JP6016326B2 - Screening method for lactic acid bacteria - Google Patents

Description

The present invention relates to a screening method of lactic acid bacteria, in particular lactic acid bacteria related to a method for screening a lactic acid bacterium having antiviral activity by measuring the polysaccharide amount, etc. produced.

  Today, human beings are constantly being exposed to new virus threats, including the new influenza that is rampant around the world. In particular, respiratory infections such as influenza, including seasonal ones, are present in everyday situations where affected individuals are present in the surrounding area. Cheap. In addition, there is a high possibility of falling seriously, and the mortality rate associated with complications is very high.

  Currently, antiviral drugs that act on the virus itself and have an effect are used against such viral respiratory infections. These antiviral drugs are extremely effective against each viral infection, and the symptoms can be quickly alleviated by taking them early after onset. In recent years, neuraminidase inhibitors have been developed that inhibit neuraminidase proteins on the surface of viruses and prevent their growth in the body. Among them, as effective against influenza A and influenza B, osetamivir (trade name: Tamiflu; Tamiflu is a registered trademark) prescribed as an oral capsule and zanamivir (trade name: Relenza; Relenza) A registered trademark is used in practice. However, these are drugs that suppress the growth of the virus and need to be taken early after infection (within about 48 hours after symptoms appear). In addition, it must be taken continuously for a certain period.

  Prevention by vaccination is also carried out. However, vaccines must be manufactured by specifying the target virus species in advance, and mass production takes a very long time. In addition, vaccination has the only effect of reducing the risk of increasing severity after infection. Furthermore, it takes a certain period of time for the antibody to become established, and it must be vaccinated before it becomes epidemic. Furthermore, if the type of vaccine inoculated differs from the type of virus that is actually infected, it can be fully effective. Can not.

  Under such circumstances, it is urgent to develop antiviral agents that are not only safe but also effective when taken in a preventive manner, and that are effective against any virus when taken. Yes.

  On the other hand, in order to cope with the threat of viruses, it is also important to increase the natural immunity that normally prevents the invasion and proliferation of viruses into the living body at the forefront. White blood cells play a particularly important role in innate immunity. Among leukocytes, NK cells (also referred to as natural killer cells) spontaneously attack infected cells while the virus infection is small, and suppress the proliferation and spread of the virus.

  In vivo, when a virus infection is detected, interferon (hereinafter referred to as IFN) is produced in immune cells and virus-infected cells. This IFN has its own antiviral activity and antitumor activity, while IFN-γ enhances innate immunity, and activation of NK cells can suppress viral growth and prevent the spread of infection. Since NK cells are effective not only for specific viruses such as vaccines but also for a wide variety of viral infections, the overall immune activity is increased by inducing the production of IFN-γ. It can also be said that it can be (immunostimulatory effect).

So far, substances that enhance the immunostimulatory effect have been searched for in various ways. For example, Patent Document 1 discloses a method in which Lactococcus lactic acid cocci are co-cultured with dendritic cells or spleen cells and selected based on the ability to induce IL-18 production.
Patent Document 2 also adsorbs specific components from the mycelium of matsutake mushrooms using an anion exchange resin, and selects a bacterial infection prevention / treatment agent based on the sugar content by the phenol sulfate method and the protein content by the copper ferrin method. Is disclosed.

JP 2005-154387 A Japanese Patent Laid-Open No. 2004-210694

  As described above, substances that enhance various immunostimulatory effects have been searched for, and among them, food microorganisms such as lactic acid bacteria used for yogurt fermentation are known to be able to produce various useful substances. . If these useful substances were effective in enhancing the immunostimulatory effect, it was considered that they can be safely and easily ingested in daily life and enhanced immunity is expected. However, a method capable of selecting an effective lactic acid bacterium capable of obtaining a sufficient activity promoting effect has not been found.

Accordingly, an object of the present invention is to provide a screening method capable of selecting lactic acid bacteria effective for enhancing the immunostimulatory effect by a simple technique.

  In view of the above-mentioned conventional problems, the present inventors have conducted extensive research and have focused on a method of measuring and comparing the amount of polysaccharide produced by lactic acid bacteria by a method such as the phenol-sulfuric acid method. As a result of examining the relationship between the measured value of the production amount and the IFN-γ production-inducing activity of the polysaccharide, it has been found that lactic acid bacteria effective for enhancing the immunostimulatory effect can be selected according to specific criteria, and the present invention. It came to complete.

