JPWO2017086451A1 - Method for producing lactic acid bacterium solid fermented product inducing and expressing the original survival ability of bacteria, and lactic acid bacterium solid fermented product produced by the method - Google Patents

Abstract

In order to induce and express the excellent survival ability of lactic acid bacteria, lactic acid bacteria are bred and activated by an original culture method in which various stresses are applied, and such active cells are obtained. And a lactic acid bacteria solid fermented product in which the survival ability inherent to the bacteria obtained by the culture is induced and expressed.
A method for producing a lactic acid bacteria solid fermentation product,
(1) A liquid culture process for culturing lactic acid bacteria using a liquid medium,
(2) A solid culture step consisting of one step or a plurality of steps in which the lactic acid bacteria cultured in step (1) is cultured in a solid medium which is a solid food mixture, wherein the solid medium comprises at least carbonate and germ, A solid culture step for growing lactic acid bacteria on the surface of the solid medium, comprising a polyphenol-containing plant and the solid medium used in the final step of the solid culture comprises a polyphenol-containing plant;
The lactic acid bacteria solid fermentation product is a lactic acid bacteria solid fermentation product in which the lactic acid bacteria are contained on the surface of the solid medium used in step (2).

Description

  The present invention relates to a method for culturing lactic acid bacteria in which the survival ability inherent to the bacteria is induced and expressed so as to increase stress tolerance.

  Lactic acid bacteria is a general term for a group of bacteria that produce lactic acid in large quantities. In addition to various fermented foods, it has been involved in the production of alcoholic beverages, brewed products, pickles, processed fruit products, etc., and humans have long benefited from lactic acid bacteria. It was.

  In recent years, lactic acid bacteria have attracted attention for various health functionalities such as an inhibitory action, enteric action, and immunostimulatory action of enteric spoilage bacteria (see Non-Patent Document 1).

  Lactic acid bacteria are known to be effective in maintaining human health as an intestinal bacterium together with bifidobacteria. Research on microbial "probiotics" that improve intestinal flora, have a beneficial effect on health, and are guaranteed to be safe has been active. In view of the growing interest in preventing lifestyle-related diseases, there is a tendency for better health functionality of foods to be pursued, and further utilization of such probiotics in foods is desired (Non-Patent Document 2).

  Currently, the most commonly used technique in culturing lactic acid bacteria is inoculating lactic acid bacteria in a liquid medium, culturing while controlling the medium pH, etc., and after reaching a stationary phase where growth has stopped, the cells are separated from the medium. There is a batch culture method to collect. In this method, since the culture environment changes due to a decrease in nutrients in the culture medium and an increase in secretions such as lactic acid, the growth of lactic acid bacteria is easily suppressed during growth, and the cell density is increased in the culture solution. It is difficult to reach more than 10 billion cfu (colony forming units) per ml, and it is difficult to give sufficient stress resistance, improving both the high density of cells and excellent stability at the same time. The technique which can be performed was anxious (nonpatent literature 3).

  From such a viewpoint, stirring and culturing using a mixture containing one or more selected from magnesium carbonate, monosaccharide or disaccharide, peptone, and koji, koji extract, embryo medium, yeast, and yeast extract as a liquid medium A method for culturing Enterococcus faecalis or Enterococcus faecium characterized by the above has been proposed (Patent Document 1).

  In addition, a method has been proposed in which filamentous fungi are cultured in plants such as wheat germ and lactic acid bacteria are cultured using a solid medium to which magnesium carbonate or the like is added (Patent Document 2).

Japanese Patent No. 3900484 Japanese Patent No. 3868346

Ali AA. Et al., Research Journal of Microbiology, 5 (12), pp.1213-1221 (2010) Naagpal R. et al., FEMS Microbiology Letters, 334 (1), pp.1-15 (2012) Lacroix C. et al., Current Opinion in Biotechnology, 18, pp.176-183 (2007)

  Since there are about 1000 types of bacteria in the human intestine and hundreds of trillions in quantity, the expression of the excellent health functionality of lactic acid bacteria is the higher the number of lactic acid bacteria that consumers consume at once. And, it is expected that the better the health functionality can be enjoyed by the more vigorous microbial cells.

  In order to further enhance the health functionality of lactic acid bacteria, the necessity of imparting excellent survival ability to bacterial cells is recognized as an important issue. Lactic acid bacteria are generally prepared by a liquid culture method, but in this case as well, it is important to impart excellent survival ability.

  The present invention is characterized in that an active cell obtained by breeding lactic acid bacteria by a unique culture method for culturing while applying various stresses in order to induce and express the excellent survival ability inherent to the fungus. It is an object of the present invention to provide a culture method capable of obtaining a body and a solid fermentation product of lactic acid bacteria in which the survival ability inherent to the bacteria obtained by the culture is induced and expressed.

  The inventors of the present invention have previously developed a method for growing lactic acid bacterial cells that have induced and expressed the original survival ability of the cells by liquid culture, and called the culture method "strain breeding culture". The present inventors further utilized a "strain breeding culture" as a core technique, and in a solid fermented product obtained by growing on a solid surface of a polyphenol-containing food mixture, the bacterial cells inducing and expressing the original viability of the fungus The culture conditions for vigorous growth were studied intensively.

  As a result, it has been found that by performing solid culture subsequent to liquid culture, the bacterial cells inducing and expressing the original survival ability of the fungus proliferate vigorously. In particular, in “strain breeding culture”, which is a liquid culture, each time the culture is repeated, a stress outside the optimal culture environment with a higher strength than that of the previous culture is applied to induce and express the original survival ability of the fungus. As compared with the case where the cells obtained by culturing in a general MRS liquid medium were further subjected to solid culture, a greater effect was obtained. Further, the present invention has found that a larger effect can be obtained by growing a solid culture on the surface of a solid medium, which is a solid food mixture, by two-stage culture of “solid environmental adaptation” and “lactic acid bacteria solid fermentation”. It came to complete.

  The method of the present invention combines liquid culture and solid culture, uses a plant containing a polyphenol capable of stressing lactic acid bacteria in a solid medium, and further step by step as each step of liquid culture and solid culture is performed. It is different from the conventional culture method in that a highly viable bacterium is obtained by increasing the stress applied to the cell.

That is, the present invention is as follows.
[1] A method for producing a lactic acid bacteria solid fermentation product,
(1) A liquid culture process for culturing lactic acid bacteria using a liquid medium,
(2) A solid culture step consisting of one step or a plurality of steps in which the lactic acid bacteria cultured in step (1) is cultured in a solid medium which is a solid food mixture, wherein the solid medium comprises at least carbonate and germ, A solid culture step for growing lactic acid bacteria on the surface of the solid medium, comprising a polyphenol-containing plant and the solid medium used in the final step of the solid culture comprises a polyphenol-containing plant;
The lactic acid bacteria solid fermentation product is a lactic acid bacteria solid fermentation product in which the lactic acid bacteria are contained on the surface of the solid medium used in step (2).
[2] The solid culture step of step (2) is performed in a plurality of steps, and the content of the polyphenol-containing plant in the solid medium used in the subsequent solid culture step is determined in the solid medium used in the previous solid culture step. The production method of [1], which is greater than the content of the polyphenol-containing plant.
[3] As the liquid culture in the step (1) and the solid culture step in the step (2) are performed, a plurality of stresses with gradually increasing strengths are loaded, and the survival ability of the lactic acid bacteria having resistance to those stresses is increased. The production method of [1] or [2], which is increased.
[4] A method for producing a lactic acid bacteria solid fermentation product,
(1) A liquid culture process for culturing lactic acid bacteria using a liquid medium,
(2-i) The lactic acid bacteria cultured in the step (1) are cultured in a solid medium that is a solid food mixture containing at least carbonate and germ, and germ buds and / or polyphenol-containing plants, and the lactic acid bacteria are cultured on the surface of the solid medium. Proliferating,
(2-ii) The lactic acid bacteria cultured in the step (2-i) is a solid medium that is a solid food mixture containing at least carbonate, germ, and a polyphenol-containing plant, and the content of the polyphenol-containing plant is the step ( Culturing in a solid medium more than the solid medium used in 2-i) and growing lactic acid bacteria on the surface of the solid medium;
The method according to [2], wherein the lactic acid bacterium solid fermented product is a lactic acid bacterium solid fermented product containing the lactic acid bacterium on the surface of the solid medium used in step (2-ii).
[5] The production method of [4], wherein in the step (2-ii), the germ medium is further contained in the solid medium.
[6] As the steps (1), (2-i) and (2-ii) are performed, the survival of lactic acid bacteria having a plurality of stresses increased in strength in stages and resistant to those stresses. The method according to [4] or [5], wherein the performance is increased.
[7] The method according to any one of [1] to [6], wherein the liquid medium used in the step (1) is a liquid medium containing at least carbonate, embryo bud, nitrogen source and carbon source.
[8] The liquid culture process of step (1) is performed in a multi-stage culture process, and in any one of the multi-stage culture processes or each of the multi-stage culture processes, at least carbonate, embryo The manufacturing method in any one of [1]-[7] using the liquid culture medium containing a candy, a nitrogen source, and a carbon source.
[9] The method according to [7] or [8], wherein the germ buds contained in the liquid medium used in step (1) include one or more of defatted rice germ, defatted wheat germ, and defatted corn germ.
[10] The method according to [7] or [8], wherein the carbonate contained in the liquid medium used in the step (1) is a mixture of calcium carbonate and magnesium carbonate.
[11] The production method according to any one of [1] to [10], wherein the germ contained in the solid medium used in the solid culture step is a defatted germ containing one or more cereal defatted germs or germs.
[12] The germ is one or two selected from the group consisting of defatted rice germ, defatted wheat germ, defatted corn germ, defatted germ derived from other grains and germs of other grains, [11] The production method.
[13] The method according to [11] or [12], wherein the defatted cereal containing the germ is defatted soybean.
[14] The polyphenol-containing plant contained in the solid medium used in the solid culture step is at least one plant selected from the group consisting of fruits, vegetables, cereals, tea leaves, seaweed and spices, [1] to [13] The production method according to any one of [13].
[15] The method according to any one of [1] to [14], wherein the embryo pods contained in the solid medium used in the solid culture step are defatted embryo pods of Aspergillus spp.
[16] The method according to any one of [4] to [15], wherein the solid medium used in step (2-ii) further contains sodium chloride.
[17] The method according to any one of [1] to [16], wherein the carbonate contained in the solid medium used in the solid culture step is a mixture of calcium carbonate and magnesium carbonate.
[18] Any one of [1] to [17], wherein the solid medium used in the solid culture step is a mixed solid medium of carbonate and embryo, and a germ mixture and / or a polyphenol-containing plant body and water. Production method.
[19] The method according to any one of [1] to [18], wherein the lactic acid bacteria obtained by liquid culture in step (1) are inoculated on the surface of the solid medium used in step (2).
[20] Lactic acid bacteria obtained by liquid culture in step (1) are inoculated on the surface of the solid medium used in step (2-i), and further lactic acid bacteria obtained by solid culture in step (2-i) The method according to any one of [4] to [19], wherein the cells are inoculated on the surface of the solid medium used in step (2-ii).
[21] A solid fermented lactic acid bacterium, wherein lactic acid bacteria are contained on the surface of a solid food mixture containing at least carbonate, embryo, and polyphenol-containing plant.
[22] The lactic acid bacterium solid fermented product according to [21], wherein the solid food mixture further includes embryo buds.
[23] The lactic acid bacterium solid fermented product according to [21] or [22], wherein the germ is a defatted cereal including one or more cereal defatted germs or germs.
[24] The germ is one or two selected from defatted rice germ, defatted wheat germ, defatted corn germ, defatted germ derived from other cereals and other cereal germs, [23] Lactic acid bacteria solid fermented product.
[25] The lactic acid bacterium solid fermented product according to any one of [21] to [24], wherein the defatted cereal containing the germ is defatted soybean.
[26] The lactic acid bacterium solid fermentation according to any one of [21] to [25], wherein the polyphenol-containing plant is at least one plant selected from the group consisting of fruits, vegetables, cereals, tea leaves, seaweed and spices. object.
[27] The solid fermented lactic acid bacterium according to any one of [21] to [26], wherein the germ bud is a defatted germ pod of Aspergillus belonging to the genus Aspergillus.
[28] The solid fermented lactic acid bacterium according to any one of [21] to [27], wherein the solid food mixture further contains sodium chloride.
[29] The lactic acid bacterium solid fermentation product according to any one of [21] to [28], wherein the carbonate is a mixture of calcium carbonate and magnesium carbonate.
[30] A cereal, a plant containing polyphenol, and a food containing lactic acid bacteria, each containing the lactic acid bacteria solid fermented product according to any one of [21] to [29].

  This specification includes the disclosure of Japanese Patent Application No. 2015-226074 which is the basis of the priority of the present application.

  According to the method for producing a lactic acid bacterium solid fermented product that induces and expresses the original survival ability of the bacterium according to the present invention, compared to the number of bacteria per 1 g or 1 ml of fermented food containing viable bacteria such as yogurt, lactic acid bacteria beverage, Korean kimchi The lactic acid bacterium solid fermented product finally obtained contains 10 to 100 times or more high-density microbial cells, and the fermented product can be ingested with a high amount of bacteria in a small amount. Further, the survival ability inherent to the bacteria can be induced and expressed according to the strength of stress applied by the polyphenol and sodium chloride contained in the food material constituting the solid food material mixture used as the solid medium. Furthermore, the obtained lactic acid bacteria solid fermented product not only contains living cells (hereinafter referred to as “active cells”) at a high cell count, but also high-functionality containing bioactive substances derived from food materials such as polyphenols and dietary fibers. It can be used as food. Furthermore, depending on the bacterial species to be used, it can be expected that in addition to biofilm, it contains excellent health functional secretions such as folic acid and ornithine.

