TWI799153B - Novel lactic acid bacteria strains and compositions containing novel lactic acid bacteria strains - Google Patents

Abstract

The invention provides a novel lactic acid bacteria strain, especially a novel milk fat FC strain, and its application. The lactic acid bacteria strain of the present invention is Lactococcus lactis subsp. cremoris FC46 strain (registration number: NITE BP-03310), or its descendants.

Description

Novel lactic acid bacteria strains and compositions containing novel lactic acid bacteria strains

The present invention relates to a novel strain of lactic acid bacteria. In addition, the present invention relates to various uses of the lactic acid bacterial strain, such as compositions (seeds, oral compositions) containing the lactic acid bacterial strain, and methods for producing fermented foods using the lactic acid bacterial strain.

In recent years, as consumers' health awareness has increased, the demand for foods containing lactic acid bacteria has increased, and lactic acid bacteria have been widely used in the manufacture of fermented milk and lactic acid bacteria beverages. Lactococcus lactis subsp. cremoris, a type of lactic acid bacteria, has been known to be used as a producer of fermented milk products such as yogurt and cheese since ancient times. The aforementioned cream bacteria have mucopolysaccharide-producing bacteria kind. Among them, the yogurt obtained by fermenting milk fat bacteria isolated by the applicant (hereinafter referred to as "milk fat FC strain") as the main fermenting lactic acid bacteria (trade name "Kasupi yogurt (カスピ海ヨーグルト)", the applicant's registered trademark, Japanese Registration No. 5761425), has a specific viscosity that is completely different from the conventional fermented milk, milk fat FC strain and the mucopolysaccharide produced by the milk fat FC strain, it is reported that it has the function of regulating the intestines, inhibiting the rise of blood sugar, and activating the immune system Various physiological effects, such as anti-influenza virus infection defense, allergy suppression, and exercise improvement (refer to non-patent literature 1~6).

Therefore, in anticipation of the above physiological effects, not only fermented milk has been developed, but also products containing milk fat FC strains such as fermented inoculum and nutritional supplements have been developed. However, in order to fully exert this physiological effect in the intestine, it is necessary to continuously ingest milk fat FC strains . However, lactic acid bacteria, which are facultative anaerobic bacteria, are known to have reduced viability under aerobic conditions and acidic (low pH) conditions, and the milk fat FC strain is no exception. Therefore, it is constantly seeking to develop a product containing milk fat FC strain that can inhibit the reduction of the number of bacteria produced in long-term storage and maintain useful physiological effects in dry powder such as yogurt and other acidic foods or lactic acid bacteria nutritional supplements. [Prior Technical Literature] [Non-patent literature]

[Non-Patent Document 1] T. Toda et al., "Effects of fermented milk with Lactococcus lactis subsp. cremoris FC on deficiency frequency and fecal microflora in healthy elderly volunteers" J. Jpn. Soc. Food. Sci. 52(6) , 243-250 (2005) [Non-Patent Document 2] 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 ) [Non-Patent Document 3] 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) [Non-Patent Document 4] 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) [Non-Patent Document 5] 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) [Non-Patent Document 6] 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)

[Technical problem to be solved by the invention]

The problem of the present invention is to provide a novel milk fat FC strain in view of the aforementioned conventional problems, which has improved acid resistance (low pH resistance) and improved viability even under aerobic conditions and dry conditions. In other words, the present invention The object is to provide a milk fat FC strain which has good viability even in acidic foods such as fermented milk or in a dry powder state. Furthermore, the object of the present invention is to provide an inoculum for fermentation, oral compositions such as nutritional supplements and fermented foods, and fermented milk fat FC strains using the milk fat FC strain excellent in acid resistance and survivability under aerobic conditions and dry conditions. Food manufacturing methods.

In order to solve the above problems, the inventors of the present invention found a novel milk fat FC strain after in-depth research. ) have the same bacteriological characteristics, but in comparison, they have excellent characteristics in terms of acid resistance and survivability under aerobic conditions and dry conditions, and further through in-depth research to complete the present invention. Furthermore, hereinafter, the conventionally known milk fat FC strain is referred to as "milk fat FC strain", "existing milk fat FC strain" or "pro-FC strain", and the newly discovered milk fat FC strain of the present invention is called It is "this milk fat FC strain", "milk fat FC46 strain" or "FC46 strain". [Technical means]

The present invention has the following embodiments. (I) Novel lactic acid bacteria milk fat strain (this milk fat FC strain) (I-1) A lactic acid bacterium, which is Lactococcus lactis subsp. cremoris FC46 strain (registration number: NITE BP-03310), or its descendants. Furthermore, the above-mentioned subculture strain has the same bacteriological properties, morphological characteristics, physiological characteristics, carbon source assimilation, mucopolysaccharide production, acid resistance, and good under dry conditions as the FC46 strain specified by the above-mentioned registration number. The survivability of oxygen conditions is the same as that of lactic acid bacteria belonging to Lactococcus lactis subsp. cremoris. In the present invention and the descriptions below, it is also the case that it is a successor strain of FC46 strain. In this specification, the FC46 strain identified by the registration number, its original strain, and these descendant strains are collectively referred to as "this milk fat FC strain", "milk fat FC46 strain" or "FC46 strain". (I-2) The lactic acid bacteria as described in (I-1) which is a dry state.

