New lactic acid bacteria strains and compositions containing the same

Abstract

The present invention provides a new lactic acid bacteria strain, in particular a new FC lactic acid bacteria strain, and its application. The lactic acid bacteria strain is Lactococcus lactis subsp. cremoris FC46 strain (registration number: NITE BP-03310) or a subculture strain thereof.

Description

Technical Field

[0001] The present invention relates to a novel lactic acid bacteria strain and to various applications of the lactic acid bacteria strain, such as a composition (starter and oral composition) comprising the lactic acid bacteria strain, and a method for producing a fermented food or drink using the lactic acid bacteria strain. Background Art

[0002] In recent years, with the growth of consumers' health awareness, the demand for foods and beverages (drinks) containing lactic acid bacteria has been increasing, and lactic acid bacteria are widely used in the production of fermented milk, lactic acid bacteria beverages (lactic acid bacteria beverages), etc. Lactococcus lactis subsp. cremoris is a lactic acid bacterium that has long been used as a bacterium for producing fermented dairy products (such as yogurt and cheese). It is known that there are species of Lactococcus lactis subsp. cremoris that produce viscous polysaccharides. The yogurt (trade name: Caspian Sea Yogurt, registered trademark of the applicant, registration NO.5761425) obtained by fermentation using the Lactococcus lactis subsp. cremoris strain (hereinafter referred to as "Lactococcus lactis subsp. cremoris FC strain") isolated by the applicant from such a species as the main fermenting lactic acid bacteria has a unique viscosity that is completely different from conventional fermented milk. It has been reported that the Lactococcus lactis subsp. cremoris FC strain and the viscous polysaccharide produced by the Lactococcus lactis subsp. cremoris FC strain have various physiological effects, such as regulating intestinal function, inhibiting increase in blood sugar level, immune stimulation, preventing influenza virus infection, allergy suppression, and improving motility regulation (see Non-patent Literature (NPL) 1 to NPL 6).

[0003] Therefore, products (such as fermented milk, and fermentation starters and supplements) containing Lactococcus lactis cremoris FC strain have been developed in anticipation of the above-mentioned physiological effects, and continuous intake of Lactococcus lactis cremoris FC strain is required to fully exert such physiological effects in the intestine. However, lactic acid bacteria are facultative anaerobic bacteria, and their viability is known to be reduced under aerobic conditions or acidic (low pH) conditions, and Lactococcus lactis cremoris FC strain is no exception. For this reason, it is necessary to develop products containing Lactococcus lactis cremoris FC strain, which maintain useful physiological effects while suppressing the reduction in the number of viable cells caused during long-term storage in acidic foods and beverages (such as yogurt) and dry powders (such as lactic acid bacteria supplements).

[0004] Reference List

[0005] Non-patent literature

[0006] Non-Patent Document 1: T. Toda et al., “Effects of fermented milk with Lactococcus lactis subsp. cremoris FC on defecation frequency and fecal micr of lorain healthy elderly volunteers” J. Jpn. Soc. Food. Sci. 52(6), 243 - 250(2005)

[0007] Non-Patent Document 2: M. M0ri 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 cal orie”” Geriatrics. 141 - 152(2012)

[0008] 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)

[0009] Non-Patent Document 4: T. Maruo et al., “Oral administration of milk fermented with Lactococcus lactis subsp. cremoris FC protects mice against inf luenzavirus infection” Lett. Appl. Microbiol. 55(2), 135 - 140(2012)

[0010] Non-patent document 5: Y. Gotoh et al., "Effect of orally administered exopolyssccharides produced by Lactococcus lactis subsp.cremoris FC on a mo usemodel of dermatitis induced by repeated exposure to 2,4,6-trinitro-1-chlorobenzene" J.Funct.Foods.35, 43-50 (2017)

[0011] Non-patent document 6: Y. Gotoh et al., "Effect of yogurt fermented with Lactococcus lactis subsp.cremoris FC on salivary secretory IgA levels in highschool-student long-distance runners" Japanese J.Phys.Fit.Sports Med.68(6), 407-414(2019) Summary of the invention

[0012] Technical issues

[0013] In view of the above-mentioned problems of the prior art, an object of the present invention is to provide a novel Lactococcus lactis subsp. cremoris FC strain having improved acid tolerance (tolerance to low pH) and improved viability under aerobic conditions and dry conditions, in other words, a Lactococcus lactis subsp. cremoris FC strain having good viability in acidic foods and drinks (e.g., fermented milk) and in dry powder form. Another object of the present invention is to provide a starter for fermentation, an oral composition such as a supplement or a fermented food or drink, and a method for producing a fermented food or drink, each of which uses a Lactococcus lactis subsp. cremoris FC strain having excellent acid tolerance and excellent viability under aerobic conditions and dry conditions.

[0014] Solution to the problem

[0015] To achieve the above-mentioned object, the present inventors conducted extensive research and found a novel Lactococcus lactis subsp. cremoris FC strain having the same bacteriological characteristics as the Lactococcus lactis subsp. cremoris FC strain (Lactococcus lactis subsp. cremoris FC, FERM P-20185) owned by the applicant as a Lactococcus lactis subsp. cremoris strain, and having more excellent characteristics than the Lactococcus lactis subsp. cremoris FC strain (Lactococcus lactis subsp. cremoris FC, FERM P-20185) in terms of both acid tolerance and viability under aerobic conditions and dry conditions. The present invention was completed by conducting further research.

[0016] In the following, the known Lactococcus lactis subsp. cremoris FC strain is referred to as the “Lactococcus lactis subsp. cremoris FC strain”, the “existing Lactococcus lactis subsp. cremoris FC strain” or the “parent FC strain”, and the newly discovered Lactococcus lactis subsp. cremoris FC strain of the present invention is referred to as the “Lactococcus lactis subsp. cremoris FC strain of the present invention”, the “Lactococcus lactis subsp. cremoris FC46 strain” or the “FC46 strain” to distinguish it from the known Lactococcus lactis subsp. cremoris FC strain.

