CN117062905A - Novel lactic acid bacteria strain and composition comprising the same - Google Patents
Novel lactic acid bacteria strain and composition comprising the same Download PDFInfo
- Publication number
- CN117062905A CN117062905A CN202180095075.0A CN202180095075A CN117062905A CN 117062905 A CN117062905 A CN 117062905A CN 202180095075 A CN202180095075 A CN 202180095075A CN 117062905 A CN117062905 A CN 117062905A
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- strain
- milk
- lactococcus lactis
- lactic acid
- fermentation
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- 229910052749 magnesium Inorganic materials 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical class [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
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- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 235000015145 nougat Nutrition 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 239000013588 oral product Substances 0.000 description 1
- 229940023486 oral product Drugs 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 238000001139 pH measurement Methods 0.000 description 1
- 235000019161 pantothenic acid Nutrition 0.000 description 1
- 229940055726 pantothenic acid Drugs 0.000 description 1
- 239000011713 pantothenic acid Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 229940127557 pharmaceutical product Drugs 0.000 description 1
- KJFMBFZCATUALV-UHFFFAOYSA-N phenolphthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2C(=O)O1 KJFMBFZCATUALV-UHFFFAOYSA-N 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000011591 potassium Chemical class 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 235000008476 powdered milk Nutrition 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 235000011962 puddings Nutrition 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- 235000020185 raw untreated milk Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000020254 sheep milk Nutrition 0.000 description 1
- 235000021055 solid food Nutrition 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 1
- 229960003495 thiamine Drugs 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
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- 238000001291 vacuum drying Methods 0.000 description 1
- 229940011671 vitamin b6 Drugs 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000011701 zinc Chemical class 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Dairy Products (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present application provides novel lactic acid bacteria strains, in particular novel milk fat FC strains, and uses thereof. The lactobacillus strain is lactococcus lactis milk fat subspecies FC46 strain (registration number: NITE BP-03310) or a passaged strain thereof.
Description
Technical Field
The present application relates to novel lactic acid bacteria strains. The application also relates to various applications of the lactic acid bacteria strain, such as compositions (starter) and oral compositions comprising the lactic acid bacteria strain, and methods of using the lactic acid bacteria strain for producing fermented food or beverage products.
Background
In recent years, with the increase of consumer health consciousness, demands for foods and drinks (food-and-drink) containing lactic acid bacteria have been increasing, and lactic acid bacteria have been widely used for producing fermented milk, lactic acid bacteria beverages (lactic acid bacteria-and-drink) and the like. Lactococcus lactis subsp.cremoris (Lactococcus lactis subsp. Cremoris) is a lactic acid bacterium which has long been used as a bacterium for producing fermented dairy products, such as yogurt and cheese. The presence of lactococcus lactis subspecies creamer species that produce viscous polysaccharides is known. Yogurt (trade name: caspian Sea Yogurt, applicant registered trademark, registered No. 5761425) obtained by fermentation using a lactococcus lactis subsp.cremoris (l. Cremoris) strain isolated by the present inventors in such a species (hereinafter referred to as "lactococcus lactis subsp.m FC strain") as a main fermenting lactic acid bacterium has unique viscosity completely different from conventional fermented milk. Lactococcus lactis subsp.butter.fc strain and the viscous polysaccharide produced by the lactococcus lactis subsp.butter.fc strain have been reported to have various physiological effects such as regulation of intestinal functions, inhibition of blood glucose level elevation, immunostimulation, prevention of influenza virus infection, allergy inhibition, and improvement of motility regulation (see Non-patent documents (Non-patent Literature, NPL) 1 to NPL 6).
Thus, products comprising lactococcus lactis subspecies milk FC strains (e.g. fermented milk, as well as primers and supplements for fermentation) have been developed to expect the above physiological effects, and continuous ingestion of lactococcus lactis subspecies milk FC strains is required to fully exert such physiological effects in the intestinal tract. However, lactic acid bacteria are facultative anaerobes, which are known to reduce their viability under aerobic or acidic (low pH) conditions, and lactococcus lactis subspecies milk FC strains are no exception. For this reason, there is a need to develop products comprising a strain of lactococcus lactis, the subspecies milk, FC strain, which retain a useful physiological effect while inhibiting the reduction of viable count during long-term storage in acidic foods and drinks (e.g. yogurt) and dry powders (e.g. lactobacillus supplements).
List of references
Non-patent literature
Non-patent document 1: T.Toda et al, "Effects of fermented milk with Lactoco ccus lactis subsp.cremoris FC on defecation frequency and fecal micr of lora in healthy elderly volunteers" J.Jpn. Soc. Food. Sci.52 (6), 243-250 (2005)
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)
Non-patent document 3: kosaka et al, "Lactococcus lactis subsp.cremoris FC triggers IFN-. Gamma. production from NK and T cells via IL-12 and IL-18" int.Immunopharmacol.14 (4), 729-733 (2012)
Non-patent document 4: maruo et al, "Oral administration of milk fermented with Lactococcus lactis subsp. Cremoris FC protects mice against inf luenza virus infection" Lett. Appl. Microbiol.55 (2), 135-140 (2012)
Non-patent document 5: gotoh et al, "Effect of orally administered exopoly ssccharides produced by Lactococcus lactis subsp. Cremori FC on a mo use 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: gotoh et al, "Effect of yogurt fermented with Lacto coccus lactis subsp.cremori FC on salivary secretory IgA levels in high school-student long-distances runners" Japanese J.Phys.Fit.sports Med.68 (6), 407-414 (2019)
Disclosure of Invention
Technical problem
In view of the above problems of the prior art, it is an object of the present invention to provide novel lactococcus lactis subsp.cremoris FC strains with improved acid tolerance (tolerance to low pH) and improved viability under aerobic and dry conditions, in other words, in acidic foods and drinks (e.g. fermented milk) and in dry powder form. It is another object of the present invention to provide a starter for fermentation, an oral composition such as a supplement or a fermented food or beverage, and a method for producing a fermented food or beverage, each of which uses a lactococcus lactis subsp.