(II) Inoculum for fermentation (II-1) An inoculum for fermentation characterized by containing Lactococcus lactis subsp. cremoris FC46 strain (registration number: NITE BP-03310), or its substrains. (II-2) The inoculum for fermentation as described in (II-1), which further contains Streptococcus thermophilus. (II-3) The inoculum for fermentation as described in (II-2), wherein it is Lactococcus lactis subsp. cremoris FC46 strain (registration number: NITE BP-03310), or its descendant strain, and Symbionts of 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 (III-1) An oral composition characterized by containing the lactic acid bacteria described in (I-1) or (I-2). (III-2) The oral composition as described in (III-1), which is a food, an oral drug, or an oral quasi-drug. (III-3) The oral composition as described in (III-1) or (III-2), which is a fermented food.

(IV) Manufacturing method of fermented food, and fermented food (IV-1) A method for producing a fermented food, which is characterized by subjecting the lactic acid bacteria described in (I-1) or (I-2), or any one of (II-1) to (II-4). The steps of planting the fermented food raw materials with the fermented inoculum described in the document and fermenting. [Effect of the invention]

Although the present milk fat FC strain of the present invention has the same mycological properties and useful physiological effects as the existing milk fat FC strain, compared with the existing milk fat FC strain, it has lower viability and viscosity under low pH conditions around pH 4. High maintenance effect, excellent acid resistance. In addition, the present milk fat FC strain has higher survivability under dry conditions and aerobic conditions, and is excellent in tolerance to dry and aerobic conditions, compared with the existing milk fat FC strains. Therefore, this milk fat FC strain has excellent stability in oral compositions such as acidic foods such as yogurt and lactic acid bacteria beverages, and nutritional supplements in a dry state, and can be maintained in the form of oral compositions for a long time and exert its usefulness. physiological effects.

(I) Novel lactic acid bacteria milk fat strain (this milk fat FC strain) This milk fat FC strain belongs to the strain of Lactococcus lactis subsp. cremoris with the following mycological properties, and a part of it is internationally deposited in the independent administrative legal person product evaluation technology foundation institution Chartered Microorganism Depository Center ( Room No. 122, 2-5-8 Kamiso Kamazu, Kisarazu City, Chiba Prefecture, Japan), November 6, 2020, identification mark: Lactococcus lactis subsp. cremoris FC46, deposit number: NITE BP-03310.

(a) Mycological properties For the milk fat FC strain, use TY-Glu medium (1% glucose, 0.5% Bacto (TM) trypsin (Thermo Fisher Scientific company), 0.5% Bacto (TM) yeast extract (Thermo Fisher Scientific company), 0.5% NaCl ) (pH 7.0) or MRS medium (manufactured by Difco), the mycological properties (morphology, physiological characteristics, carbon source assimilation) of static culture under aerobic conditions are listed in Table 1 and Table 2 below .

[Table 1]

[Table 2]

(b) Morphological features Cell shape: (strep) cocci Colony shape: round (hemispherical) diameter 0.5~1.0mm Colony color: white Sporulation: no

(c) Physiological characteristics Gram stain: positive Mobility: no Catalase activity: no Gas production in glucose metabolism: no Lactic acid optical activity: L Reproduction at 10°C: Reproducible Reproduction at 45°C: non-reproductive Optimum temperature: 25~30℃ Optimum pH: pH6~7 Lactic Acid Fermentation: Homotype

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

(e) Other, characterizing the nature of the microorganism For the above properties, refer to K.H.Schleifer et al., "Transfer of Streptococcus lactis and Related Streptococci to the Genus Lactococcus gen. nov." System. Appl. Microbiol. 6, 183-195 (1985), and according to the 16S rRNA gene As a result of homology search, this milk fat FC strain has 100% homology with the registered existing milk fat FC strain (Lactococcus lactis subsp. cremoris FC, FERM P-20185), and was identified as belonging to Lactococcus lactis subsp. cremoris ( Lactococcus lactis subsp. cremoris). The base sequence of the 16S rRNA gene of this milk fat FC strain is described in the sequence number 1 of the sequence listing.

The present milk fat FC strain produces mucopolysaccharides in the same manner as the existing milk fat FC strain except for the above-mentioned mycological properties. Therefore, it is conceivable that the present milk fat FC strain has various useful physiological effects such as intestinal regulation effect, blood sugar rise suppression effect, immune activation effect, influenza virus infection defense effect, allergy suppression effect, and exercise condition improvement similarly to the existing milk fat FC strain.

On the other hand, as shown in Examples 2 and 3 described later, the present milk fat FC strain has high viability and viscosity maintenance effect under low pH conditions around pH 4 and excellent acid resistance compared with the existing milk fat FC strain characteristic. In addition, as shown in Example 4 described later, this milk fat FC strain has the characteristics of high survivability under dry conditions and aerobic conditions, and excellent resistance to dry and aerobic conditions, compared with the existing milk fat FC strains . Therefore, this milk fat FC strain has excellent stability in oral compositions such as acidic foods such as yogurt and lactic acid bacteria beverages, and nutritional supplements in a dry state, and can be maintained for a long period of time in the form of oral compositions and exert its useful effect. Physiological effect.