That is, the present invention provides: (A) a standard value ( appropriately determined within a range of 110 to 170 mg per 1 kg of the culture polysaccharide produced by the phenol-sulfuric acid method among a plurality of strains of lactic acid bacteria ; For example, from the step of selecting a lactic acid bacteria strain of 120 mg / kg) or more, and from the lactic acid bacteria strain selected in (B) step (A), the measurement value of the amount of polysaccharide produced by purification is 1 kg of culture. From the step of selecting a lactic acid bacteria strain that is a reference value (for example, 120 mg / kg) or more determined within the range of 110 to 170 mg per day, and (C) from among the lactic acid bacteria strains selected in step (B), these strains based on measurements of the IFN-gamma production inducing activity of polysaccharide produced is the screening method of lactic acid bacteria, which comprises a step, a of selecting a strain of high antiviral activity lactobacilli

  In the screening method, the lactic acid bacterium is Lactobacillus delbrueckii ssp. bulgaricus (Lactobacillus delbruecki subspecies bulgaricus) is preferred.

According to the screening method for lactic acid bacteria of the present invention, lactic acid bacteria having high antiviral activity (hereinafter also referred to as lactic acid bacteria of the present invention) are provided.

In addition, according to the screening method for lactic acid bacteria of the present invention, for example, an antiviral agent comprising an effective amount of lactic acid bacteria (hereinafter also referred to as the antiviral agent of the present invention) is provided.

  In the antiviral agent, the lactic acid bacterium is Lactobacillus delbrueckii ssp. bulgaricus is preferred.

Moreover, according to the screening method of the lactic acid bacteria of this invention, the antiviral food-drinks composition (henceforth the food-drinks composition of this invention) containing an effective amount of lactic acid bacteria is provided, for example.

  In the antiviral food and drink composition, the lactic acid bacteria are Lactobacillus delbrueckii ssp. bulgaricus is preferred. The antiviral food / beverage composition is preferably a fermented milk food / beverage product, and particularly preferably a yogurt food / beverage product such as yogurt or yogurt drink.

The strain of lactic acid bacteria having high antiviral activity selected by the screening method for lactic acid bacteria of the present invention can enhance the natural immunity of those who have ingested the lactic acid bacteria. Possible preventive effects can be given. The selected lactic acid bacteria are highly safe, exhibit an effect when taken in a preventive manner, and can provide an immunostimulatory activity that is effective against any virus when taken.

FIG. 3 is a graph showing the amount of polysaccharide produced by Lactobacillus delbruecki subspecies bulgaricus (measured value by the phenol-sulfuric acid method) as a result of Example 1. As a result of Example 2, it is the graph which showed the polysaccharide production amount (measurement value) by refinement | purification about the top 10 strain with much polysaccharide production amount (measurement value) in Example 1. FIG. FIG. 3 is a graph showing the IFN-γ production inducing activity of the polysaccharides of the top three strains with the highest polysaccharide production (measured value) in Example 2 as a result of Example 3.

  Hereinafter, the present invention will be described in detail, but the present invention is not limited to the individual forms described below.

The phenol-sulfuric acid method used in the present invention is a generally known method used for quantification of saccharides.
Although this invention is not limited to this, For example, the following reagents are used.
-Phenol reagent (eg 5% (W / V))
Concentrated sulfuric acid / standard monosaccharide aqueous solution (eg, aqueous solution of glucose, etc.)
Examples of the measuring method include the following procedures. A predetermined amount of phenol reagent is added to a predetermined amount of the sample aqueous solution and stirred. A predetermined amount of concentrated sulfuric acid is added to this and stirred well. After cooling at room temperature, the absorbance at 490 nm is measured with a spectrophotometer. By measuring the standard monosaccharide aqueous solution in the same manner and comparing it with the result of the sample aqueous solution, the contained saccharide can be quantified.

