WO2020166708A1 - Novel lactic acid bacterium, and composition containing same - Google Patents

Abstract

The present invention relates to a Lactobacillus crispatus strain, wherein the nucleotide sequence of the 16S rRNA gene of said strain has at least 95% identity with the nucleotide sequence indicated in SEQ ID NO: 1.

Description

Novel lactic acid bacterium and composition containing the same

The present invention relates to a novel lactic acid bacterium and a composition containing the same.

Conventional technology

In the past, the intestinal microflora generally referred to the bacteria present in feces and contents. However, in recent years, the physiological significance of bacteria adhering to the host mucosa and the existence of mucoadhesive bacterial flora formed in the mucosa have become clear.

For example, it has been reported that patients with inflammatory bowel disease (IBD) and Crohn's disease form mucoadhesive bacterial flora different from that of healthy individuals (Non-patent Document 1). In addition, among bacteria that can be contained in the intestinal flora, lacnospiraceae bacteria, Acinetobacter bacteria, Bacteroides bacteria, and Prevotella bacteria are known to increase in the intestine during intestinal tract and systemic inflammation (non- Patent Documents 2 to 8).

On the other hand, breast milk is known to contain various nutrients necessary for infant development. In recent years, it has been reported that lactic acid bacteria are also contained in breast milk and contribute to the formation of intestinal bacterial flora in newborns (Non-Patent Document 9). Furthermore, the relationship between the bacterial flora of breast milk and the development of offspring has been reported (Non-Patent Document 10).

However, breastfeeding may be difficult depending on the constitution of the mother and the child-rearing environment. In addition, because there are individual differences in the bacterial flora of breast milk, if the bacterial flora of the mother is poor, the mucoadhesive flora and intestinal flora of the offspring may also be affected.

One embodiment of the present invention has an object to provide a novel lactic acid bacterium capable of effectively controlling the bacterial composition balance in the indigenous bacterial flora.

The present inventors have discovered a novel lactic acid bacterium that can effectively control the bacterial composition balance of the indigenous bacterial flora including the mucoadhesive bacterial flora or the intestinal bacterial flora. The present invention is based on such findings.

One embodiment of the present invention includes the following aspects.
[1] A Lactobacillus crispatus strain, wherein the 16s rRNA gene nucleotide sequence of the strain is at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 1.
[2] The strain according to [1], wherein the identity is 98% or more.
[3] Lactobacillus crispatas CFF No. deposited under accession number NITE BP-02869 The strain according to [1] or [2], which is 2031.
[4] A composition comprising the strain according to any one of [1] to [3].
[5] The composition according to [4], which is used as a medicine, food or drink, cosmetics or feed.
[6] The composition according to [4] or [5] for infants or minors.
[7] The composition according to any one of [4] to [6], which is used for controlling the structural balance of bacteria contained in the indigenous bacterial flora.
[8] The bacterium contained in the indigenous bacterial flora is at least one selected from Lachnospiraceae bacteria, Acinetobacter bacteria, Musispirillum bacteria, Prevotella bacteria and Bacteroides bacteria, [7] Composition.
[9] The composition according to [7] or [8], wherein the indigenous flora is a mucoadhesive flora or an intestinal flora.
[10] The composition according to any one of [4] to [9] for suppressing a disease or symptom associated with inflammation.
[11] The composition according to any one of [4] to [10] for protecting the digestive tract.
[12] The composition according to any one of [4] to [11] for suppressing weight loss.
[13] Use of a Lactobacillus crispatus strain in the production of a composition for controlling a bacterial composition balance contained in an indigenous bacterial flora, wherein the nucleotide sequence of the 16s rRNA gene of the strain is the nucleotide sequence shown in SEQ ID NO:1. Use with at least 95% identity.
[14] A method for adjusting the compositional balance of bacteria contained in an indigenous bacterial flora, comprising administering an effective amount of a Lactobacillus crispatus strain to a subject in need thereof, wherein the 16s rRNA of the strain. The method wherein the nucleotide sequence of the gene has at least 95% identity to the nucleotide sequence set forth in SEQ ID NO:1.

According to one embodiment of the present invention, it is possible to effectively regulate the bacterial composition balance in the indigenous flora including the mucoadhesive flora or the intestinal flora. According to one embodiment of the present invention, the formation of healthy indigenous bacterial flora can be advantageously promoted. The lactic acid bacterium strain of the present invention can be advantageously used for ameliorating a disease or condition caused by inflammation. Further, according to one embodiment of the present invention, the lactic acid bacterium strain can be advantageously used for preventing a disease after growth by ingesting a child such as a minor or an infant.