[0017] The present invention includes the following embodiments.

[0018] (I) Novel Lactococcus lactis subsp. cremoris (Lactococcus lactis subsp. cremoris FC strain of the present invention)

[0019] (I-1) The lactic acid bacteria are Lactococcus lactis subsp. cremoris FC46 (Registration No.: NITE BP-03310) or a subcultured strain thereof.

[0020] (I-2) The lactic acid bacteria according to (I-1), which are in a dry state.

[0021] (II) Starters for fermentation

[0022] (II-1) A starter for fermentation, comprising Lactococcus lactis subsp. cremoris FC46 (Registration No.: NITE BP-03310) or a subcultured strain thereof.

[0023] (II-2) The starter for fermentation according to (II-1), further comprising Streptococcus thermophilus.

[0024] (II-3) The starter for fermentation according to (II-2), which is a symbiotic organism of Lactococcus lactis subsp. cremoris FC46 (Registration No.: NITE BP-03310) or a subculture strain thereof and Streptococcus thermophilus.

[0025] (II-4) The starter for fermentation according to any one of (II-1) to (II-3), which is in a dry state.

[0026] (III) Oral Composition

[0027] (III-1) An oral composition comprising the lactic acid bacteria according to (I-1) or (I-2).

[0028] (III-2) The oral composition according to (III-1), which is a food or drink, an oral product, or an oral quasi-drug.

[0029] (III-3) The oral composition according to (III-1) or (III-2), which is a fermented food or drink.

[0030] (IV) Method for producing fermented food or beverage, and fermented food or beverage

[0031] (IV-1) A method for producing a fermented food or drink, comprising inoculating the lactic acid bacteria according to (I-1) or (I-2) or the starter for fermentation according to any one of (II-1) to (II-4) into the raw materials of the fermented food or drink, and fermenting the raw materials.

[0032] Advantageous Effects of the Invention

[0033] The Lactococcus lactis cremoris FC strain of the present invention has the same bacteriological characteristics and useful physiological effects as the existing Lactococcus lactis cremoris FC strain, and under low pH conditions of about pH 4, it exhibits higher viability, higher viscosity maintenance effect and more excellent acid tolerance than the existing Lactococcus lactis cremoris FC strain. The Lactococcus lactis cremoris FC strain of the present invention also exhibits higher viability under dry conditions and aerobic conditions, and is more tolerant to dry conditions and aerobic conditions than the existing Lactococcus lactis cremoris FC strain. Therefore, the Lactococcus lactis cremoris FC strain of the present invention has excellent stability in oral compositions such as acidic foods and drinks (such as yogurt and lactic acid bacteria drinks) and supplements in a dry state, and can maintain and exhibit its useful physiological effects for a long time in the form of an oral composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results of measuring the viable count (cfu / ml) of Lactococcus lactis subsp. cremoris FC strains (FC46 strain and parent FC strain) in an acidic medium over time in Example 2 are shown. DETAILED DESCRIPTION

[0035] (I) Novel Lactococcus lactis subsp. cremoris (Lactococcus lactis subsp. cremoris FC strain of the present invention)

[0036] The Lactococcus lactis cremoris FC strain of the present invention belongs to Lactococcus lactis cremoris and has the following bacteriological characteristics. Some Lactococcus lactis cremoris FC strains of the present invention were internationally deposited on November 6, 2020 with the identification "Lactococcus lactis cremoris FC46" under the registration number NITE BP-03310 at the National Institute of Technology and Evaluation (NITE, National Institute of Technology and Evaluation) Patent Microorganism Collection Center (Room 122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan).

[0037] (a) Bacteriological characteristics

[0038] When the Lactococcus lactis subsp. cremoris FC strain of the present invention is statically cultured under aerobic conditions using TY-Glu medium (1% glucose, 0.5% Bacto(TM) tryptone (Thermo Fisher Scientific), 0.5% Bacto(TM) yeast extract (ThermoFisher Scientific), 0.5% NaCl) (pH 7.0) or MRS medium (Difco), its bacteriological characteristics (morphological and physiological characteristics and carbon source assimilation characteristics) are shown in Tables 1 and 2 below.

[0039] Table 1

[0040] Cell shape cocci Gram stain + Mobility - Spore formation - Catalase activity - Grow at 10°C + Grow at 45°C - Gas produced from glucose - Optical activity of lactic acid L

[0041] Table 2

[0042]

[0043]

[0044] (b) Morphological characteristics

[0045] Cell morphology: (Streptococcus)

[0046] Colony shape: round (hemispherical), diameter 0.5 to 1.0 mm

[0047] Colony color: white

[0048] Spore formation: No

[0049] (c) Physiological characteristics

[0050] Gram stain: positive

[0051] Mobility: No

[0052] Catalase activity: No

[0053] Gas produced by glucose: None

[0054] Optical rotation of lactic acid: L

[0055] Growth at 10°C: viable

[0056] Growth at 45°C: Not viable

[0057] Optimum temperature: 25 to 30°C

[0058] Optimum pH: pH 6 to 7

[0059] Lactic acid fermentation: homogeneous

[0060] (d) Carbon source assimilation characteristics (sugar fermentation characteristics)

[0061] The Lactococcus lactis subsp. cremoris FC strain of the present invention assimilates the following carbon sources:

[0062] D-galactose, D-glucose, D-fructose, D-mannose, D-lactose, N-acetylglucosamine and esculin.

[0063] (e) Other characteristics that characterize microorganisms

[0064] According to KH Schleifer et al., "Transfer of Streptococcus lactis and Related Streptococci to the Genus Lactococcus gen.nov." System. Appl. Microbiol. 6, 183-195 (1985), based on the above characteristics and the results of homology search using 16S rRNA, the Lactococcus lactis subsp. cremoris FC strain of the present invention has 100% homology with the existing registered Lactococcus lactis subsp. cremoris FC strain (Lactococcus lactis subsp. cremoris FC, FERM P-20185) and is identified as a strain belonging to Lactococcus lactis subsp. cremoris. The base sequence of the 16S rRNA gene of the Lactococcus lactis subsp. cremoris FC strain of the present invention is shown as SEQ ID NO: 1 in the sequence table.