In the present invention, a lactic acid bacterium to be screened is cultured by a known method, a saccharide (particularly a polysaccharide) produced from the culture solution is separated by a known method, and the polysaccharide (Exopolysaccharide, EPS) outside this microbial cell is phenol. -Quantify by sulfuric acid method. In the present invention, this quantitative value is used as a measurement value of the amount of polysaccharide produced by the phenol-sulfuric acid method .
As a method for separating polysaccharide from the culture medium, for example, a method of performing step a) to d) below in this order.
a) Deproteinization using trichloroacetic acid,
b) precipitating the polysaccharide by ethanol precipitation,
c) Dialysis against water using a dialysis membrane in the obtained polysaccharide aqueous solution,
d) An aqueous solution on the polymer side is obtained.
However, the steps such as deproteinization, ethanol precipitation, and dialysis can be performed by appropriately adjusting the conditions according to lactic acid bacteria, culture solution, culture conditions and the like. Moreover, as for the dialysis membrane to be used, a fractional molecular weight (MWCO) of 3500 to 5000 daltons can be exemplified, but other fractional molecular weights can also be used.

Further, in the present invention that purification of the extracellular polysaccharide from the culture (hereinafter, also referred to as a purified polysaccharide.) Were weighed to measure the production polysaccharide amount. In the present invention, this weighed value is a measured value of the amount of polysaccharide produced by purification. Examples of the method for purifying the exopolysaccharide from the culture solution include a method of performing the following steps a) to i) in this order.
a) Deproteinization using trichloroacetic acid,
b) precipitating the polysaccharide by ethanol precipitation,
c) Dialysis against water using a dialysis membrane in the obtained polysaccharide aqueous solution,
d) Deoxyribonuclease I (Type II, derived from bovine pancreas, Enzyme Commission (EC) Number 3.1.21.1, Cas number 9003-98- in Tris-HCl buffer (pH 8.0) with respect to the aqueous solution on the polymer side. 9) and ribonuclease A (RNase A, derived from bovine pancreas, Enzyme Commission (EC) Number 3.1.27.5, Cas number 9001-99-4) and incubated at 37 ° C. for 6 hours (hr) Digestion,
e) Proteinase K (endopeptidase K, derived from Tritirachium album, Commission (EC) Number 3.4.21.64, Cas number 39450-01-6) was added and incubated at 37 ° C. for 16 hours to digest the protein.
f) The enzyme is deactivated by heating at 90 ° C. for 10 minutes (min),
g) Precipitating the polysaccharide by ethanol precipitation,
h) Dialyzing the aqueous solution of the obtained polysaccharide against water using a dialysis membrane to obtain an aqueous solution on the polymer side,
i) Dry the aqueous solution on the polymer side and weigh it to obtain a measurement of the amount of polysaccharide produced by purification.
However, the steps such as deproteinization, ethanol precipitation, dialysis, nucleic acid digestion, protein digestion and the like can be performed by appropriately adjusting the conditions according to lactic acid bacteria, culture solution, culture conditions and the like. Moreover, as for the dialysis membrane to be used, a fractional molecular weight (MWCO) of 3500 to 5000 daltons can be exemplified, but other fractional molecular weights can also be used. In addition, deoxyribonuclease, ribonuclease and proteinase are not limited to the above examples, and other ones can be used .

In the present invention, from the lactic acid bacteria whose measured value of the amount of produced polysaccharide by the phenol-sulfuric acid method is a reference value (for example, 120 mg / kg) or more, the measured value of the amount of produced polysaccharide by purification (for example, 120 mg as the reference value). Lactic acid bacteria are selected based on the magnitude of the measured value of the IFN-γ production-inducing activity of the polysaccharide from the selected lactic acid bacteria. singles.
In this case, a lactic acid bacterium having a polysaccharide amount determined by the phenol-sulfuric acid method of preferably 120 mg / kg or more, more preferably 170 mg / kg or more is selected. Note that the reference value here is not limited to 120 mg / kg and 170 mg / kg. In the present invention, a value appropriately determined within the range of 110 to 170 mg / kg (for example, 110, 120, 130, 140, 150, 160 or 170 mg / kg) can be employed.
In addition, lactic acid bacteria having a polysaccharide amount determined by the purification of preferably 120 mg / kg or more, more preferably 170 mg / kg or more are selected. In addition, the reference value here is not limited to 120 mg / kg and 170 mg / kg. In the present invention, values appropriately determined within the range of 110 to 170 mg / kg (for example, 110, 120, 130, 140, 150, 160 or 170 mg / kg) can be employed.
The reference value in the phenol-sulfuric acid method and the reference value in the purification may be the same or different. For example, when the reference value in the phenol-sulfuric acid method is 120 mg / kg, the reference value in purification may be 120 mg / kg or another value (for example, 140 mg / kg).