In Example 1, the control group (C group) and the Lactobacillus crispatus CFF No. It is a graph (vertical axis parameter: Axis 2, horizontal axis Axis 1) which shows the result of having performed main coordinate analysis by the metagenome analysis of the mucous-adherent bacterial flora of the cecum regarding the 2031 strain administration group (L group). In Example 1, the control group (C group) and the Lactobacillus crispatus CFF No. It is a graph (vertical axis parameter: Axis 2, horizontal axis Axis 3) which shows the result of having performed main coordinate analysis by the metagenomic analysis of the mucous membrane flora of the cecum for the 2031 strain administration group (L group). In Example 1, it is a graph which shows the result of having performed the microflora structure analysis based on the metagenomic analysis of the mucoadhesive microbiota of the cecum. In Example 1, with respect to the mucosal bacterial flora of the cecum, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupation rate of Unclassified f_Lachnospiraceae in a 2031 strain administration group (L group), and the said occupation rate in a control group (C group). In Example 1, with respect to the mucosal bacterial flora of the cecum, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupancy rate of the bacterium of the genus Musispirillum (Mushispirillum) in the 2031 strain administration group (L group) and the occupancy rate in the control group (C group). In Example 1, it is a graph which shows the result of having carried out the microflora structure analysis based on the metagenome analysis of the mucous membrane flora of the colon. In Example 1, regarding the mucoadhesive bacterial flora of the colon, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupation rate of the Acinetobacter genus bacteria (Acinetobacter) in the 2031 strain administration group (L group), and the said occupation rate in a control group (C group). In Example 1, it is a graph which shows the result of having performed the microflora structure analysis based on the metagenome analysis of the bacterial flora of the cecal contents. In Example 1, regarding the bacterial flora of the cecal contents, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupation rate of Bacteroides genus bacteria (Bacteroides) in the 2031 strain administration group (L group), and the said occupation rate in a control group (C group). In Example 1, regarding the bacterial flora of the cecal contents, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupancy rate of Prevotella bacteria ([Prevotella]) in the 2031 strain administration group (L group), and the occupancy rate in the control group (C group). In Example 1, regarding the bacterial flora of the cecal contents, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupancy rate of a Lachnospiraceae bacterium (Unclassified Lachnospiraceae) in a 2031 strain administration group (L group), and the said occupancy rate in a control group (C group). In Example 1, it is a graph which shows the result of having performed the microflora structure analysis based on the metagenome analysis of the microbiota of the colon content. In Example 1, regarding the bacterial flora of the colon contents, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupation rate of Bacteroides genus bacteria (Bacteroides) in the 2031 strain administration group (L group), and the said occupation rate in a control group (C group). In Example 1, regarding the bacterial flora of the colon contents, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupation rate of Prevotella genus bacteria (Prevotella) in the 2031 strain administration group (L group), and the said occupation rate in a control group (C group). In Example 1, regarding the bacterial flora of the colon contents, Lactobacillus crispatus CFF No. It is a graph which shows the comparison result of the occupation rate of the Prevotella bacterium ([Prevotella]) in the 2031 strain administration group (L group), and the said occupation rate in a control group (C group). 5 is a graph showing changes in body weight of test mice in Example 2. The body weight on the first day (d1) of the DSS administration is set to 100.00%, and the body weight transition (%) until the 11th day (d11) of the DSS administration is shown. 5 is a graph showing the transition of the Disease Activity Index (DAI) of the test mouse in Example 2. The DAI score from the first day (d1) to the 11th day (d11) of the DSS administration start is shown.

Hereinafter, the present invention will be described in detail.
According to one embodiment of the present invention, a new strain of lactic acid bacteria belonging to Lactobacillus crispatus, Lactobacillus crispatus CFF No. 2031 strains are provided.

Lactobacillus crispatas CFF No. The 2031 strain (hereinafter, also simply referred to as “CFF No. 2031 strain” or “deposited strain”) was isolated using human breast milk as a separation source. The mycological properties of the strain are shown in the examples below. On January 29, 2019, the strain will be registered at the Patent Microorganism Depositary Center, National Institute of Technology for Product Evaluation (Japan), Microorganism Depositary Center (Room 2-5-8, Kazusa, Kamasa, Kisarazu, Chiba Prefecture, 292-0818, Room 122), with the accession number NITE BP. Deposited under -02869.

The lactic acid bacterium strain of the present invention may be the above-mentioned deposited strain or a strain substantially equivalent thereto. The deposited strain or a strain substantially equivalent thereto is a strain belonging to Lactobacillus crispatus, and the nucleotide sequence of the 16S rRNA gene thereof is at least the nucleotide sequence of the 16S rRNA gene of the deposited strain shown in SEQ ID NO: 1. It has 95% identity. The above-mentioned identity is preferably 98% or more, more preferably 99% or more, even more preferably 99.9% or more, and even more preferably 100%.

In the present invention, "identity" between two nucleotide sequences means the percent of identical nucleotides obtained after best alignment (optimal alignment) between the two sequences being compared, this percentage being It is merely statistical and the differences between the two sequences are randomly distributed over their entire length. The comparison of sequences between two nucleotide sequences is usually done by aligning them in an optimal way and then comparing these sequences, in which case the comparison is performed segment by segment or by "comparison window". You may be cut off. Optimal alignment of sequences for comparison can be performed by computer software (BLAST P comparison software).

The above-mentioned substantially equivalent strain is preferably functionally equivalent to the above-mentioned deposited strain. Whether or not the above-mentioned substantially equivalent strains are functionally equivalent to the above-mentioned deposited strains is within a range in which there is no significant difference by a statistical method by performing the same test as in Examples 1 and 2 of Examples described later. It can be determined by

The above-mentioned substantially equivalent strain preferably has the same function of regulating the bacterial composition balance contained in the indigenous bacterial flora as the deposited strain. Moreover, according to a more preferred embodiment, the bacteria contained in the above-mentioned indigenous bacterial flora are Lactonospiraceae bacteria (including Lachnospiraceae and Unclassified Lachnospiraceae), Acinetobacter spp. (Including Prevotella, [Prevotella]), or a bacterium belonging to the genus Bacteroides (Bacteroides). Here, the classification of the above-mentioned strains is based on the classification of software phyloseq (https://joey711.github.io/phyloseq) and Greengenes referred to by the software. Further, according to a further preferred embodiment, the substantially equivalent strain is equivalent to the deposited strain in the same manner as in the deposited bacterial strain, the activity of reducing the activity of Lachnospiraceae bacteria, Acinetobacter bacteria, Prevotella bacteria or Bacteroides bacteria in the indigenous bacterial flora. Have. Further, according to still another preferred embodiment, the substantially equivalent strain has an activity of increasing the bacterium of the genus Musispirillum in the indigenous bacterial flora in the same manner as the deposited strain. Further, according to another preferred embodiment, the substantially equivalent strain has an activity of suppressing weight loss caused by diarrhea and the like during intestinal inflammation in a subject to which it is administered, in the same manner as the deposited strain. Further, according to another more preferred embodiment, the substantially equivalent strain has an activity of suppressing a DAI score at the time of onset of enteritis in a subject to which it is administered, similarly to the deposited strain. Moreover, the above-mentioned indigenous flora is preferably a mucoadhesive flora or an intestinal flora.

