JP2022053525A - Agent for keeping lactic acid-producing bacterial flora, oral composition including the same, and method for keeping lactic acid-producing bacterial flora - Google Patents

Abstract

To provide an agent for keeping lactic acid-producing bacterial flora, an oral composition including the same, and a method for keeping lactic acid-producing bacterial flora, which can hold the balance of viable cells in lactic acid-producing bacterial flora.SOLUTION: A agent for keeping lactic acid-producing bacterial flora contains at least one compound selected from the group consisting of organic acids and pharmacologically acceptable salts thereof as an active ingredient. The agent for keeping is useful as constituent materials of oral compositions such as general foods, supplements, health-promoting foods, or pharmaceuticals.SELECTED DRAWING: None

Description

The present invention relates to a lactic acid-producing flora retainer, an oral composition using the same, and a method for retaining a lactic acid-producing flora.

It is said that the majority of intestinal bacteria inhabit the human intestine. A wide variety of intestinal bacteria grow in the human intestine, and a bacterial flora called the intestinal flora is formed. The intestinal bacteria that form this intestinal flora are roughly classified into good bacteria such as lactic acid bacteria and bifidobacteria (sometimes called lactic acid-producing bacteria); bad bacteria such as Clostridium perfringens; opportunistic bacteria such as Bacteroides spp. Will be done. It is known that when the intestinal flora is out of balance, the intestinal environment changes, and as a result, it adversely affects the physical condition and may cause various diseases.

In recent years, there has been an increasing need to actively ingest foods containing such lactic acid-producing bacteria in order to improve the intestinal environment.

Here, Non-Patent Document 1 states that the growth of bifidobacteria can be promoted by coexisting a predetermined amount of short-chain fatty acids (for example, acetic acid, propionic acid or butyric acid) with bifidobacteria. It has been reported.

By using such a technique, it is possible to increase the growth efficiency of the ingested lactic acid-producing bacteria and counteract the above-mentioned changes in the intestinal environment. However, even if lactic acid-producing bacteria are ingested as they are, only a few can reach the intestine as live bacteria. It is difficult to construct a good intestinal environment that maintains the balance of the intestinal flora only by promoting the growth of lactic acid-producing bacteria.

Kaneko T et al., J. Dairy Sci., 1994, 77 (2), pp.393-404

An object of the present invention is to solve the above-mentioned problems, and an object thereof is to use a lactic acid-producing flora retainer capable of maintaining a viable cell balance in the lactic acid-producing flora and a retainer thereof. It is an object of the present invention to provide a method for retaining an oral composition and a lactic acid-producing flora.

The present invention is a lactic acid-producing flora retainer containing at least one compound selected from the group consisting of organic acids and pharmaceutically acceptable salts thereof as an active ingredient.

In one embodiment, the organic acid is a fatty acid having 2 to 10 carbon atoms.

In one embodiment, the organic acid is a fatty acid having 2 to 6 carbon atoms.

In one embodiment, the organic acid is at least one fatty acid selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, and succinic acid.

In one embodiment, the lactic acid-producing bacteria constituting the lactic acid-producing flora are Bifidobacterium, Lactobacillus, Enterococcus, Staphylococcus, Streptococcus, Lactococcus, Pediococcus, and Leuco. It is at least one microorganism selected from the group consisting of the genus Nostock.

The present invention is also an oral composition containing the preservative for the lactic acid-producing flora.

The present invention is also a method for retaining a lactate-producing flora, which comprises a step of combining a system containing the lactate-producing flora with a preserving agent for the lactate-producing flora.

According to the present invention, it is possible to increase the retention efficiency of the entire bacterial flora composed of lactic acid-producing bacteria. This can be expected, for example, to form a predominant intestinal environment in the intestine. It can also be expected to suppress the growth of harmful bacteria (so-called bad bacteria) in the intestine through the predominant intestinal environment. The retainer of the present invention is also composed of a material general-purpose in the field of food manufacturing, and the risk of adverse effects on the body due to ingestion by humans is reduced in advance.

3 is a graph showing changes in the number of bacteria with respect to the culture time for the culture solutions obtained in Example 1 and Comparative Example 1.

Hereinafter, the present invention will be described in detail.

(Retaining agent for lactic acid-producing flora)
The retainer of the lactic acid-producing flora of the present invention contains at least one compound selected from the group consisting of an organic acid and a pharmaceutically acceptable salt thereof as an active ingredient.

As used herein, the term "lactic acid-producing bacterial flora" refers to a bacterial flora composed of one or more types of lactic acid-producing bacteria described below, and cross-talk between lactic acid-producing bacteria or between them and a host. Includes those that make up a stable and complex ecosystem through. If a lactating flora is present in the intestine, it may constitute an intestinal flora or intestinal flora. In the present invention, the lactic acid-producing flora is present in a living body (for example, in an organ such as the intestine) such as humans, pet animals, livestock, poultry, and cultured fish; foods and drinks, cosmetics, pharmaceuticals, and feeds. It includes any of those present in industrial products such as; those present in a culture environment with a medium in a predetermined culture means (eg, petri dish).

Lactic acid-producing bacteria constituting the lactic acid-producing flora are microorganisms capable of producing lactic acid by metabolism, and can be generally used in the manufacturing field of foods, cosmetics, pharmaceuticals, etc. (for example, gram-positive bacilli and cocci). ) Is included. Examples of lactic acid-producing bacteria include Bifidobacterium, Lactobacillus, Enterococcus, Staphylococcus, Streptococcus, Lactococcus, and Lactococcus. Microorganisms belonging to the genus Pediococcus, Leuconostoc, Fructobacillus, Oenococcus, Tetragenococcus or Bacillus, And combinations thereof.