Further, in the present invention, the phenol - lactic acid bacteria with the polysaccharide amount is therefore determined in sulfuric acid method and purification is not less than a predetermined amount and said polysaccharide material is IFN-gamma production inducing activity is selected.
The IFN-γ production-inducing activity of the exopolysaccharide can be measured, for example, by the following method.
According to a conventional method, spleen cells of C3H / HeJ mice are suspended in a medium, and a polysaccharide obtained from the cell culture supernatant is added thereto. The spleen cells are cultured under predetermined conditions, the culture supernatant is collected, and IFN-γ contained in the culture supernatant is added to a predetermined kit (for example, trade name: BD OptEIA ^™ Mouse IFN-γ ELISASet (BD OptEIA is The production amount of IFN-γ induced by the polysaccharide can be obtained by measurement according to BD Pharmingen, catalog number 555138)). By comparing this with the control, IFN-γ production-inducing activity can be determined. In this invention, what refine | purified the exopolysaccharide, the said refinement | purification polysaccharide, etc. can be mentioned as a measuring object of IFN-gamma production induction activity.

The lactic acid bacterium used in the present invention is not limited as long as it is a lactic acid bacterium producing a polysaccharide. In particular, as lactic acid bacteria, Lactobacillus delbrucky subspecies bulgaricus, Lactococcus lactis ssp. Cremoris, etc. are preferable, and selected from these. Examples of lactic acid bacteria selected by the method of the present invention include Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (deposit number: FERM P-17227, international deposit number: FERM BP-10741) (hereinafter also referred to as 1073R-1 strain) was found to be particularly useful as an antiviral agent.
The inventors have reported that Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 was deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. The contents for specifying the deposit are described below.
(1) Depositary institution name: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (2) Contact: 305-8566
Tsukuba City, Ibaraki Pref.
(3) Accession number: FERM BP-10741
(4) Display for identification: Lactobacillus delbrueckii subsp. Bulgaricus OLL 1073R-1
(5) Date of original deposit: February 22, 1999 (6) Date of transfer to deposit under the Budapest Treaty: November 29, 2006 In addition, the strain described as JCM in the strain name is RIKEN The reference strains obtained from the Laboratory for Microbial Materials at the Institute for Bioresources are shown below.

  The lactic acid bacteria used in the antiviral agent of the present invention are its culture, concentrate, pasted product processed into a paste, spray dried product, freeze dried product, vacuum dried product, drum dried product, liquid dispersed in a medium. Product, a diluted product diluted with a diluent, or a crushed product obtained by crushing a dried product with a mill or the like. The treatment process may be performed alone or in combination.

The dosage of the antiviral agent containing the lactic acid bacterium of the present invention as an active ingredient can be appropriately set in consideration of various factors such as the route of administration and the age, weight, and symptoms of animals to be administered including humans.
Although the present invention is not limited thereto, it is preferable to administer at least 9 × 10 ⁹ cfu / body / day as an active ingredient. However, when it is taken for the purpose of prevention and / or treatment over a long period of time, the amount may be smaller than the above range, and this active ingredient has a problem with respect to safety as a normal yogurt food. There is no problem even if the dose is higher than the above range.

The antiviral agent comprising the lactic acid bacterium of the present invention as an active ingredient can be administered either orally or parenterally (intramuscular, subcutaneous, intravenous, suppository, transdermal, etc.).
The dosage form of the antiviral agent comprising the lactic acid bacterium of the present invention as an active ingredient can be selected depending on the therapeutic purpose, administration route, etc., for example, tablets, coated tablets, pills, capsules, granules , Powders, solutions, suspensions, emulsions, syrups, injections, suppositories, dip agents, decoction, tinctures and the like. These various preparations are prepared according to conventional methods as necessary with respect to the active ingredient, such as fillers, extenders, excipients, binders, humectants, disintegrants, surfactants, lubricants, coloring agents, flavoring agents. It can be formulated using known adjuvants (hereinafter, also referred to as formulation adjuvants) that can be generally used in the pharmaceutical formulation technical field, such as solubilizing agents, suspension agents, and coating agents. Moreover, you may contain a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, etc. and other pharmaceuticals in this pharmaceutical formulation.