The lactic acid bacterium strain of the present invention also includes strains bred by mutation treatment, gene recombination, selection of natural mutant strains, etc. from the deposited strain or a strain substantially equivalent thereto, as long as the effect of the present invention is not impaired. To be done.

The lactic acid bacterium strain of the present invention can be easily grown, for example, by culturing the same strain. CFF No. There is no particular limitation as long as the 2031 strain can grow, and a method usually used for culturing lactic acid bacteria can be appropriately modified and used as necessary. For example, the culture temperature may be 25 to 50°C, preferably 35 to 42°C. The culture may be carried out under either aerobic conditions or anaerobic conditions, but it is preferable to carry out the cultivation under anaerobic conditions. For example, the culture can be carried out while aerating an anaerobic gas such as carbon dioxide gas. Moreover, you may culture|cultivate under microaerobic conditions, such as liquid stationary culture.

The medium for culturing the lactic acid bacterium strain of the present invention is not particularly limited, and a medium usually used for culturing lactic acid bacteria can be appropriately modified and used as necessary. That is, as the carbon source, for example, saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, hydrolyzed starch, molasses and the like can be used depending on the assimilability. As the nitrogen source, for example, ammonia, ammonium salts such as ammonium sulfate, ammonium chloride, ammonium nitrate and nitrates can be used. As the inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, etc. can be used. Further, organic components such as peptone, soybean powder, defatted soybean meal, meat extract, yeast extract and the like may be used. Further, as the prepared medium, for example, MRS medium can be preferably used.

As the lactic acid bacterium strain of the present invention, the obtained culture after culturing may be used as it is, diluted or concentrated, or the microbial cells recovered from the culture may be used. In addition, various additional operations such as heating and freeze-drying can be performed after the culture unless the effects of the present invention are impaired.

The lactic acid bacterium strain of the present invention is preferably a live bacterium, but may be a dead bacterium.

The lactic acid bacterium strain of the present invention may be used as it is, or may be used in the form of a composition together with natural or other additives, if desired.

The lactic acid bacterial strain of the present invention can be used as a regulator for the bacterial composition balance contained in the indigenous bacterial flora in a subject. Therefore, the composition comprising the lactic acid bacterium strain of the present invention is preferably used as a composition for controlling the bacterial composition balance contained in the indigenous bacterial flora in a subject. Further, according to a more preferred embodiment, the composition is used for reducing Lactonospiraceae bacteria, Acinetobacter bacteria, Prevotella bacteria, or Bacteroides bacteria in the indigenous flora. According to another more preferred embodiment, the composition is used for increasing the bacterium of the genus Musispirillum in the indigenous flora. Moreover, according to still another preferred embodiment, the composition is used for suppressing weight loss due to diarrhea at the onset of enteritis in a subject to which the composition is administered. According to still another preferred embodiment, the composition is used for suppressing the DAI score at the time of onset of enteritis in the subject to which the composition is administered. In yet another more preferred embodiment, the composition is used for suppressing mucosal inflammation and suppressing diarrhea or fecal bleeding in a subject to which it is administered.

Also, the lactic acid bacterium strain of the present invention can be used to reduce the amount of bacteria involved in inflammation in the indigenous bacterial flora. Thus, in another embodiment, the compositions of the invention are used to control diseases or conditions associated with inflammation.

The resident flora in the present invention is preferably a mucoadhesive flora or an intestinal flora.

The composition of the present invention can be preferably used as a medicine, food or drink, cosmetics or feed.

The medicine is not particularly limited as long as it contains the lactic acid bacterium strain of the present invention. As a medicine, the lactic acid bacterium strain of the present invention may be formulated and used with a pharmaceutically acceptable carrier.

The dosage form of the medicament of the present invention is not particularly limited, and specifically, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, ointments. , Patch, eye drop, nasal drop and the like. In the case of formulation, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, solvents for injections, etc. that are usually used as formulation carriers. Can be used.

The content of lactic acid bacteria in the medicament of the present invention is appropriately set depending on the dosage form, usage, patient's age, sex, type of disease, degree of disease, other conditions, etc., but is usually 1×10 ⁶ to 1 It is preferably in the range of ×10 ¹² cfu/g or 1×10 ⁶ to 1×10 ¹² cfu/ml, and preferably 1×10 ⁷ to 1×10 ¹¹ cfu/g or 1×10 ⁷ to 1×10 ¹¹ More preferably, it is within the range of cfu/ml. CFF No. If the 2031 strain is killed, cfu/g or cfu/ml can be replaced with individual cells/g or individual cells/ml.