The milk fat FC strain includes not only the international deposit number: NITE BP-0331 milk fat FC strain (deposited bacteria) and its original strain, but also its descendants. The subculture of the milk fat FC strain can be carried out according to the established method generally used in the subculture of lactic acid bacteria, and is not particularly limited. For example, the following method can be mentioned: inoculate the milk fat FC strain into a test tube containing the above-mentioned TY-Glu medium (pH 7.0), and culture it at an optimum temperature of about 25~30°C for about 12~48 hours. For the milk fat FC strain that has been cultured and subcultured, glycerin can be added to make the final concentration 10~30w/v%, and stored at -80°C until use.

The culture medium of this milk fat FC strain, in addition to the aforementioned TY-Glu medium, can use a general medium containing carbon sources, nitrogen sources, inorganic ions, and organic micronutrients as needed. The carbon source may be any as long as it contains an assimilable carbon source. For example, carbohydrates such as D-glucose, D-galactose, D-fructose, D-mannose, and D-lactose can be preferably used. The nitrogen source may be any as long as it contains or contains assimilable nitrogen compounds, for example, ammonium sulfate, casein amino acid, protein, etc. can be used. In addition, inorganic salts such as phosphoric acid, iron, potassium, magnesium, zinc, manganese, copper, and calcium can be used appropriately. Further, vitamins such as amino acids necessary for bacterial growth, biotin, riboflavin, pyridoxine, niacin, pantothenic acid, folic acid, and thiamine can be added to the culture medium as needed.

The culture of the milk fat FC strain can be carried out at a temperature above 15°C (preferably around 25-30°C) and at a pH above 5 (preferably around pH 6-7) to promote proliferation. The milk fat FC strain can be grown by static culture under aerobic conditions, but preferably can be grown under anaerobic conditions to promote growth.

This milk fat FC strain has high viability in a dry state and aerobic conditions as mentioned above, after being separated from the culture medium, it can also be prepared into a dry product by performing drying treatments such as vacuum drying or spray drying, and can also be mixed with culture medium components or Other components such as excipients are dried together to prepare a dry product.

(II) Inoculum for fermentation This milk fat FC strain can be used as an inoculum (inoculum for fermentation, also referred to as "lactic acid starter") used in the production of fermented foods by utilizing its fermentability. Fermented foods include: milk fermented food (yogurt, lactic acid bacteria beverage, cheese, etc.) whose main raw material is milk, soybean milk fermented food (yogurt, lactic acid bacteria beverage, etc.) whose main raw material is grain (rice, wheat, etc.), grain fermented foods (sour, bread, etc.), vegetable and fruit fermented foods (pickles, etc.).

The fermented inoculum of the present invention is preferably used in the fermentation of raw materials mainly composed of milk or soymilk, and through the fermentation of the raw materials, milk-fermented foods such as yogurt or lactic acid bacteria beverages, or soybean milk-fermented foods are produced. Here, the raw material mainly composed of milk or soybean milk (milk raw material, soybean milk raw material), as long as it contains protein derived from milk or soybean (preferably soybean), it may be milk or soybean milk itself, and other Those who remove or reduce lipids, etc. Furthermore, in the description of this case, the "milk" used in the present invention refers to those containing animal milk, such as cow's milk, goat's milk, goat's milk, horse's milk, etc., and its form is not particularly limited. Milk in any form such as milk, skimmed milk, whole milk powder, or skimmed milk powder. In addition, the "soymilk" used in the present invention refers to soymilk obtained by grinding soybeans with water and then squeezing the juice, but it is not limited thereto, and may be an emulsion obtained by mixing soybeans with water and liquefying them. (Soybean emulsion), the type of beans used as a raw material is not particularly limited, and may be any beans such as soybeans, red beans, peas, broad beans, kidney beans, and mung beans.

The fermented inoculum of the present invention is preferably inoculated until the bacterial count of the milk fat FC strain mentioned above is about 1×10 ⁵ ~1×10 ⁹ (cfu/g) when producing milk fermented food or soybean milk fermented food. Therefore, when using it, it is preferable to culture this Milk Fat FC strain in advance using, for example, milk raw materials or soybean milk raw materials, and adjust the number of bacteria in the culture to be about 1×10 ⁷ ~1×10 ⁹ (cfu/g) (inoculum composition). Specifically, although not limited, it can be, for example, inoculating the Milk Fat FC strain to fermented raw materials (milk raw materials, soymilk raw materials) that have been sterilized in advance, or the aforementioned fermented raw materials to which yeast extract is added (for example, 5 ~10% skimmed milk powder, etc.), cultured at the optimum temperature, and adjusted to the above bacterial concentration. In addition, the measurement of the number of viable bacteria of this milk fat FC strain can be carried out by performing the mixed dilution culture method (25°C) using TY-Glu medium (pH 7.0) and counting the number of colonies propagated (colony count) . Since the number of bacteria is related to turbidity, if the correlation between the number of bacteria and turbidity (absorbance at a wavelength of 600nm) is obtained in advance, the number of bacteria can be calculated by measuring the turbidity.

