JP6744542B2 - Method for culturing Lactobacillus Lactobacillus - Google Patents

Description

The present invention relates to a method for culturing Lactobacillus lactic acid bacteria, and particularly to a method for culturing Lactobacillus paracasei (Lactobacillus paracasei) or Lactobacillus paracasei (Lactobacillus paracasei).

As lactic acid bacteria, Lactobacillus rhamnosus (Lactobacillus rhamnosus) KO3 strain (NITE BP-771), Lactobacillus casei (Lactobacillus casei) YU3 strain (NITE BP-772pIacUlusPUaclusLacBluacLus paraIsei paracasei). -775) is known (Patent Document 1). These lactic acid bacteria have a very high antibacterial activity against oral bacteria such as caries bacteria, periodontal disease bacteria, and Candida bacteria. It is described that these lactic acid bacteria may be cultivated in a fruit juice medium, a vegetable juice medium, a milk medium, a skimmed milk powder medium, or a medium containing milk components, but actually, they were cultured in a synthetic medium.

However, considering that the culture is used for foods and the like, it has been required to culture in a medium composed of natural components instead of an artificial medium.

International Publication No. 2011/007584

The present invention aims to provide a new method for culturing Lactobacillus rhamnosus, Lactobacillus casei, or Lactobacillus paracasei.

The present inventors have conducted a study on a natural medium, which is a natural component of Lactobacillus rhamnosus, Lactobacillus casei, or Lactobacillus paracasei. Among them, the present invention has been completed by discovering a new finding that the obtained culture has a very high antibacterial activity when cultured using cereals.

That is, the present invention is to cultivate Lactobacillus lactobacillus in a medium containing a grain lactobacillus, in which Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus casei, or Lactobacillus paracasei is cultivated in a medium containing a grain lactobacillus. The present invention relates to a method for culturing lactic acid bacteria.

The grain is preferably soybean or rice.

It is preferable that the medium further contains a saccharide.

In the culturing method of the present invention, the lactic acid bacterium of the genus Lactobacillus is cultivated in a medium containing grains, so that the culture has a very high bactericidal effect.

1 shows the medium and the amount of ATP of the culture of the Lactobacillus rhamnosus KO3 strain (NITE BP-771) cultured in a milk medium or an MRS medium. A is the result of culture in 12% skim milk powder + 0.5% yeast extract medium, and B is the result of culture in MRS medium. 1 shows the medium and ATP amount of the culture of the Lactobacillus rhamnosus KO3 strain (NITE BP-771) cultured in a grain medium. C is the result of culturing with 10% soybean peptide + 2% glucose, D is the result of culturing with 10% soybean peptide + 2% arabinose, and E is the result of culturing with 10% rice peptide + 2% glucose.

The method for culturing a lactic acid bacterium of the genus Lactobacillus of the present invention comprises a grain containing a medium containing Lactobacillus paracasei (Lactobacillus paracasei) or a culture medium containing Lactobacillus paracasei (Lactobacillus paracasei). It is characterized by having a process.

Among the genus Lactobacillus rhamnosus, the KO3 strain (NITE BP-771) is a type of lactic acid bacterium isolated from human saliva, as described in International Publication No. 2011/007584. , Those that have been internationally deposited. The nucleotide sequence of 16S rRNA exhibits 100% homology between the nucleotide sequence of Lactobacillus rhamnosus strain IDCC3201 and 1485/1485, and exhibits a Gram-positive rod-like appearance under a microscope after Gram staining. It has been identified as Lactobacillus rhamnosus. The main bacteriological properties of the KO3 strain are shown below.
1) Gram-positive lactobacillus 2) Homo-type lactic acid fermentation 3) Negative catalase 4) No ability to form spores 5) Cultivation under aerobic conditions 6) Production of extracellular polysaccharides

Among the genus Lactobacillus casei, the YU3 strain is isolated from human saliva as described in WO 2011/007584, and the nucleotide sequence of 16S rRNA is Lactobacillus. -Lactobacillus casei (Lactobacillus casei) shows 100% homology between the nucleotide sequence of Lactobacillus casei ATCC 334 and 1485/1485 and exhibits the appearance of a clam-positive bacillus under a microscope after Gram staining. casei). The main mycological properties of the YU3 strain are shown below.
1) Gram-positive lactococcus 2) Homo-type lactic acid fermentation 3) Negative catalase 4) No spore-forming ability 5) Can be cultured under aerobic conditions 6) Produces extracellular polysaccharides

