JP7035283B1 - A novel lactic acid bacterium strain and a composition containing the novel lactic acid bacterium strain. - Google Patents

Abstract

The present invention provides a novel lactic acid bacterium strain, particularly a novel Cremoris FC strain, and uses thereof. The lactic acid bacterium strain of the present invention is Lactococcus lactis subsp. Cremoris FC46 strain (accession number: NITE BP-03310) or a successor strain thereof.

Description

NPMD NITE BP-03310

The present invention relates to a novel lactic acid bacterium strain. The present invention also relates to various uses of the lactic acid bacterium strain, such as a composition containing the lactic acid bacterium strain (inoculum, oral composition) and a method for producing a fermented food or drink using the lactic acid bacterium strain.

In recent years, with the increasing health consciousness of consumers, the demand for foods and drinks containing lactic acid bacteria has increased, and lactic acid bacteria are widely used in the production of fermented milk and lactic acid bacteria beverages. Lactococcus lactis subsp. Cremoris, which is a type of lactic acid bacterium, has long been used as a bacterium for producing fermented dairy products such as yogurt and cheese. It is known that there are bacterial species that produce. Among them, yogurt (trade name "Caspian Sea yogurt", applicant registered trademark, registered) obtained by fermenting using Cremoris bacterium isolated by the applicant (hereinafter referred to as "Clemoris FC strain") as the main fermented lactic acid bacterium. No. 5761425) has a peculiar viscosity completely different from that of conventional fermented milk, and the viscous polysaccharides produced by the Cremoris FC strain and the Cremoris FC strain have an intestinal regulating effect, an effect of suppressing an increase in blood glucose level, and an immunostimulatory effect. It has been reported that it has various physiological actions such as action, protection against influenza virus infection, suppression of allergies, and improvement of exercise conditions (see Non-Patent Documents 1 to 6).

For this reason, not only fermented milk but also products containing Cremoris FC strains such as inoculum and supplements for fermentation have been developed in anticipation of the above-mentioned physiological effects, but in order to fully exert such physiological effects in the intestine. Is required to continuously ingest the Cremoris FC strain. However, lactic acid bacteria, which are facultative anaerobes, are known to have reduced survival under aerobic conditions and acidic (low pH) conditions, and Cremoris FC strains are no exception. Therefore, in acidic foods and drinks such as yogurt and dry powders such as lactic acid bacteria supplements, it is required to develop a product containing Cremoris FC strain that suppresses the decrease in the number of viable bacteria that occurs during long-term storage and maintains a useful physiological action. There is.

T. Toda et al., “Effects of fermented milk with Lactococcus lactis subsp. Cremoris FC on defecation frequency and fecal microflora in healthy elderly volunteers” J. Jpn. Soc. Food. Sci. 52 (6), 243-250 (2005) ) M. Mori et al., “Beneficial effect of viscous fermented milk on blood glucose and insulin responses to carbohydrates in mice and healthy volunteers: preventive geriatrics approach by“ slow calorie ”” Geriatrics. 141-152 (2012) A. Kosaka et al., “Lactococcus lactis subsp. Cremoris FC triggers IFN-γ production from NK and T cells via IL-12 and IL-18” Int. Immunopharmacol. 14 (4), 729-733 (2012) T. Maruo et al., “Oral administration of milk fermented with Lactococcus lactis subsp. Cremoris FC protects mice against influenza virus infection” Lett. Appl. Microbiol. 55 (2), 135-140 (2012) Y. Gotoh et al., “Effect of orally administered exopolysaccharides produced by Lactococcus lactis subsp. Cremoris FC on a mouse model of dermatitis induced by repeated exposure to 2,4,6-trinitro-1-chlorobenzene” J. Funct. Foods. 35, 43-50 (2017) Y. Gotoh et al., “Effect of yogurt fermented with Lactococcus lactis subsp. Cremoris FC on salivary secretory IgA levels in high school-student long-distance runners” Japanese J. Phys. Fit. Sports Med. 68 (6), 407 -414 (2019)

In view of the above-mentioned conventional problems, the present invention is a novel Cremoris FC strain having improved acid resistance (low pH resistance) and improved survival under aerobic and dry conditions, in other words, fermentation. An object of the present invention is to provide a Cremoris FC strain having good survivability even in an acidic food or drink such as milk or in a dry powder state. Further, the present invention comprises an inoculum for fermentation using the Cremoris FC strain having excellent survival under dry conditions under the acid resistance and aerobic conditions, an oral composition such as a supplement or a fermented food or drink, and fermentation. The subject is to provide a method for producing food and drink.

The present inventors have been diligently studying to solve the above-mentioned problems, and found that they have the same mycological properties as the Lactococcus lactis subsp. Cremoris FC, FERM P-20185, which is owned by the applicant as a cremoris strain. By discovering a new Cremoris FC strain that has excellent properties in terms of both acid resistance and survival under dry conditions under more acid resistance and aerobic conditions, and by further study. The present invention has been completed.
Hereinafter, the conventionally known Cremoris FC strain is referred to as "Clemoris FC strain", "existing Cremoris FC strain" or "parent FC strain", and the newly found Cremoris FC strain of the present invention is referred to as "Clemoris FC strain". It is referred to as "Clemoris FC strain", "Clemoris FC46 strain" or "FC46 strain".