The seed bacteria for fermentation of the present invention may also be formed only from the milk fat FC strain, and other microorganisms with fermentation ability may also be used in combination as long as the effects of the present invention are not hindered. Other microorganisms are not limited, but for example, Lactococcus lactis subsp. cremoris and Lactococcus lactis subsp. lactis biovar. diacetylactis belong to Lactococcus Lactic acid bacteria of the genus; Streptococcus thermophilus and other lactic acid bacteria belonging to the genus Streptococcus; and Lactobacillus casei subsp. casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus gasseri (Lactobacillus gasseri), Lactobacillus acidophilus (Lactobacillus acidophilus) and other lactic acid bacteria belonging to the genus Lactobacillus. One or two of these lactic acid bacteria may be used in any combination.

The microorganism used in combination with the milk fat FC strain is preferably Streptococcus thermophilus (hereinafter referred to as "S. thermophilic bacteria"). As shown in Example 3 below, the milk fat FC strain, by co-fermenting with S. thermophiles under low pH conditions, can maintain a high bacterial count (increased survivability) and inhibit post-storage The viscosity is reduced. In the fermented inoculum of the present invention (the symbiont of the milk fat FC strain and S. thermophile) that uses this milk fat FC strain and S. thermophilic bacteria in combination, although there is no limit to the ratio of the number of living bacteria between the two, it can be An example of this milk fat FC strain: S. thermophiles=1:0.001~1000. More preferably, it can be exemplified as present milk fat FC strain: S. thermophiles=1:0.01~100, more preferably, it can be exemplified as 1:0.1~10. Furthermore, the measurement of the viable count of S. thermophiles can be carried out by colony counting by the mixed dilution culture method (37° C.) using BCP plate count agar medium (Nissui Pharmaceutical Co., Ltd.).

In addition, acetic acid bacteria can also be used as microorganisms used in combination with the present milk fat FC strain. The acetic acid bacteria are not limited, but examples thereof include acetic acid bacteria belonging to the genus Acetobacter, Gluconobacter, and Gluconacetobacter.

The shape of the inoculum for fermentation of the present invention is not particularly limited as long as it exhibits fermentation ability when used in the production of fermented foods. From the viewpoints of sanitation, quality maintenance, and handling, it is preferably a dried solid form, more preferably a dry powder or dry granule. It may also be prepared as a dry product by drying it together with other components such as medium components or excipients.

The fermentation conditions (cultivation conditions) of the seed bacteria for fermentation of the present invention include a culture temperature of more than 15°C, preferably about 25-30°C; a culture pH of more than 5, preferably about pH 6-7; a culture time of more than 3 hours, preferably Preferably about 8 to 24 hours.

(III) Oral composition The oral composition of the present invention is characterized in that it contains this milk fat FC strain. The oral composition is prepared in a food form using an appropriate edible carrier (food material) similarly to general food. In addition, the oral composition is prepared into a preparation form (a nutritional supplement form, a pharmaceutical form, a quasi-drug form) using appropriate pharmaceutically acceptable excipients, diluents and other raw materials for preparation.

This milk fat FC strain is expected to exert various physiological effects in the body (intestinal regulation effect, blood sugar rise suppression effect, immune activation effect, influenza virus infection defense effect, allergy suppression effect) through oral ingestion similar to the existing milk fat FC strain and improvement of exercise status, etc.). This effect can be considered to be the effect of the present milk fat FC strain itself and/or the mucopolysaccharide produced by the present milk fat FC strain. Therefore, the oral composition of the present invention preferably contains the milk fat FC strain in a raw state. However, it is desirable to contain as much as possible this milk fat FC strain as live bacteria, and there is no problem in including this milk fat FC strain in a dead state.

The oral composition of the present invention only needs to contain this milk fat FC strain, and within this limit, it can also be a culture of this milk fat FC strain, a crude or refined product of the culture, or a freeze-dried product or spray thereof dry matter. The above-mentioned cultures are not limited, but can be obtained, for example, by using a medium suitable for the milk fat FC strain, such as TY-Glu medium (pH 7.0), etc., and culturing at an optimum temperature of about 25-30°C for about 12-48 hours. . In addition, the bacterial cells of the Milk Fat FC strain can be obtained by, for example, centrifuging the culture at 3,000 rpm for about 10 minutes after the above-mentioned cultivation, and collecting the bacterial cells. These can also be purified according to general methods. Furthermore, the above-mentioned bacterial cells can also be freeze-dried or spray-dried. The dried bacterial cells thus obtained can also be used as an active ingredient of the oral composition of the present invention. In this specification, the present milk fat FC strain, its culture, the crude product to the refined product of the culture, and these dried products are collectively referred to as "the present milk fat FC strain and its culture".

The oral composition of the present invention may further contain an appropriate amount of nutritional components suitable for the maintenance or proliferation of the milk fat FC strain as needed. Specific examples of the nutrient components include: carbon sources such as glucose, galactose, fructose, lactose, mannose, etc. used in the culture medium of microorganisms; nitrogen sources such as yeast extract and protein; vitamins, minerals, etc. species, trace metal elements, other nutrients and other components.