Among Lactobacillus paracasei (Lactobacillus paracasei), the YU4 strain was isolated from human saliva as described in WO 2011/007584, and the nucleotide sequence of 16S rRNA was Lactobacillus paracasei. Paracasei (Lactobacillus paracasei) strain shows the homology of Partial Sequence of DJ1 16S ribosomal RNA gene with 1477/1477 (100%) and shows the appearance of a Gram-positive bacillus under the microscope after Gram staining. (Lactobacillus paracasei). The main mycological properties of the YU4 strain are shown below.
1) Gram-positive lactococcus 2) Homo-type lactic acid fermentation 3) Negative catalase 4) No spore-forming ability 5) Can be cultured under aerobic conditions 6) Produces extracellular polysaccharides

The culturing method is not particularly limited as long as a medium containing cereals is used as the medium, and the lactic acid bacterium culturing method well known to those skilled in the art can be applied as it is. Examples of grain media include rice, beans, wheat, corn and the like. Not only the grains themselves, but also processed products such as rice can be used. Examples of the beans include soybean, red beans, peas, and beans, and examples of the wheat include wheat, barley, rye, and the like. Grain-containing media, unlike synthetic media such as milk media, give clear cultures. The content of grains in the medium is preferably 0.1 to 10% by weight, more preferably 2 to 10% by weight.

The culture method is not particularly limited as long as the medium has grains, and examples thereof include static culture, neutralization culture at a constant pH, batch culture, continuous culture, and the like.

If the medium has grains, it may be mixed with a fruit juice medium, a vegetable juice medium, a milk medium, a skimmed milk powder medium or a milk component. Examples of such a medium include a reduced skim milk medium obtained by reducing skim milk by heat sterilization, a skim milk powder medium containing yeast extract, an MRS medium, a GAM medium and the like. The culturing method is not particularly limited as long as the cells can grow well, such as static culturing or neutralization culturing at a constant pH, batch culturing and continuous culturing. If necessary, a fermentation promoting substance, for example, a carbon source such as sugar, starch, dextrin, a nitrogen source such as yeast extract and peptone, vitamins, minerals and the like can be added. Examples of saccharides include glucose, arabinose, sucrose, lactose, sorbitol, fructose and the like. Above all, it is preferable to contain glucose, arabinose and the like. When saccharides are used, the amount used is preferably 0.1 to 10% by weight in the medium, more preferably 0.2 to 5% by weight.

The culture temperature is not particularly limited, but is preferably 20 to 42°C, more preferably 25 to 37°C. If it exceeds 42°C, the culture tends to be impossible, and if it is less than 20°C, the culture time tends to be long.

Although the culture time is not particularly limited, it is preferably 24 to 500 hours, more preferably 24 to 240 hours, and further preferably 72 to 240 hours. A long period of culture is required as compared with the culture time of yogurt or the like.

The atmosphere during culturing is not limited, but aerobic conditions are preferable.

The culture obtained by the culturing method of the present invention can be used as it is, or various solvent extracts obtained by solvent extraction of bacterial cells or bacterial cell cultures, diluted solutions thereof, concentrated solutions thereof, or dried solutions thereof. It can also be used as a powder.

As the extraction solvent used for obtaining the extract, either a polar solvent or a non-polar solvent can be used, and these can also be mixed and used. For example, water; alcohols such as methanol, ethanol, propanol, butanol; polyhydric alcohols such as propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; tetrahydrofuran, diethyl ether Chain-like and cyclic ethers such as; polyethers such as polyethylene glycol; hydrocarbons such as hexane, cyclohexane and petroleum ether; aromatic hydrocarbons such as benzene and toluene; pyridines and the like. Esters such as ethyl acetate and alcohols such as ethanol are preferable.

The extraction conditions vary depending on the solvent used, but for example, 1 to 10 parts by mass of the solvent is used with respect to 1 part by mass of the culture solution, at a temperature of 0 to 50° C., preferably 25 to 37° C. It is preferable to extract for 3 hours to 3 hours.

The extract can be used as it is, but after the extract is diluted, concentrated or lyophilized, it can be used in the form of powder or paste, if necessary. Further, it can be used after being appropriately purified by a technique such as liquid-liquid distribution.