The present invention has the following embodiments.
(I) New lactic acid bacterium Cremoris strain (this Cremoris FC strain)
(I-1) Lactococcus lactis subsp. Cremoris FC46 strain (accession number: NITE BP-03310) or its subspecies lactic acid bacterium.
(I-2) The lactic acid bacterium according to (I-1), which is in a dry state.

(II) Fermentation inoculum (II-1) Fermentation inoculum containing Lactococcus lactis subspecis Cremoris FC46 strain (accession number: NITE BP-03310) or its successor strain.
(II-2) The inoculum for fermentation according to (II-1), which further contains Streptococcus thermophilus.
(II-3) Lactococcus lactis subspecies Cremoris FC46 strain (accession number: NITE BP-03310) or its successor strain,
The fermenting inoculum according to (II-2), which is a symbiotic organism with Streptococcus thermophilus.
(II-4) The inoculum for fermentation according to any one of (II-1) to (II-3), which is in a dry state.

(III) Oral Composition An oral composition containing the lactic acid bacterium according to (III-1) (I-1) or (I-2).
(III-2) The oral composition according to (III-1), which is a food or drink, an oral drug, or a quasi-drug.
(III-3) The oral composition according to (III-1) or (III-2), which is a fermented food or drink.

(IV) A method for producing a fermented food or drink, and a fermented food or drink (IV-1) as a raw material for a fermented food or drink, the lactic acid bacterium described in (I-1) or (I-2), or (II-1) to (II). -4) A method for producing a fermented food or drink, which comprises inoculating and fermenting the inoculum for fermentation according to any one of 4).

The present Cremoris FC strain of the present invention has the same mycological properties and useful physiological effects as the existing Cremoris FC strain, but is raw under low pH conditions near pH 4 as compared with the existing Cremoris FC strain. It has a high residual and viscosity-maintaining effect and is excellent in acid resistance. Further, the present Cremoris FC strain has higher survival under dry conditions and aerobic conditions and is excellent in resistance to dry and aerobic conditions as compared with the existing Cremoris FC strain. Therefore, this Cremoris FC strain is excellent in stability in acidic foods and drinks such as yogurt and lactic acid bacteria beverages and oral compositions such as supplements in a dry state, and is useful in the form of oral compositions. It can maintain and exert its physiological action for a long period of time.

In Example 2, the results of measuring the viable cell count (cfu / ml) of each Cremoris FC strain (FC46 strain, parent FC strain) in an acidic medium over time are shown.

(I) New lactic acid bacterium Cremoris strain (this Cremoris FC strain)
This Cremoris FC strain is a strain belonging to Lactococcus lactis subsp. Cremoris, which has the following mycological properties, and some of them are patented microorganisms of the National Institute of Technology and Evaluation. At the Deposit Center (Room 122, Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan), as of November 6, 2020, identification display: Lactococcus lactis subsp. Cremoris FC46, deposit number: NITE BP-03310 It has been deposited internationally.

(A) Mycological properties This Cremoris FC strain was used in TY-Glu medium (1% glucose, 0.5% Bacto (TM) Tryptone (Thermo Fisher Scientific), 0.5% Bacto (TM) Yeast Extract (Thermo Fisher Scientific)). , 0.5% NaCl) (pH 7.0) or MRS medium (manufactured by Difco), mycological properties (morphological and physiological characteristics, carbon source assimilation) when statically cultured under aerobic conditions. Gender) is as shown in Tables 1 and 2 below.

(B) Morphological characteristics Cell morphology: (Streptococcus) Colony shape: Circular (hemispherical) diameter 0.5 to 1.0 mm
Colony color: White Spore formation: None

(C) Physiological features Gram stain: Positive Motility: None
Catalase activity: None Gas production from glucose: None Lactic acid optical rotation: L
Growth at 10 ° C: Possible to grow Growth at 45 ° C: Not possible Optimal temperature: 25 to 30 ° C
Optimal pH: pH 6-7
Lactic acid fermentation: homozygous

(D) Carbon source assimilation (sugar fermentability)
Assimilating to the following carbon sources D-galactose, D-glucose, D-fructose, D-mannose, D-lactose, N-acetylglucosamine, esculin.

(E) Other properties that characterize the microorganism
KH Schleifer et al., “Transfer of Streptococcus lactis and Related Streptococci to the Genus Lactococcus gen. Nov.” System. Appl. Microbiol. 6, 183-195 (1985) From the results of the sex search, this Cremoris FC strain has 100% homology with the existing registered Cremoris FC strain (Lactococcus lactis subsp. Cremoris FC, FERM P-20185). It was identified as a strain belonging to Lactococcus lactis subsp. Cremoris. The nucleotide sequence of the 16S rRNA gene of this Cremoris FC strain is shown in SEQ ID NO: 1 in the sequence listing.

In addition to the above-mentioned mycological properties, this Cremoris FC strain produces viscous polysaccharides like the existing Cremoris FC strain. Therefore, this Cremoris FC strain, like the existing Cremoris FC strain, has various useful physiology such as intestinal regulation effect, blood glucose level increase inhibitory effect, immunostimulatory effect, influenza virus infection protection effect, allergy suppression and exercise condition improvement. It is thought to have an effect.

On the other hand, as shown in Examples 2 and 3 described later, this Cremoris FC strain has a higher survival and viscosity maintenance effect under low pH conditions near pH 4, and is acid resistant, as compared with the existing Cremoris FC strain. It has the characteristic of being excellent in. Further, as shown in Example 4 described later, this Cremoris FC strain has higher survival under dry and aerobic conditions than the existing Cremoris FC strain, and is dry or aerobic. It has the property of being excellent in resistance to. Therefore, this Cremoris FC strain is excellent in stability in acidic foods and drinks such as yogurt and lactic acid bacteria beverages and oral compositions such as supplements in a dry state, and is useful in the form of oral compositions. It can maintain and exert its physiological action for a long period of time.

