JP2015177748A - Methods for producing lactic acid bacterial producing material, lactic acid bacterial producing material and inhibitors of allergic dermatitis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for efficiently producing a lactic acid bacterial producing material by co-cultivation of plural lactic acid bacteria.SOLUTION: According to the invention, lactic acid bacteria of the genus Lactobacillus, lactic acid bacteria of the genus Lactococcus and the lactic acid bacteria of genus Enterococcus are co-cultivated to produce a lactic acid bacterial product of interest. Two, three or four or more lactic acid bacteria selected from the above lactic acid bacteria are combined and made into plural groups of different members and each group is pre-cultured for efficient production of the lactic acid bacterial product.

Description

  The present invention relates to a method for producing a lactic acid bacterium-producing substance using a plurality of lactic acid bacteria and a lactic acid bacterium-producing substance.

With the recent diversification and mass consumption of eating habits, improvements in eating habits and eating habits are sought. In particular, attention has been focused on lifestyle-related diseases and metabolic syndrome caused by overnutrition and lack of exercise, but effective preventive methods for these have not been established, and effective breakthroughs other than lifestyle improvements have been established. The situation does not exist.
In this situation, attention has been focused on health foods that, when ingested, lead to health promotion, and those containing lactic acid bacteria have attracted attention as such health foods. Lactic acid bacteria are expected to have beneficial effects in various health maintenance such as improving immunity and preventing adult diseases. This is considered to be an effect obtained from the fact that the intestinal environment is adjusted by lactic acid bacteria taken from outside and the intestinal tract immune function is improved (see, for example, Patent Document 1).
As a method of ingesting lactic acid bacteria, for example, a method of ingesting fermented foods such as yogurt containing live bacteria of lactic acid bacteria, beverages, health foods, and the like is known. However, in fact, even when lactic acid bacteria are ingested from the outside, it is said that many of them are killed by the action of, for example, gastric acid and digestive enzymes, and few reach the small intestine. Therefore, the present inventors focused on ingesting a substance (lactic acid bacteria-producing substance) obtained by culturing lactic acid bacteria rather than ingesting live bacteria of lactic acid bacteria.

JP2004-18469

  In order to lead to health promotion effects such as improving immunity by ingesting the above-mentioned lactic acid bacteria-producing substance, it is first necessary to establish a method for efficiently cultivating lactic acid bacteria and producing a large amount of lactic acid bacteria-producing substance. Hundreds of types of lactic acid bacteria have been discovered, but their properties are different from each other, and there is a great difference in the effect of the resulting lactic acid bacteria-producing substance. Therefore, the present inventors have selected various kinds of lactic acid bacteria used for lactic acid fermentation, combinations, nutrient sources (medium), etc., and conducted various studies in combination.

  An object of this invention is to provide the manufacturing method of the lactic-acid-bacteria production substance which has a more efficient and high health promotion effect.

The present invention relates to a method for producing a lactic acid bacterium-producing substance, wherein a lactic acid bacterium belonging to the genus Lactobacillus , a lactic acid bacterium belonging to the genus Lactococcus, and a lactic acid bacterium belonging to the genus Enterococcus , A method for producing a lactic acid bacterium-producing substance comprising a pre-culturing step of combining two, three or four or more lactic acid bacteria selected from the above to form a plurality of groups in which the lactic acid bacteria included in the combination are different and co-culturing each group To do.

It said group present invention, containing lactobacilli (Lactobacillus) create a group comprising lactic acid bacteria belonging to Lactobacillus and Lactococcus (Lactococcus) genus belonging to the genus Lactococcus (Lactococcus) Lactobacillus lactic acid bacteria belonging to Enterococcus (Enterococcus) genus belonging to the genus And a group containing lactic acid bacteria belonging to the genus Enterococcus and lactic acid bacteria belonging to the genus Lactobacillus, and a preculture step of coculturing in each group.

The present invention provides L. lactic acid bacteria belonging to the genus Lactobacillus as described above. Lactobacillus acidophilus , L. Brevis ( Lactobacillus brevis ), L. Lactobacillus casei , L. Lactobacillus delbrueckii , L. Lactobacillus delbrueckii subsp. Bulgaricus , L. Gasseri ( Lactobacillus gasseri ), L. Lactobacillus helveticus , L. Paracasei subspecies paracasei (Lactobacillus paracasei subsp. Paracasei), L. Plantarum ( Lactobacillus plantarum ), L. Lmonobasus ( Lactobacillus rhamnosus ), L. Lactobacillus salivarius is used, and lactic acid bacteria belonging to the genus Lactococcus are L. Lactococcus garvieae , L. Lactococcus lactis subsp. Lactis is used as a lactic acid bacterium belonging to the genus Enterococcus. Enterococcus durans , E.I. Enterococcus faecalis , E. Any of fesium ( Enterococcus faecium ) can be used.

  The present invention relates to the E.I. A pre-culturing step of forming a group containing fesium and the lactic acid bacteria belonging to the genus Lactobacillus and co-culturing can be included.

  The present invention relates to the E.I. It may include a pre-culturing step of forming a group containing Durance and the lactic acid bacteria belonging to the genus Lactobacillus and co-culturing.

  The present invention relates to the E.I. Fesium and the aforementioned E.I. It is possible to include a pre-culture process in which a group including Durance is formed and co-cultivated.

  The present invention relates to the E.I. Fesium and the aforementioned E.I. A group including Durance is created, and separately from the above E.E. Fesium and the aforementioned E.I. Durance and L. A pre-culturing step of forming a group containing galviae and coculturing each of them can be included.

  The present invention relates to the E.I. It may include a pre-culturing step in which a plurality of groups containing different lactic acid bacteria including fesium and different from each other are formed and cocultured.

  In the present invention, the preliminary culturing step is carried out as described in E. above. Fesium and the aforementioned E.I. Durance and L. Groups that include Galviae can be done without creating them.

  The present invention may include a step of fermenting soy milk.

  In the present invention, soybeans stored at an ice temperature can be used as a raw material.