  Therefore, the lactic acid bacteria solid fermented product produced by the method of the present invention has not only a function as a culture method for producing active bacterial cells inducing and expressing the original survival ability of bacteria, but also functions such as lactic acid bacteria and polyphenols. It also has a function as a fermented product that can be ingested with excellent health functional ingredients.

  As an effect of the present technology, it becomes possible to induce and express the survival ability inherent to the bacterium, and this factor is presumed to be the effect of stabilizing the cell membrane due to the change in the surface layer structure of the bacterium. Therefore, microbial storage is excellent for stress caused by polyphenols and sodium chloride, as well as for microbial damage caused by refrigerated storage, high osmotic pressure, high concentration sodium chloride, freezing / thawing, heating, low pH, bile, or drying. It is expected that resistance is given.

  When drying bacterial cells obtained by liquid culture, it is essential to use a protective agent or stabilizer having an appropriate composition. In contrast, a lactic acid bacteria solid fermented product containing active cells produced by the method of the present invention is a biofilm having a stabilizing function comparable to that of a protective agent or stabilizer as a metabolite during solid fermentation. It is presumed that these components are secreted and produced abundantly outside the cells, so that no protective agent or stabilizer is required. Therefore, in a general “fermentation scene” of fermented foods, polyphenol-rich foods were not considered to be fermented by lactic acid bacteria due to their high antibacterial stress, but according to the method of the present invention, A solid fermented lactic acid bacterium containing high-density bacterial cells by inducing and expressing the residual ability is obtained, and a solid fermented product obtained by a drying treatment such as freeze-drying treatment or warm air can also be provided.

It is a schematic diagram of the state in which each component of the solid culture medium used by the solid culture of this invention is mixing homogeneously. It is a figure which shows the characteristic of each process in the method of 3 processes which perform liquid culture and solid culture of 2 processes. It is a figure which shows the number of bacteria of the microbial cell obtained by culture | cultivating at each culture process, and a heat resistant result. It is a figure which shows the survival microbe number and heat resistance after freeze-drying of the microbial cell obtained by culture | cultivating at each culture | cultivation process. It is a figure which shows the "survival rate x heat resistance" (%) after ventilation drying of the microbial cell obtained by culture | cultivating at each culture | cultivation process.

Hereinafter, the present invention will be described in detail.
1. Cultivation method The present invention is to inoculate a fresh medium environment again with microbial cells that have reached the stationary phase by growing lactic acid bacteria, and to increase the stress intensity to be applied each time it reaches the stationary phase through the logarithmic growth phase. The culture method is characterized by undergoing a liquid culture step and a solid culture step for inducing and expressing the original survival ability of the fungus. Here, the stationary phase means that the number of viable cells in the medium is maintained at a constant level after the logarithmic growth phase, that is, the cell division rate is reduced, the division rate and the death rate reach equilibrium, It means a state where the number of bacteria is constant, also called the stationary phase. The solid culture step may be a single step, but preferably a multi-step solid culture of two or more steps is performed.

  Here, the survival ability inherent to the bacteria refers to the ability of the bacteria to survive after a certain period of time under specific conditions. By the culture method of the present invention, the degree of stress load increases as the liquid culture and the solid culture process are performed, for example, the liquid culture process and the plurality of solid culture processes are performed. The bacteria having high resistance to the stress environment can be obtained. As a result, cells with high growth ability can be obtained.

  By the culturing method of the present invention, it is possible to obtain bacterial cells in which excellent survival ability is induced and expressed. Further, by the culture method of the present invention, lactic acid bacteria grow on the surface of the solid medium, and a “lactic acid bacteria solid fermented product” containing the lactic acid bacteria on the surface of the solid medium can be obtained. Therefore, the method of the present invention is also a method for producing a “lactic acid bacteria solid fermented product”.

  In the culture method of the present invention, stress is applied to the lactic acid bacteria to be cultured in each step, and the stress intensity to be applied is increased as the culture step proceeds. Here, the stress refers to a culture condition that deviates from the optimum range as a culture environment for lactic acid bacteria species or strains. Examples of the culture conditions that can cause stress include pH, osmotic pressure, temperature, and the amount of an assimilating substance contained in the medium. That is, examples of stress applied in the series of culture steps of the present invention include osmotic pressure stress, alkaline pH stress, acidic pH stress, oxidative stress, temperature stress, nutritional stress, and stress caused by polyphenol. The greater the deviation from the optimum range, the higher the intensity of stress applied. The bacterial cells obtained by applying stress change to a state having sufficient stress resistance, and the stability is further improved. Changing to a state with sufficient stress resistance by stress loading is referred to as “inducing and expressing the original survival ability of bacteria”. In the culturing process of the present invention, a plurality of these stresses are combined and loaded on the lactic acid bacteria.

  For example, alkaline / slightly soluble carbonate used as a medium component in the culture process of the present invention increases the density of cells in the culture and increases the amount of organic acid that is secreted and produced to release carbon dioxide. However, it changes to water-soluble salts. Along with this, the ionic strength of the culture solution gradually increases, so that osmotic stress can be applied to the cells, which is necessary for improving the properties of the cells in liquid culture that makes the cells dense. The effect of increasing the types of stress to be loaded is expected. An increase in the ionic strength of the culture solution can be evaluated by measuring electrical conductivity. On the other hand, the release of carbon dioxide from the culture solution has the effect of reducing dissolved oxygen, and the production of hydrogen peroxide can be attenuated. This means that even when the cell density has increased, the amount of hydrogen peroxide produced does not increase in proportion to the increase in cell density, but the concentration is appropriately controlled. It means that. Therefore, the load of oxidative stress caused by hydrogen peroxide can be maintained in a moderately desirable state. As a result, resistance to oxidative stress is imparted and self-cell damage caused by excessive hydrogen peroxide in the culture process is suppressed. be able to. In this way, various stress stimuli that are acceptable for the cells to be cultured can be applied at the same time, gradually and appropriately according to the culture process, so that the density of the cells can be increased, as well as the MRS liquid. Selective cultivation of cells that have been given maximum stability of cells and vigorous growth ability in low and high temperatures, compared to culture in a medium rich in nutrients such as medium. become.

  Furthermore, polyphenol-containing plants, grain germs, and germ buds used as components of the medium in the culture process of the present invention contain polyphenols, and the antibacterial action of polyphenols as ionophores stresses lactic acid bacteria. To do. In the present invention, this stress is referred to as “polyphenol stress”.

  Lactic acid bacteria used for the culture are not limited, and include Lactobacillus plantarum, Lactobacillus plantarum, Lactobacillus rhamonosus, Lactobacillus salivarius, Lactobacillus salivarius, Lactobacillus brevis ( Lactobacillus brevis), Lactobacillus casei, Lactobacillus gasseri, Lactobacillus delbrueckii, Lactobacillus fermentum Lactobacillus fermentum Lactobacillus chicil, Lactobacillus fermentum Lactobacillus paracasei, Lactococcus lactis, Lactococcus cremoris, Leuconostoc mesenteroides ), Leuconostoc citreum, Pediococcus acidilactici, Pediococcus pentosaceus and the like.

  Moreover, although one type of lactic acid bacteria may be used, two or more types of lactic acid bacteria may be included.

  The liquid culture process is a liquid culture in which lactic acid bacteria are cultured using a liquid medium. The lactic acid bacteria obtained in this liquid culture process are called “liquid species”. In the present invention, the liquid culture step is referred to as “step (1)”. The liquid culture process may further include a plurality of processes.

  The solid culture step is a step of culturing lactic acid bacteria by solid culture using a solid medium, and is a step of inoculating a solid medium with a liquid species containing lactic acid bacteria obtained in the liquid culture step. Solid culture is a culture that mimics the natural environment, and is a culture in which lactic acid bacteria are grown in an environment close to the original habitat environment of lactic acid bacteria. The solid culture process adapts lactic acid bacteria from a liquid environment to a solid environment. In this respect, the solid culture process is referred to as an adaptation process from liquid culture to solid culture, or a “solid environment adaptation” process. Further, in the solid culture process, the solid food mixture is loaded with a higher stress on the lactic acid bacteria than in the liquid culture process, and the survival ability inherent to the bacteria is induced and expressed, and the bacteria having high resistance to the stress environment can be obtained. . A solid culture process in which bacteria having high resistance to a stress environment are grown at a high density is called a “lactic acid bacteria solid fermentation” process. Preferably, the “solid environment adaptation” step and the “lactic acid bacterium solid state fermentation” step for growing bacteria highly resistant to a stress environment are performed in separate steps.

  In general, lactic acid bacteria are cultured in liquid culture. In liquid culture, lactic acid bacteria can easily grow if nutrients that diffuse in the culture medium can be taken into the cells. On the other hand, since the solid culture of the present invention uses a solid medium containing defatted germ as a main component, it has a lower nutrient environment and a lower moisture environment than liquid culture. The environment of solid culture is closer to the natural environment than liquid culture. Therefore, the expression rate of the genes inherent to the bacteria is increased, and as a result, cells with high survival ability can be obtained.

  The solid culture process includes one or more processes. For example, the solid culture step includes two steps of step (2-i) and step (2-ii), and step (2-i) is a step of culturing lactic acid bacteria by solid culture using a solid medium, In this step, a liquid medium containing lactic acid bacteria obtained by liquid culture in step (1) is inoculated into a solid medium and cultured. In step (2-i), lactic acid bacteria are adapted from a liquid environment to a solid environment. In this respect, the step (2-i) is referred to as an adaptation step from liquid culture to solid culture or a solid environment adaptation step. In addition, the bacteria adapted to the solid culture obtained in the step (2-i) are referred to as “solid species”.

  Step (2-ii) is a step of cultivating lactic acid bacteria by solid culture using a solid medium. A part of the solid species obtained by solid culture in step (2-i) is further inoculated into the solid medium and cultured. It is a process to do. In the step (2-ii), a higher stress is applied to the lactic acid bacterium than the stress applied to the lactic acid bacterium in the solid culture of the step (2-i). In addition, the step (2-ii) is referred to as a “lactic acid bacteria solid fermentation” step.

  Conventional culture of lactic acid bacteria is a liquid culture using a liquid medium, and is a method of growing cells in a liquid medium that is an aqueous solution containing nutrients necessary for growth such as a carbon source, a nitrogen source, and salts. . In this method, the water activity (Water Activity) of the growth region where lactic acid bacteria grow is 1.0. Here, the water activity of the growth region refers to the ratio of free water in the culture solution in which lactic acid bacteria are cultured, and the water vapor pressure (P) and the temperature when the liquid medium is placed in a closed culture vessel It is determined by the ratio of pure water vapor pressure (PO) (P / PO).

  As a culture method using a solid medium, an agar plate culture method used for research purposes or the like for separating microorganisms is known. The solid culture in the present invention is different from the conventional agar plate culture, in which food materials such as solid plants are mixed, and the water activity is less than 1.0 by adding 45-60% water to the resulting mixture, preferably A culture method in which cells are grown on the surface of a solid medium in a solid medium of 0.95 to 0.98. Water activity can vary depending on the species.

Hereinafter, the liquid culture process and the solid culture process will be described in detail.
1-1. Liquid culture process (1)
Step (1), which is a liquid culture step, can be performed using a known liquid medium used for culturing lactic acid bacteria. For example, a low-selectivity medium that promotes the growth of all types of lactic acid bacteria may be used. The medium composition may be an MRS (de Man, Rogosa, Sharpe) liquid medium that complies with the international standard method NF_ISO 15214. In the present invention, liquid culture performed using a known liquid medium is referred to as “general liquid culture”. This “general liquid culture” is usually performed in one step.

  Further, the liquid culture in step (1) may be performed in a plurality of steps. That is, a multi-stage culture is performed from the pre-culture to the pre-culture and the main culture. In each case, lactic acid bacteria can be subjected to temperature stress by changing the culture temperature by several degrees Celsius compared to the previous culture, or by gradually reducing the concentration of carbon source and nitrogen source in the medium. Nutrient stress can be applied to lactic acid bacteria. Furthermore, you may set the conditions which raise a citric acid density | concentration and sodium chloride in steps. In the liquid culture in the step (1), the culture in which a plurality of stages of culture are performed to increase the stress intensity to be applied is also referred to as “strain breeding culture”. As a result of applying stress to lactic acid bacteria by “strain breeding culture”, bacterial cells that have induced and expressed the survival ability of the bacteria are selected, and bacterial cells that proliferate vigorously in subsequent solid culture can be obtained.

  The method of “strain breeding culture” is described in detail below.

  In “strain breeding culture”, stress intensity such as undernutrition to cells and changes in culture temperature is set higher while repeating liquid culture in the presence of antibacterial stress-bearing substances such as polyphenols To do. That is, together with carbon source and nitrogen source, etc., polyphenol-containing defatted germ cake obtained by solid culture of Aspergillus oryzae and its extract fraction or defatted germ and dried fruit and tea leaf extract fraction, and poorly soluble calcium carbonate and carbonate When lactic acid bacteria are aerobically cultured with shaking or stirring using a liquid medium characterized by containing magnesium, a plurality of stresses are simultaneously applied to the cells. Furthermore, in the “strain breeding culture”, every time the culture process is repeated from the pre-culture to the pre-culture and the main culture, the temperature of the culture is changed by 3 to 5 ° C. compared to the previous culture and a temperature stress is applied. The nutrient stress that lowers the concentration of the carbon source and nitrogen source in the medium is loaded, and the citric acid concentration is increased stepwise.