[0065] In addition to the above-mentioned bacteriological characteristics, the Lactococcus lactis subsp. cremoris FC strain of the present invention produces a variety of viscous polysaccharides like the existing Lactococcus lactis subsp. cremoris FC strain. Therefore, the Lactococcus lactis subsp. cremoris FC strain of the present invention is considered to have a variety of useful physiological effects, such as regulating intestinal function, inhibiting the increase of blood sugar level, immune stimulation, preventing influenza virus infection, allergic reaction inhibition, and improving motility regulation, like the existing Lactococcus lactis subsp. cremoris FC strain.

[0066] In addition, as shown in Examples 2 and 3 described later, under low pH conditions of about pH 4, the Lactococcus lactis subsp. cremoris FC strain of the present invention exhibits higher viability, higher viscosity maintenance effect, and more excellent acid tolerance than the existing Lactococcus lactis subsp. cremoris FC strain. Moreover, as shown in Example 4 described later, the Lactococcus lactis subsp. cremoris FC strain of the present invention exhibits higher viability under dry conditions and aerobic conditions than the existing Lactococcus lactis subsp. cremoris FC strain, and is more tolerant to dry conditions and aerobic conditions. Therefore, the Lactococcus lactis subsp. cremoris FC strain of the present invention has excellent stability in oral compositions such as acidic foods and beverages (such as yogurt and lactic acid bacteria beverages) and supplements in a dry state, and can maintain and exhibit its useful physiological effects for a long time in the form of an oral composition.

[0067] The Lactococcus lactis subsp. cremoris FC strain of the present invention includes not only the Lactococcus lactis subsp. cremoris FC strain (deposited strain) deposited internationally under the registration number NITE BP-03310 and the original strain thereof, but also subcultured strains thereof.

[0068] The subculture of the Lactococcus lactis cremoris FC strain of the present invention can be carried out according to the common method generally used for the subculture of lactic acid bacteria and is not particularly limited. Examples include a method in which the Lactococcus lactis cremoris FC strain of the present invention is inoculated into a test tube containing the above-mentioned TY-Glu medium (pH 7.0) and cultured at an optimal temperature of about 25 to 30°C for about 12 to 48 hours. Glycerol can be added to the subcultured Lactococcus lactis cremoris FC strain to a final concentration of 10w / v% to 30w / v%, and it can be stored at -80°C until use.

[0069] In addition to the above-mentioned TY-Glu culture medium, a common culture medium containing a carbon source, a nitrogen source, an inorganic ion and additional (if necessary) organic micronutrients can be used as a culture medium for the Lactococcus lactis subspecies FC strain of the present invention. The carbon source can be any carbon source containing an assimilable carbon source, and for example, sugars, such as D-glucose, D-galactose, D-fructose, D-mannose and D-lactose, can be preferably used. The nitrogen source can be any nitrogen source that is an assimilable nitrogen compound or contains an assimilable nitrogen compound, and for example, ammonium sulfate, casamino acids, peptone, etc. can be used. In addition, inorganic salts of phosphoric acid, iron, potassium, magnesium, zinc, manganese, copper, calcium, etc. can also be suitably used. In addition, amino acids, vitamins such as biotin, riboflavin, pyridoxine, nicotinic acid, pantothenic acid, folic acid and thiamine, etc., required for bacterial growth can be added to the culture medium if necessary.

[0070] The Lactococcus lactis subsp. cremoris FC strain of the present invention can be cultured at a temperature of 15° C. or higher, preferably about 25 to 30° C., and at a pH of 5 or higher, preferably about pH 6 to 7 to enhance proliferation. The Lactococcus lactis subsp. cremoris FC strain of the present invention can be statically cultured under aerobic conditions, but is preferably statically cultured under anaerobic conditions to enhance proliferation.

[0071] The Lactococcus lactis subsp. cremoris FC strain of the present invention, which shows high viability under dry and aerobic conditions as described above, can be prepared as a dry product by subjecting the strain to a drying treatment (e.g., vacuum drying or spray drying) after being separated from the culture medium, or by drying the strain together with other components (e.g., culture medium components and excipients).

[0072] (II) Starters for fermentation

[0073] The Lactococcus lactis subsp. cremoris FC strain of the present invention can be used as a starter (a starter for fermentation, which may be referred to as a "starter culture of lactic acid bacteria") for producing fermented foods and drinks by utilizing its fermentability.

[0074] Examples of fermented foods and drinks include fermented milk foods and drinks made mainly from milk (yogurt, lactic acid bacteria drinks, cheese, etc.), fermented soy milk foods or drinks made mainly from soybeans (yogurt, lactic acid bacteria drinks, etc.), fermented grain foods and drinks made mainly from grains (rice, wheat, etc.) (amazake (a sweet drink made from fermented rice), bread, etc.), and fermented vegetable and fruit foods and drinks (pickled vegetables, etc.).

[0075] The starter for fermentation of the present invention is preferably used for the fermentation of a raw material mainly composed of milk or soy milk, and a fermented milk food or drink or a fermented soy milk food or drink, such as yogurt or a lactic acid bacteria beverage, is produced by the fermentation of the raw material. The raw material mainly composed of milk or soy milk (milk raw material or soy milk raw material) can be any material containing protein derived from milk or beans (preferably soybeans), and can be, for example, milk or soy milk itself or a milk raw material or soy milk raw material from which lipids have been removed or reduced.

[0076] In this specification, " milk " used in the present invention includes animal milk, such as cow's milk, goat's milk, sheep's milk and horse's milk. The form of milk is not particularly limited and can be used in any form, including raw milk, concentrated whole milk, skim milk, concentrated whole milk powder or skim milk powder. " Soy milk " used in the present invention includes but is not limited to the soy milk obtained by grinding soybeans and water and then squeezing. " Soy milk " is an emulsified liquid (soybean emulsion) made from beans and water. The type of beans used as raw materials is not particularly limited, and any beans such as soybeans, adzuki beans, peas, broad beans, common beans and mung beans can be used.