The oral composition of the present invention can be prepared in the form of food, drug, and quasi-drug according to general methods. In addition, the carriers suitable for preparing these various forms may be any one of edible carriers, pharmaceutically acceptable excipients, diluents and other carriers. The details of preparation into food form and the edible carrier available at this time will be described in the item "Oral composition in food form" described later.

Generally speaking, the amount of the milk fat FC strain in the oral composition of the present invention can be from 1×10 ⁷ to 1×10 in the oral composition of the present invention. ¹¹ (cfu) or so appropriate choice. The compounding amount of the above-mentioned milk fat FC strain is based on the above-mentioned amount, and can be appropriately changed according to the form of the oral composition of the present invention to be prepared, the intended effect, and the like.

Oral composition in food form The oral composition of the present invention in the form of food may be ingested as food. Although not limited, preferred specific examples include: fermented milk such as yogurt or milk-fermented foods made from milk, such as lactic acid bacteria drinks, and soybean milk made from soybeans, such as soybean yogurt and fermented soy milk drinks. Fermented foods. Others include solid preparations (tablets, pills, powders, granules, microcapsules, capsules, etc.) and liquid preparations (liquids, suspensions, syrups, emulsions, etc.) containing this milk fat FC strain, etc. Food in the form of preparations. In addition, snacks such as chewing gum, milk candy, soft candy, nougat, and chocolate containing the milk fat FC strain of the present invention; fermented foods such as fermented vegetable drinks and fermented fruit drinks; the above-mentioned fermented foods such as pudding and Bavarian cream other than dairy products, etc. Furthermore, in this specification, the terms "fermented milk" and "lactic acid bacteria beverage" are in accordance with Article 2 37 "fermented milk" and 38 "lactic acid bacteria beverage" of the former Japanese Ministry of Health and Welfare "Ministry Order Concerning Milk and Dairy Product Components, etc." definition. That is, "fermented milk" refers to a pasty or liquid form obtained by fermenting milk or dairy products with lactic acid bacteria. Accordingly, the fermented milk includes a beverage form and a yogurt form. In addition, "lactic acid bacteria beverage" refers to a beverage obtained by diluting the pasty or liquid form of milk or dairy products fermented with lactic acid bacteria as the main raw material with water.

(IV) Manufacturing method of fermented food, and fermented food The production method of the fermented food of the present invention is characterized in that it contains the milk fat FC strain. The fermented food of the present invention can be produced according to a predetermined method, for example, by inoculating the milk fat FC strain in suitable fermentation raw materials containing the nutritional source of the milk fat FC strain (for example, containing milk, soybean milk (soybean emulsion), grains, vegetables, and fruits) are cultivated to ferment the raw materials.

Fermentation using this milk fat FC strain is preferably a method of preparing bacterial elements in advance and inoculating them on the raw materials for fermentation to ferment them. Here, as the bacterial element, the above-mentioned seed bacteria for fermentation of the present invention can be mentioned. For such bacterial elements, it is preferable that the number of viable bacteria of this milk fat FC strain is about 1×10 ⁷ ~1×10 ⁹ (cfu/ml).

For the above-mentioned raw materials for fermentation, fermentation promoters for the good propagation of the milk fat FC strain can be added as needed, such as carbon sources such as glucose, galactose, fructose, lactose, and mannose; nitrogen sources such as yeast extract and protein; vitamins Classes, minerals, trace metal elements, other nutrients, etc.

The amount of inoculum of the milk fat FC strain, for example, in the case of using milk as the raw material for fermentation, the appropriate amount of the milk fat FC strain in the milk is more than 1×10 ⁵ (cfu/ml), preferably 1×10 ⁷ ~1 Choose about the amount of ×10 ⁹ (cfu/ml). Generally speaking, culture conditions are selected from a fermentation temperature of 15° C. or higher, preferably around 25 to 30° C., and a fermentation time of about 3 to 24 hours.

Furthermore, the above-mentioned fermented product obtained in this way may have a curd-like form (yogurt or pudding-like form), and this product can also be ingested as a solid food as it is. The curd-like fermented product can be further homogenized into a desired beverage form. This homogenization can be implemented using a general emulsifier (homogenizer). By such homogenization, beverages with a smooth mouthfeel can be obtained. Furthermore, for this homogenization, it may also be appropriately diluted as required, or organic acids for adjusting the pH may be added. In addition, sugars, fruit juices, thickeners, surfactants, spices, etc., which are generally used in beverage production, may also be added appropriately. of various additives. The beverage thus obtained can be aseptically filled in suitable containers and made into the final product.

The ingestion (administration) amount is appropriately determined according to the age, sex, body weight, disease degree, etc. of the organism ingesting it, and is not particularly limited. Generally speaking, it is preferable to select from the range of about 1×10 ⁷ to 1×10 ¹¹ (cfu/ml) of the milk fat FC strain. This composition can generally be adjusted so that each person can ingest about 50-1,000 mL per day.

Preferable specific examples of the oral composition of the present invention in food form include nutritional supplement forms, specifically solid preparations such as tablets, pills, powders, granules, microcapsules, and capsules; and liquid preparations. Foods in the form of preparations such as liquid preparations such as suspensions, syrups, and emulsions. Foods in the form of preparations are preferably solid preparations in a dried state.