  The stress load due to the culture temperature is increased or decreased by 3 to 5 ° C. in comparison to the temperature of the previous culture, and the temperature of the main culture is 3 to 3% higher than the temperature of the preculture. Raise and lower by 5 ° C.

  Stress load due to undernutrition is the concentration of carbon source (glucose) and nitrogen source (High New AM, Fuji Oil Co., Ltd. soybean peptide) in the liquid medium used in the pre-culture, pre-culture, and main culture. (%) Can be achieved by setting a condition that lowers the liquid culture step by step each time the liquid culture is repeated. For example, when lactobacilli such as Lactobacillus rhamnosus GG (ATCC53103) are targeted for cultivation, the glucose concentration of 6.0% by weight of the previous culture / 3.75% by weight of the high-newt AM concentration is 2.5% by the preculture. After passing through% / 1.8% by weight, it may be further reduced to 1.5% / 1.6% by weight in the main culture. In addition, when cultivating lactic acid cocci such as P. acidilactici ATCC25740 strain, pre-culture with a glucose concentration of 2.0% by weight and a high-newt AM concentration of 7.0% by weight. Can be reduced to 1.5% / 4.8% by weight, and in the main culture, 1.0% / 2.5% by weight.

  The concentration of citric acid in the liquid medium is 24 mM in the pre-culture, 37 mM in the pre-culture, and then the main culture in the pre-culture, pre-culture, and main culture. Each time the culture is repeated with 60 mM and a fresh liquid medium, the concentration may be increased by setting a higher concentration. By increasing the citric acid content, lactic acid bacteria reduce glucose consumption and assimilate citric acid.

  The culture temperature in "strain breeding culture", the type and concentration of the carbon source and nitrogen source of the liquid medium, and the concentration of citrate can be appropriately adjusted according to the species and strain used, and the above description is an example It is only what showed.

  The liquid medium used in the “strain breeding culture” in the step (1) is a liquid medium containing at least carbonate, embryo bud, nitrogen source and carbon source. Furthermore, it may contain germ bud extract, yeast dead cells, fatty acids, polysorbates as surfactants, iron, manganese and the like. Examples of fatty acids include animal and vegetable oils and fats containing oleic acid.

  As the “carbonate”, alkaline / slightly soluble carbonates such as calcium carbonate and magnesium carbonate are preferable, and both calcium carbonate and magnesium carbonate are mixed in order to realize an optimum medium pH, and an appropriate blending ratio is used. Is preferably added to the medium. In liquid culture, both light calcium carbonate and heavy calcium carbonate can be used as the carbonate, but light calcium carbonate is preferably used in terms of solubility. When magnesium carbonate is added to the liquid medium alone, the medium pH changes to the alkaline side from the normal medium pH 7.0 to 6.0, which is acceptable to the majority of lactic acid bacteria. It is desirable to increase the amount of carbonate added, but in this case the initial pH increases to the alkali side, so that the stress stress is extremely severe for bacterial species and strains that have poor resistance to alkaline pH. (Alkaline pH stress), and if vigorous growth ability is not imparted to the cells at the seed culture preparation stage, there is a possibility that the cells cannot grow. Furthermore, when saccharides containing glucose and other medium components are mixed and sterilized at the same time, hydrogen peroxide is generated along with the promotion of medium browning (Maillard reaction), thereby inhibiting bacterial growth. Therefore, in the “strain breeding culture” of the step (1) of the present invention, alkaline and sparingly soluble carbonates and germ buds are mixed, separated from other medium components and subjected to high-temperature and high-pressure sterilization treatment, thereby The initial pH can be lowered, and carbonate can be released slowly into the liquid medium, avoiding rapid medium pH fluctuations.

  These alkaline and sparingly soluble carbonates react with these organic acids when the concentration of organic acids such as lactic acid and acetic acid produced by the cells increases as the culture progresses from the logarithmic growth phase to the stationary phase. As a result, it changes to water-soluble salts and carbon dioxide gas is released into the culture solution, increasing the ionic strength of the culture solution. This is an osmotic stress load on the bacteria during the culture process, and as a result of further loading with stimuli such as alkaline pH stress, acidic pH stress, oxidative stress, or temperature stress, the resulting cells have sufficient stress resistance. The stability is further improved.

  In addition, the above-mentioned sustained release process of carbonate also acts to moderately suppress self-cell damage by lactic acid bacteria themselves from a plurality of viewpoints. That is, (A) Carbon dioxide gas is released into the medium, so that the dissolved oxygen in the culture solution decreases. For example, lactic acid bacteria belong to facultative anaerobic bacteria and molecular oxygen cannot be directly used for energy metabolism. Rather, when they come into contact with oxygen, they generate active oxygen such as hydrogen peroxide, superoxide radicals, and hydroxy radicals. Damages the cells. Therefore, due to the effect of reducing the dissolved oxygen in the culture solution, an increase in the concentration of hydrogen peroxide or the like during the culturing process is suppressed, and autologous cell damage can be suppressed. In addition, (B) by performing high-density liquid culture, the ability to produce organic acid is suppressed, and self-cell damage is suppressed. Furthermore, (C) the alkaline / slightly soluble carbonate reacts with an organic acid such as lactic acid produced by lactic acid bacteria, thereby reducing the pH reduction rate of the culture solution. As a result, the production rate of non-dissociated lactic acid is unlikely to increase, and autologous cell damage is suppressed.

  The mixing ratio of the two alkaline / slightly soluble carbonates can be appropriately set according to the optimum pH of the strain, but it is preferable that the magnesium carbonate does not exceed calcium carbonate at least in the addition amount on a weight basis. In addition, when only one of them is used, the obtained effect decreases, which is not preferable.

  The “germ bud” is a cocoon obtained by inoculating a cereal germ with Aspergillus spp. And fermenting it by solid culture. Germ buds contain polyphenols. Plants used as raw materials for “germ lees” can be appropriately selected from grains such as rice, wheat, beans, corn, and defatted germs from which oils and fats have been removed. In view of the problem of fat and oil rancidity, a solid medium composition composed of defatted rice germ, defatted wheat germ, and defatted corn germ, or further defatted soybean is desirable, and 0.5 to 1.0% heavy calcium carbonate may be added. desirable. Aspergillus belonging to the genus Aspergillus that performs solid culture on the surface of defatted germ particles, examples include Aspergillus oryzae, Aspergillus oryzae, Aspergillus sojae, Aspergillus oryzae, and Aspergillus sojae. ), Aspergillus kawachii as the Aspergillus, Aspergillus awamori as Aspergillus, Aspergillus saitoi, Aspergillus glaucus as Aspergillus and Aspergillus glaucus. “Sterilization treatment” performed by mixing heavy calcium carbonate and defatted germ is not particularly limited as long as it is autoclave sterilization (121 ° C., 15 minutes or longer) or a treatment condition for sterilization effect corresponding thereto.

  The “germ bud extract” is an extract of germ bud (50% ethanol fraction, but the extraction method is not limited to this method), and the germ fermentation component of Aspergillus genus belonging to the genus Aspergillus together with the germ bud is the liquid medium of the present invention. Add to. For example, “Rice Germ Ferment Extract-P” from Oriza Oil Chemical Co., Ltd. can be applied.

  “Nitrogen source” and “carbon source” are added as basic energy sources. Non-fat dry milk, soy peptide, meat extract, etc. can be used as the nitrogen source. Polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), High New AM and High New DC6 (manufactured by Fuji Oil Co., Ltd.), Labremuco powder (manufactured by Oxoid Co., Ltd.) and the like can be used, and these can also be mixed and used. The carbon source has different requirements depending on the strain, but one or more kinds can be selected and mixed from glucose, fructose, trehalose, cellobiose, N-acetylglucosamine, mannose, maltose, lactose, sucrose and the like. High Newt is a separated soybean protein breakdown product.

  “Fatty acids” are added as growth promoting factors, and beef fats, fish oils, animal oils and the like can be adopted as “animal and vegetable oils and fats containing oleic acid” in addition to vegetable oils such as olive oil. "Polysorbates as surfactants" are sorbitan fatty acid esters with about 20 molecules of ethylene oxide condensed. Polysorbate 20, polysorbate 60, polysorbate 65, and polysorbate 80 can be used, but unsaturated fatty acids are mainly used. Polysorbate 80 is particularly preferred for emulsifying olive oil by incorporating it into the hydrophobic molecular region of the surfactant, and a combination with polysorbate 20 is preferred when beef tallow is used. As for “iron”, it is preferable to add heme iron as a growth promoting factor of lactic acid cocci such as Lactococcus. In addition, when cultivating bacteria of natural habitat that coexist with other lactic acid bacteria or fungi among lactic acid bacteria species or strains, citric acid, acetic acid, and dry yeast cells, or self-preparation of yeast cells The liquid medium composition supplemented with nutrients such as peptide fraction FYE is appropriately devised. As dry yeast cells, either baker's yeast or beer yeast may be used. In the examples, commercially available Ebios tablets (made by Asahi Food and Healthcare Co., Ltd.) were used, but for example, frozen yeast cells (made by Oriental Yeast Co., Ltd.) There is no problem.

  “Pre-culture” refers to culture in a small amount of medium, which is performed as a pre-stage of expansion culture. In addition, by applying the liquid medium according to the present invention in the “multi-stage culture process”, the effect of improving the properties of the cells can be seen. In other words, in addition to performing the main culture expanded using the liquid culture to increase the density of the bacterial cells according to the present invention, by using the high-density liquid culture also in the preparation of the inoculum to inoculate it, depending on the harsh culture conditions It is presumed that bacterial cells having high resistance to stress are selectively cultured, and the stress resistance of the bacterial cells themselves is improved by environmental normal stress. In multi-stage culture, it is desirable to gradually increase the severity of culture conditions, that is, the degree of stress load, as the culture stage progresses.

  Unlike the general liquid culture method, the strain breeding culture that cultivates lactic acid bacteria while applying various stresses is different from the general liquid culture method in that the liquid medium contains polyphenol-containing insoluble food particles and poorly soluble carbonate as a carbon source, nitrogen source, inorganic Although it is included with the salt and polyphenol-containing food extract fractions, it is a liquid culture system, but the medium environment is different from the homogeneous environment in normal liquid culture and is in a heterogeneous state. .

Each time the liquid culture is repeated, it is viable but non-culturable even though it is alive with active cells that have been induced to express the survival ability of the fungus due to stress due to antibacterial activity of polyphenols or stress such as malnutrition. state: hereafter referred to as “VBNC”) is characterized by the presence of mixed inactive cells in the culture, increasing the types of stress stress in stages and increasing the stress intensity in stages. Setting culture conditions to be high is essential for inducing and expressing the original survival ability of bacteria.
1-2. Solid culture process The solid culture process is a process of culturing the lactic acid bacteria cultured in the liquid culture process (1) in a solid medium which is a solid food mixture, and consists of one process or a plurality of processes. The solid medium used in the solid culture step includes at least carbonate and embryo, and embryo buds and / or polyphenol-containing plants, and the solid medium used in the final step of the solid culture includes polyphenol-containing plants. That is, the solid medium used in the solid culture step includes one or both of embryo buds and polyphenol-containing plants. In addition, the solid medium used in the final step of the solid culture step always includes a polyphenol-containing plant. When the solid culture step is performed in one step, it is sufficient that the polyphenol-containing plant body is included in the step. When the solid culture process is composed of a plurality of steps, the solid medium used in the final process always contains a polyphenol-containing plant, but the solid medium used in the solid culture process in the previous stage also contains a polyphenol-containing plant. May be. However, in this case, the content of the polyphenol-containing plant in the solid medium used in the solid culture process in the subsequent stage is higher than the polyphenol-containing plant in the solid medium used in the solid culture process in the previous stage.

The solid culture step preferably includes a “solid environment adaptation” step and a “lactic acid bacteria solid fermentation” step for growing bacteria having strong resistance to a stress environment. The following two solid culture steps (2-i) and (2- The solid culture process consisting of ii) will be described in detail.
Process (2-i) (Solid environment adaptation process)
The step (2-i) is a solid culture step using a solid medium, and the solid culture of the step (2-i) includes at least a carbonate and an embryo, and further includes an embryo pod and / or a polyphenol-containing plant. Incubate in the prepared solid medium. The content of the solid medium is a substance that can be added to foods. Therefore, the solid medium is referred to as “solid food mixture”. In the solid culture process, lactic acid bacteria grow on the particle surface of the solid medium. In step (2-i), lactic acid bacteria are adapted from a liquid environment to a solid environment. In this respect, the step (2-i) is referred to as an adaptation step from liquid culture to solid culture or a “solid environment adaptation” step. Here, the term “solid environmental adaptation” refers to an environmental adaptation in which new phenotypic expression required for vigorous growth on the solid surface is induced.

  The “carbonate” is preferably an alkaline / slightly soluble carbonate such as calcium carbonate or magnesium carbonate that can be present on the particle surface. Either calcium carbonate or magnesium carbonate may be included, but in order to adjust the pH of the culture environment in solid culture to the optimum pH, both calcium carbonate and magnesium carbonate are mixed at an appropriate mixing ratio to form a solid medium. It is preferable to add. The calcium carbonate used here is preferably heavy calcium carbonate. Here, heavy calcium carbonate means calcium carbonate prepared from natural minerals. For example, heavy calcium carbonate can be obtained by pulverizing and classifying limestone. Heavy calcium carbonate has the characteristics that the particle shape is indefinite and the density is higher than that of light calcium carbonate. On the other hand, light calcium carbonate refers to chemically synthesized calcium carbonate. Carbonates such as calcium carbonate and magnesium carbonate place pH stress on lactic acid bacteria by adjusting the pH of solid culture, particularly the pH of the micro-growth region where lactic acid bacteria grow, to alkalinity.