Specific examples of microorganisms of the genus Bifidobacterium include Bifidobacterium longum (B. longum), Bifidobacterium bifidum (B. bifidum), and Bifidobacterium addresscentis (B. adolescentis), Bifidobacterium infantis (B. infantis), Bifidobacterium breve (B. breve), Bifidobacterium lactis (B. lactis), Bifidobacterium pseudolongum (B. pseudolongum), Bifidobacterium animalis (B. animalis), Bifidobacterium actinocoloniiforme, Bifidobacterium aemilianum, Bifidobacterium aero Film (B. aerophilum), Bifidobacterium aesculapii (B. aesculapii), Bifidobacterium anglatum (B. angulatum), Bifidobacterium anselis (B. anseris), Bifidobacterium apyri (B. anseris) B. apri), Bifidobacterium aquikefiri (B. aquikefiri), Bifidobacterium asteroides (B. asteroides), Bifidobacterium avesanii (B. avesanii), Bifidobacterium biavati (B. biavatii), B. bohemicum, B. bombi, B. boum, B. callimiconis , B. callitrichidarum, B. callitrichos, B. canis, B. castoris, bi B. catenulatum, B. catulorum, B. cebidarum, B. cebidarum, B. catulorum. choerinum), B. choloepi, B. commune, B. coryneforme, B. criceti , Bifidobacterium kuniculi (B. cuniculi), Bifidobacterium denticolens (B. denticolens), Bifidobacterium dentium (B. dentium), Bifidobacterium dricotidis (B. dolichotidis), B. erythrocebi, B. eulemuris, B. faecale, B. felsineum , B. gallicum, B. gallinarum, B. globosum, B. goeldii, bi B. hapali, B. imperatoris, B. indicum, B. inopinatum, bi B. italicum, B. jacchi, B. kashiwanohense, B. lemurum, Bifidobacterium B. leontopitheci, B. magnum, B. margollesii, B. merycicum, Bifidobacterium B. minimum, B. mongoliense, B. moraviense, Bifidobacterium mukarabe B. moukalabense, B. myosotis, B. oedipodis, B. olomucense, Bifidobacterium panos (B. olomucense) B. panos), B. parmae, B. parvulorum, B. porcinum, B. porcinum (B.) primatium), Bifidobacterium pseudocatenulatum, B. psychraerophilum, B. pullorum, Bifidobacterium B. ramosum, B. reuteri, B. rousetti, B. ruminale, Bifidobacterium luminantium (B. ruminale) B. ruminantium), Bifidobacterium saeculare (B. saeculare), Bifidobacterium saguini (B. saguini), Bifidobacterium samirii (B. samirii), Bifidobacterium scarigeram (B. scaligerum), B. scardovii, B. simiarum, B. stellenboschense, B. stellenboschense (B. stellenboschense) B. stercoris), B. subtile, B. suis, B. thermacidophilum, B. thermacidophilum (B. thermophilum), B. tibiigranuli, B. tissieri, Bifidobacterium tu B. tsurumiense, B. vansinderenii, B. vespertilionis, and B. xylocopae. Will be.

Specific examples of Lactobacillus microorganisms include Lactobacillus acidophilus, L. amylovorus, L. animalis, L. bulgaricus, and Lactobacillus. L. delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, Lactobacillus plantarum (L.) . plantarum), Lactobacillus sakei (L. sakei), Lactobacillus brevis (L. brevis), Lactobacillus reuteri (L. reuteri), Lactobacillus ramnosus (L. rhamnosus), Lactobacillus paracasei (L. paracasei), Lactobacillus L. gasseri, L. buchneri, L. crispatus, L. vulgatus, L. thermophilus, L. thermophilus. kefiri), Lactobacillus pentosus, Lactobacillus cellobiosus, L. divergens, L. fermentum, L. fructosus , Lactobacillus hilgardii, L. leicnmannii, L. ruminis, L. sanfancisco, and L. vaccinostrcus.

Specific examples of Enterococcus microorganisms include Enterococcus avium, E. casseliflavus, E. cecorum, E. durans, Enterococcus faecalis, E. faecium, E. gallinarum, E. hirae, Enterococcus malodoratus, Enterococcus E. mundtii), Enterococcus pseudoavium, Enterococcus raffinosus, Enterococcus saccharolyticus, Enterococcus seriolicida, Enterococcus Examples include E. solitarius and E. villorum.

Specific examples of Staphylococcus genus microorganisms include Staphylococcus carnosus, S. xylosus, and S. epidermidis. ..

Specific examples of microorganisms of the genus Streptococcus include Streptococcus thermophilus, S. faecalis, and S. salivarius.

Specific examples of microorganisms of the genus Lactococcus include Lactococcus lactis, L. raffinolactis, and L. plantarum.

Specific examples of microorganisms of the genus Pediococcus include Pediococcus acidilactici, Pediococcus claussenii, and Pediococcus pentosaceus. ..

Specific examples of Leuconostoc microorganisms include Leuconostoc mesenteroides, Leuconostoc lactis, and Leuconostoc fallax.