  The said this invention shall perform the main culture process which performs the cocultivation culture | cultivation including all the lactic acid bacteria to be used after the said preculture.

  The present invention may include a pulverization process for pulverizing the culture solution obtained in the main culture process.

  The said invention can include the filtration process which filters the culture solution obtained at the said main culture process.

  The present invention provides a lactic acid bacteria-producing substance containing the filtrate obtained by the filtration step as an active ingredient.

  In the present invention, the filtrate may contain the soybean component and the bacterial cell component of the lactic acid bacterium.

  In the present invention, the filtrate may contain a bacterial cell component derived from the killed lactic acid bacterium.

  In the present invention, the filtrate may not contain the live lactic acid bacteria.

  ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of an efficient lactic-acid-bacteria production substance can be provided. Moreover, according to this invention, the lactic acid bacteria production substance with a high health promotion effect, such as improvement of immunity, can be provided.

It is a graph which shows a time-dependent change of the thickening of an auricle. It is a graph which shows the expression level of IL-13 mRNA of auricle. It is a graph which shows the expression level of periostin mRNA of auricle. It is a graph which shows the expression level of TSLP mRNA of an auricle. It is a graph which shows the expression level of IL-4 mRNA of a spleen. It is a graph which shows the amount of IgE in blood. It is a graph which shows the amount of IgA in blood.

  The manufacturing method of the lactic acid bacteria production substance of this invention and the lactic acid bacteria production substance obtained by this are demonstrated in detail below.

The method for producing a lactic acid bacteria production substance includes a step of coculturing a plurality of lactic acid bacteria. The lactic acid bacteria used include lactic acid bacteria belonging to the genus Lactobacillus , lactic acid bacteria belonging to the genus Lactococcus, and lactic acid bacteria belonging to the genus Enterococcus .

The following lactic acid bacteria can be preferably exemplified as the lactic acid bacteria.
First, examples of bacteria belonging to the genus Lactobacillus include L. Lactobacillus acidophilus , L. Brevis ( Lactobacillus brevis ), L. Lactobacillus casei , L. Lactobacillus delbrueckii , L. Lactobacillus delbrueckii subsp. Bulgaricus , L. Gasseri ( Lactobacillus gasseri ), L. Lactobacillus helveticus , L. Paracasei subspecies paracasei (Lactobacillus paracasei subsp. Paracasei), L. Plantarum ( Lactobacillus plantarum ), L. L actobacillus rhamnosus , L. Salivarius ( Lactobacillus salivarius ) can be mentioned.
Examples of bacteria belonging to the genus Lactococcus include L. Lactococcus garvieae , L. The lactis subspecies lactis ( Lactococcus lactis subsp . Lactis ) can be mentioned.
Further, examples of bacteria belonging to the genus Enterococcus include E. coli. Enterococcus durans, E.I. Enterococcus faecalis, E. Mention may be made of fesium ( Enterococcus faecium).

  One or more lactic acid bacteria are selected and used from each genus Lactobacillus genus, Lactococcus genus, Enterococcus genus. The same kind of lactic acid bacteria may contain different strains.

These lactic acid bacteria are subjected to the following primary culture to fourth culture under aerobic conditions.
The primary culture is a culture process in which each lactic acid bacterium is cultured alone. For example, when the lactic acid bacterium is inactivated and stored, it can be activated by this primary culture. Moreover, it grows until it becomes the number of bacteria required for use in secondary culture.

  The secondary culture is a culture process in which a plurality of groups selected from two, three, or four or more types from the lactic acid bacteria are combined, and the lactic acid bacteria are cocultured in each group. The combination of lactic acid bacteria included in each of these groups is carried out by co-culturing various combinations of each lactic acid bacterium, and the proportion of each lactic acid bacterium is kept constant even when subcultured, and one of them is trapped by survival competition. It can be found from things that do not happen. Further, when a plurality of groups are formed, the same type of lactic acid bacteria may be included in the plurality of groups.

Therefore, such grouping is suitable for culturing lactic acid bacteria that are difficult to grow alone and are easy to grow by mixing with other lactic acid bacteria.
Moreover, if it divides into groups, it will become possible to culture | cultivate in culture environments, such as a raw material culture medium suitable for every group, culture | cultivation temperature, culture | cultivation time, and atmospheric conditions. Furthermore, before performing the tertiary culture described later, each lactic acid bacterium can be adapted to the conditions for culturing with other lactic acid bacteria and the transition to the tertiary culture can be performed smoothly.
Such a process up to the secondary culture is a preliminary culture process.

As a more specific grouping performed in secondary culture, the following combinations of Group A to Group I can be given as examples. The following group is a combination of the inventors in order to reproduce the state in which multiple types of lactic acid bacteria form and coexist with each other in the intestinal environment.
This combination of lactic acid bacteria can efficiently cultivate each lactic acid bacterium and obtain a lactic acid bacterium-producing substance. However, the combination is not limited to these combinations as long as the ratio of each lactic acid bacterium is kept constant even after subculture.

First, L.M. Let Acidophilus be A group alone.
L. Casei and L. Lactis subsp. Del Bricky subsp. Bulgaricus and L. Two types of paracasei subspecies paracasei are group C. Durance and E.I. Two types of fesium are designated as D group.
In addition, L. Brevis and L. Plantarum and E.I. Three types of fesium are designated as E group. Del Bricky and L. Helveticas and E. Three types of faecalis are designated as F group. Gasseri and L. Ramonausus and E. Three types of fesium are designated as G group. Gasseri and L. Sarivarius and E. Three types of Durance are grouped as H group. Galviae and E. Durance and E.I. Three types of fesium are designated as I group.
Each of these combinations is summarized in [Table 1] below. In addition, A in a table | surface shows A group and B-I shall show B group-I group similarly.
Group A is an L.I. Acidophilus is cultivated alone and not co-cultured. In order not to forget the mixing of acidophilus, it is a group.

