TW202045715A - 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 bacteria and compositions containing them

Invention field

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

Background of the invention

Conventionally, the so-called intestinal bacterial flora generally refers to bacteria present in feces or contents. However, in recent years, the physiological significance of bacteria attached to the host mucosa and the existence of mucosal attached bacterial clusters formed in the mucosa have become clear.

For example, it has been reported that patients with inflammatory bowel disease (IBD) and Crohn's disease (Crohn's disease) have a mucosal-adherent bacterial cluster that is different from those of healthy people (Non-Patent Document 1). In addition, it is known that among the bacteria that can be contained in the intestinal bacterial flora, Lachnospiraceae bacteria and Acinetobacter bacteria, Bacteroides bacteria, Prevotella bacteria ) Bacteria increase in the intestine when the intestine or the whole body is inflamed (Non-Patent Documents 2-8).

On the other hand, it is known that breast milk contains various nutrients needed for infant development. In addition, in recent years, it has been reported that breast milk also contains lactic acid bacteria and contributes to the formation of intestinal bacterial clusters in newborns (Non-Patent Document 9). Furthermore, there are reports on the correlation between the bacterial flora of breast milk and the development of children (Non-Patent Document 10).

However, depending on the physique of the mother and the childcare environment, it may be difficult to carry out breastfeeding. In addition, there are individual differences in the bacterial flora of breast milk. Therefore, when the mother's bacterial flora is poor, the child's mucosa attached bacterial flora and intestinal bacterial flora may also be affected. [Prior Technical Literature] [Non-Patent Literature]

[Non-Patent Document 1] The Japanese Society of Gastroenterology, Nishino et al., DOI 10.1007/s00535-017-1384-4, 2017 [Non-Patent Literature 2] The Journal of Nutrition, Genomics, Proteomics, and Metabolomics, Preidis et al., 940-948, First published online April 6, 2016 [Non-Patent Document 3] The ISME Journal 6, Berry et al., 2091-2106, (2012) [Non-Patent Document 4] Neuroscience, Aldy et al., 1-12, 03 August 2017 [Non-Patent Document 5] Gut Microbes, Volume 6, Issue 1, Tang et al., 48-56, 2015 [Non-Patent Document 6] Setoyama et al. Microbes Infect. 2003 Feb;5(2):115-22 [Non-Patent Document 7] Scher et al. eLife 2013;2:e01202.DOI:10.7554/eLife.01202 [Non-Patent Document 8] Arai et al. Japanese Society of Allergology, 67(1), 24-27, 2018 [Non-Patent Document 9] SCIENTIFIC Reports, 7, 9940, DOI:10.1038/s41598-017-09278 -y, Drell et al., 30 August 2017 [Non-Patent Document 10] Frontiers in Microbiology, Volume 8, Article 2100, Toscano et al., October 2017.

Summary of the invention The problem to be solved by the invention

An object of an embodiment of the present invention is to provide a novel lactic acid bacteria, which can effectively adjust the balance of bacterial composition in the resident bacterial flora. Means to solve the problem

The inventors of the present invention have discovered a novel lactic acid bacteria that can effectively adjust the balance of the bacterial composition of the resident bacterial flora, led by mucosal attached bacterial flora or intestinal bacterial flora. The present invention is based on such knowledge.

An implementation aspect of the present invention includes the following aspects. [1] A strain of Lactobacillus crispatus, the nucleotide sequence of the 16s rRNA gene 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 aforementioned identity is 98% or more. [3] The strain described in [1] or [2], which is deposited with Lactobacillus crispatas CFF No. 2031 under the deposit number NITE BP-02869. [4] A composition comprising the strain described in any one of [1] to [3]. [5] The composition as described in [4], which is used as medicine, food and drink, cosmetics or feed. [6] The composition as described in [4] or [5], which is intended for infants or minors. [7] The composition described in any one of [4] to [6] is used to adjust the composition balance of bacteria contained in the resident bacterial flora. [8] The composition according to [7], wherein the bacteria contained in the aforementioned resident bacterial flora are selected from the group consisting of bacteria of the genus Trichospiraceae, bacteria of the genus Acinetobacter, bacteria of the genus Mucispirillum, and bacteria of the genus Prevot At least one of Bacteroides and Bacteroides. [9] The composition according to [7] or [8], wherein the aforementioned resident bacterial clusters are mucosal attached bacterial clusters or intestinal bacterial clusters. [10] The composition as described in any one of [4] to [9], which is used to inhibit inflammation-related diseases or symptoms. [11] The composition described in any one of [4] ~ [10] is used to protect the digestive tract. [12] The composition as described in any one of [4] to [11], which is used to inhibit weight loss. [13] A use of a strain of Lactobacillus crispatus, which is to produce a composition for regulating the composition of bacteria contained in the resident bacterial flora, and the nucleotide sequence of the 16s rRNA gene of the strain has the same sequence identification The nucleotide sequence shown in number 1 is at least 95% identical. [14] A method for adjusting the balance of the composition of bacteria contained in the resident bacterial flora, comprising the following: administering an effective amount of Lactobacillus crispatus strain to a subject in need thereof, and the nucleus of the 16s rRNA gene of the aforementioned strain The nucleotide sequence is at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 1. Invention effect

According to one aspect of the present invention, it is possible to effectively adjust the balance of the bacterial composition in the resident bacterial colony headed by the mucosal attached bacterial colony or the intestinal bacterial colony. According to one embodiment of the present invention, the formation of healthy resident bacterial clumps can be advantageously promoted. The lactic acid strain of the present invention can be advantageously used to improve diseases or conditions caused by inflammation. In addition, according to one aspect of the present invention, the lactic acid strain can be advantageously used in the prevention of growing-up diseases by ingesting minors, infants and other children.