[0077] The initiator for fermentation of the present invention is preferably inoculated so that the viable cell count of the Lactococcus lactis subsp. cremoris FC strain of the present invention in the production of fermented milk food or beverage or fermented soy milk food and beverage is about 1×10 5 Up to 1×10 9 (cfu / g). Therefore, for use, it is preferred that the Lactococcus lactis subsp. cremoris FC strain of the present invention is cultured in advance using, for example, a milk raw material or a soy milk raw material and prepared so that the viable cell count of the culture is about 1×10 7 Up to 1×10 9 (cfu / g) (a large number of primers). In particular, although not limited to, for example, the Lactococcus lactis cremoris FC strain of the present invention can be inoculated into a pre-sterilized fermentation raw material (milk raw material or soy milk raw material) or such a fermentation raw material containing yeast extract (for example, 5% to 10% skim milk powder), and cultured at an optimal temperature to achieve the above-mentioned bacterial concentration. The number of viable cells (colony count) of the Lactococcus lactis cremoris FC strain of the present invention can be determined by counting the colonies formed using a pour plate culture method (25°C) using a TY-Glu medium (pH 7.0). The cell count and turbidity are correlated with each other. Therefore, if the correlation between the cell count and turbidity (absorbance at a wavelength of 600nm) is determined in advance, the cell count can be calculated by measuring the turbidity.

[0078] The starter for fermentation of the present invention may consist of only the Lactococcus lactis subsp. cremoris FC strain of the present invention, or may contain a combination of the Lactococcus lactis subsp. cremoris FC strain of the present invention and one or more other fermentable microorganisms, as long as the effects of the present invention are not impaired. Examples of other microorganisms include, but are not limited to, lactic acid bacteria belonging to the genus Lactococcus, such as Lactococcus lactis subsp. lactis biovar. diacetylactis; lactic acid bacteria belonging to the genus Streptococcus, such as Streptococcus thermophilus; and lactic acid bacteria belonging to the genus Lactobacillus, such as Lactobacillus casei subsp. casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus gasseri, and Lactobacillus acidophilus. One of these lactic acid bacteria may be used, or two or more of these lactic acid bacteria may be used in any combination.

[0079] The microorganism used in combination with the Lactococcus lactis cremoris FC strain of the present invention is preferably Streptococcus thermophilus (hereinafter referred to as "Streptococcus thermophilus (S.thermophilus)"). As shown in Example 3 described later, under low pH conditions, compared with the fermentation using the Lactococcus lactis cremoris FC strain of the present invention alone, the fermentation using the Lactococcus lactis cremoris FC strain of the present invention and Streptococcus thermophilus maintained a high viable count (improved viability) and suppressed the decrease in viscosity after storage. In the starter for fermentation of the present invention comprising the combined Lactococcus lactis cremoris FC strain of the present invention and Streptococcus thermophilus (a symbiont of the Lactococcus lactis cremoris FC strain of the present invention and Streptococcus thermophilus), the ratio of the number of viable cells between the two is not limited. For example, the ratio of the number of viable cells is such that the Lactococcus lactis cremoris FC strain of the present invention: Streptococcus thermophilus is 1:0.001 to 1000, preferably 1:0.01 to 100, and more preferably 1:0.1 to 10.

[0080] The number of viable cells of Streptococcus thermophilus can be determined by colony counting using a pour plate culture method (37° C.) using a plate count agar medium containing BCP (Nissui Pharmaceutical Co., Ltd.).

[0081] Acetic acid bacteria can also be used as microorganisms used in combination with the Lactococcus lactis subsp. cremoris FC strain of the present invention. Examples of acetic acid bacteria include, but are not limited to, acetic acid bacteria belonging to the genus Acetobacter, genus Gluconobacter, genus Gluconacetobacter, and the like.

[0082] The starter for fermentation of the present invention may be in any form as long as it exhibits fermentability when used to produce fermented food and beverages. In terms of hygiene, quality maintenance, ease of handling, etc., the starter for fermentation of the present invention is preferably in a dry solid form, more preferably in a dry powder form or a dry granular form. The starter for fermentation of the present invention may also be prepared into a dry product by drying the starter together with other components (e.g., culture medium components or excipients).

[0083] The fermentation conditions (culture conditions) using the starter for fermentation of the present invention are, for example, the following: the culture temperature is 15° C. or higher, preferably about 25 to 30° C.; the culture pH is 5 or higher, preferably pH is about 6 to 7; and the culture time is 3 hours or more, preferably 8 to 24 hours.

[0084] (III) Oral Composition

[0085] The oral composition of the present invention is characterized in that it contains the Lactococcus lactis subsp. cremoris FC strain of the present invention. The oral composition in the form of food or drink is prepared in the same manner as usual food and drink by using a suitable edible carrier (food or drink material). The oral composition is also prepared in a pharmaceutical form (supplement form, drug form or quasi-drug form) by using a suitable formulation raw material (e.g., a pharmaceutically acceptable excipient or diluent).

[0086] Like the existing Lactococcus lactis cremoris FC strain, when orally ingested, the Lactococcus lactis cremoris FC strain of the present invention is expected to exert a variety of physiological effects in the body (regulating intestinal function, inhibiting increased blood sugar levels, immune stimulation, preventing influenza virus infection, allergic reaction inhibition, improving motility regulation, etc.). This effect is believed to be the effect of the Lactococcus lactis cremoris FC strain itself of the present invention and / or the viscous polysaccharide produced by the Lactococcus lactis cremoris FC strain of the present invention. Therefore, the oral composition of the present invention preferably contains the Lactococcus lactis cremoris FC strain of the present invention in a living state. It is desirable that the oral composition of the present invention contains a large amount of the Lactococcus lactis cremoris FC strain of the present invention as a live bacterium, but this does not exclude the inclusion of the Lactococcus lactis cremoris FC strain of the present invention in a dead state.

