing a bacteriostatic agent and a starter according to the present invention is obtained by adding a starter obtained by cultivating a seed culture to the concentrated medium described above. In the present invention, a starter is a culture of seed culture and is also referred to as a mother starter. A culture of this is a bulk starter, but when it is not further cultured, it is called a bulk culture. The starter fungus means a seed culture contained in the starter. The bacteriostatic agent and the starter-concentrated culture medium of the present invention are suitable for a culture medium for culturing a bulk starter or a bulk culture, but may be used for other cultures. The bacteriostatic agent and the starter-concentrated concentrated medium of the present invention are preferably liquid, but may be in the form of gel or sol.

Bacteriostasis is a state in which microorganisms are alive but cannot grow, and certain substances act bacteriostatically on microorganisms, and in such cases, microorganisms recover their growth when the substances are removed or diluted. To do. Moreover, sugar, alcohol, salt, acid, preservatives, bactericides, antibiotics, etc. are known as substances having a bacteriostatic effect. The bacteriostatic agent in the present invention is preferably a substance that can be eaten and consumed by humans as food and is safe unless an abnormal amount is ingested. Moreover, although the fungus is alive but cannot be propagated by adding the substance, or shows a bactericidal effect, it means that the added microorganism grows by appropriately diluting.

The bacteriostatic agent of the present invention is characterized by comprising at least one of alcohols, acids, and sodium chloride, but preferably has a concentration that suppresses the growth of contaminating bacteria as a concentrated medium and enables distribution and storage at room temperature. Also, the concentration at which the added fungus grows by dilution with water is important. Although sugar can also be used as a bacteriostatic agent, emphasis was placed on the concentration that maintains the appropriate growth concentration of the starter fungus after dilution as a growth nutrient component. The water used in the present invention is clear water, and the microorganism content is 100 or less per ml on the basis of tap water.

The bacteriostatic agent to be added to the concentrated medium or the starter was determined using the plate medium containing the bacteriostatic agent, and the type, content and water content of the bacteriostatic agent suitable for the starter fungus were determined. Since the growth control concentrations for the bacteriostatic agents are different for the bacteria used in the starter, first, select a medium on which the starter bacteria grow, and a plate medium containing 0-30% of various bacteriostatic agents in this medium. Create and perform streak culture to select a bacteriostatic agent controlled at a higher concentration. Similarly, the upper limit of growth is selected using a plate medium containing the bacteriostatic agent controlled at this high concentration at a low concentration of 0.01% to 16%. By this operation, the bacteriostatic agent to be added can be grown by selecting a type having a higher concentration and adding water to the highest growth concentration. Of course, second and third bacteriostatic agents can also be used, but the amount of diluted water increases.

As alcohols, 70% aqueous solution of ethyl alcohol is widely used as a bactericidal agent, and it is said that lipids in cell membranes are dissolved and destroyed, and proteins are eluted to produce a bactericidal action. As the number of carbons increases, the bactericidal effect increases. When the number of carbons is the same, the effect decreases in the order of primary> secondary> tertiary. However, in consideration of human safety and test examples, an ethyl alcohol content of 1% or more and 70% is effective, but as described in Examples 1 and 2, for each type of starter used, Although it changes with the mixing ratio of other bacteriostatic agents, the addition amount of alcohols 2% to 20% is suitable for bacteriostasis of general contaminating bacteria.

The acids as the bacteriostatic agent of the present invention may be one or more of mineral acids such as hydrochloric acid, sulfuric acid and nitric acid and organic acids such as propionic acid, lactic acid, malic acid, citric acid and ascorbic acid. Use of an organic acid that is effective in a small amount is preferred, and acetic acid has the most inhibitory effect. Many bacteria are said to be able to suppress growth at a pH of 5 or less and less than pH 2, but a content of 0.1% or more and 25% or less is effective in consideration of bactericidal activity other than the pH lowering effect of organic acids. However, as explained in Examples 1 and 2, it varies depending on the bacterial species of the starter used and the mixing ratio of other bacteriostatic agents. An addition amount of 0.01% or more and 20% or less is suitable.