The form used to implement the invention

Hereinafter, the present invention will be explained in detail. According to one aspect of the present invention, there is provided a Lactobacillus crispatus CFF No. 2031 strain, which is a novel strain of lactic acid bacteria belonging to Lactobacillus crispatus.

Lactobacillus crispatus CFF No. 2031 strain (hereinafter also referred to as "CFF No. 2031 strain" or "deposited strain") is isolated from human breast milk as the source of isolation. The mycological properties of this strain are shown in the examples described later. The strain was deposited on January 29, 2019 at the National Institute of Technology and Evaluation Patent Microorganism Deposit Center (〒292-0818 2-5-8 Kazusa Kazuzu, Kisarazu City, Chiba Prefecture) Room 122), under the deposit number NITE BP-02869.

The lactic acid strain of the present invention may be the aforementioned deposited strain or a strain substantially equivalent thereto. The above-mentioned deposited strain or the substantially equivalent strain is a strain belonging to Lactobacillus crispatus, and the nucleotide sequence of its 16S rRNA gene is at least the same as that of the 16S rRNA gene of the above-mentioned deposited strain shown in SEQ ID NO: 1. 95% identity. In addition, the aforementioned identity is preferably at least 98% or more, more preferably at least 99% or more, still more preferably at least 99.9% or more, and still more preferably 100%.

In the present invention, the so-called "identity" identity between two nucleotide sequences means that the two sequences being compared are in the best alignment (optimal alignment). The percentage of identical nucleotides obtained after) is only statistical, and the difference between the two sequences is randomly distributed across the entire length. Sequence comparison between two nucleotide sequences is usually carried out by comparing these sequences after the most suitable method is used for comparison. In this case, the aforementioned comparison is based on each fragment or each "comparison window" Go ahead. The best alignment of sequences used for comparison can be performed through computer software (BLAST P comparison software).

In addition, the aforementioned substantially equivalent strains are preferably functionally equivalent to the aforementioned deposited strains. Whether the above-mentioned substantially equivalent strains are functionally equivalent to the above-mentioned deposited strains can be tested in the same manner as in Examples 1 and 2 of Examples described later, and it is determined by statistical methods whether they are within a range of no significant difference.

The above-mentioned substantially equivalent strains preferably have a function of regulating the composition balance of bacteria contained in the resident bacterial flora on the same level as the above-mentioned deposited strains. In addition, according to a better implementation, the bacteria contained in the above-mentioned resident bacterial flora are bacteria of the Lachnospiraceae family (including Lachnospiraceae and Unclassified Lachnospiraceae), Acinetobacter bacteria (Acinetobacter), Mucispirillum bacteria (Mucispirillum), Prevotella bacteria (including Prevotella, [Prevotella] ([Prevotella])) , Or Bacteroides. Here, the classification of the above-mentioned strains is regarded as the classification of the software phyloseq (https://joey711.github.io/phyloseq) and the Greengenes referenced by the software. In addition, according to a further preferred embodiment, the above-mentioned substantially equivalent strain has the same reduction in the above-mentioned resident bacterial colony of Trichospiraceae bacteria, Acinetobacter bacteria, and Prevotes. The activity of bacteria of the genus Bacteroides or Bacteroides. In addition, according to yet another preferred embodiment, the above-mentioned substantially equivalent strain is equivalent to the above-mentioned deposited strain and has the activity of increasing the Myxospirillum bacteria in the above-mentioned resident bacterial cluster. In addition, according to another preferred embodiment, the above-mentioned substantially equivalent strain is equivalent to the above-mentioned deposited strain, and has the ability to suppress weight loss caused by diarrhea during intestinal inflammation in the subject to which it is administered. The activity. In addition, according to another preferred embodiment, the above-mentioned substantially equivalent strain is equivalent to the above-mentioned deposited strain, and has the activity of suppressing the DAI score at the onset of enteritis in the subject to which it has been administered. In addition, the aforementioned resident bacterial clusters are preferably mucosal attached bacterial clusters or intestinal bacterial clusters.

As far as the lactic acid strain of the present invention is concerned, as long as the effect of the present invention is not impaired, it also includes strains bred from deposited strains or substantially equivalent strains through mutation treatment, genetic recombination, and selection of natural mutant strains.

The lactic acid strain of the present invention can be easily grown by culturing the strain, for example. The method of culturing is not particularly limited as long as the CFF No. 2031 strain can proliferate, and it can be used by appropriately modifying methods that can be used for culturing of lactic acid bacteria as needed. For example, the culture temperature may be 25-50°C, preferably 35-42°C. In addition, the culture may be performed under either aerobic conditions or anaerobic conditions, and is preferably performed under anaerobic conditions. For example, the culture can be performed while aerating an anaerobic gas such as carbon dioxide. In addition, it can also be cultured under microaerobic conditions such as liquid static culture.

The medium for cultivating the lactic acid strain of the present invention is not particularly limited, and it can be used by appropriately modifying the medium that can be used for culturing of lactic acid bacteria as necessary. That is, as a carbon source, sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and waste molasses can be used in accordance with assimilation. As the nitrogen source, for example, ammonium salts or nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate can be used. In addition, as inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate, etc. can be used. In addition, organic ingredients such as egg whites, soybean meal, defatted soybean meal, meat extract, and yeast extract can also be used. In addition, as for the prepared medium, for example, MRS medium can be suitably used.

With regard to the lactic acid strain of the present invention, after culture, the obtained culture can be used as it is, it can be diluted or concentrated for use, and bacteria recovered from the culture can also be used. In addition, as long as the effects of the present invention are not impaired, various additional operations such as heating and freeze-drying can be performed after the cultivation.