The Cremoris FC strain includes not only the Cremoris FC strain (deposited bacterium) deposited internationally under the accession number: NITE BP-03310 and its original strain, but also its successor strain.
The passage of this Cremoris FC strain can be carried out according to a commonly used method for passage of lactic acid bacteria, and is not particularly limited. For example, a method of inoculating the Cremoris FC strain in a test tube containing the TY-Glu medium (pH 7.0) and culturing at an optimum temperature of about 25 to 30 ° C. for about 12 to 48 hours can be mentioned. .. Cremoris FC strains passaged by culture can be stored at −80 ° C. until use with glycerol added to a final concentration of 10-30 w / v%.

As the medium of this Cremoris FC strain, in addition to the above-mentioned TY-Glu medium, a normal medium containing a carbon source, a nitrogen source, inorganic ions, and if necessary, organic micronutrients can be used. The carbon source may be any as long as it contains an assimilated carbon source, and for example, carbohydrates such as D-glucose, D-galactose, D-fructose, D-mannose, and D-lactose are preferably used. .. The nitrogen source may be an assimilated nitrogen compound or one containing the nitrogen compound, and for example, ammonium sulfate, casamino acid, peptone and the like are used. In addition, inorganic salts such as phosphoric acid, iron, potassium, magnesium, zinc, manganese, copper and calcium can be appropriately used. Further, if necessary, amino acids necessary for bacterial growth, biotin, riboflavin, pyridoxine, nicotinic acid, pantothenic acid, folic acid, thiamine and other vitamins can be added to the medium for use.

The growth of this Cremoris FC strain can be promoted by culturing at a temperature of 15 ° C. or higher, preferably about 25 to 30 ° C., and at a pH of 5 or higher, preferably about 6 to 7 ° C. This Cremoris FC strain can be carried out by static culture under aerobic conditions, but preferably under anaerobic conditions, the growth can be promoted.

Since this Cremoris FC strain has high survivability under dry conditions and aerobic conditions as described above, it is dried by subjecting it to drying treatment such as vacuum drying or spray drying after isolation from the medium. It may be prepared as a dried product by drying with other components such as a medium component or an excipient.

(II) Fermentation inoculum This Cremoris FC strain should be used as an inoculum (fermentation inoculum, also referred to as "lactic acid bacterium starter") used when producing fermented foods and drinks by utilizing its fermenting ability. Can be done.
Fermented foods and drinks include fermented milk foods and drinks made mainly from milk (yogurt, lactic acid bacteria beverages, cheese, etc.), fermented soymilk foods and drinks made mainly from beans (yogurt, lactic acid bacteria beverages, etc.), grains (rice, rice, etc.). Examples include fermented grain foods and drinks (sweet sake, bread, etc.) and fermented vegetables and fruits (pickles, etc.) whose main raw material is wheat).

The fermenting inoculum of the present invention is preferably used for fermentation of a raw material mainly composed of milk or soymilk, and the fermentation of the raw material produces a milk fermented food or drink such as yogurt or a lactic acid bacteria beverage, or a soymilk fermented food or drink. Fermentation. Here, the raw material mainly composed of milk or soymilk (milk raw material, soymilk raw material) may be any as long as it contains a protein derived from milk or beans (preferably soybean), and may be milk or soymilk itself. It may be the one from which lipids have been removed or reduced.
In the specification of the present application, the "milk" used in the present invention includes animal milk, and examples thereof include milk, goat milk, sheep milk, horse milk, and the like, and the form thereof is not particularly limited. , Raw milk, full-fat milk, defatted milk, full-fat milk powder, defatted milk powder, or any other form of milk can be used. Further, the "soymilk" used in the present invention includes, but is not limited to, soymilk obtained by grinding soybeans and water and then squeezing the juice, but is not limited to this, and the beans and water are combined. It is a liquefied emulsified liquid (soybean emulsified liquid), and the type of beans used as a raw material is not particularly limited, and any beans such as soybeans, small beans, red beans, soybeans, green beans, and green beans may be used.

In the production of the fermented milk food or the fermented soymilk, the inoculum for fermentation of the present invention has a viable cell count of the above-mentioned Cremoris FC strain of about 1 × 10 ⁵ to 1 × 10 ⁹ (cfu / g). It is preferable to be inoculated into. Therefore, before use, this Cremoris FC strain is cultured in advance using, for example, a dairy raw material or a soymilk raw material, and the viable cell count of the culture is about 1 × ¹⁰⁷ to 1 × 10 ⁹ (cfu / g). It is preferable to prepare soymilk (inoculum composition). Specifically, although not limited, the Cremoris is added to, for example, a fermented raw material (milk raw material, soymilk raw material) that has been sterilized in advance, or the fermented raw material (for example, 5 to 10% skim milk powder, etc.) to which yeast extract is added. Examples thereof include those prepared by inoculating the FC strain and culturing at the optimum temperature so as to have the above-mentioned bacterial concentration. The viable cell count of this Cremoris FC strain can be measured by performing a pour culture method (25 ° C.) using a TY-Glu medium (pH 7.0) and measuring the number of grown colonies. Yes (colony count). Since the number of bacteria and turbidity correlate with each other, if the correlation between the number of bacteria and the turbidity (absorbance at a wavelength of 600 nm) is obtained in advance, the number of bacteria can be counted by measuring the turbidity.