  As "germ", grain (cereal) germ may be used. Preferably, defatted germs from which fats and oils have been removed are used in order to avoid fat and oil rancidity. Grains are not limited, and examples include rice, wheat, corn, soybeans, barley, oats, rye, straw, straw, oats, and red beans. One or more germs of these cereals, preferably defatted germs are used. In addition, depending on the cereal, it is difficult to obtain only the germ portion, and for such a cereal, a whole grain including the germ may be used. Also when whole grain is used, defatted grain is preferably used. For example, since it is difficult to obtain only germ for soybeans, whole soybeans may be used. Even when whole grains are used, as long as germs are contained in the grains to be used, grain germs may be used in the present invention. For example, defatted soybeans can be said to be defatted grains containing germs. Among the grains, rice, wheat, corn, and soybean are preferable. For example, defatted soybean can be used in addition to defatted rice germ, defatted wheat germ, and defatted corn germ.

  The “germ bud” is a pod obtained by sterilizing a mixture of carbonate and grain germ, inoculating a koji mold belonging to the genus Aspergillus, growing the koji mold by solid culture, and fermenting it. As the germs of the germ buds, the above-mentioned germs can be used, preferably defatted rice germ, defatted wheat germ and defatted corn germ, or defatted rice germ, defatted wheat germ, defatted corn germ and defatted soybean. Since the defatted soybean used here contains germ, it is contained in germ. When producing an embryo pod, the above carbonate may be added, and preferably 0.5 to 1.0% of heavy calcium carbonate is added. That is, “germ bud” includes one or more germs, gonococci belonging to the genus Aspergillus and carbonate. Aspergillus spp. Is a strain belonging to the genus Aspergillus used to produce fermented foods such as sake, miso, vinegar, pickles, soy sauce, shochu, awamori, bonito, etc., with yellow koji, white koji, black koji, katsuobushi Aspergillus oryzae, Aspergillus sojae, Aspergillus kawachii, Aspergillus kawachii, Aspergillus awamori, Aspergillus awamori, Aspergillus awamori, Aspergillus awamori, Aspergillus awamori Aspergillus saitoi), Aspergillus glaucus (Aspergillus glaucus), etc. are mentioned as a bonito fungus.

  Polyphenol-containing plants include fruits (blueberries, prunes, grapes, apples, strawberries, strawberries, bananas, raspberries, mulberries, etc.), vegetables (onions, lettuce, broccoli, kale, lettuce, eggplant, sweet potatoes, burdock, etc.) , Grains (rice, wheat, corn, soybeans, etc .; buckwheat, cacao beans, coffee beans, red beans, peanuts, etc.), tea leaves (green tea, matcha, oolong tea, guava tea leaves, etc.), seaweed (brown algae ), And spices (thin, peppermint, perilla, rosemary, chamomile, turmeric, pepper, olive, etc.), among which fruits such as blueberries, apples, grapes, and matcha are preferred. . These plants may be in an undried raw state or a dried plant, but preferably dry plants are used, such as dry roux berry, dry apple, dry raisins, and matcha tea. The grain germ used in the solid medium of the present invention, the whole grain containing the germ, and the germ pod also contain polyphenol. In the components of the solid medium of the present invention, the grain germ, the whole grain containing the germ, the germ bud Apart from that, polyphenol-containing plants are included. The polyphenol-containing plant body may contain one or more species.

  Polyphenol is defined as a general term for compounds with multiple phenolic hydroxyl groups in the same molecule, and functions as a biological defense substance "Phytoalexin" that protects plants from nature, such as ultraviolet rays, pests and pathogens, and dryness and cold. To do. Compounds belonging to polyphenols are composed of phenylcarboxylic acid, lignan, curcumin, coumarin, and flavonoids. For example, flavonoids with a common structure of diphenylpropane structure include anthocyanidins and their glycosides (anthocyanins). ), Flavanones and their glycosides, flavanols (catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, theaflavin, etc.), isoflavones and their glycosides (daidzein, daidzein glycoside) And flavonols and glycosides thereof (quercetin, quercetin glycosides such as rutin, kaempferol, myricetin, etc.) and flavones (apigenin, luteolin, etc.).

  Polyphenols have antibacterial activity. For example, in the case of hydroxycinnamic acid, polyphenols are lactic acid bacteria such as Lactobacillus brevis, Escherichia coli gram-negative bacteria, Gram-positive bacteria Staphylococcus aureus and Bacillus cereus have been reported to inhibit mold growth and have a broad antibacterial spectrum ranging from bacteria to mold and yeast fungi It is characterized by that. The action mechanism of the antibacterial activity of polyphenol is an ionophore that penetrates the cell membrane of the test bacterium and makes a hole, releasing components such as nucleic acids and proteins from the cytoplasm inside the bacterium, leading to the death of the bacterium. Squeeze or lead to VBNC. When the mechanism of action of an antibiotic is ionophore, it is known that microorganisms are difficult to acquire resistance to the antibiotic. In the present invention, by using a polyphenol-containing plant as a component of a solid medium, a polyphenol whose antibacterial activity is an ionophore acts as a stress on lactic acid bacteria, and a stress due to the polyphenol is loaded. As a result, it is possible to breed lactic acid bacteria having high stress tolerance by inducing and expressing resistance to the polyphenols inherent to the bacteria.

  The solid medium used in the solid culture can be prepared by mixing the above-mentioned solid medium components and adding water so that the water content is 45 to 60% by weight. The water activity of the hydrated solid medium is less than 1.0, preferably 0.95 to 0.98. Water activity can vary depending on the species.

  The content of carbonate, germ, and polyphenol-containing plant that are contained components is 1 to 5% by weight of carbonate, 50 to 90% by weight of germ, and 2 to 20% by weight of polyphenol-containing plant, preferably 3 -10% by weight, more preferably 3-7% by weight, and if germ buds are included, 15-30% by weight germ buds are included. The total of each component becomes 100%. As an example, a solid medium containing 1.6% by weight of magnesium carbonate, 2.66% by weight of heavy calcium carbonate, 67.98% by weight of defatted germ, 22.66% by weight of defatted soybean containing germ, 5.1% by weight of polyphenol-containing plant, or 1.6% by weight of magnesium carbonate %, Heavy calcium carbonate 2.6% by weight, defatted germ 47.68% by weight, defatted soybeans with germs 22.66% by weight, polyphenol-containing plant 5.16% by weight, germ buds 20.3% by weight.

  The carbonate, germ, and polyphenol-containing plant, or further germ buds, which are the components of the solid medium, may be contained uniformly in a granular state. Here, being contained in a granular state means that each component is contained as a granular material having a certain size. For example, the embryo may be contained as a granular material having an average particle diameter of about several tens μm to several thousand μm, preferably about several hundred μm to several thousand μm, and may be used after being pulverized to some extent. It may be included as it is. In addition, when using whole grains containing germs, the whole grains may be used as they are, or granular grains containing germs having an average particle diameter of about several tens to several thousand μm, preferably about several hundreds to several thousand μm. It may be included as a substance. Similarly, germ buds may be included as a granular material having an average particle size of about several tens to several thousand μm, preferably about several hundreds to several thousand μm. In addition, the polyphenol-containing plant may be contained as a granular material having an average particle size of about several tens μm to several thousand μm, preferably about several hundred μm to several thousand μm. What is necessary is just to include, and what is necessary is just to cut or grind | pulverize large things, such as a fruit, and to process and contain as a granular substance with an average particle diameter of about several tens of micrometers to several thousand micrometers, preferably about several hundreds of micrometers to several thousand micrometers. . Although carbonate is powdery, it is hardly soluble, so it exists in a state of adhering to the surface of germ buds and polyphenol-containing plants.

  Containing uniformly means that each component is evenly distributed in the solid medium, and the average particle size of each component does not need to be uniform, and the average particle size varies from component to component. There may be.

  FIG. 1 shows a schematic diagram of a state in which the components of the solid medium are homogeneously mixed. In FIG. 1, lactic acid bacteria are also shown, but are shown larger than the actual size so that the location of the lactic acid bacteria 1 can be seen. The average diameter of lactic acid bacteria is about 1 μm to several μm.

  As shown in FIG. 1, a granular polyphenol-containing plant 2, a granular germ 3, a granular germ pod 4, and a powdered carbonate (not shown in the figure) are in contact with each other, and a gap (pore ) Is formed and air permeability is obtained in the deposited layer. Lactic acid bacteria grow and grow in a state of adhering to the surface of each granular component in the gap, but it is assumed that all water-soluble components contained in the solid medium during medium sterilization could be distributed uniformly among the particles. Is done. Therefore, the component to which lactic acid bacteria adhere and grow is not limited, and it is assumed that it can grow at the same growth rate on any surface of granular germs, germ buds, and polyphenol-containing plants. In a solid medium, each water-soluble component is homogeneously mixed, but the environment that can grow for lactic acid bacteria to be cultivated is a diverse and varied environment. Moreover, since the gap | interval formed between a granular component becomes a passage of air, lactic acid bacteria grow aerobically on the surface of a various granular component.

  The water activity of the solid medium is less than 1.0, and there is little free water in the gap between the granular components, but when added, the nutrients of each component dissolve in the water and spread throughout the solid medium, so it is called a nutrient From a point of view, any surface in a diverse surface environment is relatively uniform. For example, when lactic acid bacteria grow on the surface of defatted rice germ, lactic acid bacteria do not grow depending only on nutrients supplied from defatted rice germ. Nutrients contained in other germs and plants are distributed on the defatted rice germ surface during hydration, so that lactic acid bacteria can grow depending on various nutrients contained in the solid medium. In this respect, it can be said that the solid medium is uniform or nearly uniform from the viewpoint of nutrients required by lactic acid bacteria.

  However, in solid culture, lactic acid bacteria are exposed to an environment having a water activity of less than 1.0, in which water is present in a granular material that contains water. For this reason, although nutrients contained in embryos and polyphenol-containing plants are spread, the incorporation of nutrients into the cells becomes inconvenient, resulting in a low-nutrient environment and being loaded with nutritional stress.

  In the culture in the step (2-i), the lactic acid bacteria obtained by the liquid culture in the step (1) are inoculated into the solid medium as a “liquid species”. For the inoculation, a culture solution containing the proliferated lactic acid bacteria obtained by the liquid culture in the step (1) may be added to the solid medium. The addition may be performed by, for example, mixing the culture solution with a solid medium. At this time, the liquid medium in step (1) may be added in an amount of 1 to 10% by weight, preferably 1 to 5% by weight, more preferably 1 to 2% by weight, based on the solid medium used in step (2-i). . After the addition, it may be mixed well with a spatula or the like to homogenize the distribution of the cells. Preferably, after culturing for several hours, the mixture is further mixed with a spatula or the like to further homogenize the cell distribution.

  The components are mixed well with the above content, and deionized water may be added to a final content of 45 to 60% by weight with respect to the weight of the mixture of water and solid medium components. Then sterilize and use. After cooling, a culture solution containing lactic acid bacteria obtained by liquid culture is inoculated, and solid culture is performed. The culture can be performed at 15 to 42 ° C., but is preferably performed at the same temperature as the liquid culture process. When the culture temperature is 27 ° C. to 42 ° C., the culture is performed for 10 hours to 30 hours, preferably 20 to 30 hours, more preferably 24 hours. The relative humidity (RH) during culture is preferably around 80%. By culturing under these conditions, the fermented bacteria reach a stationary phase. When the culture temperature is lower than 27 ° C., it is necessary to extend the fermentation time as appropriate, but even in that case, the stationary phase is reached within 40 hours.

  The composition of the solid medium and the solid culture conditions can be appropriately changed depending on the bacterial species and strains used.

  In the culture solution of step (1), there are active cells in which the survival ability inherent to the bacteria is induced and expressed by stress loading, and inactive cells induced to VBNC. In the culturing process after the step (2-i), only active cells having the original survival ability of bacteria are selectively and actively proliferated. That is, the cells that grow on the surface of the granular component of the solid medium after step (2-i) are cells that have a property that is difficult to be induced to VBNC by the antibacterial activity of polyphenol.

By solid culture in the step (2-i), 1 billion or more, preferably 2 billion or more, more preferably 5 billion or more, particularly preferably 10 billion or more lactic acid bacteria grow per 1 g of the solid medium. That is, it is possible to perform culture with a high cell density by solid culture.
Step (2-ii)
The step (2-ii) is a solid culture step using a solid medium as in the step (2-i), and the solid culture in the step (2-ii) is at least a carbonate, germ, and polyphenol-containing plant. Culture in solid medium containing body. The solid medium may further contain embryo buds. The carbonate, embryo, embryo bud, and polyphenol-containing plant are the same as in step (2-i), and in step (2-ii), the content of the solid medium is a substance that can be added to food. Therefore, the solid medium is referred to as “solid food mixture”. The state of the solid medium used in the step (2-ii) is the same as that of the solid medium used in the step (2-i), and the granular components are homogeneously mixed. The solid medium used in step (2-ii) is also hydrated so that the final water content is 45 to 60% by weight, and the water activity is less than 1.0, preferably 0.95 to 0.98.

  When the polyphenol-containing plant is contained in the step (2-i), in the step (2-ii), the total content of polyphenols contained in the solid medium is larger than the solid medium used in the step (2-i). Set to. By setting a large total amount of polyphenols, lactic acid bacteria are loaded with a greater stress due to polyphenols than in step (2-i), and cells having higher survival ability are selected.

  For this purpose, the content of the polyphenol-containing plant may be increased. Moreover, since polyphenols are also contained in the germ and the germ pod, the content of the germ and the germ bud may be increased. Preferably, the content of the polyphenol-containing plant is increased.