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

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

The lactic acid strain of the present invention can be used as an agent for regulating the composition balance of bacteria contained in the resident bacterial flora in the subject. Therefore, the composition containing the lactic acid strain of the present invention can preferably be used as a composition for adjusting the balance of the bacteria contained in the resident bacterial flora in the subject. In addition, according to a better embodiment, the above composition can be used to reduce the bacteria of the Trichospiraceae, Acinetobacter, Prevotella, or Bacteroides in the above-mentioned resident bacterial flora . In addition, according to another preferred embodiment, the composition can be used to increase the Myxospirillum bacteria in the resident bacterial flora. In addition, according to yet another preferred mode of administration, the above-mentioned composition can be used to suppress the weight loss caused by diarrhea at the onset of enteritis in subjects who have been administered it. In addition, according to yet another preferred embodiment, the above-mentioned composition can be used to inhibit the DAI score of active enteritis at the onset of enteritis in a subject who has been administered it. In addition, according to another preferred embodiment, the above-mentioned composition can be used to inhibit inflammation of the mucosa in a subject to which it has been administered, and to inhibit diarrhea or fecal bleeding.

In addition, the lactic acid strain of the present invention can be used to reduce the amount of inflammation-related bacteria in the resident bacterial flora. Therefore, according to another embodiment, the composition of the present invention can be used to inhibit inflammation-related diseases or symptoms.

The resident bacterial colony in the present invention is preferably a mucosal attached bacterial colony or an intestinal bacterial colony.

Preferably, the composition of the present invention can be used as medicine, food and drink, cosmetics or feed.

The medicine is not particularly limited as long as it contains the lactic acid strain of the present invention. In terms of medicine, the lactic acid strain of the present invention can also be formulated and used together with a pharmaceutically acceptable carrier.

The dosage form of the medicine of the present invention is not particularly limited. Specifically, examples thereof include tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, and ointments. , Patches, eye drops and nose drops, etc. In addition, during formulation, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavors, diluents, surfactants, or solvents for injections that are generally used as formulation carriers can be used .

The content of lactic acid bacteria in the medicine of the present invention is appropriately set according to the dosage form, usage, age, sex of the patient, type of disease, degree of disease, and other conditions, etc., and is generally preferably 1×10 ⁶ to 1×10 ¹² cfu/g or 1×10 ⁶ ~1×10 ¹² cfu/ml, more preferably 1×10 ⁷ ~1×10 ¹¹ cfu/g or 1×10 ⁷ ~1×10 ¹¹ cfu/ml Inside. When the CFF No. 2031 strain is dead, cfu/g or cfu/ml can be replaced with cells/g or cells/ml.

Food and beverage products are not particularly limited as long as they contain the lactic acid strain of the present invention. For food and beverage products, examples include soft drinks, carbonated beverages, nutritious beverages, fruit juice beverages, and beverages such as lactic acid bacteria beverages (including concentrated stock solutions of these beverages). And preparation powder); ice cream, snow fruit frost, shaved ice and other iced desserts; candy, chewing gum, hard candy, gum, chocolate, candy ingots, snacks, biscuits, jelly, jam, fresh cream, And baked snacks and other snacks; processed milk, milk drinks, fermented milk, yogurt drinks, and cream and other dairy products; bread; enteral nutrition food, liquid food, milk for childcare, sports drinks; other functional foods. In addition, the food and drink may also be a supplement, for example, a lozenge-shaped supplement. In the case of supplements, it is possible to ingest CFF No. 2031 strain without being affected by other foods regarding the daily meal amount and calorie intake.

Foods and beverages can be produced by adding the lactic acid strain of the present invention to the raw materials of foods and beverages, and can be produced in the same manner as general food and beverages except for adding the lactic acid strain of the present invention. The addition of the lactic acid strain of the present invention can be carried out at any stage of the food and beverage production step. In addition, food and beverages can also be manufactured through fermentation steps caused by the added lactic acid strain of the present invention. Examples of such foods and beverages include lactic acid bacteria drinks and fermented milk.

As for the raw materials of food or beverages, raw materials that can be used for general beverages and foods can be used.

The food-drinks of the present invention also include raw materials for manufacturing food-drinks, food additives, etc., which are added to the food-drinks during or after the manufacturing process of the food-drinks. For example, the lactic acid strain of the present invention can be used as a bacterial cell for fermented milk production. In addition, the lactic acid strain of the present invention may be subsequently added to fermented milk that has been produced.

The content of the lactic acid strain of the present invention in the food and beverage is appropriately set according to the aspect of the food and beverage. Generally, in the food and beverage, it is preferably 1×10 ⁶ to 1×10 ¹² cfu/g or 1×10 ⁶ to 1 Within the range of ×10 ¹² cfu/ml, more preferably within the range of 1 × 10 ⁷ to 1 × 10 ¹¹ cfu/g or 1 × 10 ⁷ to 1 × 10 ¹¹ cfu/ml.

The food and beverage of the present invention can be sold as food and beverage products marked with the following uses: for adjusting the composition balance of bacteria contained in the resident bacterial flora, and for reducing the bacteria of the Trichospiraceae family in the resident bacterial flora , Acinetobacter bacteria, Bacteroides bacteria or Prevotella bacteria, used to increase the mucospirillum bacteria attached to the mucosa or in the intestinal flora, used to inhibit weight loss, used to inhibit DAI Fraction is used to inhibit diarrhea or stool bleeding, to reduce inflammation-related strains, and to inhibit inflammation-related diseases or symptoms.