The food and drink is not particularly limited as long as it contains the lactic acid bacterium strain of the present invention, and as the food and drink, drinks such as soft drinks, carbonated drinks, nutritional drinks, fruit juice drinks, and lactic acid bacterium drinks (concentrated stock solution and preparation of these drinks Ice cream, sorbet, shaved ice, etc.; candy, chewing gum, candy, gum, chocolate, tablets, snacks, biscuits, jellies, jams, creams, baked goods, etc.; processed milk, Examples thereof include dairy products such as milk drinks, fermented milk, drink yogurt, and butter; breads; enteral nutritional foods, liquid foods, baby milk, sports drinks; and other functional foods. Further, the food and drink may be a supplement, for example, a tablet-like supplement. If it is a supplement, the CFF No. 2031 strain can be ingested.

Food and drink can be produced by adding the lactic acid bacterium strain of the present invention to the raw material of food and drink, except that the lactic acid bacterium strain of the present invention is added, can be produced in the same manner as ordinary food and drink .. The addition of the lactic acid bacterium strain of the present invention may be performed at any stage of the manufacturing process of foods and drinks. Further, the food or drink may be produced through a fermentation process using the added lactic acid bacterium strain of the present invention. Examples of such foods and drinks include lactic acid bacteria beverages and fermented milk.

As raw materials for foods or beverages, the raw materials used for ordinary beverages and foods can be used.

The food and drink of the present invention include raw materials for food and drink production, food additives and the like, which are added to food and drink during or after the manufacturing process of the food and drink. For example, the lactic acid bacterium strain of the present invention can be used as a starter for producing fermented milk. Further, the lactic acid bacterium strain of the present invention can be added later to the fermented milk produced.

The content of the lactic acid bacterium strain of the present invention in the food or drink is appropriately set depending on the mode of the food or drink, but is usually 1×10 ⁶ to 1×10 ¹² cfu/g or 1×10 ⁶ to 1 in the food or drink. It is preferably in the range of ×10 ¹² cfu/ml, and more preferably in the range of 1×10 ⁷ to 1×10 ¹¹ cfu/g or 1×10 ⁷ to 1×10 ¹¹ cfu/ml.

The food or drink of the present invention is for the control of the bacterial composition balance contained in the indigenous flora, for the reduction of Lactomospiraceae bacteria, Acinetobacter bacteria, Bacteroides bacteria or Prevotella bacteria in the indigenous flora, mucoadhesive bacterial flora or For increasing the amount of Musispirillum bacteria in the intestinal flora, for suppressing weight loss, for suppressing DAI score, for suppressing diarrhea or fecal bleeding, for reducing strains involved in inflammation, for suppressing diseases or symptoms related to inflammation It can be sold as food or drink with the intended use indicated.

In addition, the food and drink of the present invention, "for the adjustment of the structural balance of bacteria contained in the indigenous flora", "decreased lacnospiraceae bacteria, Acinetobacter bacteria, Bacteroides bacteria or Prevotella bacteria in the indigenous bacterial flora" "For increasing the number of Musispirillum bacteria in the indigenous flora", "For suppressing weight loss", "For suppressing DAI score", "For suppressing diarrhea or fecal bleeding", "For the bacteria involved in inflammation" "For reduction", "for suppression of diseases or symptoms associated with inflammation", etc. can be displayed. Needless to say, other than this, any word can be used as long as it represents the effect secondarily produced by the use of the composition for the above purpose. Examples of such wording include “toning the stomach”, “improving stomach or intestinal discomfort”, and the like.

The "display" means all actions to inform the consumer of the above-mentioned use, and if it is a display that can recall or analogize the above-mentioned use, the purpose of the display, the content of the display, the display Regardless of the target object, medium, etc. to be processed, they all correspond to the "display" of the present invention. However, it is preferable to display the expression so that the consumer can directly recognize the use.

Specifically, the act of describing the above-mentioned use on the product or the product packaging related to the food or drink of the present invention, the product or the product packaging indicating the above-mentioned use is transferred, and exhibited for the purpose of delivery, transfer or delivery. However, the act of importing, advertising regarding products, displaying or distributing by listing the above-mentioned use in price lists or transaction documents, or by describing the above-mentioned use in the information containing these, electromagnetic (Internet etc.) method The actions provided by the above can be exemplified, and it is particularly preferable to display them on packaging materials, containers, catalogs, pamphlets, advertising materials at sales sites such as POP, and other documents.

Also, it is preferable that the display is a display permitted by the government (for example, a display obtained in a form based on such approval after being approved based on various systems established by the government). For example, health foods, functional foods, enteral nutritional foods, special-purpose foods, nutritionally functional foods, indications as quasi-drugs, and the like, other indications approved by the Ministry of Health, Labor and Welfare, for example, specific Examples include labeling approved for health foods and similar systems. Examples of the latter include a display as a food for specified health use, a display as a food for specified health use with conditions, a display that affects the structure and function of the body, and a display for reducing disease risk. More specifically, the label as a food for specified health use (in particular, the label for the purpose of health use) stipulated in the Health Promotion Law Enforcement Regulation (April 30, 2003, Ministry of Health, Labor and Welfare Ordinance No. 86 of Japan), and Similar displays and the like can be illustrated.

The cosmetics of the present invention may be prepared according to the above-mentioned pharmaceuticals or foods and drinks and used as a beauty agent for internal use, or may be a topical or systemic external preparation for skin. Preferable examples of the external preparation for skin include basic cosmetics such as lotion, milky lotion, cream, ointment, lotion, oil and pack, facial cleansers such as solid soap, liquid soap and hand wash, skin cleansing agents, and massage agents. , Cleansing agent, depilatory, depilatory, shaving treatment, after shave lotion, pre-show lotion, shaving cream, foundation, lipstick, blusher, eye shadow, eyeliner, mascara and other makeup cosmetics, perfume, Examples include nail enamel, nail enamel, nail enamel remover, poultice, plaster, tape, sheet, patch, aerosol and the like.