  The above I group is the same as L. It is a combination with Garviae added. This L. Garbiae is known to decompose soy isoflavone, which is a component of soymilk medium, to produce equol having a high health promoting effect. Therefore, L. Easy to cocultivate with galviae Durance, E.C. Fesium (D group combination) By adding galbiae, the above-mentioned I group that easily obtains the lactic acid bacteria-producing substance having a high equol content was provided.

According to the above grouping, all groups use the same medium, and all groups are cultured at 32 ° C. to 36 ° C. for 12 hours to 48 hours.
Moreover, according to the said grouping, the number of lactic acid bacteria obtained from each group can be made into substantially the same number. Therefore, strains that are difficult to grow in the subsequent tertiary culture and strains that want to increase the content can be included in a plurality of groups. Moreover, since it can transfer to a subsequent culture | cultivation process by mixing equal amount of each group, a mixing process is easy and can transfer between processes smoothly.

In the secondary culture, a step of further culturing by mixing several culture solutions from the culture solutions cultured for each group can be provided. That is, the group can be appropriately subdivided.
If it demonstrates using the said grouping, it will replace with the method of producing A to I group separately and cultivating each group, for example, make D group beforehand, culture | cultivate, take out one part, L. Garbiae can be added to form Group I. Alternatively, the E. coli can be cultured by preparing a group in which the G group and the I group are mixed before the third culture described below. Fesium content can be reduced.

  The tertiary culture is a culture process in which the culture solutions obtained for each group divided by the secondary culture are mixed and all lactic acid bacteria are cultured simultaneously in one incubator. This tertiary culture is to cocultivate all the selected lactic acid bacteria, and is an intermediate culture stage for making various lactic acid bacteria accustomed to the environment and culturing more efficiently in the post-process quaternary culture. .

In the tertiary culture, a culture solution cultured in each group in the secondary culture is mixed at a predetermined ratio and a mixing amount, and cultured under a predetermined medium.
Originally, if the lactic acid bacteria obtained in the secondary culture are added directly to a large amount of medium used in the fourth culture, the lactic acid bacteria solution may be diluted, and the efficiency of the culture may deteriorate. Therefore, tertiary culture can be performed as preliminary culture, and the number of bacteria can be increased to smoothly shift to quaternary culture.
In this case, the mixing ratio and the mixing amount of each group can be appropriately determined by deriving from the number of lactic acid bacteria necessary for the third culture. However, for work convenience, it is preferable to mix equal amounts in each group.
In the third culturing step, each lactic acid bacterium is cultured at a relatively low temperature of 30 ° C. to 36 ° C. for 12 hours to 72 hours in order to get used to the environment in which each lactic acid bacterium is mixed with other lactic acid bacteria.

  Moreover, various raw materials that can be fermented with lactic acid bacteria can be used as the raw material for the medium, but it is preferable to use soy milk. By carrying out like this, the lactic-acid-bacteria production substance obtained by culture | cultivating lactic acid bacteria can be made to contain the soybean origin component which is a vegetable nutrient component preferable for a biological body. Therefore, it is possible to obtain a lactic acid bacterium-producing substance that has a high nutritional value and can easily obtain a health promotion effect.

Such soy milk is also preferably soy milk obtained from a predetermined soybean. For example, preferred soybeans include soybeans that have been stored at ice temperature. The storage at ice temperature is a process of standing still at a low temperature at which the antifreeze leaks without freezing soybeans. The temperature is preferably -3 ° C to -1 ° C, and the storage period is preferably 2 days to 3 days. Various effects are known for this ice temperature preservation. For example, there is a report that the amount of free amino acids can be increased about twice as much as that of conventional products in the case of bread, and the content of amino acids such as glutamic acid can be increased as compared with conventional products in the case of natto. Soybeans have the advantage of being able to maintain the quality of soy milk medium because storage at ice temperature can suppress the reduction of harmful microorganisms and suppress the respiratory metabolism of living organisms and maintain the freshness of soybeans over a long period of time. . Therefore, in order to obtain the various effects as described above, it is preferable to use soy milk using soybeans stored at ice temperature in the present invention.
For the preparation of soy milk, when using soybeans stored at ice temperature, after leaving the soybeans taken out of the ice temperature storage at 5 ° C. to 15 ° C., which is higher than the ice temperature but lower than normal temperature, for 6 hours to 24 hours, Soybeans returned to room temperature can be used.

  In addition to soy milk, various raw materials can be added to the medium as needed. For example, sugars such as glucose and glucose, yeast extract, skim milk and the like can be mentioned.

The quaternary culture is a main culture process in which the culture solution obtained in the tertiary culture is further cultured.
The quaternary culture preferably has the following three steps. Step 1 is a stage of culturing at 32 ° C. to 37 ° C. for 25 hours to 30 hours.
Step 1 is a step of acclimatizing each lactic acid bacterium at a relatively low temperature as in the case of the tertiary culture. The reason why the fourth culture is similar to the third culture is that the medium can be changed from the third culture, and in this case, each lactic acid bacterium may not be used to the environment.

Then, as step 2, it culture | cultivates at 38 to 43 degreeC for 46 to 51 hours.
This step 2 is a step in which lactic acid bacteria are grown in earnest and lactic acid bacteria fermentation is advanced. Therefore, the most preferable temperature for the culture is set, and the time when the lactic acid bacteria increase most is set.

  Finally, as Step 3, the cells are cultured at 63 to 67 ° C. for 16 to 21 hours. This step 3 is a process of stopping the cultivation of lactic acid bacteria by sterilization by heat treatment. The culture solution obtained by performing such quaternary culture becomes a lactic acid bacteria production substance.

  The lactic acid bacteria culture solution obtained through the quaternary culture is preferably pulverized (homogenized) by, for example, a homogenizer. By this treatment, lactic acid bacteria can be crushed into dead bacteria, and culture can be stopped more reliably. As a result of Step 3 in the quaternary culture, the protein in the culture solution may be heat denatured and partially solidified. Therefore, a uniform suspension can be obtained by performing this homogenization treatment. The obtained lactic acid bacterium-producing substance, which is a uniform suspension, is a stable lactic acid bacterium-producing substance of a quality that does not contain viable bacteria and is unlikely to denature.