Specific examples of microorganisms of the genus Fructobacillus include Fructobacillus durionis, Fructobacillus ficulneus, and Fructobacillus fructosus. (Fructobacillus pseudoficulneus) and Fructobacillus tropaeoli.

Specific examples of microorganisms of the genus Oenococcus include Oenococcus Oeni, Oenococcus kitaharae and Oenococcus sicerae.

Specific examples of Tetragenococcus microorganisms include Tetragenococcus halophilus, Tetragenococcus koreensis, Tetragenococcus muriaticus, and Tetragenococcus muriaticus. Examples include Tetragenococcus osmophilus and Tetragenococcus solitarius.

Specific examples of Bacillus microorganisms include Bacillus cereus, Bacillus coagulans, Bacillus subtilis, Bacillus thuringiensis and Bacillus mecentericus. (Bacillus mesentericus).

The lactic acid-producing bacterium constituting the lactic acid-producing flora is not limited to the above, and may be any one in addition to or different from the above.

In principle, the lactic acid-producing bacteria that make up the lactic acid-producing flora are live bacteria. Therefore, the viable number of lactic acid-producing bacteria constituting the lactic acid-producing flora, regardless of whether the system containing the lactic acid-producing flora is placed in the above-mentioned living body, industrial product, or culture environment, for example. Will eventually decrease over time. The retainer of the present invention suppresses a decrease in the viable cell count of lactate-producing bacteria over time in the lactate-producing flora, whereby the duration of the lactate-producing flora, that is, the lactate-producing bacteria exists as viable bacteria. The period can be extended.

For example, when culturing lactic acid-producing bacteria, the number of lactic acid-producing bacteria does not increase during a certain period from the start of culture (this period is called the induction period). After that, the lactic acid-producing bacteria enter a period of logarithmic growth (this period is called a logarithmic growth period). When the growth of lactic acid-producing bacteria approaches a certain limit, the growth rate gradually decreases, and a period is reached in which no significant change in the number of bacteria is observed (this period is called the steady-state period). In the stationary phase, new growth and death of lactic acid-producing bacteria reach equilibrium. After that, the growth of lactic acid-producing bacteria is completely stopped, and only the killing progresses, so that the number of lactic acid-producing bacteria gradually decreases (this period is called the decay period). The retainer of the lactic acid-producing flora of the present invention functions effectively, for example, in the decay period in the culture of such a lactic acid-producing bacterium, suppresses the killing of the lactic acid-producing bacterium in the decay period, and is in a predetermined system. The lactate-producing flora present in is allowed to exist for a longer period of time.

In this sense, the term "maintenance of lactic acid-producing flora" as used herein means that the number of viable bacteria contained in the lactic acid-producing flora is maintained from a certain time to an arbitrary time thereafter. In addition, the decrease in the number of viable bacteria contained in the lactic acid-producing flora from a certain time to any time thereafter is compared with the system containing the lactic acid-producing flora without the retainer of the present invention. Including that the lactate-producing flora present in a given system can be present for a longer period of time as a result of suppression. Therefore, the retainer of the lactic acid-producing flora of the present invention can also function as an attenuation inhibitor that suppresses the attenuation of the viable cell count of the lactic acid-producing bacteria contained in the lactic acid-producing flora. The term "maintenance of lactic acid-producing flora" excludes an increase in the number of lactic acid-producing bacteria associated with the growth of the lactic acid-producing bacteria, such as the logarithmic growth phase during culturing of the lactic acid-producing bacteria.

Further, when culturing a lactic acid-producing bacterium, sugar may be added to a system in which a lactic acid-producing flora is present at the start of culturing. In that case, the lactic acid-producing flora can be retained by the retainer of the present invention even if the sugar concentration after the stationary phase is, for example, about 1% with respect to the sugar concentration at the start of culture. From this, it is expected that the lactic acid-producing bacteria after the stationary phase grow using an organic acid or a salt of an organic acid as a carbon source rather than sugar. Here, it is not generally known that lactic acid-producing bacteria such as bifidobacteria and lactic acid bacteria (for example, Lactobacillus microorganisms) have a metabolic pathway of an organic acid or a salt of an organic acid. On the other hand, other than lactic acid-producing bacteria, bacteria that can grow using an organic acid or a salt of an organic acid via the TCA cycle or β-oxidation pathway even under anaerobic conditions are known (for example, Escherichia coli). Like these bacteria, it is unclear whether lactate-producing bacteria have a TCA cycle, β-oxidation pathway or similar metabolic pathways, but if lactate-producing bacteria have organic acid metabolic pathways, lactate-producing bacteria It is considered that the organic acid or the salt of the organic acid is used as a carbon source even under anaerobic conditions and in an environment where sugar is low. The sugar is not particularly limited, and examples thereof include sucrose, glucose, fructose, galactose, and lactose.

The organic acid contained in the retainer of the present invention is preferably a fatty acid having 2 to 10 carbon atoms, more preferably 2 to 6 carbon atoms. When the number of carbon atoms constituting the organic acid is a fatty acid satisfying such a range, it is possible to suppress a decrease in the number of viable bacteria contained in the lactic acid-producing flora. In one embodiment, the organic acid constituting the retainer of the present invention is preferably a linear fatty acid having 2 to 10 carbon atoms. In one embodiment, the organic acid constituting the retainer of the present invention is preferably a saturated fatty acid having 2 to 10 carbon atoms.