The inoculum for fermentation of the present invention may consist only of the Cremoris FC strain, or may be used in combination with other microorganisms having fermentative ability as long as it does not interfere with the effects of the present invention. Other microorganisms include, but are not limited to, for example, Lactococcus lactis subsp. Cremoris, and Lactococcus lactis subsp. Lactobacillus casei subsp. Casei, Lactobacillus casei subsp. Casei, Lactobacillus casei subsp. Casei, Lactobacillus casei subsp. Lactobacillus (Lactobacillus delbrueckii subsp. Bulgaricus), Lactobacillus gasseri, Lactobacillus acidophilus and other lactic acid bacteria belonging to the genus Lactobacillus can be exemplified. These lactic acid bacteria may be used alone or in any combination of two.

The microorganism used in combination with the Cremoris FC strain is preferably Streptococcus thermophilus (hereinafter, referred to as "S. thermophilus"). As shown in Example 3 described later, this Cremoris FC strain was subjected to S. cerevisiae under low pH conditions. By co-fermenting with Streptococcus thermophilus, a higher viable cell count can be maintained (improved survivability) and a decrease in viscosity after storage can be suppressed as compared with single fermentation. This Cremoris FC strain and S. In the fermenting inoculum of the present invention (a symbiotic of the Cremoris FC strain and the S. thermophilus bacterium) in combination with the Thermophilus bacterium, the viable cell count ratio between the two is not limited, but the Cremoris FC strain: S. thermophilus FC strain: S. Thermophilus bacteria = 1: 0.001 to 1000 can be exemplified. Preferably, the Cremoris FC strain: S. Thermophilus bacteria = 1: 0.01 to 100, more preferably 1: 0.1 to 10 can be exemplified.
In addition, S. The viable cell count of Streptococcus thermophilus can be measured by colony counting by a pour culture method (37 ° C.) using a BCP-added plate count agar agar medium (Nissui Pharmaceutical Co., Ltd.).

In addition, acetic acid bacteria can also be used as a microorganism to be used in combination with the present Cremoris FC strain. Examples of acetobacter include, but are not limited to, acetobacters belonging to the genus Acetobacter, the genus Gluconobacter, the genus Gluconacetobacter, and the like.

The fermenting inoculum of the present invention may be any as long as it exhibits fermentative ability when used in the production of fermented foods and drinks, and its shape is not particularly limited. From the viewpoint of hygiene, quality retention, handleability and the like, it is preferably a dry solid form, more preferably a dry powder or a dry granule. It may be prepared as a dried product by drying with other components such as a medium component or an excipient.

Fermentation conditions (culture conditions) using the inoculum for fermentation of the present invention include a culture temperature of 15 ° C. or higher, preferably about 25 to 30 ° C.; a culture pH of 5 or higher, preferably about pH 6 to 7; a culture time of 3 hours or longer, preferably 8 It can be mentioned for about 24 hours.

(III) Oral Composition The oral composition of the present invention is characterized by containing the present Cremoris FC strain. The oral composition is prepared in the form of a food or drink by using an appropriate edible carrier (food and drink material) in the same manner as a normal food or drink. In addition, the oral composition is prepared in a pharmaceutical form (supplement form, pharmaceutical form, quasi-drug form) by using a suitable pharmaceutical raw material such as an excipient or a diluent which is pharmaceutically acceptable. Will be done.

Similar to the existing Cremoris FC strain, this Cremoris FC strain has various physiological actions (intestinal regulation action, blood glucose level increase inhibitory action, immunostimulatory action, influenza virus infection protection action) in the body when taken orally. , Allergy suppression and exercise condition improvement action, etc.) are expected to be exhibited. The action is considered to be the action of the Cremoris FC strain itself and / or the viscous polysaccharide produced by the Cremoris FC strain. Therefore, it is preferable that the oral composition of the present invention contains the Cremoris FC strain in a viable state. However, it is desirable to contain as many Cremoris FC strains as possible as viable bacteria, and this does not prevent the Cremoris FC strains from being contained in a dead state.

The oral composition of the present invention may contain the Cremoris FC strain as long as it contains the culture of the Cremoris FC strain, a crude product or refined product of the culture, and a freeze-dried product or a spray-dried product thereof. May be. Although the above culture is not limited, it is cultured for about 12 to 48 hours at an optimum temperature of about 25 to 30 ° C. using, for example, a medium suitable for this Cremoris FC strain, for example, TY-Glu medium (pH 7.0). Can be obtained by doing. In addition, the cells of this Cremoris FC strain can be obtained by centrifuging the culture at 3,000 rpm for about 10 minutes after the above culture and collecting the cells. These can also be purified according to a conventional method. Further, the cells can be freeze-dried or spray-dried. The dried cells thus obtained can also be used as an active ingredient of the oral composition of the present invention. In the present specification, the Cremoris FC strain, its culture, a crude product or a refined product of the culture, and a dried product thereof are collectively referred to as "the Cremoris FC strain and its culture".

If necessary, the oral composition of the present invention may further contain an appropriate amount of a nutritional component suitable for maintenance, growth and the like of the Cremoris FC strain. Specific examples of the nutritional components include carbon sources such as glucose, galactose, fructose, lactose and mannose used in culture media for culturing microorganisms; nitrogen sources such as yeast extract and peptone; vitamins and minerals. Each component such as class, trace metal element, and other nutritional components can be mentioned.