Sodium chloride is suitable for the addition of salts. When the salt concentration is 2% or more and 20% or less, the growth of microorganisms can be almost suppressed. Although this suppression mechanism is shown by osmotic pressure, chloride ion, etc., it varies depending on the mixing ratio with other bacteriostatic agents, but the bacteriostatic effect of salt is generally as described in Examples 1 and 2. The amount of sodium chloride added is preferably 2% to 15%.

As the types of bacteriostatic agents of the present invention, alcohols, acids, and salts have been described. However, these three types of bacteriostatic agents have different bacteria that exhibit high bacteriostatic effects. Therefore, as described in the examples, the bacteriostatic spectrum is wider when two types are combined than the one type, and the bacteriostatic spectrum is wider when three types are combined. Bacillus bacteria, lactic acid bacteria, and Escherichia coli cannot grow when the alcohol content exceeds 6%. Lactic acid bacteria are strong against acids such as 1% acetic acid, but Bacillus, E. coli, etc. cannot grow. The salt was about 5%, and Bacillus, E. coli, etc. could not grow. In addition, as described in Example 2, the bacteriostatic effect was observed in many bacteria even when the content percentage was lower than that of single use by mixing the bacteriostatic agent.

The storage at normal temperature and distribution in the present invention are those that exceed the refrigeration temperature condition of 10 ° C. or less, and preferably 35 ° C. or less. When the temperature exceeds 35 ° C., when alcohol is added as a bacteriostatic agent, the vaporization of the alcohol tends to proceed, and the bacteriostatic effect decreases due to a decrease in the content. When only other acids and salts are used, the bacteriostatic effect does not decrease even when placed in an environment around 35 ° C. In refrigeration at 10 ° C. or lower, there is no problem in bacteriostatic suppression even with all three types of bacteriostatic agents.

In the present invention, when a spore bacterium is used as a starter, the concentration at which the spore is inactivated is preferable. Inactivation refers to vegetative cells of general bacteria and spore bacteria, vegetative cells of fungi and spore yeast, actinomycetes Refers to a state in which only the spore is killed and survives. If the growth conditions improve, the spore rapidly changes to a vegetative cell and proliferates. These are described in Example 3.

The concentrated medium used in the present invention can cope with any bacteria if a medium or bacteriostatic agent suitable for a starter or seed culture is studied. Therefore, it can be used for bacteria, fungi and actinomycetes in general. Aerobic bacteria may be kept warm and oxygen supplied, and facultative anaerobes are similar, but oxygen supply is not necessary. In the case of anaerobic bacteria, carbon dioxide gas or nitrogen gas is filled and kept warm.

The concentrated medium solution containing the bacteriostatic agent and the starter fungus and the starter containing the bactericide can be used as long as they can withstand the bacteriostatic agent. What culture | cultivated culture is more suitable. Among the sporic bacteria, B. subtilis, B. coagulans, B. licheniformis, B. polymyxa, B. cereus, Alby (B) belonging to the genus Bacillus .Alvei), B. brevis, B. macerans, B. megaterium, B. stearothermophilus, B. circulans, B. firmus , B. laterosporus, B. sphaericus, B. larvae, B. lavae opilliae), it can be used, such as Ren Timo over bus (B.lentimorbus) also scan polo Lactobacillus Inurinasu (Sporolactobacillus inulinus) and Geobacillus (Geobacillus) genus.

In the spore yeast, it is a spore spore yeast, belonging to the Saccharomycesae family, belonging to the genus Saccharomyces (S. cerevisiae), Carlsbergensis (genus S. carlsbergensis) and the genus Pichia (D. cerevisiae). ), Endomycopsiae, Nematospoidae, etc. can be used.

Bacteria that can be used in the seed culture and starter of the present invention are bacteria, fungi, and actinomycetes, and these bacteria are collectively referred to as fungi or microorganisms for convenience. In addition, Bacillus subtilis JCM1465, Bacillus coagulans JCM2258, Saccharomyces cerevisiae JCM7255 used in the examples are independent administrative corporations rhamnosus) ATCC 53103 is available at the American Type Culture Center.

In the method for producing a concentrated medium of the present invention, medium components suitable for the starter to be cultured can be selected, but based on the known MRS culture solution, standard agar medium and Sabouraud medium formulation, the liquid is removed except for agar. Create a concentrated medium according to the purpose of use. For example, agar is removed from a standard agar medium that can be used to grow various bacteria, and 10 g concentrated medium is obtained by dissolving 25 g of yeast extract, 50 g of tryptone, and 10 g of glucose in 915 g of water, and 100 g of yeast extract and 200 g of tryptone. A 40-fold concentrated medium is obtained by dissolving 40 g of glucose in 660 g of water. This was sterilized at 65 ° C. to 120 ° C. for 30 minutes. It can be made later by cooling and adding the selected type and amount of bacteriostatic agent.