The food in the form of nutritional supplements is the same as the composition in the form of pharmaceuticals. As a preparation carrier, fillers, extenders, binders, wetting agents, disintegrants, and surfactants used in this field can generally be used. , lubricants and other diluents or excipients to manufacture.

The amount of the lactic acid coccus of this invention mixed in the oral composition of a food form should just be the amount which can exhibit the effect of the said lactic acid coccus, and can be suitably selected from a wide range within this limitation. In general, it is preferable to contain about 1×10 ⁷ to 1×10 ¹¹ (cfu) in 1 ingestion (administration) unit form.

The administration method of the above-mentioned composition is not particularly limited as long as it is ingested orally, and it can be determined according to various forms, age of the subject, gender, other conditions, degree of disease, and the like.

The intake amount of the above-mentioned components is appropriately selected according to its usage, age of the subject, gender and other conditions, but as mentioned above, it is preferably about 1×10 ⁷ ~1×10 ¹¹ (cfu) per 1 intake (administration) Take this creamy FC strain. The intake of this milk fat FC strain which is an active ingredient can be more than about 1×10 ⁷ (cfu) per day, and can be divided into multiple intakes per day. In addition, the oral composition of the present invention is preferably continuously ingested in order to effectively obtain the effect. For example, continuous or intermittent ingestion over two or more weeks can be illustrated.

The oral composition containing the present milk fat FC strain in a raw state is expected to exert useful physiological effects in vivo similarly to the existing milk fat FC strain after ingestion (administration). Therefore, the oral composition of the present invention can be effectively used as a health-enhancing agent based on its physiological effects. Such an oral composition of the present invention includes foods that have specific functions and are ingested and eaten for the purpose of maintaining health and the like. Specifically, the oral composition of the present invention includes foods that have intestinal regulation, blood sugar rise suppression, immune activation, influenza virus infection defense, allergy suppression, and/or exercise improvement effects. In addition, these foods include foods that are manufactured and sold by expressing or emphasizing the aforementioned functions or uses, such as health functional foods (nutritional functional foods, functionally indicated foods, foods for specific health uses), foods for special purposes (foods for specific health uses) food), and similar health foods.

Above, in this specification, the terms "comprising" and "containing" include the meanings of "made from" and "substantially made from". [Example]

Hereinafter, the present invention will be described using examples to facilitate understanding of the constitution and effects of the present invention. However, the present invention is not limited by these examples. Unless otherwise stated, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. In addition, unless otherwise specified, "%" described below means "mass %", and "part" means "mass part".

Embodiment 1 Isolation and identification of novel lactic acid bacteria milk fat FC strain The milk fat FC strain (FERM P-20185) was inoculated into sterilized 8% skim milk powder (1%), and placed at its optimum temperature (25°C) for about 10 hours to activate the milk fat FC strain (bacteria 1). On the other hand, Streptococcus thermophilus (hereinafter referred to as "thermophilus") was inoculated into sterilized 8% skim milk powder (1%), and placed at its optimum temperature (37°C) for 10 After about 1 hour, activate the thermophilic bacteria (Bacteria 2). Then, inoculate the prepared bacteria 1 and 2 into the sterilized milk prepared separately, and ferment at 30°C. Cooling (4°C) was initiated at the point at which curd formation was observed.

After storing the obtained fermented milk in a dark place at 4°C for 46 days, in 3L of TY-Gal medium (1% glucose, 0.5% Bacto (TM) trypsin (Thermo Fisher Scientific Company), 0.5% Bacto (TM) ) Yeast extract (Thermo Fisher Scientific), 0.5% NaCl) (pH 7.0) inoculated with 4%, and incubated at 26°C for 72 hours. The culture solution was streaked on TY-Gal agar medium (pH 7.0) and cultured at 25°C for 48 hours, and the propagated colony culture was preserved in sterilized 8% skim milk powder.

The screened strains with high low pH tolerance (acid resistance) were obtained by the method described in Example 1, and their bacteriological properties were evaluated according to established methods. The results are shown in Table 1. In addition, the ability to assimilate 49 kinds of carbon sources (sugar fermentation ability) was evaluated using API50CHL (manufactured by BioMérieux Japan Co., Ltd.). The results are shown in Table 2. Furthermore, Table 1 is as described in the previous paragraph [0015], and Table 2 is as described in the previous paragraph [0016].

From these results, it was confirmed that the obtained strain had the same mycological properties and sugar assimilation properties as the milk fat FC strain (hereinafter, also simply referred to as "parent FC strain") used as a parent strain. Therefore, the strain was identified as a strain belonging to Lactococcus lactis subsp. cremoris, and named Lactococcus lactis subsp. cremoris FC46 (hereinafter referred to as "cremoris"). FC46 strain", or simply referred to as "FC46 strain"). In addition, the milk fat FC46 strain is internationally deposited at the Licensed Microorganism Deposit Center of the Independent Administrative Legal Person Product Evaluation Technology Base Organization, whose address is No. 122, Kamiso Kamazu 2-5-8, Kisarazu City, Chiba Prefecture, Japan, and the date is 2020 On November 6, identification mark: Lactococcus lactis subsp. cremoris FC46, deposit number: NITE BP-03310 (date of issue of deposit certificate: November 30, 2020).