The preparation of the oral composition of the present invention into a dietary form, a pharmaceutical form, and a quasi-drug form can be carried out according to a conventional method. Further, the carrier used for the preparation into each of these forms may be any of an edible carrier or a carrier such as a pharmaceutically acceptable excipient or a diluent. Details of the preparation into the food and drink form and the edible carrier that can be used at that time are described in the section "Oral composition of the food and drink form" described later.

The blending amount of the Cremoris FC strain in the oral composition of the present invention is generally such that the viable cell count of the Cremoris FC strain in the oral composition of the present invention is 1 × 10 ⁷ to 1 × 10 ¹¹ (cfu). ) It can be appropriately selected from the amount before and after. The blending amount of the Cremoris FC strain can be appropriately changed according to the form of the oral composition of the present invention to be prepared, the desired action and effect, and the like, using the above amount as a guide.

Oral composition in the form of food and drink The oral composition of the present invention in the form of food and drink may be ingested as food and drink. Although not limited, suitable specific examples are milk fermented foods and drinks produced from fermented milk such as yogurt and milk such as lactic acid bacteria beverages, and soymilk produced from soybeans such as soybean yogurt and fermented soymilk beverages. Fermented foods and drinks can be exemplified. In addition, pharmaceutical forms such as solid formulations (tablets, pills, powders, granules, microcapsules, capsules, etc.) and liquid formulations (liquids, suspensions, syrups, emulsions, etc.) containing the Cremoris FC strain can be used. The food and drink that you have is also included. In addition, confectioneries such as gum, caramel, gummy, nougat, and chocolate containing the Cremoris FC strain of the present invention; fermented foods and drinks such as fermented vegetable drinks and fermented fruit drinks; other than the above fermented foods and drinks such as pudding and bavarois. Examples include dairy products. In this specification, the terms "fermented milk" and "lactic acid bacteria beverage" follow the definitions of Article 2, 37 "fermented milk" and 38 "lactic acid bacteria beverage" of the former Ministry of Health and Welfare "Ministry Ordinance on Ingredients of Milk and Dairy Products". It shall be. That is, "fermented milk" refers to milk or dairy products fermented with lactic acid bacteria in a paste-like or liquid form. Therefore, the fermented milk includes a yogurt form as well as a beverage form. The "lactic acid bacteria beverage" refers to a beverage obtained by diluting milk or a dairy product into a paste or liquid fermented with lactic acid bacteria as a main raw material and diluting it with water.

(IV) Fermented Food and Beverage Production Method and Fermented Food and Beverage The fermented food and drink production method of the present invention is characterized by containing the present Cremoris FC strain. Such fermented foods and drinks can be produced according to a conventional method, and include, for example, suitable fermenting raw materials (for example, milk, soymilk (soybean emulsion), grains, vegetables, fruits) containing a nutrient source of the Cremoris FC strain. It can be produced by inoculating the Cremoris FC strain into a raw material), culturing the strain, and fermenting the raw material.

For fermentation using the present Cremoris FC strain, a method in which a starter is prepared in advance and the starter is inoculated into a fermenting raw material to ferment it is preferable. Here, as the starter, the above-mentioned inoculum for fermentation of the present invention can be mentioned. Such a starter preferably contains a viable cell count of about 1 × 10 ⁷ to 1 × 10 ⁹ (cfu / ml) of the present Cremoris FC strain.

If necessary, the above fermentation raw materials include fermentation promoters for good growth of the Cremoris FC strain, such as glucose, galactose, fructose, lactose, mannose, and other carbon sources; yeast extract, peptone, and the like nitrogen. Source: Vitamin, minerals, trace metal elements, other nutrients, etc. can be added.

The inoculation amount of the Cremoris FC strain is, for example, when milk is used as a raw material for fermentation, the Cremoris FC strain is 1 × 10 ⁵ (cfu / ml) or more in the milk, preferably 1 × 10 ⁷ to 1 ×. It is appropriate to select from the amount contained around 10 ⁹ (cfu / ml). The culture conditions are generally selected from a fermentation temperature of 15 ° C. or higher, preferably about 25 to 30 ° C., and a fermentation time of about 3 to 24 hours.

The fermented product obtained as described above may have a curd-like form (yogurt or pudding-like form), which can be ingested as a solid food as it is. The curd-shaped fermented product can be made into a desired beverage form by further homogenizing it. This homogenization can be carried out using a general emulsifier (homogenizer). By such homogenization, a beverage having a smooth texture can be obtained. For this homogenization, dilute appropriately as necessary, add organic acids for pH adjustment, and manufacture beverages such as sugars, fruit juices, thickeners, surfactants, and flavors. Various additives usually used in the above can also be appropriately added. The beverage thus obtained can be aseptically filled in a suitable container to make a final product.

The amount of ingestion (administration) thereof is appropriately determined according to the age, sex, body weight, degree of disease, etc. of the living body ingesting the ingestion, and is not particularly limited. Generally, this Cremoris FC strain is preferably selected from the range of about 1 × 10 ⁷ to 1 × 10 ¹¹ (cfu / ml). The composition can generally be prepared so that about 50-1,000 mL thereof is ingested per person per day.

Suitable specific examples of the oral composition of the present invention in the form of food and drink are supplement forms, specifically solid preparations such as tablets, pills, powders, granules, microcapsules and capsules; and liquids and suspensions. Examples thereof include foods and drinks having a formulation form such as a liquid formulation such as an agent, syrup and emulsion. As a food or drink having a pharmaceutical form, it is preferably a solid pharmaceutical product in a dry state.