After cooling, a bacteriostatic agent selected from alcohols, acids, and salts, which are suitable bacteriostatic agents for the starter used, is added to this concentrated medium. For example, in the case of lactic acid bacteria, an organic acid such as acetic acid shown in Example 1 is added. Appropriate addition of 10% also suppresses the growth of acid-resistant fungi. By adding 15% ethyl alcohol to this, the growth of other lactic acid bacteria is also suppressed. Moreover, the content of the bacteriostatic agent in which lactic acid bacteria are not suppressed is 5% for ethyl alcohol and 10% or less for acetic acid.

The method for producing a concentrated medium to which a starter and a bacteriostatic agent of the present invention are added can be produced by preparing a concentrated medium to which a bacteriostatic agent is added according to the production method described above, and adding the starter thereto. The bacterium is preferably a spore bacterium such as Bacillus coagulans or Bacillus subtilis or a spore yeast. Moreover, the manufacturing method of the starter which added the bacteriostatic agent makes a starter by culture | cultivating a seed culture in the culture medium suitable for growth, and adds a bacteriostatic agent. The bacteriostatic agent added here was also produced by selecting and adding the type and amount by the previous method. The seed culture is preferably a spore bacterium or a spore yeast such as Bacillus coagulans or Bacillus subtilis. In the present invention, the seed starter was cultured and then collected by centrifugation, drying or the like and added to the liquid as a starter.

In order to cultivate a target bacterium using the concentrated medium of the present invention, the concentrated medium has been described above that a large amount of medium components are dissolved, but it is diluted to an appropriate growth concentration for starter fungi and cultured. This is important for both growth and economy. In addition, it is also performed in parallel to keep the conditions under which the target bacteria can be proliferated by adding fresh water to the concentration at which they grow without being controlled by the bacteriostatic agent. A starter is added here, and the aerobic bacteria are hygienic in the incubator (tank), and it is only necessary to stir with a warming and aeration device. The facultative anaerobic bacteria are the same container (tank) and the aeration device is unnecessary and low speed is required. The anaerobic bacteria can be easily cultured by filling them with carbon dioxide gas or nitrogen gas in a sealed incubator (tank), and keeping them warm and stirring.

Since the starter is already added to the culture method using the bacteriostatic agent and starter-concentrated medium of the present invention, the concentrated medium is diluted with clean water to an appropriate concentration as in the case of using the concentrated culture solution described above. Under the same conditions as in the previous concentrated medium, this can be kept at the growth temperature and stirred if necessary. The method of using the starter with bacteriostatic agent is to add 0.1 to 5% to a concentrated medium or a normal culture solution to which a bacteriostatic agent is added in the same manner as a general starter, and aerobic or anaerobic conditions. Is maintained at the growth temperature and stirred if necessary. Naturally, it can also be produced in large quantities, put directly into a treatment tank such as a wastewater treatment plant, and propagated in the treatment tank.

For example, in the case of a starter that does not contain a normal bacteriostatic agent, the incubator used in the present invention manually adds the starter after diluting the concentrated medium transported at room temperature to the culture site with fresh water. There is only one culture tank. Conditions such as aerobic or anaerobic conditions are prepared, and the culture is stirred and cultured while keeping the temperature at the growth temperature of the bacteria. Useful bacteria can be cultured with a very simple culture apparatus.

In order to automate culture, a sub-tank for concentrated medium and a separate sub-tank for containing a bactericide are installed, and a main culture container that matches the capacity of the starter, concentrated medium, and the appropriate dilution amount of fresh water. (Tank) should be installed, conditions such as aerobic or anaerobic are adjusted, and the culture is stirred and maintained at the growth temperature of the bacteria. At this time, the starter, concentrated culture solution, and the volume of diluted fresh water used The culture temperature, the culture time, etc. may be automatically controlled. Similarly, in the case where a bacteriostatic agent and a concentrated medium are added to a starter, automatic control may be performed by one sub tank holding a starter-containing concentrated medium and one main tank for automatic control.