In addition, in the food and beverage of the present invention, it is possible to perform "for adjusting the composition balance of bacteria contained in the resident bacterial flora" and "for reducing the bacteria of the Trichospiraceae family and Acinetobacter in the resident bacterial flora" Bacteria, Bacteroides or Prevotella bacteria", "Used to increase Myxospirillum bacteria in the resident bacterial flora", "Used to inhibit weight loss", "Used to inhibit DAI score" , "Used to inhibit diarrhea or stool bleeding", "Used to reduce inflammation-related bacteria", "Used to inhibit inflammation-related diseases or symptoms" and other labels. Furthermore, in addition, words that indicate secondary effects produced by the use of the composition for the above-mentioned purposes can also be used without further elaboration. In terms of such phrases, for example, "rectify the state of the intestines and stomach", "improve the feeling of stomach or intestinal discomfort" and so on.

The aforementioned "labeling" refers to all actions used to make the needy aware of the above-mentioned use. If it is a label that can think of/analogize the above-mentioned use, regardless of the purpose of the labeling, the content of the label, the object of the label, the media, etc. All correspond to the "label" of the present invention. However, it is better to mark it through the use-like performance that the needer can directly recognize.

Specifically, it can be exemplified: the act of describing the above-mentioned use in the product or the package of the food and beverage of the present invention, the transfer or delivery of the above-mentioned use in the product or the packaging of the product, and the display or delivery for the transfer or delivery. The act of input is displayed or published by recording the above-mentioned purposes in advertisements, price lists, or transaction documents about products, or using electromagnetic (Internet, etc.) methods to record the above-mentioned purposes in information using these as content The behaviors provided are particularly good for the labeling of promotional materials and other documents at the sales site such as packaging, containers, catalogs, brochures, and POPs.

In addition, the labeling is preferably a label approved by an administrative agency or the like (for example, labeling approved based on various systems established by the administrative agency and performed in a manner based on such approval). Examples include labels for health foods, functional foods, enteral nutrition foods, special purpose foods, nutritional functional foods, quasi-drugs, etc. Other labels approved by the Ministry of Health, Labour and Welfare, such as foods for specific health care, With a mark similar to that recognized by its system. In the latter case, examples can include: labeling as a specific health food, labeling as a conditional specific health food, labeling to affect the structure or function of the body, and labeling to reduce the risk of disease. In more detail, it can be exemplified as an example of the labeling of foods for specific health use (especially labels for health use) formulated in the Enforcement Regulations of the Health Promotion Act (Order No. 86 of the Ministry of Health, Labour and Welfare of Japan on April 30, 2015), and Similar signs and so on.

The cosmetics of the present invention can also be prepared according to the above-mentioned medicines or foods and beverages and used as cosmetics for internal administration, but can also be used as external skin preparations for topical or systemic use. Suitable examples of skin external preparations include: lotion, lotion, cream, ointment, lotion, oil, facial mask and other basic cosmetics, solid soap, liquid soap, hand soap and other facial cleansers or Cleanser, massage agent, make-up remover, hair remover, depilatory agent, shaving treatment, beard after lotion, beard lotion, shaving cream, foundation, lipstick, blush, eye shadow, eyeliner, mascara, etc. Cosmetics, perfumes, manicure, nail polish, nail polish remover, poultice, plaster, patch, tablet, patch, aerosol, etc.

The content of the CFF NO.2031 strain in the cosmetics of the present invention is appropriately set according to the state of the cosmetics and the subject of administration, preferably 1×10 ⁶ ~1×10 ¹² cfu/g or 1×10 ⁶ ~1 Within the range of ×10 ¹² cfu/ml, more preferably within the range of 1 × 10 ⁷ to 1 × 10 ¹¹ cfu/g or 1 × 10 ⁷ to 1 × 10 ¹¹ cfu/ml.

In addition, for cosmetics, it is possible to arbitrarily select and use the additives listed below to produce it. Such additives are not particularly limited, and examples include oils and fats (cocoa butter, chamomile oil, carrot oil, cucumber oil, shea fatty acid, chestnut oil, safflower oil, shea butter, etc.), waxes Types (beeswax, carnauba wax, spermaceti, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice bran wax, etc.), mineral oil (liquid paraffin, Vaseline, paraffin, ozokerite, ceresin, microcrystalline wax, etc.), fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid) , Linoleic acid, docosahexaenoic acid, eicosapentaenoic acid, 12-hydroxystearic acid, undecylenic acid, tall oil, lanolin fatty acid and other natural fatty acids, isononanoic acid, hexyl Acid, 2-ethylbutyric acid, isovaleric acid, 2-methylvaleric acid, 2-ethylhexanoic acid, isovaleric acid and other synthetic fatty acids), alcohols (ethanol, isopropanol, lauryl alcohol, cetyl alcohol) , Stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol and other natural alcohols, 2-hexyldecanol, isostearyl alcohol, 2-octyldodecanol and other synthetic alcohols, etc.) , Various vitamins (vitamin A group: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotene, lycopene (original vitamin A), vitamin B group: thiamine hydrochloride Thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acid, nicotinic acid, pantothenic acid, biotin, Choline, inositols, vitamin C group: vitamin C acid or its derivatives, vitamin D group: ergocalciferol (vitamin D2), etc.), polymer compounds (gum arabic, benzoin gum, dammar gum ), guaiac resin, chitosan, hyaluronic acid or its salt, chondroitin sulfate or its salt, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxyethyl Sodium cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone (polyvinylpyrrolidone), polyvinyl Polyalkylene oxide such as methacrylate, polyacrylate, polyethylene oxide and polypropylene oxide or its crosslinked polymer, carboxyvinyl polymer, polyethyleneimine, etc.).