The CFF NO. The content of the 2031 strain is appropriately set depending on the aspect of the cosmetic product and the administration subject, but is in the range of 1×10 ⁶ to 1×10 ¹² cfu/g or 1×10 ⁶ to 1×10 ¹² cfu/ml. It is preferable that it is in the range of 1×10 ⁷ to 1×10 ¹¹ cfu/g or 1×10 ⁷ to 1×10 ¹¹ cfu/ml.

Also, cosmetics can be manufactured by optionally selecting and using the additive components exemplified below as needed. Such additive components are not particularly limited, but oils and fats (cocoa butter, chamomile oil, carrot oil, cucumber oil, tallow fatty acid, kukui nut oil, safflower oil, shea butter, etc.), waxes (beeswax, carnauba wax, whale wax) , Lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc., mineral oil (liquid paraffin, petrolatum, paraffin, ozokeride, ceresin, microcristan wax, etc.), fatty acids (lauric acid, myristin) Acids, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, natural fatty acids such as lanolin fatty acid, isononanoic acid, capron Acids, synthetic fatty acids such as 2-ethylbutanoic acid, isopentanoic acid, 2-methylpentanoic acid, 2-ethylhexanoic acid, isopentanoic acid), alcohols (ethanol, isopropanol, lauryl alcohol, cetanol, stearyl alcohol, oleyl alcohol, lanolin alcohol) , Cholesterol, phytosterols, natural alcohols such as phenoxyethanol, synthetic alcohols such as 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol), various vitamins (vitamin A group: retinol, retinal (vitamin A1), dehydroretinal ( Vitamin A2), carotene, lycopene (provitamin A), vitamin B group: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acid, Nicotinic acids, pantothenic acids, biotins, choline, inositols, vitamin C group: vitamin C acid or its derivatives, vitamin D group: ergocalciferol (vitamin D2), etc., polymer compounds (arabic gum, benzoin gum, dammar gum) , Guaiac fat, chitosan, hyaluronic acid or its salt, chondroitin sulfate or its salt, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxyethyl cellulose sodium, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl Ether, polyvinylpyrrolid And polyvinyl methacrylate, polyacrylic acid salts, polyalkylene oxides such as polyethylene oxide and polypropylene oxide, or cross-linked polymers thereof, carboxyvinyl polymers, polyethyleneimine, etc.) and the like.

Examples of the feed of the present invention include pet food, livestock feed, fish feed and the like. The feed of the present invention can be used for general feed such as cereals, meals, bran, fish meal, bone meal, oils and fats, skim milk powder, whey, mineral feed, yeasts, etc. It can be produced by mixing 2031 strains. In addition, for example, like silage, the added CFF NO. The feed may be produced through a fermentation process using the 2031 strain. The produced feed can be orally administered to general mammals, livestock, fish farms, pet animals and the like.

The CFF NO. Although the content of the 2031 strain is appropriately set depending on the form of the feed and the administration subject, it is within the range of 1×10 ⁶ to 1×10 ¹² cfu/g or 1×10 ⁶ to 1×10 ¹² cfu/ml. It is preferable that it is in the range of 1×10 ⁷ to 1×10 ¹¹ cfu/g or 1×10 ⁷ to 1×10 ¹¹ cfu/ml.

According to another aspect, there is provided a method of adjusting the compositional balance of bacteria contained in the indigenous flora, comprising administering to a subject in need thereof an effective amount of the lactic acid bacterium strain of the present invention. A method is provided. According to another preferred embodiment of the present invention, the method is for reducing the bacteria involved in inflammation (for example, Lachnospiraceae bacteria, Acinetobacter bacteria, Bacteroides bacteria, Prevotella bacteria, etc. in the indigenous flora). Is the way. According to another more preferred aspect, the above method is a method for increasing the bacterium of the genus Musispirillum in the indigenous flora. According to another more preferred embodiment, the above method is a method for suppressing at least one symptom selected from weight loss, DAI score, diarrhea, or fecal bleeding caused by diarrhea that develops enteritis.

Further, according to another aspect, there is provided a method for suppressing a disease or symptom associated with inflammation, which comprises administering an effective amount of the lactic acid bacterial strain of the present invention to a subject in need thereof. It Here, “suppression” includes not only treating or ameliorating an established pathological condition, but also preventing a pathological condition that may be established in the future.

The above-mentioned diseases or symptoms related to inflammation include inflammatory bowel disease (IBD), specifically ulcerative colitis (UC) and Crohn's disease (CD).

Also, according to one aspect, the method of the present invention excludes medical practice. According to another aspect, the method of the invention is a non-therapeutic method.

The effective amount of the lactic acid bacterium strain of the present invention is not particularly limited as long as it does not impair the effects of the present invention, and can be, for example, 1.0×10 ⁴ cfu/dose to 1.0×10 ¹² cfu/dose. According to one aspect, CFF NO. An effective amount of 2031 strain is taken as an intake amount per meal.

The effective amount of the lactic acid bacterium strain of the present invention can be administered once or in several divided doses a day, or may be administered once every several days or weeks. Further, it is possible to appropriately select the administration time depending on the condition of the subject.

The subject of the present invention is preferably a human, more preferably a minor under the age of 15, and even more preferably an infant. The application of the lactic acid bacterium strain of the present invention to infants is particularly advantageous in controlling the composition balance of bacteria contained in the mucoadhesive flora or intestinal flora as an alternative to breast milk.