  Furthermore, in the step (2-ii), sodium chloride may be contained. Sodium chloride has an antibacterial activity at a high concentration. By containing sodium chloride, sodium chloride stresses lactic acid bacteria, and cells having higher survival ability are selected.

  That is, with respect to lactic acid bacteria adapted to solid culture while loading stress in step (2-i), in step (2-ii), further stress is applied and polyphenols contained in a solid medium at a high concentration As a result of environmental adaptation to the stress caused by sodium chloride, the survival ability inherent to the bacteria is further induced and expressed. The process (2-ii) is characterized in that it is a breeding environment that promotes induced expression of survival ability inherent to the fungus according to the stress intensity due to polyphenol added to the growth environment on the solid surface of the component. is there. Step (2-ii) can be referred to as a “lactic acid bacteria solid state fermentation” step.

  The content of carbonate, germ, and polyphenol-containing plant which are contained components is 1-5% by weight of carbonate, 50-90% by weight of germ, 5-30% by weight of polyphenol-containing plant, preferably 10 In the case of containing ~ 30% by weight of embryo pod, it contains 15 ~ 30% by weight of embryo pod. The total of each component becomes 100%. When the polyphenol-containing plant is also included in the step (2-i), the content of the polyphenol-containing plant needs to be larger than that in the step (2-i), and 1.5 in the step (2-i). The content may be double to 10 times, preferably 2 to 8 times.

  Moreover, when sodium chloride is contained in the solid medium of the step (2-ii), it may be added in an amount of 0.15 to 0.60% based on the total weight of the solid medium. Is possible.

  In the culture in the step (2-ii), the lactic acid bacteria obtained by the solid culture in the step (2-i) are inoculated into the solid medium as a “solid species”. Inoculation may be performed by adding a solid medium containing proliferated lactic acid bacteria obtained by solid culture in step (2-i) to the solid medium used in step (2-ii). For example, the addition may be performed by thoroughly mixing the solid medium containing the lactic acid bacteria in step (2-i) with a spatula or the like, adding the mixture to the solid medium used in step (2-ii), and further mixing with a spatula or the like. Good. At this time, the solid medium in step (2-i) may be added in an amount of 1 to 10% by weight, preferably 2 to 5% by weight, based on the solid medium used in step (2-ii).

  The culture may be performed under the same or approximate conditions as in step (2-i).

  In each step of the method of the present invention, the growth of lactic acid bacteria may be confirmed by microscopic observation, or by a specific method according to the bacterial species. For example, the pH of the medium can be measured, and the growth of bacteria can be confirmed using the decrease in pH as an index.

  FIG. 2 shows the features of step (1), step (2-i) and step (2-ii).

The above example is a case where the solid culture process is composed of two processes. When the solid culture process is one process, the solid culture process is cultured in a solid medium containing at least carbonate, germ, and a polyphenol-containing plant. It can be performed under culture conditions that satisfy the condition that embryo buds may be included. For example, the culture may be performed under conditions approximate to the conditions of step (2-i), or may be performed under conditions approximate to the conditions of step (2-ii). In addition, when the solid culture step is performed in three or more steps, each step may be performed in a solid medium containing at least carbonate and embryo, and embryo pods and / or polyphenol-containing plants. May be performed under culture conditions that satisfy the condition that a polyphenol-containing plant is always included. When a polyphenol-containing plant is included in the solid medium used in the multi-step culture process among the plurality of solid culture processes, the content of the polyphenol-containing plant in the solid medium used in the subsequent solid culture process is earlier than that. It is preferable that the content of the polyphenol-containing plant in the solid medium used in the solid culture step is larger. For example, the polyphenol-containing plant may be contained in the subsequent solid culture step by 1.5 to 10 times, preferably 2 to 8 times the previous solid culture step. By performing solid culture in multiple steps under these conditions, as multiple solid culture steps are performed, multiple stresses with gradually increasing strength are loaded, and the survival ability of lactic acid bacteria resistant to those stresses is increased. Can be granted.
2. Lactic acid bacteria obtained by the culture method of the present invention First, liquid culture is performed, followed by solid culture, whereby lactic acid bacteria having high heat resistance and high growth ability can be obtained. The liquid culture may be a one-step “general liquid medium” using a known medium such as an MRS liquid medium, or a “strain breeding culture” in which a multi-step liquid culture is performed while increasing the stress load. However, when “strain breeding culture” is performed, each time the culture is repeated, a higher strength stress is applied compared to the previous culture, thereby obtaining a cell body that induces and expresses the original viability of the fungus. It is done. In the case of using the bacterial cells obtained in this "strain breeding culture", compared to the case of using the bacterial cells obtained by culturing in "general liquid culture", in the solid medium step which is a subsequent step, for example, Lactic acid bacteria having a higher growth ability can be obtained in the solid culture twice in the “solid environmental adaptation” step in the step (2-i) and the “lactic acid bacteria solid fermentation” in the subsequent step (2-ii).

  In solid culture, lactic acid bacteria obtained by liquid culture are adapted to solid culture, and various stresses are applied to the lactic acid bacteria. For this reason, the microbial cell in which the original survival ability of the bacterium is induced and expressed proliferates vigorously.

  Lactic acid bacteria that grow on the surface of a solid fermentation product of lactic acid bacteria obtained by the method of the present invention are characterized by high growth ability and high resistance to various stresses.

  The resistance to various stresses may be determined by culturing under culture conditions loaded with the stress and measuring the proliferation ability.

  The evaluation that the original viability of the bacterium was induced and expressed in the microbial cells contained in the solid fermented lactic acid bacterium obtained by the culture method of the present invention can be judged from the index obtained by the following method. Examples thereof include growth ability, heat resistance, salt resistance, polyphenol resistance, survival rate after freeze-drying treatment, and the like.

  For example, the growth capacity is indicated by the number of bacteria contained per gram of the obtained fermented bacterium solid fermented product, and the upper and lower temperature ranges that are non-permissible temperatures for growth in “general liquid culture”. It can be evaluated by the effect of allowing proliferation in The heat resistance is, for example, the survival rate after heat treatment while shaking the serially diluted solution at the time of measuring the number of bacteria at a water bath temperature of 57 ° C for 15 minutes at a speed of 75 rpm. If it is, it can evaluate by setting 57 degreeC to 60 degreeC, and all can be judged by obtaining a numerical value higher than the survival rate of the microbial cell obtained by "general liquid culture". Similar to heat resistance, the salt tolerance is an index of survival rate after treating the serially diluted solution at the time of counting the number of bacteria in a solution to which a certain concentration of sodium chloride is added for 1 to 2 hours. Polyphenol resistance can be evaluated using the degree of growth when cultured in an MRS liquid medium supplemented with several percent of dried fruits containing a high amount of polyphenol as an index. The survival rate after the freeze-drying treatment can be determined using the change in the number of bacteria before and after the freeze-drying treatment as an index.

By passing through “strain breeding culture”, a bacterial cell having a markedly reduced amount of acetic acid compared to the amount of lactic acid produced can be obtained. When this is subjected to solid culture, the lactic acid bacteria solid fermented product obtained by adapting to the solid environment produces a significantly higher amount of acetic acid compared to the amount of lactic acid produced, and exhibits a phenomenon similar to heterolactic fermentation, Citric acid also has the feature of actively assimilating.
3. Lactic acid bacteria solid fermented product A solid culture containing lactic acid bacteria obtained through the culture process including the liquid culture process and the solid culture process of the present invention is referred to as "lactic acid bacteria solid fermented product".

  The solid fermented lactic acid bacteria obtained by the culturing method of the present invention can be used by thawing a cryopreserved one, and it is completely free of protective agents and stabilizers such as sugar alcohol, starch and sodium glutamate. Even if it is not added, it can be stored as it is by performing air drying or freeze drying at 45 ° C. as it is.

  The fermented product is also a solid food mixture containing lactic acid bacteria. The lactic acid bacteria solid fermented product is a lactic acid bacteria solid fermented product containing lactic acid bacteria on the surface of a solid food mixture containing at least carbonate, germ, and polyphenol-containing plant body, and further contains germ buds and sodium chloride. May be.

  The lactic acid bacteria solid fermented product contains lactic acid bacteria and polyphenols, and further contains lactic acid bacteria exocrine components.

  Lactic acid bacteria are contained at a high density in a lactic acid bacteria solid fermented product, and are 1 billion or more, preferably 2 billion or more, more preferably 5 billion or more, particularly preferably 10 billion, per 1 g of the solid food mixture as a solid medium. One or more lactic acid bacteria are included. The lactic acid bacteria contained in the lactic acid bacteria solid fermented product are lactic acid bacteria having an excellent function that has been induced to express the survival ability of the bacteria by the culture of the present invention, and the cells themselves exhibit excellent health functionality. . In particular, the health functionality when using a strain belonging to probiotics is remarkable.

  For example, it has the following functions. The beneficial effects of lactic acid bacteria on human health include (1) promotion of intestinal peristalsis and prevention and improvement of constipation, (2) improvement of rough skin, (3) improvement of intestinal microflora and suppression of harmful bacterial growth, (4) Prevention of hay fever, (5) Improvement of atopic dermatitis, (6) Suppression of oral caries bacteria, (7) Improvement of immune function, (8) Suppression of increase in blood glucose level, (9) Prevention of hypertension, (10 ) Improvement of diarrhea associated with antibiotic administration, (11) Antibacterial activity against Helicobacter pylori (H. pylori) causing gastric cancer, (12) Improvement of ulcerative colitis, (13) Folic acid And production of health functional substances such as ornithine. In addition, when an appropriate amount is administered, a living microorganism that can be expected to have a health promoting effect on the host is said to be “probiotic”.

  Polyphenol is a polyphenol contained in a polyphenol-containing plant body, embryo, and embryo bud contained in a solid medium, and has excellent health functionality. Since the phenolic hydroxyl group of polyphenol itself is easily oxidized and has a strong chelating power against metals such as iron, any compound belonging to polyphenol exhibits an antioxidant action. Catechin, which is a representative component of polyphenol, is an astringent ingredient contained in, for example, green tea. As its physiological action, (1) antioxidant action, (2) antibacterial action, improvement of intestinal flora, (3) Anti-caries action, (4) Deodorizing action, (5) Reactive oxygen scavenging action, (6) Cholesterol rise inhibitory action, (7) Blood sugar level rise inhibitory action, (8) Blood pressure rise inhibitory action, (9) Antitumor action (10) antiallergic action, (11) platelet aggregation inhibitory action, and (12) ultraviolet ray absorbing action are known. Regarding other components belonging to polyphenols, the intensity of action varies depending on the type of the ingredient, but almost the same action has been reported. On the other hand, when an isoprenoid structural unit (prenyl group) having 5 carbon atoms is bonded to polyphenol, the hydrophobicity increases and the pharmacokinetics also change. As a result, the physiological action that was very weak in the original polyphenol can be dramatically increased. It is known from experience, and in addition to antitumor activity, antibacterial activity, antiviral activity, and antioxidant activity, female hormone / estrogenic activity, immune enhancement activity, anti-inflammatory activity, blood vessel enhancement activity Is mentioned.

  Examples of the exocrine component of lactic acid bacteria include biofilms composed of polysaccharides and glycoproteins. For example, a high molecular weight polysaccharide kefiran secreted and produced by L. kefiranofaciens has a molecular weight of 1 million to 4 million daltons, (1) suppression of blood pressure increase and anti-arteriosclerosis action, (2 Various physiological actions such as () lipid metabolism improving action, (3) blood sugar level increase inhibiting action, (4) intestinal regulating action, (5) intestinal environment improving action, and (6) liver function improving action are known.

  In addition to biofilms secreted and produced by bacterial cells, lactic acid bacteria solid fermented products contain physiologically active substances such as folic acid, ornithine, and vitamins, along with active bacterial cells that induce and express the original survival of bacteria. It also has excellent health functional ingredients.

  This invention also includes the foodstuff containing said lactic-acid-bacteria solid fermented material. The food is an excellent functional food rich in nourishment and rich in lactic acid bacteria, polyphenols, grains and the like. The lactic acid bacterium solid fermented product of the present invention can be eaten as a cereal germ itself or as a cereal food containing abundant cereal grains.

The food containing the lactic acid bacteria solid fermented product of the present invention includes health food, food for specified health use, nutritional functional food, health supplement food and the like. Here, the food for specified health refers to food that is ingested for the purpose of specific health in the diet and displays that the purpose of the health can be expected by the intake.
4). Breeding method Furthermore, the present invention induces and expresses the original survival ability of bacteria by applying stress to lactic acid bacteria and increasing the stress intensity stepwise by a culture method including a liquid culture process and a solid culture process. A method for breeding lactic acid bacteria or a method for obtaining the lactic acid bacteria is also included.

The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.
Example 1 Evaluation of Essential Elements of Effect in Solid Culture Step (2-ii) When “General Liquid Culture” is Used as Liquid Culture in Step (1) 1-1 Step (1) “Liquid Culture”
Lactobacillus rhamnosus (L. rhamnosus) GG (ATCC53103) strain (hereinafter abbreviated as LGG strain) belonging to Lactobacillus, using a MRS liquid medium as a “general liquid culture” aerobically 37 ° C., Shaking culture at 100 rpm was performed for 24 hours. The data of the obtained culture solution is shown in Table 1.

The bacterial count of the cultured strain was measured under the following three conditions:
(1) Bacterial count method-I ⁺
The MRS culture solution was diluted 100 times in phosphate buffer (pH 6.8) supplemented with 0.5% lyophilized blueberry (polyphenol-containing plant) and 3% sodium chloride, and incubated at 45 ° C. for 10 minutes. Subsequently, the bacterial suspension that had been homogenized 15 times was further diluted serially with a phosphate buffer, and then 0.1 ml of the bacterial suspension was smeared onto an MRS agar medium supplemented with 0.003% of the dye bromothymol blue (BTB). The culture was anaerobically cultured for 1 to 3 days, and the number of settlements that appeared was counted.