Examples of the organic acids constituting the retainer of the present invention include acetic acid, propionic acid, butyric acid, lactic acid, and succinic acid, and combinations thereof. Acetic acid is preferred because it has sufficient eating experience, is highly safe for living organisms, is versatile, and is easily available.

Examples of the pharmacologically acceptable salt of the organic acid constituting the retainer of the present invention (hereinafter, may be abbreviated as an organic acid salt) include sodium salt, potassium salt and magnesium salt of the above organic acid. Calcium salt, ammonium salt and the like can be mentioned. The sodium salt of the organic acid is preferable because it is highly safe for living organisms, has high versatility, and is easily available.

In systems where a lactating flora is present, the retainer of the invention may be mixed with a suitable solvent. Examples of solvents include water (eg, pure water, distilled water, ion-exchanged water, RO water, tap water), and ethanol, and combinations thereof.

When the retainer of the present invention and water as a solvent are mixed, the concentration of the organic acid and / or the salt of the organic acid is, for example, more than 0 M and 200 mM or less, more preferably 10 μM to 150 mM. When the concentration of the organic acid and / or the salt of the organic acid is 200 mM or less, the decrease in the number of viable bacteria contained in the lactic acid-producing flora can be suppressed. When the retainer of the present invention is administered to the human large intestine, the concentration of the organic acid and / or the salt of the organic acid can be adjusted to, for example, more than 0 M, or preferably 10 μM or more. On the other hand, when the retainer of the present invention is used for culturing lactic acid-producing bacteria contained in a culture broth, the concentration of the organic acid and / or the salt of the organic acid can be adjusted to, for example, 100 mM or more.

The retainer of the present invention may also contain an additive in addition to the organic acid and / or the salt of the organic acid. Examples of additives include binders, disintegrants, abundant agents, excipients, colorants, flavoring agents, surfactants, preservatives, antioxidants, UV absorbers, moisturizers, pH regulators, fragrances. , And perfume oils, and combinations thereof. The content of the additive that can be contained in the retainer of the present invention is not particularly limited, and an appropriate content can be appropriately selected by those skilled in the art.

(Oral composition)
The oral composition of the present invention contains the above-mentioned lactic acid-producing flora retainer.

The oral composition of the present invention includes compositions that can be ingested from the mouth of animals such as humans, pets, livestock, poultry, and farmed fish, and examples thereof include foods and drinks, feeds, and pharmaceuticals. Examples of the dosage form of the oral composition include powders, granules, tablets, and liquids (including suspensions and emulsions).

When the oral composition of the present invention is a food and drink, examples of the food and drink include general foods; supplements; foods for specified health use, foods with nutritional function, and foods with health claims such as foods with functional claims; soft drinks; Tea beverages; coffee beverages; processed milk; milk beverages; soy milk; and alcoholic beverages;

Foods and drinks may contain food materials and other additives in addition to the above-mentioned lactic acid-producing flora retainer. Examples of the food material include, but are not limited to, plant extracts, vegetables, meat extracts, meats, fish meals, fats and oils, sugars and the like. Other additives that may be included in foods and drinks include, but are not limited to, excipients (eg, dextrin, cellulose derivatives (eg, cellulose, crystalline cellulose, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose)), sweetness. Agents, colorants, preservatives, thickeners, stabilizers, gelling agents, glues, antioxidants, color formers, bleaching agents, antifungal agents, yeast foods, gum bases, tans, bitterness agents, enzymes, brighteners , Fragrances, acidulants, seasonings, coagulants, emulsifiers, pH regulators, swelling agents, nutrient enhancers, solvents (eg, water and ethanol) and the like. The contents of food materials and other additives that can be contained in foods and drinks are not particularly limited, and appropriate contents can be appropriately selected by those skilled in the art.

When the oral composition of the present invention is a feed (feed), examples of the feed (feed) include pet food, compound feed, and concentrated feed.

In addition to the above-mentioned lactic acid-producing flora retainer, the feed may contain feed materials and other additives. Examples of the feed material include, but are not limited to, grain flour, grass flour, eggshell flour, shell flour, fish meal, plant extract, vegetables, meat extract, meat, fats and oils, sugars and the like. Other additives that may be included in the feed include, but are not limited to, preservatives, thickeners, stabilizers, gelling agents, glues, antioxidants, fungicides, enzymes, brighteners, fragrances, for example. , Acidulants, emulsifiers, pH regulators, leavening agents, nutrient enhancers, solvents (eg, water and ethanol) and the like. The contents of the feed material and other additives that can be contained in the feed are not particularly limited, and appropriate contents can be appropriately selected by those skilled in the art.

The feed also contains various lactic acid-producing bacteria in order to provide an appropriate lactic acid-producing flora in the living body (particularly in the intestine) of pet animals, livestock, poultry, farmed fish, etc., regardless of the presence or absence of each of the above components. May further contain a preparation containing lactic acid in a viable state.