The culture can be used for various purposes. For example, it may be used for oral diseases and cosmetics such as deodorant.

The oral disease is a preventive, ameliorating or therapeutic agent for oral diseases, and refers to oral diseases caused by caries bacterium, periodontitis bacterium and Candida, for example, caries; gingivitis, Periodontal diseases such as periodontitis, glossitis, oral thrush such as thrush, stomatitis and the like. Examples of the cariogenic bacteria include Streptococcus mutans, Streptococcus sobrinus, and periodontitis bacteria Porphyromonas tereplasma gingivalis (Porphyromonas terbella vulgaris).・Denticola (Treponema denticola), Tannerella forsythensis (Tanneraella forsythensis), actinobacillus actinomycetes bacillus Fructus bacillus actinomycetes bacillus, cinnamon bacillus, and so on. (Candida albicans), Candida glabrata, Candida tropicalis and the like.

The lactic acid bacteria obtained by the culturing method of the present invention include Streptococcus mutans and Streptococcus sobrinus which are caries fungi, and Porphyromonas gingivalis porgyroisma (Porphyromonas gingivalis) that are periodontitis fungi. It has a growth inhibitory effect on any of Candida albicans which is a bacterium. Also known as Fusobacterium pneumoniae known as subgingival plaque former in the oral cavity. It has an action of suppressing the growth of Fusobacterium bacterium such as nucleatum.

Therefore, the bacterial cells or bacterial cell cultures of the lactic acid bacteria or extracts thereof can be used as a preventive, ameliorating or therapeutic agent for oral diseases, or as a growth inhibitor for caries, periodontitis and Candida. Such a preventive, ameliorating or therapeutic agent for oral diseases, a caries bacterium, a periodontitis bacterium and a growth inhibitor of Candida bacterium, is itself caused by a pathogenic microorganism in the oral cavity, caries, periodontal disease , For preventing, improving or treating oral diseases such as oral candidiasis, or for inhibiting the growth of caries bacterium, periodontal disease bacterium and Candida bacterium, can be used as a pharmaceutical composition, oral composition, or the like, or It can be used as a material for incorporation into foods, pharmaceuticals, and oral compositions. In addition, foods are functional foods such as health foods, supplements or foods for specified health uses that show the concept, such as prevention and improvement of tooth decay, periodontal disease, and other oral infections, as necessary. It is also possible to do so.

Oral administration is preferable as a form for use as a pharmaceutical, and the dosage form is, for example, a liquid form; a solid form such as tablets, granules, fine granules, powders, tablets; or the liquid form or solid form enclosed therein. Various forms such as capsules, oral sprays, troches and the like can be mentioned. In order to prepare pharmaceutical formulations and supplements of various dosage forms as described above, other pharmaceutically acceptable excipients, binders, and increasing amounts are used as long as they do not interfere with the action of the cells and culture of the present invention. Agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, flavoring agents, fragrances, film-forming agents, carriers, diluents and the like can be used in appropriate combination. When used as such a formulation for oral administration, the content of the lactic acid bacterium cell or cell culture of the present invention in the formulation or an extract thereof is 1% by mass to 50% by mass, preferably It is 10% by mass to 20% by mass.

As a form when used as a food, fruit juice or vegetable juice beverage, carbonated beverage, tea-based beverage, milk beverage, fermented milk, fermented fruit juice, fermented vegetable juice, alcoholic beverage, beverage such as soft drink, jelly-like food and various Various foods such as snacks, baked goods, cakes, chocolates, jams, breads, gums, candy, soups, pickles, boiled vegetables, etc., as well as the above-mentioned oral administration preparations (tablets, capsules, syrups, etc.) Supplements and the like.

The culture obtained by the culture method of the present invention is a yogurt, cheese, miso, soy sauce, fermented food such as pickles, etc. as it is, using such fermented milk or cheese as a material, to prevent caries and periodontal disease, for improvement It can also be made into bread, snacks, cakes, etc.

The inoculum of the lactic acid bacterium obtained by the culture method of the present invention is generally such that 1 mL of the fermentation raw material-containing liquid contains about 1×10 ⁶ cells or more, preferably about 1×10 ⁷ cells. Suitably selected from the amount. Cultivation conditions are generally selected from a fermentation temperature of about 20 to 42°C, preferably about 25 to 37°C, and a fermentation time of about 5 to 72 hours. The lactic acid fermented product thus obtained has a curd-like form (yogurt-like form), which can be directly used as a solid food. The curd-like lactic acid fermented product can be made into a desired beverage form by further homogenizing it.