Example 2 Evaluation of acid resistance The FC46 strain obtained above was inoculated at a ratio of 4% in TY-Glu medium (1% glucose, 0.5% Bacto (TM) trypsin, 0.5% Bacto (TM) yeast extract, 0.5% NaCl) (pH7.0 ), and cultured at 25°C for 15 hours to prepare a pre-culture solution. The obtained previous culture solution was inoculated into 40 ml of TY-Glu medium adjusted to pH 4.0 with lactic acid, so that the final concentration was 1%. It was cultured statically at 26°C for 27 hours, and the number of FC46 strains in the culture medium (cfu/ ml), and calculate the survival rate (%) after 27 hours. As a comparative test, the pro-FC strain was used to replace the above-mentioned FC46 strain for the same culture, and the number of bacteria (cfu/ml) of the pro-FC strain in the culture medium was measured over time, and the survival rate after 27 hours was calculated ( %).

Figure 1 shows the results of time-series measurement of the bacterial count (cfu/ml) of each milk fat strain (FC46 strain, FC-friendly strain). As shown in Figure 1, the FC-friendly strain died rapidly under the low pH condition of pH 4.0, and the number of viable bacteria decreased to 6.6×10 ³ (cfu/ml) after 27 hours of culture (survival rate 0.2%). In contrast, the FC46 strain has high viability even at a low pH of pH 4.0, and maintains 1.9×10 ⁶ (cfu/ml) even after 27 hours of culture at this pH (survival rate 33.6%) The high bacterial count. Accordingly, it was confirmed that the FC46 strain is a milk fat strain having excellent acid resistance compared with the parent FC strain.

Example 3 Stability Evaluation in Fermented Milk FC46 strain was used to prepare fermented milk, and its stability in fermented milk was evaluated. In addition, as a comparative control, fermented milk was prepared in the same manner using the parent FC strain, and its stability in fermented milk was evaluated.

1 Preparation of fermented milk Four types of fermented milk 1-4 were prepared using the activated bacteria of the following strains as bacteria. (1) Fermented milk 1: pro-FC strain (bacteria 1) (2) Fermented milk 2: FC46 strain (bacteria 3) (3) Fermented milk 3: pro-FC strain (bacteria 1) + thermophilic bacteria (bacteria 2) (4) Fermented milk 4: FC46 strain (bacteria 3) + thermophilic bacteria (bacteria 2) Furthermore, bacteria 1 and 3 were prepared by inoculating (1%) the parent FC strain and FC46 strain into sterilized 8% skim milk powder, and placing them at 30°C for about 10 hours to activate them. In addition, Bacteria 2 is prepared by inoculating (1%) thermophilic bacteria into sterilized 8% skim milk powder, and placing it at the optimum temperature (37°C) for about 10 hours to activate it.

Inoculate the sterilized milk with the bacteria prepared above, and ferment at 30°C. At the point when curd formation was observed, cooling (4°C) was started to suppress fermentation to obtain fermented milk.

2 Stability evaluation of bacteria in fermented milk After preparing the fermented milk 1~4, it should be stored at 10°C in a dark place for 30 days. Measure the number of bacteria (cfu/ml) in fermented milk after preservation (fermented milk 1 and 2 are the number of bacteria of milk fat bacteria, fermented milk 3 and 4 are the number of bacteria of milk fat bacteria and thermophilic bacteria). Furthermore, the number of milk fat bacteria is determined by the mixed dilution culture method (25°C) using TY-Gal medium (pH7.0); the number of bacteria of thermophilic bacteria is determined by BCP plate count agar medium (pH7. 0) by the mixed dilution culture method (37°C) to determine.

In addition, pH, acidity, and viscosity in 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.

[Determination of acidity] Finely weigh 10g of fermented product, add the same amount of distilled water, dilute, add 0.5ml of phenolphthalein solution as indicator, titrate with 0.1mol/L sodium hydroxide aqueous solution and take the point where the light red does not disappear within 30 seconds as the end point, from From this titration, the percentage of lactic acid per 100 g of the sample was obtained as the acidity. Furthermore, 1 ml of 0.1 mol/L sodium hydroxide aqueous solution is equivalent to 9 mg of lactic acid.

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

［表4］

如表3及表4所示，親FC株在保存期間中急速死滅，在30天後每1mL僅殘留數個生菌（發酵乳1）。另一方面，FC46株在30天後仍維持1.1×10 ⁴（cfu/ml）（存活率0.0026%）之高生菌數（發酵乳2）。此外，親FC株及FC46株均藉由與嗜熱菌共發酵，而維持較單獨發酵高之生菌數（發酵乳3及4），尤其是FC46株顯示出5.8×10 ⁶（cfu/ml）（存活率2.8%）之非常高的生菌數（發酵乳4）。 Table 3 shows the results on the first day from the storage, and Table 4 shows the results on the 30th day from the storage. [table 3]

[Table 4]

As shown in Table 3 and Table 4, the FC-friendly strain died rapidly during storage, and only a few live bacteria (fermented milk 1) remained per 1 mL after 30 days. On the other hand, the FC46 strain still maintained a high bacterial count of 1.1×10 ⁴ (cfu/ml) (survival rate 0.0026%) after 30 days (fermented milk 2). In addition, the parent FC strain and the FC46 strain both maintained a higher number of bacteria (fermented milk 3 and 4) by co-fermenting with thermophilic bacteria (fermented milk 3 and 4), especially the FC46 strain showed 5.8×10 ⁶ (cfu/ml) ( Survival rate 2.8%) very high bacterial count (fermented milk 4).