Incidentally, with respect to cell count assay -I ^+, freeze-dried blueberries was added, subjected to homogenization in the conditions of sodium chloride without addition, the appearance cell count assay the cell count assay for measuring the colonies the number of -I ^- Is displayed.
(2) Bacterial count method-II
Homogenization was carried out without adding both lyophilized blueberry and sodium chloride to the above method for measuring the number of bacteria-I ⁺ , and the number of colonies that appeared was counted.
(3) Bacterial count method-III
The MRS broth obtained by culturing the strain was immediately serially diluted with phosphate buffer.

  In each method of counting the number of bacteria, the heat resistance of the bacteria was also examined. The heat resistance was expressed as the survival rate (%) after heat-treating the serially diluted solution with shaking at a speed of 75 rpm at a hot water bath temperature of 57 ° C. for 15 minutes.

Incidentally, measuring the number of bacteria method -I ⁺ and -I ^-, as well as the number of bacteria of these measurements together with heat resistance and the active cell a substantial amount of the number of bacteria induced to VBNC or bacterial cells during storage of the culture It is a method that can be measured at the same time, and is a method that can evaluate the robustness of the cell membrane of the test bacteria. These methods were originally developed by the present inventors.

Bacterial count methods characterized by stress on the cell membrane by polyphenols contained in blueberries-I ⁺ and -I ^- are similarly measured at 45 ° C for 10 minutes, compared to II, which uses polyphenols in the culture environment. We estimate the effect of promptly returning from the state induced to VBNC due to the stress load on the cell membrane to the active state capable of forming a colony on the agar plate. Furthermore, although the heat resistance by measuring the number of bacteria method -I ⁺ can be evaluated, in the cell count assay -I ^+, since sodium chloride also are added with blueberries may evaluation of salt tolerance. The robustness of the cell membrane can be evaluated by evaluating heat resistance and salt resistance.

Measuring the number of bacteria method -I ⁺ or -I in the present invention ^- while the results obtained in comparison with the results of the cell count assay -II, was analyzed results.

In Table 1, when the measurement method is changed from -III to -II, and further from -II to -I ⁺ , the heat resistance is improved from 5.9% to 11%, and further from 11% to 23%, and there is no load stress. It was suggested that the bacterial cells adapted to the environment in a short time in the number-of-bacteria measurement process as the stress load of the polyphenol and sodium chloride and further heat treatment at 45 ° C was increased as compared with Measurement Method-III. In addition, since the number of bacteria evaluated by the bacteria count method—I ⁺ and the bacteria count method—II was the same, it was suggested that the 37 ° C. shaking culture of the MRS liquid medium did not lead to VBNC.
1-2 Step (2-i) "Solid culture"
Table 2 shows the composition of the solid medium used in the step (2-i). Add 14.2 ml of deionized water (14.2 / (14.2 + 15.5) x 100 = 47.8%) as the amount of water added per 15.5 g of dry weight of the solid medium, and sterilize the growth environment of the particulate matter surface of the solid food mixture Was prepared. In Table 2, "2-i / fruit 0%" refers to solid culture process containing no fruit (polyphenol-containing plant) (2-i), "2-i / fruit 5 ^-" at about 5 fruits This refers to the solid culture in the step (2-i) containing 2% by weight and not including germ buds, and “2-i / fruit 5 ⁺ ” contains about 5% by weight fruits and about 20% by weight germ buds (2- i) refers to solid culture.

In addition, aspergillus oryzae defatted germ moth DG (-) P has a composition of a solid medium used for the preparation, 0.7 wt% heavy calcium carbonate, 45.1 wt% defatted rice germ, defatted wheat germ 38.9 Wt%, defatted corn germ (edible grade) 15.3 wt%, solid-cultured at 30 ° C for 41 hours, and then air-dried at 50 ° C (DG (-) P: Defatted Grain (-) without MgCO _3, Protease rich Koji).

  Antioxidant activity was examined as one of the properties of solid media. Antioxidant activity is performed according to the method for quantifying the total polyphenol content (Toshida Tojiro et al., Journal of the Japan Food Industry Association, 1994, 41 (9): p.611-618.), Ascorbic acid (hereinafter referred to as “ascorbic acid”) The standard curve was prepared by reacting with (Abbreviated asA)), and the measured value was calculated as the equivalent of AsA. In addition to evaluating the antioxidant activity of polyphenols, this measurement method also reacts with antioxidant amino acids such as monosaccharides having a reducing end and tryptophan contained in a solid medium. Therefore, during solid culture, since the microbial cells assimilate these antioxidant nutrients contained in the growth environment, the antioxidant activity of the lactic acid bacteria solid fermented product is reduced through solid fermentation.

As here step (2-i), freeze dried blueberries was added solid media · 2-i / fruit 5 ^- (DG ^(-) P Kojina addition group) to inoculate the culture medium 1.2ml of MRS liquid medium In a thermo-hygrostat set at 39 ° C. and 80% relative humidity, solid culture was performed in the thermo-hygrostat for 22 hours. The medium pH was measured twice in the middle of the solid culture also for the maintenance of the medium. Table 3 shows the evaluation results of the obtained active LGG-containing solid fermented product.

1-3 Step (2-ii) "Solid culture"
After thoroughly mixing the solid medium with a spatula to homogenize the distribution of the particle components, add 15.4 ml of deionized water as the amount of water to add 15.4 ml of deionized water per 15.5 g of the total weight of the solid food mixture (dry matter). A growth environment was prepared.

  Aspergillus oryzae's defatted embryo cocoon UK (UK: Umami Koji) has a solid medium composition (UK-1) used for the preparation of 0.7% heavy calcium carbonate, defatted rice Germ 39.3 wt%, defatted wheat germ 33.0 wt%, defatted corn germ (edible grade) 15.3 wt%, and defatted soybean 11.7 wt%, solid-cultured at 30 ° C, 80% RH for 41 hours, then air dried at 50 ° C This is a cocoon produced by the company. Table 4 shows the composition and antioxidant activity of the solid medium used in step (2-ii).

  Moreover, after melt | dissolving the -20 degreeC frozen preservation | save of the active LGG containing solid fermented product obtained at the process (2-i) as a process (2-ii), the composition which showed 0.90g of solid fermented products in Table 4 The solid medium was inoculated and cultured in a constant temperature and humidity chamber set at 39.5 ° C. and 80% RH for 23 hours.

  The evaluation results of the obtained active LGG-containing solid fermented product are shown in Table 5.

The active LGG-containing solid fermented product obtained by the solid culture in the step (2-ii) has the following effects in the solid food mixture-I to -VI of the present invention.
Effect-1 The degree of decrease in the pH of the medium after 23 hours of solid culture is 1.68 to -2.03, which is larger than the degree of decrease in pH of the solid fermented product in step (2-i) -1.13, and growth on the surface of the food particles It was suggested that there was a possibility that became more prosperous.
Effect-2 Lactic acid production ability was +2.07 to +2.76, which was more activated than +1.24 in step (2-i). The acetic acid production ability was +0.16 to +0.009, which was significantly lower than +0.511 in the step (2-i). On the other hand, the acetic acid-producing ability in the control solid medium in the step (2-ii) was +1.46 to +0.50, and was hardly lowered.

One of the responses when recognizing that bacteria are stressed from the culture environment is that acetic acid production is enhanced compared to lactic acid production, resulting in changes in cell structure in response to harsh environments Therefore, it is necessary to produce more ATP energy necessary for this. On the other hand, when excellent stress resistance is imparted to the bacterial cells, it is assumed that the ATP energy obtained through the normal lactic acid production pathway is sufficient. A decrease in acetic acid production ability and an increase in lactic acid production ability suggest the bacterial state of this stress response.
Comparative Example 1 Influence on Step (2-ii) by Qualitative Difference (Culture in Solid Medium not Containing Polyphenol Plant) after Step (2-i) after “General Liquid Culture” Polyphenol-containing plant After inoculating the solid seed mixture-0% to which no fruit (freeze-dried blueberry) is added with the liquid species obtained in step (1) into solid culture and then performing solid culture, the solid species obtained are treated in step (2-ii). ) And incubating the solid culture, the influence on the step (2-ii) was confirmed. The results are shown in Table 6.

The bacterial count and pH of the active LGG-containing solid fermented product in the step (2-i) were 270 × 10 ⁸ cfu / g and pH 6.15 in Table 3 of Example 1, whereas 18 × 10 ⁸ cfu. / g and pH 6.95, and the growth on the surface of the food particles was insufficient.

In the case of Comparative Example 1 in which the growth was insufficient, the productivity was enhanced in both lactic acid and acetic acid. Therefore, it was suggested that the process (2-i) of Example 1 markedly increased in proliferation due to the addition of blueberries to the solid medium.
1-3 Step (2-ii) "Solid culture"
As the step (2-ii), after thawing the -20 ° C. frozen storage product of the active LGG-containing solid fermentation product obtained in the step (2-i), 0.90 g of the solid fermentation product of the present invention shown in Table 4 was obtained. Solid food mixture-IV was inoculated. After solid culture at 40 ° C. and 80% RH for 4 hours, the culture was continued in a thermo-hygrostat set at 80% RH for 24 hours at 39 ° C. The evaluation results of the obtained active LGG-containing solid fermented product are shown in Table 7.

  The result of the step (2-ii) shown in Table 7 is data obtained in the same composition as the result of the solid food mixture-IV of the present invention in Example 1 Table 5, and by comparing both the results. The influence on the step (2-ii) due to the qualitative difference in the step (2-i) could be analyzed.

That is, the bacterial count and pH of the active LGG-containing solid fermented product in step (2-ii) are 260 × 10 ⁸ cfu / g in Example 1 Table 5 (solid medium IV of the present invention) in the method of measuring bacterial cells-II. In Comparative Example 1, it was 130 × 10 ⁸ cfu / g and pH 5.94. Thus, since the influence by the qualitative difference of the solid species in the step (2-i) was expressed in the step (2-ii), in order to obtain a sufficient effect of the present invention, in the step (2-i) Conditions that made growth more vigorous were necessary.
Example 2 Inducible expression of survival ability inherent in bacteria in step (2-ii) by “strain breeding culture” In step (1), instead of using MRS liquid culture, which is a general liquid culture, “strain breeding” The influence on the step (2-ii) when using the liquid species obtained by liquid culture by “culture” was confirmed.
2-1 Process (1) “Liquid culture”
In accordance with the liquid culture method previously developed by the present inventors (referred to as “strain breeding culture”), the LGG strain was pre-cultured, pre-cultured, and main-cultured in the three liquid medium compositions shown in Table 8. Each culture step was performed in this order. Table 8 shows the composition of these liquid media. In addition, inoculate 10 ml of 1% light calcium carbonate-added MRS liquid medium with one platinum loop of a bacterial colony formed by overnight culture at 37 ° C in MRS agar medium supplemented with 0.003% BTB, and perform static culture at 37 ° C overnight. The culture was started in advance using 0.15 ml of the obtained culture solution.

Preculture was performed by inoculating 1% of the culture solution obtained in the previous culture, and further inoculating 1% of the culture solution obtained in the preculture. Each culture step was performed for 24 hours, and the culture solution was evaluated.
Table 9 shows the evaluation results.

  The heat resistance of the bacteria obtained from the pre-culture to the pre-culture was low. However, as a result of further increasing the stress applied to the cells each time the culture is repeated for the main culture, the main culture solution obtained by “strain breeding culture” (1) improves the heat resistance of the culture to 28%. In addition to the effects found, (2) the degree of decrease in the medium pH is smaller, (3) the amount of lactic acid produced is decreased, the amount of acetic acid produced is significantly increased, and (4) the ability to assimilate citrate is remarkably developed. The feature that was done was recognized.

The phenotypic change when the cells adapt to the stress environment of the liquid culture is to secure new ATP energy for the change in the cell structure necessary for the adaptation, for example, acetic acid production while suppressing lactic acid production Increase the amount. Also in this example, the ability to produce acetic acid is remarkably enhanced, which is understood as proof that the bacterium has tried to adapt to the environment. Similarly, since ATP energy is also produced in the citric acid metabolism process, citrate assimilation ability is enhanced, so that ATP energy necessary for imparting excellent survival ability can be generated abundantly and consumption of ATP energy. It becomes possible to respond to the amount increase. In addition, when the increased acetic acid-producing ability is reduced in the subsequent solid culture step (2-ii), it is understood that the cell body has acquired an excellent survival ability, and other metabolic pathways that are not linked to organic acid production are considered. The possibility of induced expression is considered.
2-2 Step (2-i) "Solid culture"
Step a (2-i), a polyphenol-containing plant lyophilized blueberries 5.2 wt% and defatted germ koji DG (-) solid medium and the P contained 20.3 wt% · 2-i / fruits 5 ⁺ to step (1 Inoculated with 1.2 ml of the main culture solution obtained in (1) above, and solid culture was performed for 24 hours in a thermo-hygrostat set at 39 ° C. and 80% RH.

Furthermore, in order to examine the influence of by qualitative difference in step (1) to the step (2-i), solid medium · 2-i / fruit 5 ^- also inoculated with the culture liquid to a solid under the same conditions Culture was performed. Table 10 and Table 11 show the evaluation results of the active LGG-containing solid culture in the raw state (the state before lyophilization) obtained by measuring the medium pH twice in the middle of the culture and also for the maintenance of the solid medium. .