[0087] As long as the oral composition of the present invention contains the Lactococcus lactis cremoris FC strain of the present invention, the oral composition may be a culture of the Lactococcus lactis cremoris FC strain of the present invention, a crude product or a purified product of the culture, or a freeze-dried product or a spray-dried product thereof. The culture is not limited and can be obtained, for example, by culturing for about 12 to 48 hours at an optimal temperature of about 25 to 30° C. using a culture medium suitable for the Lactococcus lactis cremoris FC strain of the present invention (e.g., TY-Glu culture medium, pH 7.0). The cells of the Lactococcus lactis cremoris FC strain of the present invention can be obtained by centrifuging the culture, for example, at 3,000 rpm for about 10 minutes after culturing to collect the cells. These can also be purified according to ordinary methods. In addition, the cells can be freeze-dried or spray-dried. The dried cells thus obtained can also be used as an active ingredient in the oral composition of the present invention. In the present specification, the Lactococcus lactis subsp. cremoris FC strain of the present invention, the culture thereof, the crude product or purified product of the culture, and the dried product thereof are collectively referred to as “the Lactococcus lactis subsp. cremoris FC strain of the present invention and the culture thereof”.

[0088] The oral composition of the present invention may further contain an appropriate amount of nutritional components, if necessary, which are suitable for the maintenance and proliferation of the Lactococcus lactis subsp. cremoris FC strain of the present invention. Specific examples of nutritional components include those used in the culture medium for culturing microorganisms, such as carbon sources such as glucose, galactose, fructose, lactose and mannose; nitrogen sources such as yeast extract and peptone; vitamins; minerals; trace metal elements; and other nutritional components.

[0089] The oral composition of the present invention can be formulated into the form of food or drink, pharmaceutical product and quasi-drug according to common methods. The carrier used in producing these forms can be an edible carrier or a pharmaceutically acceptable carrier, such as an excipient and a diluent. The details of producing the food or drink form and the details of the edible carrier used in producing the food or drink form are described later in the following "Oral Composition in Food or Drink Form" section.

[0090] The amount of the Lactococcus lactis subsp. cremoris FC strain of the present invention contained in the oral composition of the present invention can generally be selected so that the viable cell count of the Lactococcus lactis subsp. cremoris FC strain of the present invention in the oral composition of the present invention is about 1×10 7 Up to 1×10 11 (cfu). The amount of the Lactococcus lactis subsp. cremoris FC strain of the present invention can be appropriately changed depending on the form of the oral composition of the present invention to be prepared, the desired effect, etc., using the above-mentioned amount as a guide.

[0091] Oral composition in the form of food or drink

[0092] The oral composition of the present invention in the form of food or drink is ingested as food or drink. Preferred specific examples include, but are not limited to, fermented milk foods and drinks produced using milk as raw materials, such as fermented milk (e.g., yogurt) and lactic acid bacteria beverages; and fermented soy milk foods and drinks produced using soybeans as raw materials, such as soy yogurt and fermented soy milk beverages. Examples also include food and drinks in the form of medicines containing the Lactococcus lactis subspecies FC strain of the present invention, such as solid preparations (e.g., tablets, pills, powders, granules, microcapsules and capsules) and liquid preparations (e.g., fluids, suspensions, syrups and emulsions). Examples also include those containing the Lactococcus lactis subspecies FC strain of the present invention, such as sweets, such as chewing gum, caramel, soft candy, nougat and chocolate; fermented foods and drinks, such as fermented vegetable beverages and fermented fruit beverages; dairy products other than the above-mentioned fermented foods and drinks, such as pudding and bavarois, etc. The terms "fermented milk" and "lactic acid bacteria beverage" used in this specification conform to the definitions in Article 2-40 "Fermented milk" and Article 2-41 "Lactic acid bacteria beverage" in the "Ministerial Regulations on the Composition Standards of Milk and Dairy Products, etc." of the former Japanese Ministry of Health and Welfare. That is, "fermented milk" refers to a paste or liquid product prepared by fermenting milk or dairy products with lactic acid bacteria. Therefore, "fermented milk" includes not only products in the form of beverages, but also products in the form of yogurt. "Lactic acid bacteria beverage" refers to a beverage made by using a paste or liquid product as the main raw material, and the paste or liquid product is prepared by fermenting milk or dairy products with lactic acid bacteria and diluting with water.

[0093] (IV) Method for producing fermented food or beverage, and fermented food or beverage

[0094] The method for producing the fermented food or drink of the present invention is characterized in that the fermented food or drink comprises the Lactococcus lactis cremoris FC strain of the present invention. The fermented food or drink can be produced according to a common method. For example, the fermented food or drink can be produced by inoculating the Lactococcus lactis cremoris FC strain of the present invention into a suitable raw material for fermentation (e.g., a raw material comprising milk, soy milk (soy emulsion), grains, vegetables or fruits) containing a nutrient source of the Lactococcus lactis cremoris FC strain of the present invention and culturing it to cause the raw material to ferment.

[0095] A preferred method of using the Lactococcus lactis subsp. cremoris FC strain of the present invention for fermentation is to prepare a starter in advance and inoculate it into a raw material for fermentation, and ferment the raw material. The starter may be, for example, the starter for fermentation of the present invention described above. The starter preferably contains the Lactococcus lactis subsp. cremoris FC strain of the present invention so that the viable cell count of the Lactococcus lactis subsp. cremoris FC strain of the present invention is about 1×10 7 Up to 1×109 (cfu / ml).

[0096] The raw materials used for fermentation may optionally be supplemented with fermentation-promoting substances for good growth of the Lactococcus lactis subsp. cremoris FC strain of the present invention (e.g., carbon sources such as glucose, galactose, fructose, lactose, and mannose; nitrogen sources such as yeast extract and peptone; vitamins; minerals; trace metal elements; and other nutritional components).