Thereafter, a further step of filtering the suspension can be included. Specifically, there may be mentioned a step of putting the lactic acid bacteria culture solution after the homogenization treatment into a filter cloth bag and filtering it over several days (3 to 4 days) in a state where the weight is placed. Thereby, said lactic-acid-bacteria culture solution is isolate | separated into a filtrate and a fermentation residue.
The lactic acid bacterium-producing substance as the filtrate is excellent in appearance because it is obtained as a transparent liquid containing no solid content, and can give a sense of security that impurities are not included in those who take it. On the other hand, the lactic acid bacteria production substance as a fermentation residue contains the lactic acid bacteria production substance and soybean component of the filtrate, and can be used as a health food for external use and taking.

The filtrate can be further led to the next step. In other words, it is a filter filtration process that performs filtration using a filter provided with small through holes. This filter filtration process is a process of removing lactic acid bacteria components that have not been sufficiently pulverized, and is a process of completely removing live bacteria if any remain. At this time, the size of the through hole is set to such a size as to remove lactic acid bacteria, and more specifically, a filter of about 0.2 μm to 0.3 μm can be used. This is because most of the lactic acid bacteria can be removed from the filtrate if the thickness is 0.2 μm to 0.3 μm, and if it is smaller than 0.2 μm, the filtration efficiency is deteriorated, and if it exceeds 0.3 μm, the lactic acid bacteria cannot be separated. It is. By performing this filter filtration treatment repeatedly a plurality of times, lactic acid bacteria can be more reliably removed from the filtrate.
The final filtrate thus obtained is the highest quality product among the lactic acid bacteria producing substances, and can be called a high-grade lactic acid bacteria producing substance or a pure lactic acid bacteria producing substance that the inventor can provide with peace of mind.

Experimental example

1. Production of lactic acid bacteria production material [primary culture]
In order to produce the lactic acid bacteria-producing substance according to the present invention, first, lactic acid bacteria belonging to the genus Lactobacillus, the genus Lactococcus, and the genus Enterococcus were cultured. Acidophilus, L.H. Brevis, L. Casei, L. Delbricky, L. Delbricky subsp. Bulgaricus, L. Gasseri, L.M. Helveticas, L. Paracasei subspecies Paracasei, L. Plantarum, L.C. Ramonausus, L.M. Eleven types of lactic acid bacteria of Sarivarius were used.
As lactic acid bacteria belonging to the genus Lactococcus, L. Garviae, L. Two types of lactic acid bacteria of Lactis subspecies Lactis were used.
Further, examples of lactic acid bacteria belonging to the genus Enterococcus include E. coli. Durance, E.C. Fecaris, E.E. Three types of lactic acid bacteria of fesium were used. Each of these lactic acid bacteria has various lactic acid bacteria obtained by conventional methods stored at -80 ° C.

Each of the 16 types of lactic acid bacteria was inoculated individually in MRS medium as a primary culture, and cultured alone at 34 ° C. in an aerobic environment, so that OD ₆₆₀ nm = about 1.3.

[Secondary culture]
Subsequently, as the secondary culture, the above 16 types of lactic acid bacteria were made into 9 groups consisting of one or a plurality of types of lactic acid bacteria. And the group which consists of said 1 type of lactic acid bacteria culture | cultivated only 1 type of lactic acid bacteria independently, and the group which consists of the said several lactic acid bacteria group cocultivated these several lactic acid bacteria.

The combination of the lactic acid bacteria contained in each group in secondary culture is shown.
First, L.M. Acidophilus was group A.
L. Casei and L. Lactis subsp. Del Bricky subsp. Bulgaricus and L. Two types of paracasei subspecies paracasei are group C. Durance and E.I. Two types of fesium were made into D group.
In addition, L. Brevis and L. Plantarum and E.I. Three types of fesium are designated as E group. Del Bricky and L. Helveticas and E. Three types of faecalis are designated as F group. Gasseri and L. Ramonausus and E. Three types of fesium are designated as G group. Gasseri and L. Sarivarius and E. Three types of Durance are grouped as H group. Galviae and E. Durance and E.I. Three types of fesium were designated as Group I.
Each of these combinations is summarized in [Table 2] below. In addition, A in a table | surface shows A group and B-I shall show B group-I group similarly.

  In this secondary culture, the primary culture solution was added to and mixed with the soymilk medium described later, and then cultured at 32 ° C. to 36 ° C. for about 1 day in an aerobic environment. The culture solution thus obtained is referred to as “secondary culture solution”.

Here, the manufacturing method of the soymilk culture medium used by secondary culture is demonstrated.
Soy milk was prepared using soybeans that had been stored at −3 ° C. to −1 ° C. for 2 to 3 days and stored at ice temperature for a while and then returned to room temperature. The soy milk thus obtained was subjected to high-pressure steam sterilization at 121 ° C. for 15 minutes to obtain “medium soy milk”.
And after diluting 100 ml of this soymilk for culture medium with water twice and mixing skim milk powder, glucose, and yeast extract and dissolving them, high-pressure steam sterilization is performed at 115 ° C. for 15 minutes, thereby “secondary culture” Soymilk medium "was obtained.

Each secondary culture solution was diluted and then seeded on an MRS agar plate (φ90 mm). From the number of colonies formed after culturing at 34 ° C. for 24 hours, the number of lactic acid bacteria contained in each of the secondary culture solutions of the A group to I group was 2.6 × 10 ⁹ cfu / ml on average.

[Tertiary culture]
All of the 16 types of lactic acid bacteria were mixed and co-cultured. Specifically, equal amounts of each of the secondary culture solutions of Group A to Group I were mixed. This mixed solution was added to the following “third culture soymilk medium” and cultured at 32 ° C. to 36 ° C. in an aerobic environment for 1 day to obtain a “tertiary culture solution”. This tertiary culture solution was inoculated on an MRS agar plate, and the number of lactic acid bacteria contained in the tertiary culture stock solution was about 2.6 × 10 ⁹ cfu / ml from the number of colonies formed after culturing at 34 ° C. for 24 hours. It was.