  The food / beverage product composition of the present invention comprises an effective amount of lactic acid bacteria which are antiviral agents. The form of the food / beverage product composition can be applied to fermented milk such as yogurt, beverages, etc., as well as health functional foods and foods for the sick. The health functional food system was established not only for regular foods but also for foods in the form of tablets, capsules, etc., based on domestic and foreign trends and consistency with the conventional food system for specified health use. It consists of two types of food for specified health use (individual permission type) and functional food for nutrition (standard type). By directly ingesting as a special-purpose food such as a food for specified health use containing a lactic acid bacterium, which is an antiviral agent of the present invention, or a functional food, it is possible to prevent and / or treat viral infection.

  Typical examples of the food and beverage composition of the present invention include various foods and beverages (for example, milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crusts, prepared milk powder, liquid foods, food for the sick. Foods such as infant milk powder, foods such as milk powder for nursing women, nutritional foods, nutritional supplements, etc.) to which lactic acid bacteria as antiviral agents are added. The active ingredient can be used as it is, or can be used in accordance with conventional methods in ordinary food compositions such as mixing with other foods or food ingredients. Moreover, about the property, the state of the food / beverage products normally used, for example, any of solid (powder, granule, etc.), paste, liquid or suspension may be sufficient.

Other ingredients are not particularly limited, but the food and beverage composition containing lactic acid bacteria that are antiviral agents of the present invention include water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, Fruit juice, flavors, etc. can be used as a main component.
Examples of the protein or its degradation product include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein. , Β-lactoglobulin, α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and plant proteins, hydrolysates thereof, whey, cream, non-protein nitrogen and other milk-derived components .
Examples of the saccharide include saccharides, processed starch (in addition to text phosphorus, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and lactose.
Examples of lipids include various milk-derived components such as butter, cream, and phospholipid; animal oils such as lard, fish oil, fractionated oils, hydrogenated oil, transesterified oil; palm oil, safflower oil, corn Examples thereof include vegetable oils such as oil, rapeseed oil, coconut oil, fractionated oils thereof, hydrogenated oils and transesterified oils.
Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. And folic acid.
Examples of the minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, and whey mineral.
Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid.
These components can be used in combination of two or more. Moreover, the main component of the food-drinks composition of this invention may be a synthetic product, and may contain many synthetic products.

The amount of the lactic acid bacterium that is the main component of the antiviral agent of the present invention can be arbitrarily determined according to its purpose and use, and is not particularly limited, but is usually based on the total amount of the antiviral agent or the food and beverage composition. 1 × 10 ⁸ cfu / g or more is preferable.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated, this invention is not limited by this. In addition, in this specification,% display shows weight%, when not showing clearly.

Example 1 [Measurement of amount of produced polysaccharides of various lactic acid bacteria by phenol-sulfuric acid method]
The amount of polysaccharide produced from various lactic acid bacteria (140 strains) was measured by the following method.
First, a 10 w / v% nonfat dry milk medium without yeast extract was prepared, and 4 ml was dispensed into a 15 ml conical tube. Next, after each lactic acid bacterium was activated twice, the dispensed medium was inoculated with an amount of 1 v / v% (40 μl) and statically cultured at 37 ° C. for 18 hours under aerobic conditions. After completion of the culture, 400 μl of 100 w / v% trichloroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) is added to the culture solution (10 v / v% of the culture, the final concentration of trichloroacetic acid after addition to the culture is about 10 w / v) %) And stirred until the viscosity disappeared to remove proteins. The supernatant was obtained by centrifugation (12000 g, 4 ° C., 20 min), and the whole amount was transferred to a new 15 ml conical tube. MilliQ water was added to the collected supernatant, and the volume was increased to 4 ml using a conical tube scale. 8 ml (2 volumes) of cold ethanol was gradually added and mixed thoroughly, and then allowed to stand overnight at 4 ° C. to precipitate the polysaccharide. The polysaccharide was recovered as a precipitate by centrifugation, 4 ml of MilliQ water was added, and completely dissolved by voltex. Dispos 96-well dialyzer (trade name: 96-well Dispo DIALYZER ^™ (96-well Dispo DIALYZER is a registered trademark), 25-300 μl / well; MWCO 5000 Dalton dialysis membrane, manufactured by HARVARD, Model No. 74 -0902), the aqueous polysaccharide solution was dialyzed against MilliQ water for 2 days. After completion of dialysis, the polysaccharide concentration of the aqueous solution on the polymer side was measured by the phenol-sulfuric acid method, and the amount (mg) of polysaccharide per kg of culture was calculated.