When the oral composition of the present invention is a pharmaceutical product, the pharmaceutical product may contain other pharmaceutical ingredients or other additives in addition to the above-mentioned lactic acid-producing flora retainer. Examples of other pharmaceutical ingredients are not particularly limited, and include various pharmaceutical ingredients that can be adopted as oral pharmaceuticals. Other additives that may be included in pharmaceutical products include, but are not limited to, for example, binders, disintegrants, moisturizers, excipients, colorants, flavoring agents, surfactants, preservatives, antioxidants, etc. Examples include UV absorbers, moisturizers, pH regulators and the like. Specific examples of other additives that may be included in pharmaceuticals include dextrin, cellulose derivatives (eg, cellulose, crystalline cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose), gum arabic powder, polyvinylpyrrolidone, polyethylene glycol, talc. , Silicon Dioxide (eg, light anhydrous silicic acid or hydrous silicon dioxide), magnesium stearate, stearic acid, stearyl alcohol, starches, hydroxypropyl starch, sodium carboxymethyl surfactant, agar powder, ester oil, wax, higher fatty acids, higher Examples thereof include alcohols, anionic surfactants, cationic surfactants, nonionic surfactants, amphoteric surfactants, polyhydric alcohols, water and ethanol. The contents of other pharmaceutical ingredients and other additives that can be contained in the pharmaceutical product are not particularly limited, and appropriate contents can be appropriately selected by those skilled in the art.

The above-mentioned pharmaceutical products also contain various lactic acid-producing bacteria in a viable state in order to provide an appropriate lactic acid-producing flora in the human body (particularly in the intestine) regardless of the presence or absence of each of the above-mentioned components. It may be further contained.

By having the above-mentioned form, the oral composition of the present invention can efficiently ingest the above-mentioned lactic acid-producing flora retainer into a living body.

(Retention method of lactic acid-producing flora)
Next, a method for retaining the lactic acid-producing flora of the present invention will be described.

In the method of the present invention, a system containing a lactate-producing flora is treated with the above-mentioned lactate-producing flora retainer.

The treatment can be performed, for example, by combining a system containing a lactate-producing flora with a preservative for the lactate-producing flora. Here, the term "system containing a lactic acid-producing flora" used in the present specification refers to a system in which a lactic acid-producing flora is formed in advance, and for example, food and drink, cosmetics, pharmaceuticals, feeds, etc. A system in which a predetermined lactic acid-producing flora is formed in advance in order to obtain an industrial product of It also includes systems in which a predetermined lactic acid-producing flora is previously formed in a living body (for example, in an organ such as the intestine) such as humans, pet animals, domestic animals, poultry, and cultured fish. Further, in the present invention, "combining" the system containing the lactate-producing flora and the retainer of the lactate-producing flora means, for example, first arranging (for example, adding) the system containing the lactate-producing flora in a certain environment. ), And then the retainer of the lactate-producing flora is placed (eg, added); the preservative of the lactate-producing flora is first placed (eg, added) under a certain environment, and then the lactate-producing flora is included. It includes both a mode of arranging (for example, adding) a system; and a mode of simultaneously arranging (for example, adding) a system containing a lactic acid-producing flora and a retainer of the lactic acid-producing flora in a certain environment.

When culturing lactic acid-producing bacteria using the method of the present invention, the survival ratio, which is the ratio of the viable cell count for 72 hours of culture to the viable cell count for 48 hours of culture, is, for example, 15% or more, preferably 20% or more. Can be enhanced. When the survival rate is 15% or more, it is possible to suppress a decrease in the number of viable bacteria contained in the lactic acid-producing flora during the decay period.

In the method of the present invention, when the system containing the lactic acid-producing flora and the retainer of the lactic acid-producing flora are combined, the amount of the retainer is not particularly limited. An appropriate amount of the preservative can be selected by a person skilled in the art according to the type of lactic acid-producing bacteria constituting the lactic acid-producing flora.

In the method of the present invention, by coexisting the lactate-producing flora and the retainer of the lactate-producing flora in the same system as described above, the lactate-producing flora existing in the system can be maintained for a longer period of time. Can exist.

According to the retainer of the lactic acid-producing flora of the present invention, the viability of the lactic acid-producing bacterium is maintained longer in the presence of the organic acid and / or the salt of the organic acid, for example, in the living body ingesting the lactic acid-producing bacterium. It is expected that a predominant intestinal environment can be formed in the intestine. By forming a predominant intestinal environment with lactic acid-producing bacteria, it can be expected to suppress the growth of harmful bacteria (so-called bad bacteria) in the intestine. Further, even if the retainer of the present invention is added to fermented foods or the like, or added to a cell storage medium or a cell culture medium, the survival state of lactic acid-producing bacteria is maintained for a longer period of time, and storage of products that secure viable bacteria is preserved. You can also expect to extend the deadline.

Next, the present invention will be further described with reference to examples, but the present invention is not limited to these examples.

(Example 1 and Comparative Example 1: Survival maintenance confirmation test (I) of Bifidobacterium longum)
First, the medium and the anaerobic diluted solution used in this example were prepared as described in (1) to (5) below.

(1) Preparation of acetic acid-added standard medium (Example 1) A 1 M aqueous acetic acid solution was sterilized by filtration through a filter (ADVANTEC, DISMIC 25CS020AS, pore size 0.20 μm manufactured by Toyo Filter Paper Co., Ltd.). A standard medium was prepared by dissolving 0.25 g of yeast extract, 0.5 g of peptone, and 0.1 g of glucose in 85 mL of distilled water in a vessel of a multifermenter (BME-25NC manufactured by Biot Co., Ltd.), and the entire vessel was heated to 121 ° C. It was autoclaved for 15 minutes. Then, after confirming that the liquid temperature had dropped to 37 ° C., 15 mL of the above sterilized 1M acetic acid aqueous solution was added into the vessel. This gave an acetic acid-added standard medium having a final concentration of acetic acid of 150 mM.