  The soymilk medium for tertiary culture is diluted by adding 1/4 amount of water to 100 ml of the soymilk for medium, mixed with skim milk powder, glucose and yeast extract, and then dissolved at 115 ° C. It was obtained by performing autoclaving for 15 minutes.

[Fourth culture]
Subsequently, quaternary culture was performed. Specifically, the tertiary culture solution was added to the following “fourth culture soymilk medium” and cultured in an aerobic environment. This quaternary culture consists of Step 1 for culturing for 25 to 30 hours at 32 ° C. to 37 ° C., Step 2 for culturing for 46 to 51 hours at 38 ° C. to 43 ° C., and Step 16 for 63 ° C. to 67 ° C. It is composed of three stages including Step 3 for culturing for 21 hours. First, step 1 is a step in which each lactic acid bacterium is acclimatized at a relatively low temperature as in the third culture. The reason why the fourth culture is similar to the third culture is that the medium can be changed from the third culture, and in this case, each lactic acid bacterium may not be used to the environment. In subsequent step 2, lactic acid bacteria can be grown in earnest and lactic acid bacteria fermentation can proceed. Finally, in step 3, the culture of lactic acid bacteria can be stopped while preventing the protein from being denatured. Further, about 24 hours after the start of Step 1, a rod-like instrument such as a spatula was inserted into the bottom of the tertiary culture solution and mixed. By doing so, lactic acid bacteria that are located away from the surface of the tertiary culture solution and are not easily exposed to air were placed in an aerobic environment. By this operation, lactic acid bacteria can be cultured more efficiently. As a result of these operations, a “quaternary culture solution” was obtained.
Step 1 and step 2 cultures were serially diluted and seeded on MRS agar plates (φ90 mm). The number of colonies formed after culturing at 34 ° C. for 24 hours was about 1.5 × 10 ⁹ cfu / ml in Step 1 and about 2.1 × 10 ⁹ cfu / ml in Step 2. In addition, since the step 3 is the inactivation process of the microbe by processing at high temperature, the viable count was not measured.

  The soymilk medium for quaternary culture is prepared by diluting the soymilk for medium with about 1/4 amount of water, mixing skim milk powder, glucose and yeast extract, dissolving, and then 15 minutes at 115 ° C. It was obtained by performing high-pressure steam sterilization.

[Homogenization]
The quaternary culture solution was pulverized (homogenized) at 6,000 rpm for 15 minutes using a homogenizer to obtain a “quaternary culture suspension”. By this work, lactic acid bacteria can be crushed and the culture can be completely stopped. In addition, heat denaturation occurs due to the high-temperature treatment in Step 3, and the obtained culture solution is partially solidified. By this homogenization, the culture solution can be made into a uniform suspension.

[Filtration]
The whole amount of the quaternary culture suspension was put in a filter cloth bag, and the bag mouth was sealed. This was loaded with weight (6 kg) and filtered for about 3 days to obtain a filtrate and fermentation residue.

  The filtrate was further filtered twice using a 0.3 μm filter to obtain “lactic acid bacteria-producing substance” as the final filtrate. The lactic acid bacterium-producing substance contains microbial cell components derived from killed lactic acid bacteria, products of lactic acid bacteria, and liquid components derived from soybeans and soy milk, which are raw materials for the medium.

2. Sample preparation
2-1. Preparation of experimental animals In order to verify the effect of improving immunity by ingesting the lactic acid bacteria-producing substance according to the present invention, model mice in which allergic dermatitis was induced were prepared.
Specifically, 4-week-old BALB / c male mice are prepared, and acetone-dinitrofluorobenzene (hereinafter referred to as DNFB: manufactured by SIGMA-ALDRICH) is twice a week (3 days or 4 days) in both ears of each mouse. It was applied a total of 16 times once a day). By doing so, allergic dermatitis showing symptoms of contact dermatitis can be induced.

  During the breeding period, the mice were subjected to animal experiments in a light and dark environment with artificial lighting (light period 8:00 to 20:00, dark period 20:00 to 8:00), constant temperature (22 ° C. ± 2 ° C.), and humidity uncontrolled. They were raised indoors and allowed to freely take water and feed. A normal feed (CE-2: manufactured by Nippon Claire Co., Ltd.) and a feed containing the lactic acid bacterium-producing substance of the present invention were given to this normal feed.

2-2. Preparation Method of Feed Containing Lactic Acid Bacterial Producing Substance The powdered lactic acid bacteria producing substance according to the present invention was mixed with the normal feed so as to have a mass ratio of 3% to obtain a feed containing 3% lactic acid bacteria producing substance. Similarly to this, the above-mentioned normal feed was mixed with the powdered lactic acid bacteria-producing substance according to the present invention at a mass ratio of 5% to obtain a feed containing a 5% lactic acid bacteria-producing substance.

2-3. Treatment group 3 given a feed containing 3% of the lactic acid bacteria-producing substance obtained as above from the 20th day to the 52nd day from the start of application of DNFB as animals for administration experiments of lactic acid bacteria-producing substances to allergic dermatitis model animals 5% of the treatment group given the feed containing 5% of the lactic acid bacteria-producing substance, and the prevention containing the feed containing 3% of the lactic acid bacteria-producing substance from the 14th day before starting the DNFB application to the 52nd day of the application. 3% of the group, 5% of the prevention group given the feed containing 5% of the lactic acid bacteria-producing substance of the present invention, the control group which applied the DNFB but applied only the normal feed, and the DNFB application First, a total of five groups were prepared (n = 5), including the DNFB (−) group fed only with normal feed.
The average body weight of all mice used in this experiment was 27.3 g, and the average food intake was about 4.2 g / day. Therefore, it is considered that 3% of the treatment group and 3% of the prevention group ingested about 0.126 g of lactic acid bacteria-producing substance per day. On the other hand, it is considered that 5% of the treatment group and 5% of the prevention group ingested about 0.210 g of lactic acid bacteria-producing substance per day.
In the treatment group 3%, the treatment group 5%, the prevention group 3%, the prevention group 5%, and the control group, blood was collected on the 52nd day from the start of DNFB application, and the auricle and spleen were collected. In the DNFB (−) group, blood was collected in the same manner as the other groups at the same age, and the auricle and spleen were collected.