The concentrated medium containing a bacteriostatic agent, the concentrated medium containing a bacteriostatic agent and the starter containing a bacteriostatic agent of the present invention can be distributed, stored and stored at room temperature in a plastic or metal container. Although various volumes may be used, the capacity is preferably about 10 ml to 20 l in consideration of transportation, but the capacity is not specified if a tank and a pump are used.

In accordance with the present invention, in a facility with manufacturing facilities excellent in microbial management, a concentrated medium with a bacteriostatic agent added in advance is manufactured, transported to the site where it is used at room temperature, an appropriate amount of water is added, After adjusting to the aerobic and anaerobic conditions of the fungus, the proliferating bacterial solution can be produced by a simple culture method of culturing at the growth temperature, and the necessary amount can be used at the necessary place when necessary. According to the present invention, it can be easily cultured in large quantities at the site where microorganisms are used, used for processing organic matter in wastewater treatment plants, used for deodorization in barns and garbage collection sites, and used as biopesticides on farms. Easy to use. In addition, by using a food material as a bacteriostatic agent, it is expected to be used in food as a concentrated medium that is more resistant to contamination.

Hereinafter, the present invention will be specifically described with reference to examples.

Selection of the bacteriostatic agent in the starter bacteria used In order to select the type of bacteriostatic agent in the concentrated medium according to the starter bacteria used, ethyl alcohol, acetic acid and sodium chloride were used. Plates were made by adding width to SPC medium. Adjust Bacillus subtilis JCM1465 (S), Lactobacillus rhamnosus ATCC 53103 (R), Saccharomyces cerevisiae JCM7255 (Ce), drainage + soil fungus (H) to 10 ⁵ to 10 ⁶ cfu / ml, respectively. And streaked at 30 ° C. or 35 ° C. for 48 to 72 hours to confirm growth on the plate. In addition, drainage + soil fungus (S) was used after 3 ml of wastewater from a wastewater treatment plant of a food factory and 0.5 g of soil in the field were diluted and diluted with 10 ml of diluted water.

The results are shown in Table 1 and show that the initials of each test bacterium are grown under the indicated concentrations. Bacillus subtilis was found to be very weak against acetic acid and moderately strong against ethyl alcohol (ethanol) and salt. Lactobacillus rhamnosus was found to be strong against acetic acid and moderately strong against ethyl alcohol and salt. Saccharomyces cerevisiae was found to be resistant to ethyl alcohol and salt. As a comparative example, drainage + soil fungi were cultured, but growth was suppressed within 10% in all bacteriostatic agents, and no colonies were generated.

Examination of the maximum content of bacteriostatic agent in which starter fungus grows The concentration of starter bacteriostatic agent that can be used was examined. Since it was found from Example 1 that bacteria other than special environmental bacteria can be bacteriostatic if two types of bacteriostatic agents are added, Bacillus subtilis is 0, 2, 4, 6, and 8%, respectively. Was adjusted according to the orthogonal coordinates shown in Table 2 to prepare a plate medium added to the SPC medium. Lactobacillus rhamnosus was adjusted to 0, 2, 4, 6, 8% alcohol concentration and 0, 4, 8, 12, 16% acetic acid concentration according to the Cartesian coordinates shown in Table 3, and added to the SPC medium. A plate medium was prepared. For Saccharomyces cerevisiae, ethyl alcohol and salt, 0, 4, 8, 12, and 16%, respectively, were adjusted according to the orthogonal coordinates shown in Table 4 to prepare a plate medium added to the SPC medium. A test solution of 10 ⁵ to 10 ⁶ cfu / g of the test bacteria was streaked on the flat plate with a platinum loop and cultured at 35 ° C. or 30 ° C. for 48 to 72 hours to confirm growth on the plate. As the test strain, Bacillus subtilis JCM1465, Saccharomyces cerbuscie JCM7255, Lactobacillus rhamnosus ATCC 53103 and contaminated bacteria (drainage + soil fungus) were used.

Since growth of Bacillus subtilis was suppressed at a moderate concentration with ethyl alcohol or sodium chloride from Example 1, the concentration to be grown with these bacteriostatic agents and the concentration to be suppressed were examined. According to the results shown in Table 2, the growth of ethyl alcohol or sodium chloride was suppressed at 6% or more, and growth was at 4% or less. Moreover, when both were mixed, it became clear that it grew at the% by which ethyl alcohol and salt content fell somewhat.