Examples of the feed of the present invention include pet food, livestock feed, and fish feed. The feed of the present invention can be mixed with CFF NO.2031 strains into general feeds, such as cereals, meal, bran, fish meal, bone meal, oils and fats, skimmed milk powder, whey, mineral feed, or yeast, etc. And manufacturing. In addition, for example, like silage, it is also possible to manufacture feed through the fermentation process caused by the added CFF NO. 2031 strain. The feed produced can be orally administered to general mammals, livestock, fish breeding, and play animals.

The content of the CFF NO.2031 strain in the feed of the present invention is appropriately set according to the state of the feed and the subject of administration, but it is preferably 1×10 ⁶ ~1×10 ¹² cfu/g or 1×10 ⁶ ~1 Within the range of ×10 ¹² cfu/ml, more preferably within the range of 1 × 10 ⁷ to 1 × 10 ¹¹ cfu/g or 1 × 10 ⁷ to 1 × 10 ¹¹ cfu/ml.

In addition, according to another aspect, a method is provided, which is a method of adjusting the composition balance of bacteria contained in the resident bacterial flora, including administering an effective amount of the lactic acid strain of the present invention to a subject in need thereof. According to another preferred aspect of the present invention, the above method is used to make bacteria related to inflammation (for example, bacteria of the genus Trichospira, bacteria of the genus Acinetobacter, bacteria of the genus Bacteroides or Prevotella bacteria, etc.) reduction methods. In addition, according to another better aspect, the above method is a method for increasing the Myxospirillum bacteria in the resident bacterial flora. In addition, according to another further preferred aspect, the above method is a method for suppressing at least one symptom selected from the group consisting of weight loss, DAI score, diarrhea, or stool bleeding due to diarrhea caused by the onset of enteritis.

In addition, according to another aspect, a method is provided, which is a method for suppressing inflammation-related diseases or symptoms, including a method of administering an effective amount of the lactic acid strain of the present invention to a subject in need thereof. Here, in terms of "inhibition", it is not only to treat or improve an established pathology, but also to prevent a pathology that may be established in the future.

Regarding the above-mentioned inflammation-related diseases or symptoms, inflammatory bowel disease (IBD) can be mentioned, specifically, ulcerative colitis (UC) and Crohn's disease (CD) can be mentioned.

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

The effective amount of the lactic acid strain of the present invention is not particularly limited as long as it does not hinder the effects of the present invention. For example, it can be 1.0×10 ⁴ cfu/time to 1.0×10 ¹² cfu/time. According to one aspect, the effective amount of CFF NO.2031 strain is the intake of each meal.

The effective amount of the lactic acid strain of the present invention can be administered once a day or divided into multiple times, and it may be administered once every few days or weeks. In addition, according to the status of the subject, etc., the timing of the injection can be appropriately selected.

The object of the present invention is preferably humans, more preferably minors under 15 years of age, and still more preferably infants. The application of the lactic acid strain of the present invention to infants and young children is particularly advantageous in that it is used as a substitute for breast milk to adjust the composition balance of the bacteria contained in the mucosa attached bacterial colony or the intestinal bacterial colony.

In addition, the lactic acid strain of the present invention may not be limited to be administered continuously for a period of time, and may also be administered in a period of infants and toddlers. The administration of the lactic acid strain of the present invention in infants and young children is also particularly advantageous in preventing diseases after growth.

In addition, the subject may be a physically and mentally sound person, a person with a disease or symptom related to inflammation in the gastrointestinal tract, or a person with poor physical condition caused by the former.

In addition, the administration method in the present invention is preferably oral. However, as long as the effect of the present invention is not impaired, it is not particularly limited. For example, the lactic acid strain of the present invention may be administered to a subject by other methods such as via tube, intestine, or transderm (coating, etc.).

In addition, according to another aspect, a use of the lactic acid strain of the present invention is provided, which is used in the manufacture of a composition for adjusting the composition of bacteria contained in the resident bacterial flora. According to a preferred aspect, the above-mentioned composition is used to reduce inflammation-related bacteria (for example, bacteria of the Trichospiraceae family, bacteria of the genus Acinetobacter, bacteria of the genus Prevotella or Bacteroides, etc.). In addition, according to another preferred aspect, the above-mentioned composition is a composition for increasing Myxospirillum bacteria in the resident bacterial flora. In addition, according to another better aspect, the above-mentioned composition is a composition for suppressing at least one symptom selected from weight loss due to diarrhea caused by the onset of enteritis, DAI score, diarrhea, or stool bleeding.

In addition, according to another preferred aspect, a use of the lactic acid strain of the present invention is provided, which is used in the manufacture of a composition for inhibiting inflammation-related diseases or symptoms. [Example]

The following describes embodiments of the present invention, but the present invention is not limited to these embodiments.

Example 1: Confirmation test of the effect of administering lactic acid bacteria on mucosal attached bacterial flora and intestinal bacterial flora Two-week-old C57BL/6N mice purchased from Japanese SLC were acclimated in a facility for 7 days, and then weaned at 3 weeks of age. Next, the mice were divided into two groups (n=6) of a control group (C group) and a Lactobacillus crispatus CFF No. 2031 strain administration group (L group) so that their body weight was equalized, and they were fed and managed. The mice after weaning were allowed to freely ingest Labo MR Stock for mice/rats/hamsters (Nippon Agricultural Industry Co., Ltd.) and to freely ingest unsterilized water. The feeding room has a 12-hour light-dark cycle with lights on at 8 o'clock and lights off at 20 o'clock, and the room temperature is set at 25±2°C.