(2) Preparation of standard medium (Comparative Example 1; control) In a vessel of a multifermentor (BME-25NC manufactured by Biot Co., Ltd.), 0.25 g of yeast extract, 0.5 g of peptone, and 0.1 g of glucose were added to 100 mL of distilled water. The standard medium was lysed and the entire vessel was autoclaved at 121 ° C. for 15 minutes.

(3) Preparation of MRS (de Man, Rogosa and Sharpe) liquid medium An MRS liquid medium (manufactured by Becton Dickinson) is prepared at a solid content concentration of 0.55 w / v% and sterilized by autoclave at 121 ° C. for 15 minutes. did.

(4) Preparation of Blood-Free BL Agar Medium A BL agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.) was prepared at a solid content concentration of 0.58 w / v%. No horse defibrinated blood was added during this preparation. This was autoclaved at 115 ° C. for 20 minutes and then poured into a sterilized petri dish to solidify.

(5) Preparation of anaerobic diluted solution 6.0 g of disodium hydrogen phosphate, 4.5 g of potassium dihydrogen phosphate, 0.75 g of Tween 80, 0.5 g of L-cysteine hydrochloride monohydrate, Each 0.5 g of Bacto Agar was weighed and 1000 mL of distilled water was added to these mixtures. An anaerobic diluted solution was prepared by heating and dissolving at 100 ° C. for 3 hours and then autoclaving at 121 ° C. for 15 minutes.

Next, the cells were prepared, pre-cultured in a liquid medium, washed, and main-cultured in a liquid medium as described in (6) to (9) below.

(6) Preparation of cells Platinum from a glycerol stock containing cryopreserved Bifidobacterium longum subsp.longum JCM1217 ^T (reference strain of B. longum; hereinafter, JCM1217 ^T ) cells. Partially scraped off with an ear, the image was drawn on a blood-free BL agar medium, and the cells on the agar medium were cultured at 37 ° C. for 48 hours under anaerobic conditions. Then, a single colony of ^JCM1217T grown on an agar medium was caught, striphered on a new blood-free BL agar medium, and cultured at 37 ° C. for 48 hours under anaerobic conditions.

(7) Preculture in liquid medium JCM1217 ^T single colonies on the above blood-free BL agar medium are caught with a loopful and inoculated with JCM1217 ^T in 5 mL MRS liquid medium in a 15 mL volume centrifuge tube. Then, the MRS liquid medium after inoculation was shaken. After culturing at 37 ° C. for about 8 hours under anaerobic conditions, JCM1217 ^T was inoculated by adding 50 μL of the bacterial solution (culture solution) to 5 mL of a new MRS liquid medium. Then, the cells were cultured at 37 ° C. for about 18 hours under anaerobic conditions. As a result, a preculture solution was obtained.

(8) Washing (removal of MRS liquid medium)
First, a PBS tablet (manufactured by Takara Bio Inc.) was dissolved in 100 mL of distilled water per tablet to obtain a PBS aqueous solution. This aqueous PBS solution was autoclaved at 121 ° C. for 15 minutes.

Then, the preculture solution obtained above was centrifuged at 3000 rpm for 5 minutes in a desktop centrifuge (Model 4000 manufactured by Kubota Shoji Co., Ltd.), and the supernatant was removed. As a result, the cells in the preculture solution were obtained as a precipitate. Then, 5 mL of a previously sterilized PBS aqueous solution was added, and the precipitate was suspended in this PBS aqueous solution to wash the cells. 1 mL of the washed bacterial solution was added to 9 mL of sterile PBS aqueous solution and diluted 10-fold.

(9) Main culture in a liquid medium A vessel containing a sterilized acetic acid-added standard medium (Example 1) or a standard medium (Comparative Example 1; control) is set in a multifermentor (BME-25NC manufactured by Biot Co., Ltd.). Then, CO ₂ gas was aerated at a flow rate of 10 mL / min. Stirrer of the medium was started by rotating the stirrer in the vessel at 300 rpm. The pH of the liquid medium was adjusted to pH 6.0 with a 2N aqueous sodium hydroxide solution, and it was confirmed that the pH was stable. 100 μL of the washed bacterial solution was added to each vessel. During the culture, a 2N aqueous sodium hydroxide solution was used to control the pH to always be 6.0. In addition, the temperature of the culture solution was controlled to be always 37 ° C. with a heater of the multi-fermentor.

Then, each of the following measurements (10) to (12) was carried out using the culture solution obtained by the above main culture.

(10) Measurement of bacterial count 1 mL of the culture solution was collected from the vessel during the culture of (9) above, added to 9 mL of an anaerobic diluted solution, and sufficiently stirred to prepare a uniform diluted solution. Then, 10 ^{-fold serial dilution was performed using the same method to prepare a sample diluted to 10-2-10-5 .} Then, the sample was applied to a blood-free BL agar medium so that 30 to 300 colonies could be obtained per plate, and the cells on the agar medium were cultured at 37 ° C. for 48 to 72 hours under anaerobic conditions. The number of colonies corresponding to the culture broth collected at predetermined time intervals from the start of the main culture was counted, and the viable cell count (CFU / mL) per 1 mL of the culture broth was calculated from the number of colonies. The obtained results are shown in FIG.

Furthermore, from the results of the numbers of the bacteria at 48 hours and 72 hours of culture, the survival ratio (%) from 48 hours to 72 hours of culture was calculated from the following formula:
Survival ratio (%) = (number of bacteria in 72 hours of culture (CFU / mL)) / (number of bacteria in 48 hours of culture (CFU / mL)) x 100

The results obtained are shown in Table 1.