[0097] For example, when milk is used as a raw material for fermentation, the amount of the Lactococcus lactis subsp. cremoris FC strain of the present invention to be inoculated is selected so that the amount of the Lactococcus lactis subsp. cremoris FC strain of the present invention contained in the milk is 1×10 5 (cfu / ml) or more (preferably about 1×10 7 Up to 1×10 9 (cfu / ml)) is suitable. As for the culture conditions, the fermentation temperature is usually selected from the range of 15°C or higher, preferably about 25 to 30°C and the fermentation time is usually selected from the range of about 3 to 24 hours.

[0098] The fermentation product thus obtained can be a curd form (yogurt sample or pudding sample form), and such a product can be directly taken in as solid food. The fermentation product in the curd form can be further homogenized to be prepared into the desired beverage form. This homogenization can be carried out using a common emulsifier (homogenizer). By such homogenization, a beverage with a pure and sense can be obtained. In this homogenization process, if necessary, suitable dilution can also be carried out, and organic acid is added to regulate pH, or multiple additives commonly used in the production of the beverage are added, such as sugar, fruit juice, thickener, surfactant and flavoring agent. The beverage thus obtained can be aseptically poured into a suitable container to provide a finished product.

[0099] The intake (dosage) of the fermented food or drink (oral composition) is appropriately determined according to the age, sex, weight, severity of disease, etc. of the organism that ingests the composition, and is not particularly limited. The intake (dosage) of the oral composition is usually selected from the Lactococcus lactis subsp. cremoris FC strain of the present invention in an amount of about 1×10 7 Up to 1×10 11 The oral composition can be prepared so that the composition is usually ingested in an amount of about 50 to 1,000 mL per person per day.

[0100] Specific preferred examples of the oral composition of the present invention in the form of food or drink include food and drink in the form of supplements, particularly food and drink in the form of medicines, such as solid preparations such as tablets, pills, powders, granules, microcapsules and capsules; and liquid preparations such as fluids, suspensions, syrups and emulsions. Food or drink in the form of medicines is preferably a dry solid preparation.

[0101] Food or drink in the form of a supplement can be produced by using a diluent or excipient generally used in the art as a pharmaceutical carrier, such as a filler, a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, and a lubricant.

[0102] The amount of the lactic acid bacteria of the present invention contained in the oral composition in the form of food or drink can be appropriately selected from a wide range as long as it is sufficient to exert the effect of the lactic acid bacteria. Preferably, the lactic acid bacteria are generally contained in an amount of about 1×10 7 Up to 1×10 11 (cfu) / per ingested unit (dose).

[0103] The method for administering the composition is not particularly limited as long as the composition is orally ingested, and is determined depending on the form, age, sex and other conditions of the patient, severity of the disease and the like.

[0104] The intake amount of the composition is appropriately selected according to the method of use, the age, sex and other conditions of the patient. As described above, it is preferred that the Lactococcus lactis subsp. cremoris FC strain of the present invention is taken at a dosage of about 1×10 7 Up to 1×10 11 (cfu) / each intake (dose). The Lactococcus lactis subsp. cremoris FC strain of the present invention is used as an active ingredient, and its intake is preferably about 1×10 7 (cfu) or more / day, and multiple servings may be taken in one day. It is preferred to take the oral composition of the present invention continuously to effectively obtain its effect. For example, the oral composition of the present invention may be taken continuously or intermittently over a period of 2 weeks or more.

[0105] Like the existing Lactococcus lactis cremoris FC strain, when the oral composition containing the Lactococcus lactis cremoris FC strain of the present invention in a live state is ingested (administered), it is expected to play a useful physiological role in the body. Therefore, the oral composition of the present invention can be effectively used as a health enhancer based on its physiological effect. Therefore, the oral composition of the present invention includes foods and drinks that have specific functions and are ingested for health maintenance, etc. In particular, the oral composition of the present invention includes such foods and drinks, which have, for example, effects of regulating intestinal function, effects of inhibiting the increase in blood sugar levels, immunostimulatory effects, effects of preventing influenza virus infection, allergic reaction inhibition effects, and / or effects of improving motility regulation. These foods and drinks include, for example, foods and drinks produced and sold together with labels or declarations of the above-mentioned effects or uses, such as foods with health claims (foods with nutritional function claims, foods with functional claims, and foods for specific health uses), foods for special dietary uses (foods for specific health uses) and similar health foods.

[0106] In the present specification, the terms "comprising" and "including" encompass the meanings "consisting essentially of" and "consisting of.

[0107] Example

[0108] In order to promote the understanding of the structure and function of the present invention, the present invention is described below with reference to examples. However, the present invention is not limited to these examples. Unless otherwise stated, the following experiments are carried out under room temperature (25 ± 5 ° C) and atmospheric pressure conditions. In the following, unless otherwise stated, "%" means "mass %", and "parts" means "parts by mass".

[0109] Example 1: Isolation and identification of a novel Lactococcus lactis subsp. cremoris FC strain

[0110] Lactococcus lactis subsp. cremoris FC strain (FERM P-20185) was inoculated (1%) into a sterilized 8% skim milk powder liquid and placed at its optimal temperature (25°C) for about 10 hours to activate the Lactococcus lactis subsp. cremoris FC strain (Introducer 1). Streptococcus thermophilus (hereinafter referred to as "Thermophilus (S. thermophilus)") (1%) was inoculated into a sterilized 8% skim milk powder liquid and placed at its optimal temperature (37°C) for about 10 hours to activate the thermophilus (Introducer 2). Subsequently, the above-prepared primers 1 and 2 were inoculated into separately prepared sterilized milk and fermented at 30°C. When curd formation was observed, cooling (4°C) was started.

[0111] The obtained fermented milk was stored at 4°C in the dark for 46 days, inoculated into 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) at a ratio of 4%, and then cultured 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 grown colonies were each cultured in a sterilized 8% skim milk powder liquid and stored.