The oral administration of each test substance started from the day after the weaning day, and the oral administration was carried out for 14 days. Group C was orally administered PBS(-), and group L was orally administered Lactobacillus crispatus CFF No. 2031 strain. The volume of the liquid at the time of oral administration is adjusted depending on the body weight of the mouse. If the body weight is less than 12.5 g, it is made 100 l, and if it is 12.5 or more and less than 17.5 g, it is made 150 l.

As for the device for oral administration, a 1ml syringe (TERUMO) was used. In addition, for oral administration, the 1ml syringe is cut to an appropriate length, and the tip is baked into a circular CHUKOH FLO PTFE tube AWG-26 (Zhongxing Chemical Industry) for installation, and the Autoclaved 23G needle (TERUMO).

The liquid containing Lactobacillus crispatus CFF No. 2031 strain orally administered to mice was prepared as follows. The Lactobacillus crispatas CFF No. 2031 strain stored at -80°C was planted in 10 ml of MRS medium, and cultured at 37°C for 24 hours under anaerobic conditions to make a pre-culture solution. The pre-culture solution is stored in a refrigerator, and is used to prepare a lactic acid strain-containing solution every day. The strain-containing liquid was subcultured in a new 10 ml MRS medium by removing 200 μl from the preculture medium on the day before oral administration, and cultured under the same conditions as the preculture. After the incubation, centrifuge at 10,000×g at room temperature for 3 minutes. After the bacteria are made into pellets, they are washed with PBS(-) and the turbidity (OD595) is used as the standard to make it 5×10 Suspended in PBS(-) at ⁹ cfu/ml to make a liquid containing lactic acid strains.

The washout period (stop period) of the test substance was made 7 days after the end of the oral administration, and the oral administration was not carried out.

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

Use QuickGene DNA tissue kit S (KURABO) to extract bacterial DNA from the mucosa and contents of the collected tissues. 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 (NIPPON Genetics). In this reaction, the sequence for 2ndPCR is to use a tailed forward primer 341F (sequence identification number 2: 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3'), and a reverse primer 805R (sequence Identification number 3: 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3'). PCR was performed by using 12.5 μl of KAPA HIFI HS Ready Mix, 2.5 μl of template DNA, each 0.05 μl of primers, and 9.9 μl of free water to 25 μl as a reaction solution. After initial denaturation at 95°C for 3 minutes, 25 cycles were performed: 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, followed by an elongation reaction at 72°C for 5 minutes. After the PCR reaction was completed, 1% agarose gel (tris acetate EDTA: TAE) was used for electrophoresis to confirm that the target gene was amplified. Afterwards, the PCR product was purified using NucleoFast 96 PCR Clean up plate (TAKARA BIO). Through the second PCR reaction with primers loaded with Index and Illumina sequencing adapter, the purified product was tagged.

SequalPrepTM Normalization Plate Kit (Invitrogen) was used for purification and concentration adjustment of the product obtained by PCR. Next, the concentration of the purified product was quantified by real-time PCR using KAPA SYBR Fast qPCR Kit (NIPPON Genetics). The samples with sufficient concentration were mixed with 10μl per sample and purified/concentrated using Agencouet AMPure XP (BECKMAN COULTER).

The concentration of the obtained library was calculated according to the standard procedure of KAPA Library Quantification Kit (NIPPON Genetics). Then, according to the calculated concentration, the sample was diluted to 4nM with 10 mM Tris Buffer. The subsequent operation is the final preparation according to 16S Metagenomic Sequencing Library Preparation (Illumina). The prepared sample was loaded into the reagent cartridge of Miseq Reagent Kit V3 (600PE), and sequenced by MiSeq (Illumina). The sequenced data obtained is based on QIIME's primary analysis, followed by a secondary analysis using STAMP.

The result is shown in Figure 1~4. Figures 1A and 1B are the results of principal coordinate analysis based on the overall genome analysis of the cecal mucosal attached bacterial cluster. In the above analysis, phyloseq is used (refer to https://joey711.github.io/phyloseq/). Therefore, the classification of bacteria shown in Figure 2A, Figure 3A, and Figure 4A to Figure 4H is based on the classification of phyloseq and Greengenes referenced by the software. Based on the results of the principal coordinate analysis of the overall genome analysis, compared with the control group (C group), in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group), it is the mucous membrane of the cecum that adheres to the bacterial clusters. The composition confirmed the change (PERMANOVA p=0.002).

The result of bacterial flora analysis based on the overall genome analysis of the mucosal attached bacterial flora of the cecum is shown in Figure 2A. In addition, with regard to the mucosa adherent bacterial flora of the cecum, the occupancy rate of Lachnospiraceae bacteria (Unclassified Lachnospiraceae) in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) The occupancy rate (proportion of sequences (%)) in the control group is significantly lower than that (Figure 2B, Wilcoxon/Kruskal-Wallis test (rank sum)), p<0.05 ). On the other hand, the occupancy rate of Myxospirillum bacteria in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) was significantly higher than that in the control group (Figure 2C, Wilcoxon/Kruskal-Wallis test (grade sum), p<0.05).

The colony structure analysis result based on the overall genome analysis of the colon mucosal attached bacterial colony is shown in Figure 3A. Regarding the colonic mucosa-adherent bacterial clusters, the occupancy rate of Acinetobacter bacteria in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) is significant compared with the occupancy rate in the control group (C group) The ground is low (Figure 3B, Wilcoxon/Kruskal-Wallis test (grade sum), p<0.05).