Since Lactobacillus rhamnosus from Example 1 is suppressed at a high concentration of acetic acid and a moderate concentration of ethyl alcohol, the concentration at which these bacteriostatic agents are grown and the concentration to be suppressed were examined. According to the results shown in Tables 1 and 3, the growth of ethyl alcohol was suppressed at 6% or more and grew at 4% or less, while acetic acid was grown at 10% and suppressed at 12% or more.

Since Saccharomyces cerevisiae was suppressed to high concentrations in sodium chloride and ethyl alcohol from Example 1, the concentration at which these bacteriostatic agents are grown and the concentration to be suppressed were examined. According to the results shown in Table 4, the growth of both ethyl alcohol and acetic acid was suppressed at 16% and grew at 12%.

Confirmation of maintenance of bacterial count of starter containing bacteriostatic agent As a medium component of the starter, yeast extract (2.5 g), tryptone (5 g) and glucose (1 g) were dissolved in water to make 1 L. This was placed in an Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and then cooled to a culture temperature of 37 ° C. To the flask, 2% of a culture solution pre-cultured in a medium similarly treated with Bacillus subtilis JCM1465 was added and cultured at 37 ° C. for 24 hours. A starter containing a bacteriostatic agent was prepared by adding 10 parts of ethyl alcohol and 2 parts of sodium chloride to 92 parts of this cultured starter, and a storage test was performed at 30 ° C. Presence of spores was confirmed by diluting the stock solution and heating at 85 ° C. for 10 minutes, followed by pour culture in an SPC medium according to a conventional method.

The results of storage are shown in Table 5. From this result, it was confirmed that by adding ethyl alcohol and sodium chloride as a bacteriostatic agent, the vegetative cells die, but the spores (10 ⁶ levels) retain the number of bacteria over a long period of time. confirmed. In addition, since the spores were inactivated, they did not become vegetative cells even when stored at room temperature.

Growth of diluted concentrated medium containing bacteriostatic agent after dilution with fresh water (including comparative examples)

In order to prepare a 30-fold concentrated medium as a medium (SPC) component, 60 g of glucose, 150 g of yeast extract and 300 g of tryptone were dissolved in water to prepare 1800 g, and sterilized at 120 ° C. for 20 minutes to prepare a concentrated medium. To this concentrated medium, 200 g of ethyl alcohol was added to prepare 2000 g of a concentrated medium containing a bacteriostatic agent. To this concentrated medium, 58 kg of sterilized water was added to make 60 kg. Three 20 kg medium solutions were prepared in three incubators, and 2% each of starters of Bacillus coagulans JCM2258 and Lactobacillus rhamnosus ATCC 53103 were added. Further, 2% of Bacillus subtilis JCM1465 containing a bacteriostatic agent shown in Example 3 was added, and the mixture was stirred at 24 ° C. and cultured for 24 hours. As a comparative example, the cells were cultured in a normal SPC culture solution (without agar) without adding bacteriostatic agent ethyl alcohol.

From the results shown in Table 6, even if a bacteriostatic agent is present, if it is appropriately diluted with still water (ethyl alcohol concentration 0.33% or less), it starts growing, and with proper dilution, ethyl alcohol (ethanol) and sodium chloride are added. It was clarified that the number of bacteria grew as well as no-addition conditions and was adapted as a bulk (starter) medium.

Claims (7)

Concentrated medium characterized by adding bacteriostatic agent 2. The concentrated medium according to claim 1, further comprising a starter. Starter characterized by adding bacteriostatic agent The concentrated medium according to claim 1 or 2, and the starter according to claim 3, wherein the bacteriostatic agent comprises at least one of alcohols, acids and salts. The concentrated medium according to claim 2 and the starter according to claim 3, which comprise a spore bacterium or / and a spore yeast. 4. The concentrated medium according to claim 1, comprising a method for determining the type, content and water content of a bacteriostatic agent suitable for a starter bacterium using a plate medium containing a bacteriostatic agent. Starter A method for producing the concentrated medium according to claim 1, a method for producing the starter according to claim 3, and a method for growing microorganisms