Also, the lactic acid bacterium strain of the present invention may be administered continuously not only for a period of time, but may be administered for a period of infancy. The administration of the lactic acid bacterium strain of the present invention during infancy is particularly advantageous in preventing post-growth diseases.

The subject may also be a healthy person, a person who has a disease or condition associated with inflammation in the gastrointestinal tract, or a person who has a physical disorder due to it.

Also, the administration method in the present invention is preferably oral. However, it is not particularly limited as long as it does not impair the effects of the present invention, and the lactic acid bacterium strain of the present invention may be administered to a subject by other methods such as tube, enteral, transdermal (application, etc.). ..

According to another aspect, there is provided use of the lactic acid bacterium strain of the present invention in the production of a composition for adjusting a bacterial composition balance contained in an indigenous bacterial flora. According to a preferred embodiment, the above composition is a composition for reducing bacteria involved in inflammation (for example, Lacnospiraceae bacteria, Acinetobacter bacteria, Prevotella bacteria, or Bacteroides bacteria in the indigenous flora). According to another preferred embodiment, the composition is a composition for increasing the bacterium of the genus Musispirillum in the indigenous bacterial flora. According to another more preferred embodiment, the above composition is a composition for suppressing at least one symptom selected from weight loss, DAI score, diarrhea or fecal bleeding caused by diarrhea that develops enteritis. ..

According to another preferred embodiment, there is provided use of the lactic acid bacterium strain of the present invention in the manufacture of a composition for suppressing a disease or symptom associated with inflammation.

Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1: Confirmation test of effect of administration of lactic acid bacteria on mucoadhesive flora and intestinal flora Two-week-old C57BL/6N mice purchased from Japan SLC were acclimatized for 7 days, and then weaned at three weeks. Then, the mice were made to have an equal weight so that the control group (C group) and Lactobacillus crispatus CFF No. The 2031 strain was administered (L=group) to two groups (n=6) for breeding management. After weaning, the mice, rat and hamster lab MR stock (Nippon Nosan Kogyo) were allowed to freely ingest and unsterilized water was allowed to freely ingest. The breeding room was lit at 8 o'clock and turned off at 8 o'clock in a 12-hour light-dark cycle, and the room temperature was set to 25±2°C.

Oral administration of each test substance was started from the day after the weaning day, and was orally administered for 14 days. PBS (-) for group C and Lactobacillus crispatus CFF No. for group L. The 2031 strain was orally administered. The volume of the liquid for oral administration was adjusted according to the body weight of the mouse, and was 100 μl if the body weight was less than 12.5 g and 150 μl if the body weight was 12.5 or more and less than 17.5 g.

A 1 ml syringe (TERUMO) was used as a device for oral administration. For oral administration, a Chuko Flow PTFE tube AWG-26 (Chuko Kasei Kogyo Co., Ltd.), which had been cut to an appropriate length and burned at the tip to be rounded, was attached to a 1 ml syringe and autoclaved sterilized 23G needle (TERUMO). ) Was attached.

Orally administered to mice, Lactobacillus crispatus CFF No. The 2031 strain-containing solution was prepared as follows. L. sp. stored at -80°C. crispatas CFF No. The 2031 strain was inoculated into 10 ml of MRS medium and cultured under anaerobic conditions at 37° C. for 24 hours to obtain a preculture liquid. This pre-cultured liquid was stored under refrigeration and used for daily preparation of a liquid containing a lactic acid bacterium strain. As the strain-containing liquid, 200 μl of the preculture liquid was taken out the day before oral administration, subcultured in a new 10 ml of MRS medium, and cultured under the same conditions as the preculture. After completion of the culture, the cells were centrifuged at 10,000 xg at room temperature for 3 minutes to pellet the cells, which were then washed with PBS(-) and 5x10 ⁹ cfu based on the turbidity (OD595). The suspension was suspended in PBS(-) to give a lactic acid bacterium strain-containing solution.

7 days after the end of oral administration, the test substance was washed out (resting period), and the animals were raised without oral administration.

After the washout period was completed (after the mice reached the age of 6 weeks), all mice were dissected and the cecal mucosa and cecal contents and the colon mucosa and colon contents were collected, respectively.

Bacterial DNA was extracted from the mucous membranes and contents of each collected tissue using QuickGene DNA tissue kit S (KURABO). Using the extracted bacterial genomic DNA as a template, the V3-V4 region of the 16S rRNA gene was amplified by a PCR reaction using KAPA HiFi HS Ready Mix (Japan Genetics). The reaction, the forward primer 341F which sequences for 2ndPCR are attached as a tail (SEQ ID NO 2: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 '): use of a reverse primer 805R (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3 SEQ ID NO: 3') I was there. PCR was performed using a total of 25 μl of KAPA HIFI HS Ready Mix 12.5 μl, template DNA 2.5 μl, primers each 0.05 μl, and free water 9.9 μl. After initial denaturation at 95°C for 3 minutes, 25°C temperature cycles of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 30 seconds, and finally extension reaction at 72°C for 5 minutes were performed. After completion of the PCR reaction, electrophoresis was performed using a 1% agarose gel (tris acetate EDTA: TAE), and it was confirmed that the target gene was amplified. After that, the PCR product was purified using NucleoFast 96 PCR Clean up plate (Takara Bio). The purified product was tagged by the second PCR reaction using the primers loaded with Index and Illumina sequencing adaptor.