Similar to the pharmaceutical form composition, the food or drink in the supplement form is a filler, a bulking agent, a binder, a humidifying agent, a disintegrant, a surface active agent, or a lubricant which is usually used as a pharmaceutical carrier in this field. It can be produced by using a diluent such as an agent or an excipient.

The amount of the lactic acid bacillus of the present invention to be blended in the oral composition in the form of food and drink may be an amount as long as the action of the lactic acid bacillus is exerted, and can be appropriately selected from a wide range as long as it is the amount. Usually, it is preferably contained in about 1 × 10 ⁷ to 1 × 10 ¹¹ (cfu) in one ingestion (administration) unit form.

As long as the composition is orally ingested, the administration method thereof is not particularly limited, and is determined according to various forms, the subject's age, sex and other conditions, the degree of disease, and the like.

The intake amount of the above composition is appropriately selected depending on the usage, age, sex and other conditions of the subject, but as described above, 1 × 10 ⁷ to 1 × per ingestion (administration) of this Cremoris FC strain. It is preferably taken at a rate of about 10 ¹¹ (cfu). The intake of this Cremoris FC strain, which is the active ingredient, is preferably about 1 × ¹⁰⁷ (cfu) or more per day, and can be taken in several divided doses per day. In addition, the oral composition of the present invention is preferably continuously ingested in order to effectively obtain the effect. For example, an embodiment of continuous or intermittent ingestion for 2 weeks or more can be exemplified.

When the oral composition containing the present Cremoris FC strain in a viable state is ingested (administered), it is expected to exert a useful physiological action in vivo as in the existing Cremoris FC strain. Therefore, the oral composition of the present invention can be effectively used as a health-promoting agent based on its physiological action. Thus, the oral composition of the present invention includes foods and drinks having a specific function and being eaten for the purpose of maintaining health and the like. Specifically, the oral composition of the present invention is a food or drink having an intestinal regulating effect, an effect of suppressing an increase in blood glucose level, an immunostimulatory effect, an effect of protecting against influenza virus infection, an effect of suppressing allergies, and / or an effect of improving exercise conditions. Is included. In addition, these foods and drinks include foods and drinks manufactured and sold with the above-mentioned actions and uses labeled or sung, for example, foods with health claims (foods with nutritional function, foods with functional claims, foods for specified health use), foods for special use (specified). Health foods) and similar health foods are included.

As described above, in the present specification, the terms "contains" and "contains" include the meanings of "consisting of" and "substantially consisting of".

Hereinafter, the present invention will be described with reference to examples in order to help understanding the structure and effects of the present invention. However, the present invention is not limited by these examples. The following experiments were performed under room temperature (25 ± 5 ° C.) and atmospheric pressure conditions, unless otherwise noted. Unless otherwise specified, "%" described below means "% by mass" and "part" means "part by mass".

Example 1 Isolation and identification of a novel lactic acid bacterium Cremoris FC strain Cremoris FC strain (FERM P-20185) was inoculated (1%) into an 8% skim milk powder emulsion that had been sterilized and at the optimum temperature (25 ° C). The strain was left for about 10 hours to activate the Cremoris FC strain (starter 1). On the other hand, Streptococcus thermophilus (hereinafter referred to as "thermophilus") is inoculated (1%) into a sterilized 8% skim milk powder emulsion, and the optimum temperature (37 ° C) is about 10 hours. It was left to activate the Streptococcus thermophilus (Starter 2). Next, starters 1 and 2 prepared above were inoculated into sterilized milk prepared separately, and fermented under the condition of 30 ° C. Cooling (4 ° C.) was started when card formation was observed.

The obtained fermented milk was stored in a dark place at 4 ° C. for 46 days, and then 3 L of TY-Gal medium (1% galactose, 0.5% Bacto (TM) Tryptone (Thermo Fisher Scientific), 0.5% Bacto ( TM) Yeast Extract (Thermo Fisher Scientific), 0.5% NaCl) (pH 7.0) was inoculated with 4% and cultured at 26 ° C. for 72 hours. The culture solution was cultivated on a TY-Gal agar medium (pH 7.0) at 25 ° C. for 48 hours, and the grown colonies were cultured in a sterilized 8% defatted powder emulsion and stored.

From these, strains with high low pH resistance (acid resistance) screened by the method described in Example 1 were obtained, and their mycological properties were evaluated according to a conventional method. The results are shown in Table 1. Moreover, the assimilation ability (sugar fermentation ability) for 49 kinds of carbon sources was evaluated using API50CHL (manufactured by BioMérieux Japan Co., Ltd.). The results are shown in Table 2. It should be noted that Table 1 is as described above in paragraph [0015], and Table 2 is as described above in paragraph [0016].

From these results, it was confirmed that the obtained strain has the same mycological properties and saccharifying property as the Cremoris FC strain (hereinafter, also simply referred to as “parent FC strain”) used as the parent strain. rice field. Therefore, the strain was identified as a strain belonging to Lactococcus lactis subsp. Cremoris (Lactococcus lactis subsp. Cremoris FC46) (hereinafter referred to as Lactococcus lactis subsp. Cremoris FC46). It was named "Clemoris FC46 strain", or simply abbreviated as "FC46 strain"). Then, the Cremoris FC46 strain was transferred to the Patent Microbiology Depositary, National Institute of Technology and Evaluation, which has an address in Room 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan, on November 6, 2020. , Identification display: Lactococcus lactis subsp. Cremoris FC46, Deposit number: NITE BP-03310, international deposit (Deposit certificate issuance date: November 30, 2020).