It is known that bacteria of the Trichospiraceae family and bacteria of the genus Acinetobacter increase when the intestinal tract becomes inflamed (Non-Patent Documents 2 to 5). Therefore, from the result that the bacteria were reduced by administering Lactobacillus crispatus CFF No. 2031 strain, it is believed that Lactobacillus crispatus CFF No. 2031 strain has an effect of suppressing inflammation of the digestive tract. In addition, Mucispirillum is a bacteria that assimilates mucus. Based on the results that the bacteria are increasing, it is believed that by administering Lactobacillus crispatus CFF No. 2031 strain, the mucous membrane of the digestive tract is sufficiently filled with mucus. From the above, it is considered that Lactobacillus crispatus CFF No. 2031 strain has the effect of improving the barrier function of the gastrointestinal mucosa.

The results of the bacterial colony structure analysis based on the total genome analysis of the cecal contents are shown in Figure 4A. In addition, regarding the bacterial clusters in the cecum, as shown in Figure 4B, the occupancy rate of Bacteroides in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) was compared with the control group. Significantly low (student t test, p=0.0484). In addition, as shown in FIG. 4C, compared with the control group, the occupancy rate of [Prevotella] bacteria ([Prevotella]) in the L group was significantly lower (student t test, p=0.0430). In addition, as shown in Figure 4D, compared with the control group, the occupancy rate of the Lachnospiraceae bacteria (Unclassified Lachnospiraceae) in the L group is significantly lower (student t test, p <0.0001). Furthermore, compared with the control group, the occupancy rate of Lactobacillus in the L group was significantly higher (student t test, p=0.0409).

The result of the analysis of the structure of the flora based on the overall genome analysis of the bacterial flora of the colon contents is shown in Figure 4E. Regarding the bacterial clusters of the colon contents, as shown in FIG. 4F, compared with the control group, the occupancy rate of Bacteroides in the L group is significantly lower (Student t test, p=0.0004). In addition, as shown in Figure 4G, compared with the control group, the occupancy rate of Prevotella in the L group is significantly lower (Wilcoxon/Kruskal-Wallis test (rank sum), p= 0.0131). In addition, as shown in FIG. 4H, compared with the control group, the occupancy rate of [Prevotella] bacteria ([Prevotella]) in the L group is significantly lower (Student t test, p=0.0072).

It is known that prevotella bacteria and Bacteroides bacteria increase when the intestines and the whole body become inflamed (Non-Patent Documents 6 to 8). Therefore, from the result that the bacteria are reduced by administering Lactobacillus crispatus CFF No. 2031 strain, it is believed that Lactobacillus crispatus CFF No. 2031 strain has the effect of suppressing inflammation of the digestive tract. From the above, it is believed that the Lactobacillus crispatus CFF No. 2031 strain has the effect of suppressing inflammation of the digestive tract mucosa.

Example 2: Test of enhancing the function of digestive tract obstruction caused by the administration of lactic acid bacteria Two-week-old C57BL/6N mice with two fetuses (6 in each fetus), which were acquired from Charles River Laboratories Japan, Inc., and female mice, were introduced into this laboratory for breeding Room, the facility was adapted for 7 days. The young mice were weaned at the age of 3 weeks and divided into two groups (N=6): a control group (group C) and a Lactobacillus crispatus CFF No. 2031 strain administration group (group L) so that their body weights became equal Carried out feeding management. In the same manner as in Example 1, from the day following the weaning day, PBS(-) was started for oral administration in the DC group, and the initial Lactobacillus crispatus CFF No. 2031 strain was orally administered to the DL group, and It was administered orally for 14 days. The 7 days from the end of the oral administration was set as a wash-out period of the administered bacteria, and the oral administration was not carried out for breeding. At the end of the wash out period and the mice reached 6 weeks of age, in Example 2, 1.5% DSS (Dextran Sulfate Sodium (DSS) salt was added at 15 g/l). ), Colitis grade (MP Biomedicals MW 36-50kDa) dissolved in distilled water) is replaced with tap water as drinking water, so that it can be taken freely. All mice were submitted for dissection on the 11th day of DSS administration.

In addition, during the period after weaning that there is no special description, free intake of Labo MR Stock for mice/rats/hamsters (Nippon Agricultural Industry Co., Ltd.) and unsterilized tap water are allowed freely. The feeding room uses a 12-hour light-dark cycle with lights on at 8 o'clock and lights off at 20 o'clock. The room temperature is set at 25±2°C, and the humidity is set at 40~70%.

From the start day of DSS administration (d22) to the day before the final day (d31), the weight of the mice and the scoring of stool properties (hardness and bleeding) were performed every day. However, the final day (d32) is the weight measurement and fecal character score of the mice just before the dissection. DAI, which is an index indicating the severity of enteritis, was calculated from the weight loss rate and stool characteristics, and the symptoms of enteritis were evaluated. Furthermore, this evaluation method is based on Mennigen et al. (2009) (Table 2-1).

[Table 1]

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

When the time of administration and age of the mice used in this experiment are converted to humans (the lifespan of mice is set to 2 years, and the lifespan of humans is set to 60 to 80 years for conversion). In the test, the results of administering lactic acid bacteria to children between one and a half to two and a half years old for a period of one to one and a half years have prevented inflammatory diseases that may occur around 4 to 5 years old. From these results, it is considered that the administration of the lactic acid strain of the present invention in infants and young children can be used to prevent diseases after growth. [Industrial availability]

According to the present invention, a novel lactic acid bacteria is provided, which can effectively adjust the balance of the bacterial composition in the resident bacterial colony headed by the mucosal attached bacterial colony or the intestinal bacterial colony.