The lactic acid bacterium obtained by the culture method of the present invention, as a specific form when using a preventive, ameliorating or treating agent for oral diseases as an oral composition, a mouthwash, mouthwash, toothpaste, powder Toothpaste, water toothpaste, oral ointment, gel, tablets, granules, fine granules, gummy jelly, troches, tablets, capsules, candies, chewing gum and the like, preferably toothpaste, mouthwash, gummy jelly, There is a troche.

The content of the lactic acid bacterium obtained by the culture method of the present invention to the above-mentioned pharmaceuticals and foods is not particularly limited, and may be appropriately adjusted according to the daily dose etc., for example, when the dosage form is a liquid. The lactic acid bacterium concentration is preferably 1×10 ⁶ cells/ml to 1×10 ⁸ cells/ml, and in the case of a solid, 1×10 ⁷ cells/g to 1×10 ¹⁰ cells/g. Is preferred.

When the lactic acid bacterium obtained by the culture method of the present invention is administered as a viable bacterium, it is preferably administered at 1×10 ^{8 to} 5×10 ¹⁰ cfu/day per adult.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Example 1
Rice protein "Product name Profine, manufactured by Yaegaki Fermentation Giken Co., Ltd." is contained in 2-10%, and glucose or arabinose is added as carbon source in an amount of 0.2-5% (the rest is water). A sterilized medium is inoculated with Lactobacillus rhamnosus KO3 strain (NITE BP-771), and after culturing at 30 to 45°C for 24 to 240 hours, an excipient such as dextrin is added to the liquid, dried and ground. The obtained product was obtained as a lactic acid bacterium fermentation powder.

Examples 2-3
Soy protein "Product name High New, manufactured by Fuji Oil Co., Ltd." is contained in 2 to 10%, and glucose or arabinose as a carbon source is added in an amount of 0.2 to 5% as needed (the balance is water). A sterilized medium is inoculated with Lactobacillus rhamnosus KO3 strain (NITE BP-771), and after culturing at 30 to 45°C for 24 to 240 hours, an excipient such as dextrin is added to the liquid, dried and ground. The obtained product was obtained as a lactic acid bacterium fermentation powder.

Comparative Examples 1-2
As a medium, 10% skim milk powder, a medium sterilized by heat with 0.5% yeast extract, or an MRS medium was inoculated with Lactobacillus rhamnosus KO3 strain (NITE BP-771), and the mixture was inoculated at 30 to 45°C. After culturing for 24 to 240 hours, an excipient such as dextrin was added to the liquid, dried and pulverized to obtain a lactic acid bacterium fermentation powder.

<Evaluation method of antibacterial activity>
The amount of ATP was used as an index of antibacterial activity and evaluated by the following method. ATP extraction reagent (900 μl) was added to 100 μl of 25% solution of each powder, and the mixture was allowed to stand at room temperature for 30 minutes to extract ATP. The ATP amount of each sample was measured using an ATP analyzer (TOA Electronics Ltd, Tokyo, Japan). Eight similar samples were prepared for each condition, and the average value ±SD was calculated.

In FIG. 1, the ATP amount of the cultures obtained in Comparative Examples 1 and 2 was plotted, and even for the culture in MRS medium, it was about 1/10 of the control value of 1100000. The ATP amount of the cultures obtained in Examples 1 to 3 was plotted in FIG. 2, and in all cases, the ATP amount decreased so that it could not be detected.

The culturing method of the present invention is suitable for culturing Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus casei, or Lactobacillus paracasei, and the obtained antibacterial activity is suitable as an antibacterial culture. Since the culture is high and the culture is performed using a naturally-derived medium, the obtained culture can be applied to various uses including food and drink.

Claims (2)

A method for culturing Lactobacillus genus Lactobacillus, comprising the step of culturing Lactobacillus rhamnosus KO3 strain in a medium containing 0.1 to 10% by weight of a peptide derived from rice or soybean . Medium further method for culturing lactic acid bacteria of the genus Lactobacillus according to claim 1 comprising a saccharide.

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