  Each group was anesthetized with diethyl ether, and after cervical dislocation, blood was collected from the heart using an injection needle. The collected blood was allowed to stand overnight at 4 ° C. and then centrifuged (5 ° C., 10,000 rpm × 10 minutes), and serum was collected as a supernatant. The serum thus obtained was stored at −20 ° C. until analysis.

  The auricle and spleen were stored in RNA Stabilization Solution (product name: RNA later Soln (registered trademark), manufactured by Ambion) for use in RT-PCR described below, the auricle was 4 ° C., and the spleen was −80 Stored at ° C until analysis.

3. Verification of allergic inflammation inhibitory effect In order to measure the allergic inflammation inhibitory effect of the lactic acid bacterium-producing substance according to the present invention, the time course of ear thickening of each group, various cytokines and inflammatory mediator mRNA expression levels were determined. It was measured.

3-1. Measurement of auricle thickening Using the auricle thickening as an index of allergic symptoms for each of the above-mentioned mice, the relationship between the lactic acid bacteria-producing substance and allergic symptoms was verified by measuring changes in auricular thickening after application of DNFB. This measurement was performed 24 hours after each application of DNFB, for a total of 16 times. In addition, for the DNFB (−) group, the auricle thickening was measured at the same time.

  FIG. 1 shows the results of measurement of auricle thickening on allergic dermatitis caused by DNFB sensitization. In the control group, the thickening of the auricle progressed rapidly after the 11th day from the start of the application of DNFB, and the peak value (0.575 mm) was shown on the 20th day. However, after that, it decreased slightly and maintained a substantially constant thickness (around 0.55 mm). On the other hand, in the treatment group 3% and the treatment group 5%, the thickening of the auricle progressed rapidly on and after the 11th day from the start of the application of DNFB, but on the 19th day the feed containing the lactic acid bacteria-producing substance was added. As soon as I started taking, the thickness started to decrease. As a result, the treatment group 3% showed a peak value (0.532 mm) on the 20th day, and the treatment group 5% showed a peak value (0.543 mm) on the 20th day. Therefore, in both cases, thickening of the auricle was suppressed more than the peak value of the control group.

  Further, in the prevention group 3% and the prevention group 5%, as in the other groups, the thickening started to progress rapidly after the 11th day from the start of the application of DNFB, but the progress was in the control group, the treatment group 3% and the treatment group. Compared with 5%, it was moderate, and the thickening was further eased after the 13th day. Moreover, although the peak value was shown on the 27th day in both the prevention group 3% and the prevention group 5%, the prevention group 3% showed 0.500 mm and the prevention group 5% showed 0.457 mm, which was more peak than the other groups. The value was low.

  From the above results, it was suggested that the intake of the lactic acid bacterium-producing substance according to the present invention is effective in suppressing inflammation at the site of allergic dermatitis. At the same time, it was suggested that a higher inflammation-inhibiting effect can be obtained by continuing to take the above-mentioned lactic acid bacteria-producing substance before sensitization.

3-2. Measurement of mRNA expression levels of cytokines and inflammatory mediator proteins in the auricle Currently, periostin and TSLP are attracting attention as inflammatory mediator proteins. Periostin is a protein highly expressed at the site of chronic dermatitis. When the allergen enters the skin tissue, type 2 helper T cells (hereinafter abbreviated as Th2 cells) are activated, and the activated Th2 cells produce IL-4 and IL-13. IL-4 and IL-13 stimulate fibroblasts and promote periostin production. This periostin stimulates epidermal cells, and inflammatory mediators such as TSLP are produced from the epidermal cells. TSLP stimulates dendritic cells and Th2 cells are activated again, and the above cycle is repeated, causing chronic allergic dermatitis to persist. Therefore, by measuring the mRNA expression levels of periostin and TSLP in the auricles of each group, the effect of lactic acid bacteria-producing substances on chronic allergic dermatitis can be achieved. Therefore, in order to further influence on allergic dermatitis, mRNA expression levels of IL-13, periostin and TSLP in the auricle were measured.

3-2-1. Extraction of RNA Total RNA was prepared from the collected mouse pinna as described above.
Specifically, first, the auricles stored in the RNA Stabilization Solution were transferred into a container containing ceramic beads (1.4 mm: manufactured by MS Equipment Co., Ltd.) and TRIZOL (registered trademark) Reagent (manufactured by Invitrogen). . Then, the tissue was crushed by performing centrifugation treatment (6.300 rpm, 23 seconds) 6 times.
From the pinna thus obtained, total RNA was extracted according to the protocol attached to TRIZOL and stored at -20 ° C.

3-2-2. Preparation of cDNA for RT-PCR cDNA was prepared from the total RNA obtained above using the Reverse Tra Ace qPCR RT kit (manufactured by TOYOBO).
Specifically, first, the absorbance of the total RNA obtained above was measured, and diluted with milliQ water so that the total RNA concentration became 1 μg / μl. 1 μl of this total RNA was added to 6 μl of H ₂ O attached to the kit, incubated at 65 ° C. for 5 minutes, and then allowed to stand on ice. To this was added 3 μl of a Mix solution consisting of 2 μl of 5 × Buffer attached to the kit, 0.5 μl of RT Enzyme Mix and 0.5 μl of Primer Mix, and then incubated at 37 ° C. for 15 minutes and further at 98 ° C. for 5 minutes, The reaction was stopped by standing on ice. To 10 μl of the cDNA having a concentration of 10 ng / μl thus obtained, 90 μl of milli-Q water was added to make a total 100 μl cDNA solution, which was stored at −20 ° C.