[Figure 1A] is a graph showing the results of principal coordinate analysis: In Example 1, the control group (C group) and the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) were subjected to cecal mucosa Principal coordinate analysis of metagenome analysis of attached bacterial clusters (parameters on the vertical axis: Axis 2, horizontal axis Axis 1). [Figure 1B] is a graph showing the results of principal coordinate analysis: In Example 1, the control group (C group) and the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) were subjected to cecal-based mucosa The principal coordinate analysis of the overall genome analysis of the attached bacterial clusters (vertical axis parameters: Axis 2, horizontal axis Axis 3). [Fig. 2A] is a graph showing the results of the analysis of the flora structure based on the overall genome analysis of the caecal mucosal attached bacterial flora in Example 1. [Figure 2B] is a graph showing the comparison results: In Example 1, regarding the mucosa adherent bacterial clusters of the cecum, the unclassified Lactobacillus crispatus CFF No. 2031 strain administration group (L group) was unclassified in the Unclassified Trichospira family (Unclassified). The occupancy rate of f_Lachnospiraceae) is compared with the occupancy rate in the control group (group C). [Figure 2C] is a graph showing the results of comparison: In Example 1, regarding the mucosa adherent bacterial clusters of the cecum, in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group), the bacteria of the genus Mucispirillum The occupancy rate is the result of comparison with the occupancy rate in the control group (group C). [Fig. 3A] is a graph showing the results of flora structure analysis based on the overall genome analysis of the colon-attached bacterial flora in Example 1. [Figure 3B] is a graph showing the results of comparison: In Example 1, regarding the colonic mucosa adherent bacterial cluster, the bacteria belonging to the genus Acinetobacter (Acinetobacter) in the Lactobacillus crispatus CFF No. 2031 strain administration group (group L) The occupancy rate is the result of comparison with the occupancy rate in the control group (group C). [Fig. 4A] is a graph showing the results of the analysis of the flora structure based on the overall genome analysis of the bacterial flora of the cecal contents in Example 1. [Figure 4B] is a graph showing the results of comparison: In Example 1, regarding the bacterial flora of the cecal contents, the bacteria of the genus Bacteroides in the Lactobacillus crispatus CFF No. 2031 strain administration group (group L) The occupancy rate is compared with the occupancy rate in the control group (group C). [Figure 4C] is a graph showing the results of comparison: In Example 1, regarding the bacterial flora of the cecal contents, [Prevotella] bacteria in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) The occupancy rate of ([Prevotella]) is compared with the occupancy rate in the control group (Group C). [Figure 4D] is a graph showing the results of comparison: In Example 1, regarding the bacterial flora of the cecal content, in the Lactobacillus crispatus CFF No. 2031 strain administration group (group L), bacteria of the Trichospiraceae family (unclassified The occupancy rate of Lachnospiraceae (Unclassified Lachnospiraceae) is compared with the occupancy rate in the control group (group C). [Figure 4E] is a graph showing the results of bacterial flora analysis based on the overall genome analysis of the bacterial flora of the colon content in Example 1. [Figure 4F] is a graph showing the comparison results: In Example 1, regarding the bacterial flora of the colon contents, the occupancy of Bacteroides in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) Rate, the result of comparison with the occupancy rate in the control group (Group C). [Figure 4G] is a graph showing the results of comparison: In Example 1, regarding the bacterial flora of the colon contents, in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) Prevotella bacteria (Prevotella The occupancy rate of) is compared with the occupancy rate in the control group (Group C). [Figure 4H] is a graph showing the results of comparison: In Example 1, regarding the bacterial flora of the colon contents, [Prevotella] bacteria in the Lactobacillus crispatus CFF No. 2031 strain administration group (L group) The occupancy rate of ([Prevotella]) is compared with the occupancy rate in the control group (Group C). [Fig. 5A] is a graph showing the weight change of the test mice in Example 2. [Fig. It shows the weight change (%) from the first day (d1) of the initial DSS administration to 100.00% to the 11th day (d11) of the initial DSS administration. [Fig. 5B] is a graph showing the changes in the disease activity index (DAI) of the test mice in Example 2. The DAI scores from the first day (d1) to the 11th day (d11) of the initial DSS administration are shown.

Claims (14)

A Lactobacillus crispatus strain, the nucleotide sequence of the 16s rRNA gene of the strain is at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 1. Such as the strain of claim 1, wherein the aforementioned identity is 98% or more. For example, the strain of claim 1 or 2 is the Lactobacillus crispatus CFF No. 2031 deposited under the deposit number NITE BP-02869. A composition comprising the strain of any one of claims 1 to 3. Such as the composition of claim 4, which is used as medicine, food, cosmetics or feed. Such as the composition of claim 4 or 5, it is for infants or minors. Such as the composition of any one of claims 4 to 6, which is used to adjust the composition balance of bacteria contained in the resident bacterial flora. The composition of claim 7, wherein the bacteria contained in the aforementioned resident bacterial flora are selected from the group consisting of bacteria of the genus Trichospiraceae, bacteria of the genus Acinetobacter, bacteria of the genus Mucispirillum, bacteria of the genus Prevotella, and At least one of Bacteroides bacteria. The composition of claim 7 or 8, wherein the aforementioned resident bacterial clusters are mucosal attached bacterial clusters or intestinal bacterial clusters. The composition of any one of claims 4 to 9, which is used to inhibit inflammation-related diseases or symptoms. Such as the composition of any one of claims 4 to 10, which is used to protect the digestive tract. The composition of any one of claims 4 to 11, which is used to inhibit weight loss. A use of a Lactobacillus crispatus strain, which is to produce a composition for regulating the composition of the bacteria contained in the resident bacterial flora, and the nucleotide sequence of the 16s rRNA gene of the strain is as shown in the sequence identification number 1. The nucleotide sequence is at least 95% identical. A method for adjusting the balance of the composition of bacteria contained in the resident bacterial flora, comprising: administering an effective amount of a Lactobacillus crispatus strain to a subject in need thereof, and the nucleotide sequence of the 16s rRNA gene of the aforementioned strain It is at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 1.

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