Example 2 Evaluation of Acid Resistance The FC46 strain obtained above was subjected to TY-Glu medium (1% glucose, 0.5% Bacto (TM) Tryptone, 0.5% Bacto (TM) Yeast Extract, 0.5% NaCl) (pH 7.0). ) Was inoculated at a rate of 4% and cultured at 25 ° C. for 15 hours to prepare a preculture solution. The obtained preculture solution was inoculated into 40 ml of TY-Glu medium adjusted to pH 4.0 using lactic acid so that the final concentration was 1%. This was statically cultured at 26 ° C. for 27 hours, and the viable cell count (cfu / ml) of the FC46 strain in the culture medium was measured by a pour culture method using a BCP-added plate count agar agar medium (Nissui Pharmaceutical Co., Ltd.). The survival rate (%) after 27 hours was calculated by measuring over time. As a comparative test, the parent FC strain was similarly cultured using the parent FC strain instead of the FC46 strain, and the viable cell count (cfu / ml) of the parent FC strain in the culture medium was measured over time, and the viable cells were grown 27 hours later. The residual rate (%) was calculated.

FIG. 1 shows the results of measuring the viable cell count (cfu / ml) of each Cremoris strain (FC46 strain, parent FC strain) over time. As shown in FIG. 1, the parent FC strain died rapidly under a low pH condition of pH 4.0, and the viable cell count was 6.6 × 10 ³ (cfu / ml) (survival rate) after 27 hours of culturing. It decreased to 0.2%). On the other hand, the FC46 strain has high survivability even under a low pH condition of pH 4.0, and even after culturing under the pH condition for 27 hours, 1.9 × ¹⁰⁶ (cfu / ml) (survival rate 33.6). %) Maintained a high viable cell count. From this, it was confirmed that the FC46 strain is a Cremoris strain having excellent acid resistance as compared with the parent FC strain.

Example 3 Evaluation of stability in fermented milk Fermented milk was prepared using the FC46 strain, and the stability in fermented milk was evaluated. In addition, as a comparative control, fermented milk was similarly prepared using the parent FC strain, and the stability in the fermented milk was evaluated.

1 Preparation of fermented milk Four types of fermented milk 1 to 4 were prepared using the activated bacteria of the following strains as a starter.
(1) Fermented milk 1: Parent FC strain (starter 1)
(2) Fermented milk 2: FC46 strain (starter 3)
(3) Fermented milk 3: Parent FC strain (starter 1) + Thermophilus bacterium (starter 2)
(4) Fermented milk 4: FC46 strain (starter 3) + Thermophilus bacterium (starter 2)
The starters 1 and 3 were prepared by inoculating (1%) the parent FC strain and the FC46 strain into a sterilized 8% skim milk powder emulsion and leaving them at 30 ° C. for about 10 hours to activate them. The starter 2 was prepared by inoculating Streptococcus thermophilus (1%) in a sterilized 8% skim milk powder emulsion and leaving it at an optimum temperature (37 ° C.) for about 10 hours to activate it.

Each starter prepared above was inoculated into sterilized milk and fermented at 30 ° C. When the formation of curds was observed, cooling (4 ° C.) was started and fermentation was suppressed to obtain fermented milk.

2 Evaluation of Bacterial Stability in Fermented Milk After preparing fermented milks 1 to 4, they were left to stand for 30 days in a dark place at 10 ° C.
The viable cell count (cfu / ml) in the fermented milk after storage (fermented milk 1 and 2 were the viable cell counts of Cremoris bacteria, and fermented milks 3 and 4 were the viable cell counts of Cremoris bacteria and Thermophilus bacteria). The viable cell count of Cremoris was obtained by a pour culture method (25 ° C.) using TY-Gal medium (pH 7.0), and the viable cell count of Thermophilus was obtained by BCP-added plate count agar agar medium (pH 7.0). It was measured using the pour culture method (37 ° C.) using the above.

The pH, acidity, and viscosity of the fermented milk were measured by the following methods.
[Measurement of pH]
The pH in the fermented product was measured using a pH meter F-52 (manufactured by HORIBA, Ltd.) after adjusting the fermented product to 10 ° C.

[Measurement of acidity]
Weigh 10 g of the fermented product, add the same amount of distilled water, dilute it, add 0.5 ml of phenolphthalein solution as an indicator, and use a 0.1 mol / L sodium hydroxide aqueous solution for 30 seconds to eliminate the slight red color. Titration is performed up to the point where it does not occur, and the percentage amount of lactic acid per 100 g of the sample is obtained from the titration amount and used as the acidity. In addition, 1 ml of a 0.1 mol / L sodium hydroxide aqueous solution corresponds to 9 mg of lactic acid.

[Measurement of Viscosity] The absolute viscosity in the fermented product was measured using a B-type viscometer (Spindle No. 4 manufactured by Brooksfield Co., Ltd.) after adjusting the fermented product to 10 ° C. The rotation time of the rotor was set to 1 minute.