3-2-3. Quantitative RT-PCR
The cDNA was amplified by RT-PCR method.
Specifically, 2 μl of the cDNA solution obtained above was dropped into each well of a 384-well plate, and 6.4 μl of milli-Q water, 2 × SYBR GREEN (product name: THUNDERBIRD SYBR (registered trademark) qPCR Mix ( 10 μl, 10 μM forward primer 0.6 μl, 10 μM reverse primer 0.6 μl and 50-fold diluted ROX (TOYOBO) 0.4 μl added 18 μl of Master Mix solution, and the final volume was 20 μl. The base sequences of the forward primer and the reverse primer used were as follows: each sample was heat-denatured at 95 ° C. for 60 seconds, then annealed at 95 ° C. for 15 seconds and 60 ° C. for 60 seconds. Extension was repeated for 40 cycles.
Based on the cDNA thus obtained, the expression levels of various cytokines and inflammatory mediator mRNA in the auricle were measured.
-Base sequence of the primer used (IL-13)
F: 5'-AGACCAGAACTCCCCTGTGCA-3 '
R: 5'-TGGGTCCCTGTAGATGGCATTG-3 '
(Periostin)
F: 5'-GAACGAATCATTACAGGTCC-3 '
R: 5'-GGAGACCCTTTTTGCAAGA-3 '
(TSLP)
F: 5'-CTGAGAGAAAATGACGGTACTCAGG-3 '
R: 5'-GGAGATTGCATGAAGGAATAACCAC-3 '

3-3. IL-13 mRNA expression inhibitory action IL-13 mRNA expression level in the pinna of IL-13, which is an inflammatory cytokine, is shown in FIG. The expression level of IL-13 mRNA in the control group was 0.167 in the treatment group 3%, 0.250 in the treatment group 5%, 0.078 in the prevention group 3%, and 0.101 in the prevention group 5%. It was. Therefore, it was found that the expression of IL-13 mRNA was dramatically suppressed as compared with the control group that did not take the lactic acid bacteria production substance.

3-3-1. Periostin and TSLP mRNA Expression Inhibitory Actions The expression level of periostin mRNA in allergic dermatitis (auricle) of each group is shown in FIG. 3, and the expression level of TSLP mRNA is shown in FIG. If the expression level of periostin mRNA in the control group is 1, it was 0.901 in the treatment group 3%, 0.120 in the treatment group 5%, 0.472 in the prevention group 3%, and 0.355 in the prevention group 5%. It was. In addition, the TSLP mRNA expression level was 0.941 in the treatment group 3%, 0.614 in the treatment group 5%, 0.506 in the prevention group 3%, and 0.457 in the prevention group 5% with respect to the control group 1. Met.
Therefore, it was found that mRNA expression was suppressed in both periostin and TSLP as compared with the control group that did not take the lactic acid bacteria-producing substance of the present invention.

  From the results obtained above, the lactic acid bacterium-producing substance according to the present invention suppresses the production of cytokines and inflammatory mediators such as IL-13, periostin and TSLP at the site of allergic dermatitis, and has an inhibitory effect on allergic inflammation. It has been suggested.

4). Verification of systemic allergic reaction inhibitory effect Based on the above results, it was found that the lactic acid bacterium-producing substance according to the present invention has an inflammation-inhibiting effect at the site of allergic dermatitis. On the other hand, in order to measure the effect of lactic acid bacteria production substances on systemic allergic reaction, the expression levels of IL-4, blood IgE and blood IgA, which are inflammatory cytokines, were measured.

4-1. Spleen IL-4 mRNA expression level The spleen was collected from the mouse, crushed and stored in TRIZOL. In addition, total RNA extraction, cDNA preparation, and RT-PCR were performed as described above. The base sequences of the forward primer and reverse primer used in RT-PCR were as follows.
(IL-4)
F: 5'-TCTCGAAGTTACCAGGAGCATCAT-3 '
R: 5′-AGCACCCTTGGAAGCCCTACAGA-3 ′

  The spleen IL-4 mRNA expression level in the DNFB (−) group is 1, and the relative value of the mRNA expression level in other groups is shown in FIG. The DNFB (-) group was 1, whereas the control group was 5.757, indicating that IL-4 production was promoted at sites other than the inflammation site of allergic dermatitis due to DNFB sensitization. . On the other hand, the treatment group 3% is 2.438, the treatment group 5% is 2.310, the prevention group 3% is 3.110, and the prevention group 5% is 2.164. A dramatic reduction in expression was seen.

4-2. Quantification of blood IgE and blood IgA by ELISA The blood IgE and blood IgA were quantified by ELISA.
As an ELISA kit, Levis IgE-ELISA kit (mouse) AKRIE-10 (Shiba Goat Co., Ltd.) was used for blood IgE quantification, and Mouse IgA Ready-SET-Go! Measurement was performed using ELISA 88-50450 (eBioscience) according to the protocol attached to each kit.

4-2-1. Quantitative result of blood IgE concentration FIG. 6 shows the measurement result of the blood IgE amount by the ELISA method. The blood IgE concentration was 1.18 (μg / ml) in the DNFB (−) group, while it was 12.23 (μg / ml) in the control group, and the blood IgE concentration was significantly increased by DNFB sensitization. Confirmed to rise. On the other hand, 10.82 (μg / ml) in the treatment group 3%, 8.60 (μg / ml) in the treatment group 5%, 8.81 (μg / ml) in the prevention group 3%, and 5% in the prevention group In 10.00 (microgram / ml), it was falling in all compared with the control group.

4-2-2. Quantitative results of blood IgA concentration FIG. 7 shows the results of measuring blood IgA concentration by ELISA. The blood IgA concentration was 50.13 (μg / ml) in the DNFB (−) group, whereas it was 29.69 (μg / ml) in the control group, and DNFB sensitization was contrary to blood IgE. As a result, it was confirmed that the blood IgA concentration significantly decreased. On the other hand, 36.80 (μg / ml) in the treatment group 3%, 43.01 (μg / ml) in the treatment group 5%, 48.03 (μg / ml) in the prevention group 3%, and 5% in the prevention group In 49, it was 49.63 (microgram / ml), and it increased significantly in all.