Purification and concentration adjustment of the product obtained by PCR were performed using the SequalPrepTM Normalization Plate Kit (Invitrogen). Then, the concentration of the purified product was quantified by real-time PCR using KAPA SYBR Fast qPCR Kit (Japan Genetics). Samples of sufficient concentration were mixed in an amount of 10 μl per sample, and purified and concentrated using Agencoet AMPure XP (BECKMAN COULTER).

The concentration of the obtained library was calculated according to the standard protocol of KAPA Library Quantification Kit (Japan Genetics). After that, the sample was diluted to 4 nM with 10 mM Tris Buffer based on the obtained concentration. For the subsequent operations, the final preparation was performed in accordance with 16S Metagenic Sequencing Library Preparation (Illumina). The prepared sample was applied to a reagent cartridge of Miseq Reagent Kit V3 (600PE), and sequenced by MiSeq (Illumina). The obtained sequence data was subjected to a primary analysis by QIIME and then a secondary analysis using STAMP.

The results were as shown in Figures 1-4.
1A and 1B are the results of principal coordinate analysis by metagenomic analysis of cecal mucoadhesive flora. Phyloseq (https://joey711.github.io/phylloseq/) was used for the above analysis. Therefore, the classification of the bacteria shown in FIGS. 2A, 3A, and 4A to 4H follows the classification of Phyloseq and Greengenes referred to by the software. As a result of principal coordinate analysis by metagenomic analysis, Lactobacillus crispatus CFF No. In the 2031 strain-administered group (L group), a change was confirmed in the composition of the caecal mucoadhesive bacterial flora compared to the control group (C group) (PERMANOVA p=0.002).

The results of the bacterial flora structure analysis by metagenomic analysis of the mucous bacterial flora of the cecum were as shown in FIG. 2A. Also, regarding the mucoadhesive bacterial flora of the cecum, Lactobacillus crispatus CFF No. The occupancy rate of the Unclassified Lachnospiraceae in the 2031 strain administration group (L group) was significantly lower than the occupancy rate in the control group (proportion of sequences (%)) (FIG. 2B, Wilcoxon/Kruskrus/Kruskal/Kruskron/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskron/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskal/Kruskron/Kruskal/Kruskal/Kruskal/Kruskalk, respectively, respectively. -Wallis test (sum of ranks), p<0.05). On the other hand, Lactobacillus crispus CFF No. The occupancy rate of the bacterium of the genus Musispirillum in the 2031 strain-administered group (L group) was significantly higher than that in the control group (FIG. 2C, Wilcoxon/Kruskal-Wallis test (rank sum), p<0. 05).

The results of the bacterial flora structure analysis by metagenomic analysis of the mucoadhesive bacterial flora of the colon were as shown in Fig. 3A. Regarding colonic mucosa flora, Lactobacillus crispatus CFF No. The occupancy rate of Acinetobacter bacteria in the 2031 strain-administered group (L group) was significantly lower than that in the control group (C group) (FIG. 3B, Wilcoxon/Kruskal-Wallis test (rank sum)). , P<0.05).

It is known that lacnospiraceae bacteria and Acinetobacter bacteria increase during inflammation of the intestinal tract (Non-patent Documents 2 to 5). Therefore, these bacteria were identified as Lactobacillus crispatus CFF No. From the result that it was decreased by the administration of strain 2031, Lactobacillus crispatus CFF No. The 2031 strain is considered to have an effect of suppressing inflammation of the digestive tract. In addition, Mucisspirillum is a bacterium that assimilates mucus. From the result that this bacterium increased, Lactobacillus crispatus CFF No. It is considered that the gastrointestinal mucosa was sufficiently filled with mucus by the administration of strain 2031.
From the above, Lactobacillus crispatus CFF No. Strain 2031 is considered to have the effect of enhancing the barrier function of the digestive tract mucosa.

The results of the bacterial flora structure analysis by metagenomic analysis of the bacterial flora of the cecal contents were as shown in FIG. 4A. Regarding the bacterial flora of the cecal contents, as shown in FIG. 4B, Lactobacillus crispatus CFF No. 2031 strain-administered group (L group) Bacteroides in occupancy (Bacteroides) was significantly lower compared to the control group (Student's t-test, p = 0.0484). Further, as shown in FIG. 4C, the occupancy rate of Prevotella bacteria ([ Prevotella ]) in the L group was significantly lower than that in the control group (Student's t-test, p=0.0430). Further, as shown in FIG. 4D, the occupancy rate of the Lachnospiraceae bacterium ( Unclassified Lachnospiraceae ) in the L group was significantly lower than that in the control group (Student's t-test, p<0.0001). Furthermore, the occupancy of Lactobacillus in the L group was significantly higher than that in the control group (Student's t-test, p=0.0409).

The results of the bacterial flora structure analysis by metagenomic analysis of the bacterial flora of the colon contents were as shown in FIG. 4E. Respect flora of the colon contents, occupancy of Bacteroides bacteria (Bacteroides) as, in the L group shown in FIG. 4F, was significantly lower (Student's t-test compared to the control group, p = 0.0004 ). Moreover, occupancy as shown in FIG. 4G, Prevotella spp in L group (Prevotella) was significantly lower compared to the control group (Wilcoxon / Kruskal-Wallis test (rank sum), p = 0.0131 ). In addition, as shown in FIG. 4H, the occupancy rate of Prevotella bacteria ([ Prevotella ]) in the L group was significantly lower than that in the control group (Student's t-test, p=0.0072).

Prevotella and Bacteroides bacteria are known to increase during inflammation of the intestinal tract and whole body (Non-Patent Documents 6 to 8). Therefore, these bacteria were identified as Lactobacillus crispatus CFF No. From the result that it decreases by administration of 2031 strain, Lactobacillus crispatus CFF No. The 2031 strain is considered to have an effect of suppressing inflammation of the digestive tract. From the above, Lactobacillus crispatus CFF No. Strain 2031 is considered to have the effect of suppressing inflammation of the gastrointestinal mucosa.