As shown in FIG. 1, even if acetic acid is added to the medium, the logarithmic growth phase of the growth curve (the period generally shown by (a) in FIG. 1) and the stationary phase (generally shown by (b) in FIG. 1) are shown. During the period), there was no significant difference in the numbers of each of Example 1 and Comparative Example 1. On the other hand, in the decaying period of the growth curve (the period generally shown by (c) in FIG. 1), the number of bacteria in Comparative Example 1 decreased particularly sharply after 48 hours of culturing, whereas the number of bacteria in Example 1 decreased. The number did not decrease much even after 48 hours of culture, and there was a large difference between the number of bacteria in Example 1 and the number of bacteria in Comparative Example 1 at 72 hours of culture (see also Table 1). matter). Then, as shown in Table 1, the acetic acid-containing standard medium used in Example 1 was compared with the standard medium used in Comparative Example 1 (without acetic acid) in the culture of JCM1217 ^T at 48 to 72 hours. The survival rate (%) was significantly improved.

From this, the acetic acid added to the standard medium in Example 1 suppresses the decrease in the number of bacteria during the decay period of the growth curve of JCM1217 ^T used in Example 1, and has an effect on maintaining the bacterial flora as a whole. You can see that it is exerting.

(11) Measurement of Glucose Concentration Glucose concentration was measured using Glucose Assay Kit-WST (manufactured by Dojin Chemical Laboratory Co., Ltd.) according to the instruction manual (Technical Manual) in the kit. The culture broth was collected from the vessel, standard medium (control) and acetic acid-added standard medium of (9) above at 0 hours of culture, 24 hours of culture, 48 hours of culture, and 72 hours of culture. The culture solution for 0 hours of culture was diluted 20-fold with ultrapure water so that it was within the calibration curve range of glucose, and the culture solutions for 24 hours, 48 hours, and 72 hours of culture were used as measurement samples as undiluted solutions. .. In this way, the glucose concentration in each sample was determined from the calibration curve.

Further, the percentage of the glucose concentration at 24 hours of culture, 48 hours of culture and 72 hours of culture when the glucose concentration at 0 hours of culture was set to 100% was calculated as the glucose residual rate.

The results obtained are shown in Table 2.

As shown in Table 2, the culture broths of Example 1 and Comparative Example 1 were both 1% compared to the glucose concentration at 0 hours of culture during the continuation of culture (24, 48 and 72 hours of culture). Only the glucose before and after was left. Here, when these are considered in combination with the results of FIGS. 1 and 1 above, it can be seen that the effect of acetic acid on retaining the bacterial flora occurred in an environment in which glucose was hardly present in the culture medium.

(12) Measurement of acetic acid concentration in the culture solution The following measurements were made using the HPLC method.

Approximately 160 mg of sodium acetate (manufactured by Wako Pure Chemical Industries, Ltd.) was measured, and this sodium acetate was dispensed to 10 mL using ultrapure water in a measuring flask. An aqueous sodium acetate solution was prepared by dissolving sodium acetate in pure water in this way. Next, 5 mL of this sodium acetate aqueous solution was measured, and the volume was increased to 10 mL using ultrapure water in a measuring flask. As a result, the original aqueous sodium acetate solution was diluted 2-fold with ultrapure water. Then, the above operation was repeated a plurality of times to obtain a standard solution for a calibration curve having a stepwise different sodium acetate concentration. The culture broth was collected from the vessel, standard medium (control) and acetic acid-added standard medium of (9) above at 0 hours of culture, 24 hours of culture, 48 hours of culture, and 72 hours of culture. The collected culture broth was diluted 2-fold with ultrapure water and filtered through a filter (Ekikurodisk 25SP manufactured by Nippon Pole Co., Ltd., pore size 0.45 μm). The filtered sample was subjected to analysis under the following conditions. Finally, the acetic acid concentration in each sample was determined from the calibration curve.
(HPLC analysis conditions)
Detector: Differential refractive index detector Column: Aminex HPX-87H (Bio-Rad Laboratories, φ7.8 mm x 300 mm)
Guard column: Micro-Guard Cation H + Refill Cartridges (manufactured by Bio-Rad Laboratories, φ4.6 mm x 30 mm)
Column temperature: 60 ° C
Mobile phase: 5 mM H ₂ SO ₄
Flow rate: 0.6 mL / min Injection volume: 10 μL

The results obtained are shown in Table 3.

As shown in Table 3, in the culture solution of Comparative Example 1, the acetic acid concentration in the culture solution gradually increased after the start of the culture, whereas in the culture solution of Example 1, the culture solution was used up to 48 hours of culture. Although the acetic acid concentration in the culture was increased, the acetic acid concentration in the culture solution decreased between 48 hours and 72 hours of culture. Here, considering these in combination with the results of FIGS. 1 and 1 above, it is considered that the effect of acetic acid on retaining the bacterial flora may be caused by using acetic acid in the culture solution as a carbon source. ..

(Example 2 and Comparative Example 2: Survival maintenance confirmation test of Bifidobacterium addresscentis)
This Example is the same as in Examples 1 (2) to (5) except that an acetic acid-added standard medium was prepared as shown in (1') below instead of (1) in Example 1. The medium and anaerobic diluent used in the above were prepared.