4-3. Based on the results of the above-described verification of the systemic allergic reaction inhibitory effect, the suppression of IL-4 expression in the spleen suppressed the class switch from IgM to IgE, and conversely promoted the class switch to IgA. This suggested that the blood IgE concentration decreased.
Usually, allergic symptoms are caused by excessive IgE production, but it has been shown that by ingesting a lactic acid bacteria-producing substance, the allergic symptoms can be suppressed even at the whole body level.

5. Statistical processing and test method In each graph shown in FIGS. 1 to 7, all measured values are shown as mean ± standard deviation. A t-test was used as a statistical test method, and a two-sided test was performed with the control group. In each graph, the case where p <0.05 was determined to be significant, p <0.05 was indicated by *, and p <0.01 was indicated by **.

Claims (18)

A lactic acid bacterium belonging to Lactobacillus (Lactobacillus) genus, and lactic acid bacteria belonging to the Lactococcus (Lactococcus) genus, a Enterococcus (Enterococcus) method for producing a lactic acid producing substances that Force culturing lactic acid bacteria belonging to the genus
A method for producing a lactic acid bacterium-producing substance comprising a pre-culturing step of combining two, three or four or more types of lactic acid bacteria selected from the lactic acid bacteria to form a plurality of groups in which the lactic acid bacteria contained in the combination are different and co-culturing each group . To create a group that contains the Lactobacillus (Lactobacillus) lactic acid bacteria belonging to the lactic acid bacteria and Lactococcus (Lactococcus) species belonging to the genus, to create a group that contains the Lactococcus (Lactococcus) lactic acid bacteria and lactic acid bacteria belonging to Enterococcus (Enterococcus) genus belonging to the genus Enterococcus ( The method for producing a lactic acid bacterium-producing substance according to claim 1, further comprising a pre-culturing step in which a group containing lactic acid bacteria belonging to the genus Enterococcus ) and a lactic acid bacterium belonging to the genus Lactobacillus is formed, and co-cultured in each group. As lactic acid bacteria belonging to the genus Lactobacillus, L. Lactobacillus acidophilus , L. Brevis ( Lactobacillus brevis ), L. Lactobacillus casei , L. Lactobacillus delbrueckii , L. Lactobacillus delbrueckii subsp. Bulgaricus , L. Gasseri ( Lactobacillus gasseri ), L. Lactobacillus helveticus , L. Paracasei subspecies paracasei (Lactobacillus paracasei subsp. Paracasei), L. Plantarum ( Lactobacillus plantarum ), L. Lmonobasus ( Lactobacillus rhamnosus ), L. Lactobacillus salivarius is used, and lactic acid bacteria belonging to the genus Lactococcus are L. Lactococcus garvieae , L. Lactococcus lactis subsp. Lactis is used as a lactic acid bacterium belonging to the genus Enterococcus. Enterococcus durans , E.I. Enterococcus faecalis , E. The method for producing a lactic acid bacterium-producing substance according to claim 1 or 2, wherein any one of fesium ( Enterococcus faecium ) is used.   E. above. The method for producing a lactic acid bacterium-producing substance according to any one of claims 1 to 3, further comprising a pre-culturing step for forming a group containing fesium and the lactic acid bacterium belonging to the genus Lactobacillus, and coculturing.   E. above. The method for producing a lactic acid bacterium-producing substance according to any one of claims 1 to 4, further comprising a pre-culturing step in which a group containing Durance and the lactic acid bacteria belonging to the genus Lactobacillus is formed and co-cultivated.   E. above. Fesium and the aforementioned E.I. The method for producing a lactic acid bacterium-producing substance according to any one of claims 1 to 5, further comprising a pre-culturing step of forming a group containing Durance and coculturing.   E. above. Fesium and the aforementioned E.I. A group including Durance is created, and separately from the above E.E. Fesium and the aforementioned E.I. Durance and L. The method for producing a lactic acid bacterium-producing substance according to any one of claims 1 to 6, further comprising a pre-culturing step of forming a group containing galviae and cocultivating each group.   E. above. The method for producing a lactic acid bacterium-producing substance according to any one of claims 1 to 7, further comprising a pre-culturing step in which a plurality of groups containing different lactic acid bacteria including fesium and different from each other are formed and cocultured.   The pre-culturing step is the same as the E. Fesium and the aforementioned E.I. Durance and L. The method for producing a lactic acid bacteria-producing substance according to any one of claims 1 to 8, which is carried out without forming a group containing galvia.   The method for producing a lactic acid bacterium-producing substance according to any one of claims 1 to 8, comprising a step of fermenting soy milk.   The method for producing a lactic acid bacterium-producing substance according to any one of claims 1 to 10, wherein soybeans stored at ice temperature are used as a raw material.   The method for producing a lactic acid bacterium-producing substance according to any one of claims 1 to 11, wherein a main culturing step in which co-culturing including all lactic acid bacteria to be used is performed after the preliminary culture.   The method for producing a lactic acid bacterium-producing substance according to claim 12, comprising a pulverization treatment step of pulverizing the culture solution obtained in the main culture step.   The method for producing a lactic acid bacterium-producing substance according to claim 12 or 13, further comprising a filtration step of filtering the culture solution obtained in the main culturing step.   A lactic acid bacteria-producing substance containing a filtrate obtained by the filtration step as an active ingredient.   The lactic acid bacterium-producing substance according to claim 15, wherein the filtrate contains the soybean component and the bacterial component of the lactic acid bacterium.   The lactic acid bacteria production substance of Claim 15 or Claim 16 in which the said filtrate contains the microbial cell component derived from the dead bacteria of the said lactic acid bacteria. The lactic acid bacteria-producing substance according to claim 15, wherein the filtrate does not contain viable bacteria of the lactic acid bacteria.

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