Example 2 The result of step (2-i) shown in Table 11 is data obtained using a solid food mixture having the same composition as the result of step (2-i) shown in Example 1 Table 3. By comparing the two, the influence on the step (2-i) due to the qualitative difference of the liquid species in the step (1) could be analyzed as in the following (A) and (B).
(A) When inoculating a solid food mixture with the liquid species obtained in “Breed Breeding Culture”, start culture compared to solid culture inoculated with the “general liquid culture” liquid species After 2.5 hours, the pH of the medium reached -0.26, and the pH at 6.5 hours of culture reached almost the same level as at 22 hours of culture. Moreover, the difference in pH at the end of the culture was significantly different from -0.17, and the solid culture that passed through the “strain breeding culture” reached a lower pH. Therefore, when the bacterial cells obtained by the “strain breeding culture” are inoculated into the solid food material mixture in the step (2-i), the induction period until the resumption of growth is almost zero, and the solid surface thereafter Since the growth in was very vigorous, the stationary phase was reached with a culture time of 6.5 hours. It was suggested that the provision of vigorous growth ability on the surface of food particles was one of the properties that could be modified by “strain breeding culture”. By the way, when the concentration of sodium citrate in the liquid medium is set higher, depending on the concentration, the glucose consumption of the medium is decreased, the lactic acid production is decreased, the acetic acid production is increased, the bacteria per glucose consumption Lactococcus lactis CRL264 strain has been reported to increase body mass and increase in the amount of bacterial cells per ATP production (Sanchez C. et al., Applied and Environmental Microbiology, 2008 Feb., Vol. 74) (No. 4): p.1136-1144.). Therefore, it may be possible that the above-described results have been obtained by sufficiently learning the activity of assimilating citric acid contained in the liquid medium and vigorously metabolizing in step (1).
(B) The amount of citric acid assimilated in the solid culture in the step (2-i) that has undergone the “strain breeding culture” is equivalent to that in the solid culture in the step (2-i) that has undergone the “general liquid culture” However, it exhibited acetic acid production corresponding to 49% of lactic acid production, and acetic acid production was slightly increased compared to 41% in the case of “general liquid culture”. Therefore, it was suggested that the cells that had undergone the “strain breeding culture” produced more ATP energy necessary for adaptation in the solid culture in the step (2-i), and as a result, the structure could be changed more sufficiently. It was done.
2-3 Step (2-ii) "Solid culture"
As a step (2-ii), after thawing a -20 ° C. cryopreserved product of the active LGG-containing solid fermentation product of the step (2-i) obtained using the solid food mixture · 2-i / fruit 5 ⁺ , 0.90 g of the solid fermented product was inoculated into the solid food mixture-IV and -VII of the present invention shown in Table 4. After solid culture at 40 ° C. for 4 hours, the culture was continued in a thermo-hygrostat set at 80% RH for 24 hours at 39 ° C. Table 12 shows the evaluation results of the obtained active LGG-containing solid culture.

Example 2 When the active LGG solid fermented product described in Table 12 was compared with the solid fermented product described in Example 1 Table 5, all the steps (2-i) (Example 1, solid food mixture · 2-i / fruits 5 ^-; example 2, since the set to substantially the same culture conditions in solid food mixture · 2-i / fruits 5 ⁺⁾ and step (2-ii) (-IV both solid food mixture), the step (1 ) The influence on the step (2-ii) exerted by the difference in () can be analyzed as in the following (A) and (B).
(A) The solid culture in the step (2-ii) that has undergone the “strain breeding culture” is lower in the pH of the solid food mixture than the solid culture in the step (2-ii) that has undergone the “general liquid culture”. The rate, the number of bacteria reached, heat resistance, changes in the amount of lactic acid and acetic acid produced by solid fermentation, and the ability to produce lactic acid and acetic acid were all at the same level.
(B) However, as in the case of the solid fermented product obtained in the step (2-i) after the “strain breeding culture”, the citric acid was assimilated in the step (2-ii) when the “strain breeding culture” was passed. It was suggested that these specific changes may be related to the effect of induced expression of the original survival ability of bacteria (details are shown in Example 3).

FIG. 3 shows the number of bacteria and heat resistance of the cells obtained by culturing in each culture step. In FIG. 3, the table | surface (FIG. 3A) which shows the culture conditions of each culture | cultivation process (test group) 1-5, and the number of bacteria and a graph of heat resistance (FIG. 3B) are shown. FIG. 3A also shows a table number including data of each condition. Test plots 3 and 4 are test plots that satisfy the culture conditions of the present invention. If the culture medium of step (2-i) and step (2-ii) of the present invention is used, the number of bacteria is remarkably high, and the heat resistance is other. Higher than the test area.
Example 3 Lyophilization of active LGG solid fermented product and storage stability evaluation Evaluation of the lactic acid bacteria solid fermented product in the step (2-i) or the step (2-ii) was performed.
3-1 Freeze-drying Shelf-type freeze-dryer FD-550P (Tokyo Rika Co., Ltd.) without any protective agents or stabilizers added to the solid fermented product containing active LGG, as it is after solid culture. The lyophilization was carried out for 24 hours using an instrument). In general, when freeze-drying, spray-drying, or cryopreserving bacterial cells of lactic acid bacteria obtained by liquid culture, the cells are suspended with a protective agent or stabilizer with an appropriate composition before starting these treatments. This is essential, and the original composition of protective agents and stabilizers is an important know-how for manufacturers of lactic acid bacteria.

  On the other hand, the lactic acid bacteria solid fermented product obtained by the method of the present invention, in which the lactic acid bacteria secrete and produce polysaccharides and glycoproteins called biofilms on the particle surface of the solid food mixture, the solid food mixture contains polyphenol. Since it contained a considerable amount and had high antioxidant activity, it seemed that a protective agent and a stabilizer were unnecessary at the time of freeze-drying and freezing preservation of a solid fermented product. Therefore, Table 13 shows the results obtained by lyophilization without adding any protective agent or stabilizer to the LGG activity-containing solid fermented product.

  FIG. 4 shows the number of surviving bacteria and the heat resistance after lyophilization of the cells obtained by culturing in each culture step. In FIG. 4, the table | surface (FIG. 4A) which shows the culture conditions of each culture | cultivation process (test group) 1-5, and the graph (FIG. 4B) of survival cell count and heat resistance are shown. High survival cell count and heat resistance after lyophilization were obtained in test sections 3 and 4. In particular, it was good in test section 4. This result shows that in the solid culture step (step (2-i) and step (2-ii)), there is an effect of the presence of the polyphenol-containing plant. It is preferable that it is contained in both of 2-ii). Moreover, it shows that it is preferable to perform "strain breeding culture" in the liquid culture step.

As a result of freeze-drying, the step (2-ii) (both according to the present invention) due to the qualitative difference between the “strain breeding culture” (Example 2) and the “general liquid culture” (Example 1) in the step (1) The impact on the solid food mixture-IV) is that the number of surviving bacteria is 220 x 10 ⁸ cfu / g and heat resistance is 26% when the strain is bred and cultured, and the result is that when the general liquid culture is passed. The number of remaining bacteria was 97 × 10 ⁸ cfu / g, and the heat resistance was 14%. That is, as an effect obtained through the “strain breeding culture” in the step (1), active cells in which the survival ability inherent to the bacteria was induced and expressed in the solid fermentation of the step (2-ii) were obtained with a higher number of bacteria. .

Furthermore, the effect of obtaining a solid fermented lactic acid bacterium containing a high number of active cells inducing and expressing the original survival ability of the bacterium of the present invention is the polyphenol contained in the solid food mixture in the step (2-i). The importance of inoculating step (2-ii) with a solid species imparted with resistance to stress by lyophilization was suggested from the results of freeze-drying. That is, when the solid medium in the step (2-i) does not contain a polyphenol plant, the solid fermentation product of the step (2-ii) and the solid food mixture-IV shown in Table 7 of Comparative Example 1 is freeze-dried. As a result, as shown in Table 13, the number of surviving bacteria was 59 × 10 ⁸ cfu / g and the heat resistance was as low as 2.0%. On the other hand, when the solid medium in the step (2-i) contains a polyphenol-containing plant, the solid fermentation product of the step (2-ii) and the solid food mixture-IV shown in Table 13 is freeze-dried. As a result, the number of surviving bacteria increased to 97 × 10 ⁸ cfu / g and heat resistance 14%. From this result, in order to obtain a lactic acid bacteria solid fermented product containing a high number of active cells inducing and expressing the original survival ability of the bacteria in the step (2-ii), the solid medium of the step (2-i) It was suggested that increasing the polyphenol content of the rice by adding dried fruits and / or germ buds is an essential element.

On the other hand, in the method for measuring the number of bacteria in Table 13-I ⁺ , the number of surviving bacteria in the solid fermentation product using the solid food mixture-VI of the present invention was 90 × 10 ⁸ cfu / g. Compared to 200 × 10 ⁸ cfu / g, it was halved. This phenomenon is due to the fact that, when homogenizing in the bacterial count method-I ⁺ , the high concentration polyphenols in the solid fermentation product are combined with the antibacterial activity of blueberry and sodium chloride in the bacterial count evaluation system. It may have been caused by being killed.
3-2 Storage stability of freeze-dried product Freezing of solid fermented material containing active LGG using I ⁺ , a bacterial count method that can evaluate membrane robustness due to damage to cell membranes caused by polyphenols and sodium chloride in the evaluation system The number of bacteria and heat resistance after storing the dried product at 4 ° C. for 42 days were evaluated. The obtained results are shown in Table 14A and Comparative Example 14B.

As a result, in Table 14A, even after the freeze-dried solid fermented product was stored at 4 ° C. for 42 days, the high survival cell count and heat resistance were maintained at substantially the same level, and the cells were significantly destabilized. Compared with Table 14B, an effect of inducing and expressing the original survival ability of the fungus was shown.
Example 4 Commonality of Effects Obtained by the Present Invention when Lactobacillus is Changed to Lactococcus 4-1 Step (1) ("General Liquid Culture" and "Strain Breeding Culture") Using PA Strain
Using Pediococcus acidilactici (ATCC25740) strain (hereinafter abbreviated as PA strain) belonging to lactic acid cocci, the culture temperature is stepped using MRS liquid medium as "general liquid culture" In addition to the above, the liquid culture method previously developed by the present inventors described in Example 2 was used as a “strain breeding culture”, and the culture was pre-cultured and pre-cultured with 33 modified liquid medium composition specifications. And each culture | cultivation process of main culture was performed in this order. The composition of the liquid medium is shown in Table 15. Inoculate 10 ml of 1% light calcium carbonate-added MRS liquid medium with 37 ml of MRS agar medium supplemented with 0.003% BTB and incubate overnight at 37 ° C. The culture was started in advance using 0.15 ml of the obtained culture solution.

  Preculture was performed by inoculating 1% of the culture solution obtained by the pre-culture, and further, main culture was performed by inoculating 1% of the culture solution obtained by the preculture. Each culture step was performed for 24 hours, and the culture solution was evaluated. The evaluation results of the obtained culture solution are shown in Table 16A and Table 16B.

PA strain liquid medium No.1 is the result of simultaneous loading of at least three stress factors, "Cultural breeding culture", in which the culture temperature is increased stepwise, the concentration of carbohydrates and nitrogen sources is decreased, and the concentration of citric acid is increased. In the main culture, proliferation was remarkably suppressed. In the case of the medium No. 2 in which the stress element of the increase in citric acid concentration was not applied as compared with the liquid medium No. 1, the medium pH was changed in the same manner as the response in the “strain breeding culture” of the LGG strain in Example 2. The degree of reduction was very small and the amount of lactic acid produced decreased. However, contrary to the LGG strain, the amount of acetic acid produced was significantly reduced, and the ability to assimilate citrate was not induced. On the other hand, in the case of the liquid medium No. 3 in which the stress of reducing the concentration of the saccharide and nitrogen source was not applied as compared with the liquid medium No. 1, the response of the LGG strain in Example 2 to “strain breeding culture” Conversely, the number of reached bacteria increased slightly, the decrease in acetic acid production was small, and citrate assimilation ability was not induced and expressed. As described above, in the “strain breeding culture” in the step (1), the PA strain and the LGG strain exhibited significantly different responses.
4-2 Step (2-i) “solid culture” using PA strain
As the step (2-i), the solid food mixture having the composition shown in Table 17 was inoculated with 1.2 ml of the culture solution obtained in the main culture of the step (1) and set at 42 ° C. and 80% RH. The solid culture was performed in the vessel for 24 hours. During the solid culture, the pH of the solid medium was measured twice for maintenance. Table 18 shows the evaluation results of the obtained active PA-containing solid fermented product.

  The active PA-containing solid fermented product obtained in the step (2-i), 2-i (A), was proliferated in the main culture medium No. 1 of “Strain breeding culture”, although it was greatly suppressed. From the results of the evaluation of pH and the number of bacteria reached, it was suggested that the growth delay was recovered by 8 hours after the culture and reached the same level as other culture conditions by 24 hours after the culture.

The active PA-containing solid fermented product obtained in the step (2-i) has a higher production amount of acetic acid and a smaller amount of citric acid than the amount of lactic acid, but is secreted and produced. It was different from the solid fermented material containing active LGG obtained in (1). The solid fermented product 2-i (C) of the step (2-i) of the PA strain which has undergone No. 3 of the culture conditions in which the citric acid concentration of the liquid medium is increased in the step (1) has a slight decrease in heat resistance. .
4-3 Step (2-ii) “solid culture” using PA strain
As a step (2-ii), after thawing the -20 ° C. frozen storage product of the active PA-containing solid fermentation product obtained in the step (2-i), 0.90 g of the solid fermentation product was obtained as a solid having the composition shown in Table 19 Medium 2-ii sodium chloride was inoculated into 0 to 1.2% by weight. Solid culture was performed in a thermo-hygrostat set at 80% RH for 24 hours at 42 ° C. The evaluation results of the obtained active PA-containing solid fermented product in the raw state (the state before drying) are shown in Table 20A and Table 20B.