[0112] Among them, a strain with high tolerance to low pH (acid tolerance) screened by the method described in Example 1 was obtained, and its bacteriological characteristics were evaluated according to ordinary methods. Table 1 shows the results. In addition, the ability of the strain to assimilate 49 carbon sources (sugar fermentation ability) was evaluated using API 50CHL (produced by bioMérieux Japan Ltd.). Table 2 shows the results. Table 1 is as described in paragraph

[0015] , and Table 2 is as described in paragraph

[0016] .

[0113] These results demonstrated that the obtained strain had the same bacteriological characteristics and sugar assimilation characteristics as the Lactococcus lactis cremoris FC strain (hereinafter referred to as the "parent FC strain") used as the parent strain. Therefore, the strain was identified as a strain belonging to Lactococcus lactis cremoris and named Lactococcus lactis cremoris FC46 strain (hereinafter referred to as "Lactococcus lactis cremoris FC46 strain" or simply "FC46 strain"). Lactococcus lactis cremoris FC46 strain was internationally deposited at the Patent Microorganism Depository of the National Institute for Technology Evaluation (Room 122, 2-5-8 Kazusakamatari, Kisarazu-shi, Chiba, Japan) on November 6, 2020 (date of issuance of the deposit certificate: November 30, 2020) with the identity mark "Lactococcus lactis cremoris FC46" under the registration number NITE BP-03310.

[0114] Example 2: Acid tolerance evaluation

[0115] The FC46 strain obtained above was inoculated into TY-Glu medium (1% glucose, 0.5% Bacto (TM) tryptone, 0.5% Bacto (TM) yeast extract, 0.5% NaCl) (pH 7.0) at a ratio of 4% and cultured at 25°C for 15 hours to prepare a pre-culture solution. The obtained pre-culture solution was inoculated into 40 mL of TY-Glu medium adjusted to pH 4.0 with lactic acid to reach a final concentration of 1%, and then statically cultured at 26°C for 27 hours. The viable count (cfu / ml) of the FC46 strain in the culture solution was measured over time using a plate count agar medium containing BCP (Nissui Pharmaceutical Co., Ltd.) by the pour plate culture method, and the survival rate after 27 hours was calculated. As a comparative test, the FC parent strain was used to replace the FC46 strain and cultured in the same manner, and the viable count (cfu / ml) of the parent FC strain in the culture solution was measured over time, and the survival rate (%) after 27 hours was calculated.

[0116] Figure 1 The results of viable count (cfu / ml) measurements of Lactococcus lactis subsp. cremoris strains (FC46 strain and parent FC strain) over time are shown. Figure 1 As shown, the parent FC strain was rapidly killed under low pH conditions of pH 4.0, and the number of viable bacteria decreased to 6.6 × 10 after 27 h of culture. 3 (cfu / ml) (survival rate: 0.2%). In contrast, the FC46 strain showed high viability even under low pH conditions of pH 4.0, and maintained 1.9×10 6 The results showed that the FC46 strain was a Lactococcus lactis subsp. cremoris strain with excellent acid tolerance compared with the parent FC strain.

[0117] Example 3: Evaluation of stability in fermented milk

[0118] Fermented milk was prepared using the FC46 strain, and its stability in the fermented milk was evaluated. As a control, fermented milk was prepared in the same manner using the parent FC strain, and its stability in the fermented milk was evaluated.

[0119] 1. Preparation of fermented milk

[0120] Four types of fermented milks (fermented milks 1 to 4) were prepared using the following activated bacterial strains as starters:

[0121] (1) Fermented milk 1: parent FC strain (introduction 1)

[0122] (2) Fermented milk 2: FC46 strain (Introduction 3)

[0123] (3) Fermented milk 3: parent FC strain (primer 1) + Streptococcus thermophilus (ST strain) (primer 2)

[0124] (4) Fermented milk 4: FC46 strain (primer 3) + Streptococcus thermophilus (primer 2).

[0125] Primer 1 was prepared by inoculating the parent FC strain (1%) into a sterilized 8% skim milk powder liquid and leaving it at 30°C for about 10 hours for activation. Primer 3 was prepared by inoculating FC46 strain (1%) into a sterilized 8% skim milk powder liquid and leaving it at 30°C for about 10 hours for activation. Primer 2 was prepared by inoculating Streptococcus thermophilus (1%) into a sterilized 8% skim milk powder liquid and leaving it at an optimal temperature (37°C) for about 10 hours for activation.

[0126] Each of the starters prepared above was inoculated into sterilized milk, and fermentation was performed at 30° C. When curd formation was observed, cooling (4° C.) was started to suppress fermentation, thereby obtaining fermented milk.

[0127] 2. Evaluation of bacterial stability in fermented milk

[0128] After preparing fermented milks 1 to 4, they were stored in the dark at 10°C for 30 days. After storage, the number of viable bacteria (cfu / ml) in the fermented milks was determined (the number of viable bacteria of Lactococcus lactis subsp. cremoris in fermented milk 1 and fermented milk 2, and the number of viable bacteria of Lactococcus lactis subsp. cremoris and the number of viable bacteria of Streptococcus thermophilus in fermented milk 3 and fermented milk 4). The number of viable bacteria of Lactococcus lactis subsp. cremoris was determined by pour plate culture method (25°C) using TY-Gal medium (pH 7.0), and the number of viable bacteria of Streptococcus thermophilus was determined by pour plate culture method (37°C) using plate count agar medium (pH 7.0) containing BCP.

[0129] The pH, acidity, and viscosity of each fermented milk were measured by the following method.

[0130] pH Measurement

[0131] After the fermentation product was adjusted to 10°C, the pH of each fermentation product was measured using an F-52 pH meter (manufactured by Horiba, Ltd.).