In FIG. 1, the obtained result is shown as a measured value of the produced polysaccharide by the phenol-sulfuric acid method.
As a result of quantification by the phenol-sulfuric acid method, Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (hereinafter also referred to as 1073R-1 strain) was found to have the second largest production of polysaccharides.

Example 2 [Purification of polysaccharides from cultures of the top 10 producing polysaccharides]
In order to examine the immunostimulatory activity of the produced polysaccharide for the top 10 strains among the production amounts of 140 strains of lactic acid bacteria obtained by measuring the amount of produced polysaccharide by the method of Example 1, the polysaccharide was obtained by the following method. Was isolated and purified.
First, each lactic acid bacterium was activated twice and then cultured in 600 ml of each medium. The medium was a 10% nonfat dry milk medium containing no yeast extract, and cultured at 37 ° C. for 18 hours under aerobic conditions. After completion of the culture, 500 g of each culture solution is weighed and 50 ml of 100 w / v% trichloroacetic acid (manufactured by Wako Pure Chemical Industries, Ltd.) (10 v / w% of the culture solution in the final amount after addition to the culture). The concentration was about 10 w / w%), and the mixture was stirred until the viscosity disappeared to remove proteins. The supernatant was collected by centrifugation (12000 g, 4 ° C., 20 min), twice the amount of cold ethanol was added thereto, and allowed to stand at 4 ° C. overnight to precipitate the polysaccharide. The polysaccharide was recovered as a precipitate by centrifugation (12000 g, 4 ° C., 20 min), and was completely dissolved by adding MilliQ water.
The polysaccharide aqueous solution was placed in a MWCO 3500 Dalton dialysis tube and dialyzed against MilliQ water for 3 days. Thereafter, a Tris-HCl aqueous solution, an MgCl ₂ aqueous solution, and water were added to the aqueous solution on the polymer side to obtain a 0.05 M Tris-HCl (pH 8.0), 1 mM MgCl ₂ solution. Here, deoxyribonuclease I (DNase I, Type II, derived from bovine pancreas, Enzyme Commission (EC) Number 3.1.21.1, Cas number 9003-98-9, manufactured by Sigma), and ribonuclease A (RNase A, RNase A, at a final concentration of 2 μg / ml) Nucleic acid was digested by adding bovine pancreas-derived Enzyme Commission (EC) Number 3.1.27.5, Cas number 9001-99-4, manufactured by Sigma) and incubating at 37 ° C. for 6 hr. Then, proteinase K (Proteinase K, also known as endopeptidase K, derived from Tritirachium album, Commission (EC) Number 3.4.21.64, Cas number 39450-01-6, manufactured by Sigma) was added at a final concentration of 0.1 mg / ml at 37 ° C. The protein was digested by incubating for 16 hr. After completion of the enzyme treatment, the enzyme was inactivated by heating at 90 ° C. for 10 minutes.
A double amount of cold ethanol was added to the polysaccharide aqueous solution after the enzyme treatment, and the polysaccharide was precipitated again by allowing it to stand at 4 ° C. overnight. The polysaccharide was recovered as a precipitate by centrifugation (12000 g, 4 ° C., 20 min) and dissolved in MilliQ water. This polysaccharide aqueous solution was dialyzed against MilliQ water for 4 days using a MWCO 3500 Dalton dialysis membrane, and then the aqueous solution on the polymer side was freeze-dried (hereinafter also referred to as a purified polysaccharide). This was weighed.