The solid fermented product of the PA strain obtained in the step (2-ii) had a lower number of bacteria compared to the results described in Table 12 of Example 2 obtained using the LGG strain. It was significantly higher. It should be noted that the active PA-containing solid fermented product obtained in step (2-ii) has a significantly enhanced lactic acid-producing ability compared to the solid fermented product obtained in step (2-i) of the PA strain. On the other hand, the ability to produce acetic acid was almost halved, and like the LGG strain, the fermentation scene was similar in effect to malolactic lactic acid fermentation.
Example 5 Effect of step (1) on blow drying of active PA-containing solid fermented product obtained in step (2-ii) and survival ability of dried cells Cells obtained in step (2-ii) The PA-containing solid fermented product was thinly spread on a filter paper, and air-dried at 45 ° C. for 6 hours in an industrial thermostat VTFH-216-2T (manufactured by Isuzu Seisakusho). In addition to evaluating the number of surviving bacteria and heat resistance of the obtained dried product with the method of measuring the number of bacteria-II, put the dried product in a sealed glass bottle, and after 18 days at 4 ° C, similarly, the number of surviving bacteria and heat resistance Sex was evaluated. At that time, the value obtained by dividing the number of surviving bacteria by the number of bacteria obtained immediately after culturing shown in Table 20A and multiplying by the heat resistance (%) is “survival rate × heat resistance ( %) ”.

  In addition, the reason expressed by “survival rate × heat resistance (%)” is that PA strain originally showed excellent resistance to blast drying unlike LGG strain, and also has excellent heat resistance. This is because it becomes a scale that can evaluate the state in which the survival ability inherent to the bacteria is induced and expressed with higher sensitivity. In other words, if it was not killed at all by air drying, a survival rate of 100% was obtained, and if the cells dried by air were firmly maintained by air drying, the viable state (before drying) Heat resistance of the same value as the cells in the state) is obtained. Therefore, the higher the value of “survival rate × heat resistance (%)”, the more stable the cell membrane, and the stabilization effect of the state in which the survival ability inherent to the bacteria is induced and expressed is assumed.

  The results obtained are shown in Table 21. Solid fermentation product 2 of step (2-i) obtained by inoculating the liquid species of medium No. 1 in which growth stress was suppressed due to excessive stress in the main culture of “strain breeding culture” in step (1) -As a result of performing solid culture in step (2-ii) using solid (i) as a solid species, if there is no sodium chloride added to the solid food mixture, in step (1) "general liquid" The results similar to those of the solid fermented product 2-i MRS inoculated with the liquid species obtained in “Culture”, that is, “survival rate × heat resistance (%)” are immediately after air drying and the dried product at 4 ° C. for 18 days. Both after storage, 21-34% and 11-12% were both low. However, in the solid obtained by adding 0.3% by weight of sodium chloride in terms of dry matter to the solid food mixture of the present invention in the step (2-ii), the solid fermented product 2-i (A) in the step (2-i) is used as a solid species. Inoculated solid fermented product 2-ii “survival rate × heat resistance (%)” of 0.3% by weight of sodium chloride is 78% both immediately after blow drying and after storing the dried product at 4 ° C. for 18 days and It was suggested that a solid fermented product in a state in which the survival ability inherent to the bacteria was induced and expressed was obtained, which was as high as 32%.

  On the other hand, in the “strain breeding culture” of the step (1), the liquid medium No. 1 loaded with both stresses that gradually increase the malnutrition and the culture temperature. When the solid culture of step (2-ii) was performed using the solid fermented product 2-i (B) of step (2-i) obtained by inoculating the liquid species of 2 as a solid species, sodium chloride in terms of dry matter The solid fermented product grown on a solid medium supplemented with 1.2% by weight of 2-ii sodium chloride “survival rate × heat resistance (%)” of 1.2% by weight is obtained immediately after blow drying and at 4 ° C., 18% It was as high as 42% and 35% after both days of storage.

  The above results show that in the “strain breeding culture” of the step (1), the stress of the stress is very strong, and the bacterial cells that have undergone the stress load to the extent that growth is suppressed in the main culture are the solid culture process (2- In ii), even if the load intensity of stress by sodium chloride was slightly low, the survival ability inherent to the bacteria could be induced and expressed. That is, it is assumed that the sensor for response to load stress may have changed to a “sensitive type”.

  On the other hand, in the “strain breeding culture” of the step (1), when inoculating the liquid species of the liquid medium No. 2 from which the stepwise increase of the citric acid concentration is removed from the above stress, the step (2-i) In the solid culture in the step (2-ii) after passing through the above, it was suggested that when the stress intensity with a high sodium chloride concentration was set higher, the inherent survival ability of the fungus could be induced and expressed. That is, it was estimated that the sensor for response to load stress may have changed to a “normal type” that is slightly inferior to the above.

  On the other hand, in the “strain breeding culture” of the step (1), the step (2-i) obtained by inoculating the microbial cells through the condition in which the malnutrition stress is deleted from the above three types of load stress. ) Solid fermented product 2-i (C) obtained by culturing in a solid medium supplemented with 1.2% by weight of sodium chloride 2-ii sodium chloride 1.2% by weight “survival rate × heat resistance (%)” ”Was as high as 43% just after drying, but decreased to 23% after the dried product was stored at 4 ° C. for 18 days. It was higher than 11-12% of solid fermented product 2-ii of 0% by weight of sodium chloride. This finding shows that in the liquid culture of step (1), as many kinds of stress as possible are applied and their resistance is induced and expressed, so that even when solid culture is passed twice, excellent survival ability is induced. This suggests that it is important for obtaining a solid fermented product in an expressed state.

  FIG. 5 shows the “survival rate × heat resistance” (%) of the cells obtained by culturing in each culturing step after air drying. In FIG. 5, the table | surface (FIG. 5A) which shows the culture conditions of each culture process (test group) 1-5, and the graph (FIG. 5B) of "survival rate x heat resistance" (%) are shown. “Survival rate × heat resistance” (%) indicates a value after drying by blowing and a value after storage at 4 ° C. for 18 days.

  The difference between the test groups 1 to 5 is the sodium chloride concentration in the solid culture step (2-ii) and the culture method (general liquid culture or strain breeding culture) in the liquid culture step (1). The results of FIG. 5 show that the survival rate x heat resistance (%) was better when the strain breeding culture was performed as the liquid culture process and sodium chloride was added in the solid culture process (2-ii). It shows that.

  Lactic acid bacteria grown by liquid culture are cultured using a solid food mixture containing an appropriate amount of solid foods as a solid medium, thereby inducing and expressing the original survival of the bacteria and containing high-density cells A solid fermented product can be obtained, and a solid fermented product obtained by a drying process such as freeze-drying or hot air can also be provided.

DESCRIPTION OF SYMBOLS 1 Lactic acid bacteria 2 Granular polyphenol containing plant 3 Granular germ 4 Granular germ bud All the publications referred by this specification, a patent, and a patent application shall be integrated in this specification as it is.

Claims (30)

A method for producing a lactic acid bacteria solid fermentation product,
(1) A liquid culture process for culturing lactic acid bacteria using a liquid medium,
(2) A solid culture step consisting of one step or a plurality of steps in which the lactic acid bacteria cultured in step (1) is cultured in a solid medium which is a solid food mixture, wherein the solid medium comprises at least carbonate and germ, A solid culture step for growing lactic acid bacteria on the surface of the solid medium, comprising a polyphenol-containing plant and the solid medium used in the final step of the solid culture comprises a polyphenol-containing plant;
The lactic acid bacteria solid fermentation product is a lactic acid bacteria solid fermentation product in which the lactic acid bacteria are contained on the surface of the solid medium used in step (2).   The solid culture step of step (2) is performed in a plurality of steps, and the content of the polyphenol-containing plant in the solid medium used in the subsequent solid culture step is the polyphenol content in the solid medium used in the previous solid culture step The manufacturing method of Claim 1 with more content of a plant body.   As the liquid culture in the step (1) and the solid culture step in the step (2) are performed, a plurality of stresses whose strength is increased stepwise is loaded, and the survival rate of the lactic acid bacteria having resistance to those stresses is increased. The manufacturing method of Claim 1 or 2. A method for producing a lactic acid bacteria solid fermentation product,
(1) A liquid culture process for culturing lactic acid bacteria using a liquid medium,
(2-i) The lactic acid bacteria cultured in the step (1) are cultured in a solid medium that is a solid food mixture containing at least carbonate and germ, and germ buds and / or polyphenol-containing plants, and the lactic acid bacteria are cultured on the surface of the solid medium. Proliferating,
(2-ii) The lactic acid bacteria cultured in the step (2-i) is a solid medium that is a solid food mixture containing at least carbonate, germ, and a polyphenol-containing plant, and the content of the polyphenol-containing plant is the step ( Culturing in a solid medium more than the solid medium used in 2-i) and growing lactic acid bacteria on the surface of the solid medium;
The method according to claim 2, wherein the lactic acid bacteria solid fermented product is a lactic acid bacteria solid fermented product containing the lactic acid bacteria on the surface of the solid medium used in step (2-ii).   The production method according to claim 4, wherein in the step (2-ii), the germ medium is further contained in the solid medium.   As the steps (1), (2-i), and (2-ii) are performed, a plurality of stresses with gradually increasing strength are loaded, and the survival rate of lactic acid bacteria having resistance to those stresses is increased. The manufacturing method according to claim 4 or 5.   The production method according to any one of claims 1 to 6, wherein the liquid medium used in the step (1) is a liquid medium containing at least carbonate, embryo bud, nitrogen source and carbon source.   The liquid culture process of step (1) is performed in a multi-stage culture process, and in any one of the multi-stage culture processes or each of the multi-stage culture processes, at least carbonate, germ bud, nitrogen The manufacturing method of any one of Claims 1-7 using the liquid culture medium containing a source and a carbon source.   The production method according to claim 7 or 8, wherein the germ bud contained in the liquid medium used in step (1) comprises one or more of defatted rice germ, defatted wheat germ, and defatted corn germ.   The production method according to claim 7 or 8, wherein the carbonate contained in the liquid medium used in step (1) is a mixture of calcium carbonate and magnesium carbonate.   The production method according to any one of claims 1 to 10, wherein the germ contained in the solid medium used in the solid culture step is a defatted cereal including one or more defatted germs or germs.   The germ is one or two selected from the group consisting of defatted rice germ, defatted wheat germ, defatted corn germ, defatted germ derived from other cereals, and defatted cereals including other cereal germs. The manufacturing method as described.   The production method according to claim 11 or 12, wherein the defatted cereal containing the germ is defatted soybean.   The polyphenol-containing plant contained in the solid medium used in the solid culture step is one or more kinds of plants selected from the group consisting of fruits, vegetables, cereals, tea leaves, seaweed and spices. The production method according to claim 1.   The production method according to any one of claims 1 to 14, wherein the germ pod contained in the solid medium used in the solid culture step is a defatted germ pod of Aspergillus belonging to the genus Aspergillus.   The manufacturing method according to any one of claims 4 to 15, wherein the solid medium used in the step (2-ii) further contains sodium chloride.   The production method according to any one of claims 1 to 16, wherein the carbonate contained in the solid medium used in the solid culture step is a mixture of calcium carbonate and magnesium carbonate.   The production according to any one of claims 1 to 17, wherein the solid medium used in the solid culturing step is a mixed solid medium of carbonate and embryo, and a germ mixture and / or a homogenous mixture of polyphenol-containing plants and water. Method.   The manufacturing method of any one of Claims 1-18 which inoculates the surface of the solid culture medium used at a process (2) the lactic acid bacteria obtained by the liquid culture of a process (1).   The lactic acid bacteria obtained by the liquid culture in the step (1) are inoculated on the surface of the solid medium used in the step (2-i), and further the lactic acid bacteria obtained by the solid culture in the step (2-i) The manufacturing method of any one of Claims 4-19 inoculated on the surface of the solid culture medium used at a process (2-ii).   A lactic acid bacteria solid fermented product in which fermenting bacteria are contained on the surface of a solid food mixture containing at least carbonate, embryo, and polyphenol-containing plant.   Furthermore, the lactic-acid-bacteria solid fermented material of Claim 21 in which a solid foodstuff mixture contains an embryo bud.   The lactic acid bacterium solid fermented product according to claim 21 or 22, wherein the germ is a defatted cereal including one or more kinds of defatted germ or germ.   The lactic acid bacterium according to claim 23, wherein the germ is one or two selected from defatted rice germ, defatted wheat germ, defatted corn germ, defatted germ derived from other cereals and other cereal germs. Solid fermented product.   The lactic acid bacterium solid fermented product according to any one of claims 21 to 24, wherein the defatted cereal containing germ is defatted soybean.   The lactic acid bacterium solid fermented product according to any one of claims 21 to 25, wherein the polyphenol-containing plant is one or more plants selected from the group consisting of fruits, vegetables, cereals, tea leaves, seaweeds and spices. .   27. The lactic acid bacterium solid fermented product according to any one of claims 21 to 26, wherein the germ bud is a defatted germ bud of Aspergillus belonging to the genus Aspergillus.   The solid fermented lactic acid bacterium according to any one of claims 21 to 27, wherein the solid food mixture further contains sodium chloride.   The lactic acid bacterium solid fermentation product according to any one of claims 21 to 28, wherein the carbonate is a mixture of calcium carbonate and magnesium carbonate.   A food containing a cereal, a plant containing polyphenol, and a lactic acid bacterium, comprising the lactic acid bacterium solid fermented product according to any one of claims 21 to 29.

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