表３及び４に示すように、親ＦＣ株は保存期間中に急速に死滅し、３０日後には１ｍＬあたり数個しか生菌が残らなかった（発酵乳１）。一方、ＦＣ４６株は３０日後も１．１×１０^４（cfu／ml）（生残率０．００２６％）という高い生菌数を維持していた（発酵乳２）。また、親ＦＣ株およびＦＣ４６株はいずれも、サーモフィラス菌と共発酵させることで、単独発酵よりも高い生菌数を維持し（発酵乳３及び４）、特にＦＣ４６株では５．８×１０^６（cfu／ml）（生残率２．８％）という非常に高い生菌数を示した（発酵乳４）。Table 3 shows the results on the first day from the start of storage, and Table 4 shows the results on the 30th day from the start of storage.

As shown in Tables 3 and 4, the parent FC strains died rapidly during the storage period, and after 30 days, only a few viable bacteria remained per 1 mL (fermented milk 1). On the other hand, the FC46 strain maintained a high viable cell count of 1.1 × 10 ⁴ (cfu / ml) (survival rate 0.0026%) even after 30 days (fermented milk 2). In addition, both the parent FC strain and the FC46 strain maintain a higher viable cell count (fermented milk 3 and 4) than the single fermentation by co-fermenting with the thermophilus bacterium, and in particular, the FC46 strain is 5.8 × ¹⁰⁶ . It showed a very high viable cell count of (cfu / ml) (survival rate 2.8%) (fermented milk 4).

Furthermore, the decrease in viscosity of the parent FC strain was observed after storage for 30 days, but the decrease in viscosity of the FC46 strain was suppressed as compared with the parent FC strain, and the same was true when co-fermented with Thermophilus. The tendency of was obtained.
The fermented milks 1 to 4 produced in this example had good appearance (color, presence / absence of water separation), aroma, and taste, and no difference was observed in these points.

Example 4 Preparation and Stability Evaluation of Lactic Acid Bacteria-Containing Composition A lactic acid bacterium-containing composition was prepared using FC46 strain, and the storage stability in a dry state was evaluated. For comparison, a lactic acid bacterium-containing composition was prepared using the parent FC strain instead of the FC46 strain, and the storage stability in a dry state was similarly evaluated.

1 Preparation of lactic acid bacteria solution FC46 strain or parent FC strain was inoculated into Difco M17 medium (BD) to which glucose was added so that the final concentration was 0.5% at a ratio of 4%, and the cells were inoculated at 25 ° C. for 16 hours. The preculture solution was prepared by culturing. The same medium was inoculated with 4% of the preculture solution prepared above and cultured at 25 ° C. for 16 hours to obtain 10 L of the culture solution. This was subjected to centrifugation (3,000 rpm × 10 minutes), the obtained supernatant was removed by decantation, and then the residue was washed twice with physiological saline to concentrate the FC46 strain or the parent FC strain, respectively. A lactic acid bacterium solution was prepared.

To each of the prepared lactic acid bacteria solutions, defatted milk powder is added at a ratio of 10%, and this is further freeze-dried using a freeze-dryer (FD-81, Tokyo Rika Kikai Co., Ltd.) according to a conventional method to obtain about 50 g. Freeze-dried lactic acid bacteria (FC46 strain cells, parent FC strain cells) were obtained.

0.5 g of the obtained freeze-dried lactic acid bacteria (FC46 strain cells, parent FC strain cells) was placed in a sterilized laminated film bag with a chuck (Lamizip [registered trademark]: manufactured by Japan Co., Ltd.). It was put in and stored under a dark condition of 37 ° C. (forced deterioration test). The viable cell count of the FC46 strain or the parent FC strain in the freeze-dried lactic acid bacteria cells was measured over time from the start of the test (first day), and the survival rate (%) was calculated.

The results are shown in Table 5.

The parent FC strain rapidly died when stored under a temperature condition of 37 ° C, and the viable cell count after ³ weeks was 3.2 × 109 (cfu / g) (survival rate: about 1.6%). In contrast, the FC46 strain showed a very high viable cell count of 1.1 × 10 ¹⁰ (cfu / g) (survival rate: about 14.7%). From this, it was confirmed that the FC46 strain is superior in storage stability as compared with the parent FC strain even in a dry powder state under aerobic conditions.

SEQ ID NO: 1 shows the base sequence of the 16S rRNA gene of this Cremoris FC strain.

Claims (9)

Lactococcus lactis subsp. Cremoris FC46 strain (accession number: NITE BP-03310) or its subspecies lactic acid bacterium .
The passage strain has the same mycological properties, morphological characteristics, physiological characteristics, carbon source assimilation property, viscous polysaccharide-producing property, acid resistance, and dry conditions and aerobic conditions as those of the FC46 strain. It is a lactic acid bacterium belonging to Lactococcus lactis, subspecies, and cremoris, which has survivability . The lactic acid bacterium according to claim 1, which is in a dry state. A fermenting inoculum containing the lactic acid bacterium according to claim 1 or 2. The fermenting inoculum according to claim 3, further comprising Streptococcus thermophilus. The fermenting inoculum according to claim 4, which is a symbiotic organism of Lactococcus subspecies Cremoris FC46 strain (accession number: NITE BP-03310) or its successor strain and Streptococcus thermophilus. .. The fermenting inoculum according to claim 4 or 5, which is in a dry state. An oral composition containing the lactic acid bacterium according to claim 1 or 2. The oral composition according to claim 7, which is a fermented food or drink. A method for producing a fermented food or drink, which comprises a step of inoculating a fermented food or drink raw material with the lactic acid bacterium according to claim 1 or 2 or the fermenting inoculum according to any one of claims 3 to 6 and fermenting the inoculum. ..

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