The amount of the purified polysaccharide obtained is shown in FIG.
As is clear from FIG. 2, the amount of the purified polysaccharide is the highest in the 1073R-1 strain. Hereinafter, the strain 1 shown as “A” in FIG. 2 and the strain 2 shown as “G” in FIG. It was found that the amount of polysaccharide was large.

Example 3 [Measurement of IFN-γ production inducing activity of spleen cells of mouse C3H / HeJ by purified polysaccharides produced by each of the top three strains having a large amount of the purified polysaccharide of Example 2]
Spleen cells were prepared from 5 mice C3H / HeJ (8 weeks old, male, SLC) and suspended in RPMI 1640 medium at 1 × 10 ⁷ cells / ml. Polysaccharides were lyophilized at 1 mg / ml dissolved in RPMI 1640 medium, sterilized with a 0.45 μ filter, and then diluted to various concentrations with RPMI 1640 medium. Spleen cells were applied to a 96-well microplate (Corning; model number 3596) at 5 × 10 ⁵ cells / well (50 μl / well) and a polysaccharide solution was applied at 150 μl / well at 37 ° C. under 5% CO ₂ conditions. The culture was performed for 72 hours. At this time, the final action concentration of the polysaccharide was 10, 50, 100 μg / ml.
After completion of the culture, the culture supernatant was collected. IFN-γ in the culture supernatant was measured using a mouse IFN-γ measurement kit (trade name: BD OptEIA ^™ Mouse IFN-γ ELISA Set, manufactured by BD Pharmingen, catalog number 555138).

As for the polysaccharides, the top three strains (“A” in FIG. 2 (strain 1 in FIG. 3) and “G” (strain in FIG. 3) in which the amount of the purified polysaccharide obtained in Example 2 was large. 2) and “1073R-1 strain” (same name in FIG. 3)).
The obtained results are shown in FIG. As is clear from FIG. 3, a high IFN-γ production-inducing activity was observed in the polysaccharide produced by the 1073R-1 strain.

According to the screening method for lactic acid bacteria of the present invention, by selecting a strain having high IFN-γ production-inducing activity, innate immunity is enhanced, and a prophylactic effect possible before virus infection without a virus specificity such as a vaccine is obtained. Can be found easily and quickly. This selected lactic acid bacterium exhibits an effect by preventive intake, can exert an effect on any virus by taking it, and can provide immunostimulatory activity, and has high industrial utility value.

Claims (4)

(A) from a plurality of strains of lactic acid bacteria, phenol - measure production polysaccharides body weight by sulfuric acid method, per culture 1 kg, the strain of lactic acid bacteria is appropriately determined standard value or more in the range of 110~170mg A process of selecting,
From among the selected lactic acid bacteria strains in step (B) (A), measured value production polysaccharides body weight by purification, per culture 1 kg, of lactic acid is equal to or greater than a reference value determined in the range of 110~170mg Selecting a strain ;
(C) From the strains of lactic acid bacteria selected in step (B), a step of selecting a strain of lactic acid bacteria based on the measured value of the IFN-γ production inducing activity of the polysaccharides produced by these strains ;
A screening method for lactic acid bacteria characterized by comprising: The method for screening lactic acid bacteria according to claim 1 , wherein the measured value of the amount of produced polysaccharide by purification is obtained by performing the following steps a) to i) in this order.
a) Step of deproteinization using trichloroacetic acid b) Step of precipitating polysaccharides by ethanol precipitation method c) Step of dialysis against water using a dialysis membrane in the aqueous solution of the obtained polysaccharides d) High A step of digesting nucleic acids by adding deoxyribonuclease and ribonuclease to the aqueous solution on the molecular side; e) a step of further digesting proteins by adding proteinase; f) a step of inactivating the enzyme; g) by ethanol precipitation Step h) Precipitating polysaccharides Step of dialysis of the aqueous solution of the obtained polysaccharides against water using a dialysis membrane to obtain an aqueous solution on the polymer side i) Drying the aqueous solution on the polymer side, The process of weighing this The method for screening lactic acid bacteria according to claim 1 or 2 , wherein a reference value of each measurement value of the amount of produced polysaccharide in the step (A) and the step (B) is 120 mg / kg. The lactic acid bacterium is Lactobacillus delbrueckii ssp. The method for screening lactic acid bacteria according to any one of claims 1 to 3 , wherein the method is bulgaricus.

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