Example 2: Digestive Tract Barrier Function Enhancement Test by Administration of Lactic Acid Bacteria Two-week-old C57BL/6N mice (2 bellies (6 per belly)) purchased from Japan Charles River together with mother mice were introduced into the breeding room of this laboratory We got accustomed to the facility for 7 days. The pups were weaned at 3 weeks of age, and the control group (Group C), Lactobacillus crispatus CFF No. The 2031 strain was administered (L=group) to two groups (N=6) for breeding management. Similar to Example 1, from the day after the weaning day, PBS (-) was added to the DC group, and Lactobacillus crispatus CFF No. was added to the DL group. Oral administration of strain 2031 was started, and oral administration was performed for 14 days. 7 days after the end of the oral administration, it was a washout period of the administered bacteria, and the animals were bred without oral administration. When the wash out period was completed and the mice reached 6 weeks of age, in Example 2, 1.5% DSS (Dextran Sulfate Sodium (DSS) salt, Colitis grade (MP Biomedicals M.W 36-50 kDa) was added to distilled water 15 times. (dissolved at g/l) was replaced with tap water and freely taken as drinking water. On day 11 of DSS administration, all mice were subjected to dissection.

During the period after weaning, there was no special mention, and the laboratory MR stock for mice, rats, and hamsters (Japan Agricultural Industry Co., Ltd.) was allowed to freely ingest, and unsterilized tap water was freely ingested. The breeding room was set to a room temperature of 25±2° C. and a humidity of 40 to 70% under a 12-hour light-dark cycle of lighting at 8 o'clock and extinguishing at 20:00.

From the DSS administration start day (d22) to the day before the last day (d31), the mice were weighed daily and the fecal properties (hardness and bleeding) were scored. However, on the final day (d32), the mouse was weighed and the fecal characteristics were scored immediately before dissection. From the weight loss rate and the stool properties, DAI, which is an index showing the severity of enteritis, was calculated, and the intestinal inflammatory condition was evaluated. This evaluation method is described in Mennigen et al. (2009) (Table 2-1).

Also, as shown in FIG. 5A, on the 11th day (d11) after DSS administration, Lactobacillus crispatus CFF No. was compared with the control group (C group: 86.87%). In the 2031 strain-administered group (L group: 92.26%), a weight loss suppressing effect was observed (t-test, p=0.05). Further, as shown in FIG. 5B, on the 11th day (d11) after DSS administration, Lactobacillus chrispatas CFF No. was compared with the control group (C group: DAI score 9.07). In the 2031 strain administration group (L group :: DAI score 7.00), deterioration of the DAI score was also suppressed (t-test, p=0.07).

When the administration time and age of mice used in this study are converted to humans (when the lifespan of mice is converted to 2 years and the lifespan of humans is calculated to be 60 to 80 years old), 1 and a half to two and a half years are used in this test. As a result of administration of lactic acid bacteria to children of a certain age for one to one and a half years, it means that inflammatory diseases that may occur around the age of 4 to 5 years have been prevented. From this result, it is considered that the administration of the lactic acid bacterium strain of the present invention during infancy can be used to prevent post-growth diseases.

According to the present invention, there is provided a novel lactic acid bacterium capable of effectively controlling the bacterial composition balance in the indigenous bacterial flora including the mucous bacterial flora or the intestinal bacterial flora.

Claims (14)

1. Lactobacillus chrispatas strain, wherein the 16s rRNA gene nucleotide sequence of the strain has at least 95% identity with the nucleotide sequence shown in SEQ ID NO:1.
2. The strain according to claim 1, wherein the identity is 98% or more.
3. Lactobacillus chrispatas CFF No. deposited under the deposit number NITE BP-02869. The strain according to claim 1 or 2, which is 2031.
4. A composition comprising the strain according to any one of claims 1 to 3.
5. The composition according to claim 4, which is for use as a medicine, food and drink, cosmetics or feed.
6. Composition according to claim 4 or 5 for infants or minors.
7. The composition according to any one of claims 4 to 6 for controlling the bacterial composition balance contained in the indigenous bacterial flora.
8. The composition according to claim 7, wherein the bacterium included in the indigenous flora is at least one selected from lacnospiraceae bacteria, Acinetobacter bacteria, Musispirillum bacteria, Prevotella bacteria and Bacteroides bacteria.
9. The composition according to claim 7 or 8, wherein the indigenous flora is a mucoadhesive flora or an intestinal flora.
10. The composition according to any one of claims 4 to 9 for suppressing a disease or condition associated with inflammation.
11. A composition according to any one of claims 4 to 10 for protecting the digestive tract.
12. The composition according to any one of claims 4 to 11 for suppressing weight loss.
13. Use of a Lactobacillus chryspatus strain in the manufacture of a composition for controlling the bacterial composition balance contained in an indigenous bacterial flora, wherein the nucleotide sequence of the 16s rRNA gene of the strain is at least 95% with the nucleotide sequence shown in SEQ ID NO:1. Use, having the identity of.
14. A method for adjusting the structural balance of bacteria contained in an indigenous flora, comprising administering an effective amount of a Lactobacillus chryspatus strain to a subject in need thereof, wherein the nucleotides of 16s rRNA gene of said strain are administered. The method wherein the sequence has at least 95% identity to the nucleotide sequence set forth in SEQ ID NO:1.

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