Furthermore, although it was determined that the viscosity of the FC-friendly strain decreased due to 30-day storage, the FC46 strain inhibited the decrease in viscosity compared with the FC-friendly strain, and the same tendency was also obtained in the case of co-fermentation with thermophilic bacteria. Furthermore, the fermented milks 1-4 produced in this example were all good in appearance (color, water-free), aroma, and taste, and there was no difference in these points.

Embodiment 4 The preparation and stability evaluation of the composition containing lactic acid bacteria The lactic acid bacteria-containing composition was prepared using FC46 strain, and the storage stability in a dry state was evaluated. Furthermore, for comparison, the parent FC strain was used instead of the FC46 strain to prepare a lactic acid bacteria-containing composition, and the storage stability in a dry state was similarly evaluated.

1 Preparation of lactic acid bacteria solution The FC46 strain or the FC-friendly strain was inoculated at a ratio of 4% to Difco M17 medium (BD) to which glucose had been added so that the final concentration was 0.5%, and cultured at 25°C for 16 hours to prepare a pre-culture solution. In the same medium, inoculate 4% of the culture solution before the above-mentioned preparation, and culture at 25° C. for 16 hours to obtain 10 L of culture solution. By centrifuging it (3,000rpm×10 minutes), decanting to remove the obtained supernatant, and washing the residue twice with normal saline to prepare concentrated lactic acid bacteria solutions of FC46 strain or FC-friendly strain.

Add skimmed milk powder at a ratio of 10% to the prepared lactic acid bacteria, and further use a freeze dryer (FD-81, Tokyo Rikaki Co., Ltd.) to freeze-dry according to a known method to obtain about 50 g of freeze-dried lactic acid bacteria ( FC46 strain, pro-FC strain).

Put 0.5 g of the obtained freeze-dried lactic acid bacteria (FC46 strain, pro-FC strain) into a sterilized laminated film bag (LAMIZIP (ラミジップ) [registered trademark]: (stock) ) produced by a Japanese company), and stored in a dark place at 37°C (forced deterioration test). From the beginning of the test (the first day), the number of FC46 strains or parent FC strains in the freeze-dried lactic acid bacteria was measured sequentially, and the survival rate (%) was calculated.

The results are shown in Table 5. [table 5]

The parent FC strain died rapidly under the temperature condition of 37°C, and the number of viable bacteria after 3 weeks was 3.2×10 ⁹ (cfu/g) (survival rate: about 1.6%), compared with FC46 strain which showed 1.1 Very high bacterial count of ×10 ¹⁰ (cfu/g) (survival rate: about 14.7%). Therefore, it was confirmed that the FC strain is superior in storage stability compared to the pro-FC strain even in a dried powder state under aerobic conditions. [Sequence Listing]

SEQ ID NO: 1 represents the base sequence of the 16S rRNA gene of the milk fat FC strain.

[ Fig. 1 ] shows the results of time-series measurement of the bacterial count (cfu/ml) of each milk fat FC strain (FC46 strain, parent FC strain) in the acidic medium in Example 2.

Overseas Deposit Information: JP Japan, Independent Administrative Legal Person Product Evaluation Technology Base Organization, Licensed Microbiology Center, November 30, 2020, NITE BP-03310

Claims (10)

A lactic acid bacterium, which is Lactococcus lactis subsp. cremoris FC46 strain (registration number: NITE BP-03310), or its descendant strain, its characteristic is The subcultured strain has the same mycological properties, morphological characteristics, physiological characteristics, carbon source assimilation, mucopolysaccharide production, acid resistance, and survival under dry conditions and aerobic conditions as the FC46 strain, and belongs to lactic acid Lactococcus subsp. cremoris. The lactic acid bacteria as described in Claim 1, wherein it is in a dry state. A fermenting seed bacterium characterized by containing the lactic acid bacteria described in claim 1 or 2. The inoculum for fermentation according to Claim 3, further comprising Streptococcus thermophilus. The inoculum for fermentation as described in Claim 4, wherein it is Lactococcus lactis subsp. cremoris FC46 strain (registration number: NITE BP-03310), or its substrains, and the symbiosis of Streptococcus thermophilus (Streptococcus thermophilus) body. The seed bacteria for fermentation as claimed in claim 4, wherein it is in a dry state. The seed bacteria for fermentation as claimed in claim 5, wherein it is in a dry state. An oral composition characterized in that it contains the lactic acid bacteria described in claim 1 or 2. The oral composition according to Claim 8, which is a fermented food. A method for producing a fermented food, characterized in that it comprises the steps of inoculating the lactic acid bacteria described in claim 1 or 2, or the fermented seed bacteria described in any one of claims 3 to 7, on fermented food raw materials to ferment step.

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