[0132] Acidity Measurement

[0133] Weigh 10 g of each fermentation product accurately and dilute with the same amount of distilled water. Add 0.5 ml of phenolphthalein solution as an indicator and titrate with 0.1 mol / L sodium hydroxide aqueous solution until a light pink color persists for 30 seconds. The percentage of lactic acid in each 100 g sample is determined as the acidity. Note that 1 mL of 0.1 mol / L sodium hydroxide aqueous solution is equivalent to 9 mg of lactic acid.

[0134] Viscosity measurement

[0135] After the fermentation product was adjusted to 10° C., the absolute viscosity of each fermentation product was measured using a B-type viscometer (manufactured by Brookfield, spindle No. 4). The rotor rotation time was 1 minute.

[0136] Table 3 shows the results on the 1st day after the start of storage, and Table 4 shows the results on the 30th day after the start of storage.

[0137] Table 3

[0138]

[0139] Table 4

[0140]

[0141]

[0142] As shown in Tables 3 and 4, the parent FC strain was killed rapidly during storage, and only a few viable cells / mL (fermented milk 1) remained after 30 days. In contrast, the FC46 strain maintained 1.1×10 4 When fermented with thermophilic Streptococcus, both the parent FC strain and the FC46 strain maintained a higher viable count than when fermented with the parent FC strain or the FC46 strain alone (fermented milk 3 and fermented milk 4), and in particular, the FC46 strain showed a very high viable count of 5.8×10 6 (cfu / ml) (survival rate: 2.8%) (fermented milk 4).

[0143] Furthermore, the parental FC strain showed a decrease in viscosity after 30 days of storage, whereas the decrease in viscosity was suppressed in the FC46 strain compared to the parental FC strain. The same trend was observed when fermentation was performed with S. thermophilus.

[0144] All of the fermented milks 1 to 4 produced in this Example had good appearance (color, presence or absence of syneresis), good smell, and good taste, and there were no differences in these aspects.

[0145] Example 4: Preparation of a composition containing lactic acid bacteria and evaluation of its stability

[0146] A composition containing lactic acid bacteria was prepared using the FC46 strain, and its storage stability was evaluated in a dry state.For comparison, a composition containing lactic acid bacteria was prepared using the parent FC strain instead of the FC46 strain, and its storage stability in a dry state was also evaluated.

[0147] 1. Preparation of lactic acid bacteria solution

[0148] The FC46 strain or the parent FC strain was inoculated into a Difco M17 medium (BD) supplemented with glucose at a ratio of 4% to achieve a final concentration of 0.5%, and cultured at 25°C for 16 hours to prepare a pre-culture solution. 4% of the pre-culture solution was inoculated into a culture medium of the same kind as the above culture medium, and cultured at 25°C for 16 hours to obtain 10L of culture solution. The culture solution was centrifuged (3,000rpm, 10 minutes), and the resulting supernatant was removed by decantation. Thereafter, the residue was washed twice with physiological saline to prepare a lactic acid bacteria liquid in which the FC46 strain or the parent FC strain was concentrated.

[0149] To each of the prepared lactic acid bacteria liquids, skim milk powder was added at a ratio of 10%, followed by freeze drying treatment using a freeze dryer (FD-81, Tokyo Rikakikai Co., Ltd.) according to a common method, thereby obtaining freeze-dried lactic acid bacteria (FC46 strain and parent FC strain, about 50 g each).

[0150] The obtained freeze-dried lactic acid bacteria (FC46 strain and parent FC strain, about 0.5 g each) were placed in a sterilized laminated film zipper bag (LAMIZIP (registered trademark); manufactured by Seisannipponsha Ltd.) and stored at 37° C. under dark conditions (accelerated deterioration test). The number of viable cells of the FC46 strain or the parent FC strain in the freeze-dried lactic acid bacteria was measured over time from the start of the test (day 1), and the survival rate (%) was calculated.

[0151] Table 5 shows the results.

[0152] Table 5 Survival rate after storage at 37°C (%)

[0153] Day 1 After 3 weeks After 4 weeks FC46 strain 100.0 14.67 6.03 Parental FC strain 100.0 1.58 0.07

[0154] The parental FC strain was rapidly killed during storage at 37°C, and the viable count of the parental FC strain after 3 weeks was 3.2 × 10 9 (cfu / g) (survival rate: about 1.6%). In contrast, the FC46 strain showed a very high viable count of 1.1×10 10(cfu / g) (survival rate: about 14.7%). These results confirmed that the FC46 strain in the dry powder form had excellent storage stability even under aerobic conditions compared to the parental FC strain.

[0155] Sequence Listing Free Text

[0156] SEQ ID NO: 1 shows the base sequence of 16S rRNA of the Lactococcus lactis subsp. cremoris FC strain of the present invention.

Claims

1. Lactic acid bacteria, which is Lactococcus lactis subsp. cremoris FC46 with registration number NITE BP-03310 or a subculture strain thereof, The subculture strain is a lactic acid bacterium that belongs to Lactococcus lactis subsp. cremoris and has the same bacteriological characteristics, morphological characteristics, physiological characteristics, carbon source assimilation characteristics, sticky polysaccharide production characteristics, acid tolerance, and viability under dry conditions and aerobic conditions as those of the Lactococcus lactis subsp. cremoris FC46. The lactic acid bacteria according to claim 1 , which are in a dry state.

3. A starter for fermentation, comprising the lactic acid bacteria according to claim 1 or 2.

4. The starter for fermentation according to claim 3, further comprising Streptococcus thermophilus.

5. The fermentation starter according to claim 4, which is a symbiotic product of Lactococcus lactis subsp. cremoris FC46 with registration number NITE BP-03310 or a subculture strain thereof and Streptococcus thermophilus.

6. The starter for fermentation according to claim 4 or 5, which is in a dry state.

7. An oral composition comprising the lactic acid bacteria according to claim 1 or 2. The oral composition according to claim 7 , which is a fermented food or drink.

9. A method for producing a fermented food or drink, comprising inoculating the lactic acid bacteria according to claim 1 or 2 or the starter for fermentation according to any one of claims 3 to 6 into a raw material of the fermented food or drink, and fermenting the raw material.

Images