(1') Preparation of acetic acid-added standard medium (Example 2) A 1 M aqueous acetic acid solution and a 1 M aqueous sodium acetate solution were mixed to prepare an acetic acid-sodium acetate aqueous solution having a pH of 7.0. Then, this acetic acid-sodium acetate aqueous solution was sterilized by filtration with a filter (ADVANTEC, DISMIC 25CS020AS, pore size 0.20 μm manufactured by Toyo Filter Paper Co., Ltd.). A standard medium was prepared by dissolving 0.25 g of yeast extract, 0.5 g of peptone, and 0.1 g of glucose in 85 mL of distilled water in a vessel of a multifermenter (BME-25NC manufactured by Biot Co., Ltd.), and the entire vessel was heated to 121 ° C. It was autoclaved for 15 minutes. Then, after confirming that the liquid temperature had dropped to 37 ° C., 15 mL of the above sterilized aqueous solution of acetic acid-sodium acetate was added into the vessel. As a result, an acetic acid-added standard medium having a total final concentration of acetic acid and sodium acetate of 150 mM was obtained.

Next, instead of the strain used in (6) of Example 1, the cryopreserved Bifidobacterium adolescentis JCM1275 ^T (reference strain of B. adolescentis; hereinafter, JCM1275 ^T ) strain. Example 1 except that the body was used and the sterilized acetic acid-added standard medium obtained in (1') above was used instead of the sterilized acetic acid-added standard medium in (9) of Example 1. In the same manner as in (6) to (9), the cells were prepared, pre-cultured in a liquid medium, washed, and main-cultured in a liquid medium to prepare a culture solution.

For this culture solution, the number of bacteria was measured in the same manner as in (10) of Example 1 above, and the survival ratio (%) from 48 hours to 72 hours of culture was calculated. The results obtained are shown in Table 4.

As shown in Table 4, the number of bacteria in Comparative Example 2 decreased sharply after 48 hours of culture, whereas the number of bacteria in Example 2 did not decrease much even after 48 hours of culture, and in 72 hours of culture, the number of bacteria did not decrease so much. At least, there was a large difference between the number of bacteria in Example 2 and the number of bacteria in Comparative Example 2. The acetic acid-containing standard medium used in Example 2 had a survival ratio of JCM1275 ^T from 48 hours to 72 hours in comparison with the standard medium used in Comparative Example 2 (without acetic acid and sodium acetate). %) Was significantly improved.

(Example 3 and Comparative Example 3: Survival maintenance confirmation test (II) of Bifidobacterium longum)
This is the same as in (2) to (5) of Example 1 except that the acetic acid-added standard medium described in (1') of Example 2 was used instead of (1) of Example 1. The medium and anaerobic diluent used in the examples were prepared.

Next, the same cryopreserved JCM1217 ^T as in Example 1 was used, and instead of the sterilized acetic acid-added standard medium in (9) of Example 1, it was obtained in (1') of Example 2. Preparation of cells, preculture in liquid medium, washing, and main culture in liquid medium in the same manner as in (6) to (9) of Example 1 except that a sterilized standard medium containing acetic acid was used. Each was carried out to prepare a culture medium.

For this culture solution, the number of bacteria was measured in the same manner as in (10) of Example 1 above, and the survival ratio (%) from 48 hours to 72 hours of culture was calculated. The results obtained are shown in Table 5.

As shown in Table 5, the number of bacteria in Comparative Example 3 decreased significantly after 48 hours of culture, whereas the number of bacteria in Example 3 did not decrease much even after 48 hours of culture, and in 72 hours of culture, the number of bacteria did not decrease so much. At least, there was a large difference between the number of bacteria in Example 3 and the number of bacteria in Comparative Example 3. Then, in the acetic acid-containing standard medium used in Example 3, the survival ratio (survival ratio) of JCM1217 ^T from 48 hours to 72 hours was compared with the standard medium used in Comparative Example 3 (without acetic acid and sodium acetate). %) Was significantly improved.

According to the present invention, it is useful in various technical fields such as a food field, a pharmaceutical field, and a cosmetic field, which handle products using lactic acid-producing bacteria.

Claims (7)

A preservative for a lactic acid-producing flora containing at least one compound selected from the group consisting of an organic acid and a pharmaceutically acceptable salt thereof as an active ingredient. The retainer for a lactic acid-producing flora according to claim 1, wherein the organic acid is a fatty acid having 2 to 10 carbon atoms. The retainer for a lactic acid-producing flora according to claim 1 or 2, wherein the organic acid is a fatty acid having 2 to 6 carbon atoms. The lactic acid-producing flora according to any one of claims 1 to 3, wherein the organic acid is at least one fatty acid selected from the group consisting of acetic acid, propionic acid, butyric acid, lactic acid, and succinic acid. Preservative. A group in which the lactic acid-producing bacteria constituting the lactic acid-producing flora consist of the genus Bifidobacterium, the genus Lactobacillus, the genus Enterococcus, the genus Staphylococcus, the genus Streptococcus, the genus Lactococcus, the genus Pediococcus, and the genus Leukonostock. The lactic acid-producing flora retainer according to any one of claims 1 to 4, which is at least one microorganism selected from. An oral composition comprising the preservative for the lactic acid-producing flora according to any one of claims 1 to 5. A method for maintaining a lactate-producing flora,
A method comprising the step of combining the system containing the lactic acid-producing flora with the retainer of the lactic acid-producing flora according to any one of claims 1 to 5.

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