Solution to the problem
The present inventors have conducted extensive studies in order to achieve the above-mentioned object, and found that a novel lactococcus lactis subspecies FC strain having the same bacteriological characteristics as that of the lactococcus lactis subspecies FC strain (lactococcus lactis subsp FC, lactococcus lactis subsp. Cremoris FC, FERM P-20185) possessed by the applicant as a lactococcus lactis subspecies milk strain, and which has more excellent characteristics than the lactococcus lactis subspecies FC strain (lactococcus lactis subsp FC, FERM P-20185) in terms of both acid resistance and viability under aerobic conditions and drying conditions. The present invention has been completed by further studies.
Hereinafter, the known lactococcus lactis subspecies milk FC strain is referred to as "lactococcus lactis subspecies milk FC strain", "existing lactococcus lactis subspecies milk FC strain" or "parent FC strain", and the newly discovered lactococcus lactis subspecies milk FC strain of the present invention is referred to as "lactococcus lactis subspecies milk FC strain of the present invention", "lactococcus lactis subspecies milk FC46 strain" or "FC46 strain", to distinguish it from the known lactococcus lactis subspecies milk FC strain.
The invention includes the following embodiments.
(I) Novel lactococcus lactis subspecies cremoris (lactococcus lactis subspecies cremoris FC strain of the invention)
(I-1) lactic acid bacteria are lactococcus lactis subspecies milk fat FC46 (accession number: NITE BP-03310) or a subcultured strain thereof.
(I-2) the lactic acid bacterium according to (I-1), which is in a dry state.
(II) primers for fermentation
(II-1) a primer for fermentation comprising lactococcus lactis subspecies milk fat FC46 (accession number: NITE BP-03310) or a subcultured strain thereof.
(II-2) the primer for fermentation according to (II-1), which further comprises Streptococcus thermophilus (Streptococcus thermophilus).
(II-3) the primer for fermentation according to (II-2), which is lactococcus lactis subspecies milk fat FC46 (accession number: NITE BP-03310) or a co-occurrence of a subcultured strain thereof with Streptococcus thermophilus.
(II-4) the primer for fermentation according to any one of (II-1) to (II-3), which is in a dry state.
(III) oral compositions
(III-1) an oral composition comprising the lactic acid bacterium according to (I-1) or (I-2).
(III-2) the oral composition according to (III-1), which is a food or drink, an oral product, or an oral quasi drug.
(III-3) the oral composition according to (III-1) or (III-2), which is a fermented food or drink.
(IV) method for producing a fermented food or beverage, and fermented food or beverage
(IV-1) a method for producing a fermented food or drink, comprising inoculating the lactic acid bacterium according to (I-1) or (I-2) or the starter for fermentation according to any one of (II-1) to (II-4) into a raw material of a fermented food or drink, and subjecting the raw material to fermentation.
Advantageous effects of the invention
The lactococcus lactis subspecies milk fat FC strain of the invention has the same bacteriological characteristics and useful physiological effects as existing lactococcus lactis subspecies milk FC strains, and at low pH conditions of about pH4, it exhibits higher viability, higher viscosity maintenance and superior acid tolerance than existing lactococcus lactis subspecies milk FC strains. The lactococcus lactis subspecies milk FC strains of the invention also exhibit higher viability under dry and aerobic conditions and are more tolerant to dry and aerobic conditions than existing lactococcus lactis subspecies milk FC strains. Thus, the lactococcus lactis subspecies milk FC strain of the present invention has excellent stability in oral compositions such as acidic foods and drinks (e.g., yogurt and lactobacillus drinks) and supplements in a dry state, and can maintain and exhibit useful physiological effects thereof for a long period of time in the form of oral compositions.
Drawings
FIG. 1 shows the measurement results of the viable count (cfu/ml) of lactococcus lactis subspecies milk fat FC strains (FC 46 strain and parent FC strain) in acidic medium over time in example 2.
Detailed Description
(I) Novel lactococcus lactis subspecies cremoris (lactococcus lactis subspecies cremoris FC strain of the invention)
The lactococcus lactis subsp.cremoris FC strain of the invention belongs to the lactococcus lactis subsp.cremoris and has the following bacteriological characteristics. Some of the lactococcus lactis milk fat subspecies FC strains of the invention were identified as "lactococcus lactis milk fat subspecies FC46" at 11/6/2020 and were internationally deposited with the national institute of technology and evaluation (NITE, national Institute of Technology and Evaluation) at the patent microbiological deposit center (122 Chamber, 2-5-8Kazusakamatari, kisarazu-shi, chiba, japan).
(a) Bacteriological characteristics
When the lactococcus lactis subspecies milk fat FC strain of the present invention was subjected to stationary culture under aerobic conditions using TY-Glu medium (1% glucose, 0.5% Bacto (TM) tryptone (Thermo Fisher Scientific), 0.5% Bacto (TM) yeast extract (Thermo Fisher Scientific), 0.5% NaCl) (pH 7.0) or MRS medium (Difco), the bacteriological characteristics (morphological and physiological characteristics and carbon source assimilation characteristics) thereof are shown in tables 1 and 2 below.
TABLE 1
| Cell shape | Cocci (coccus) |
| Gram staining | + |
| Mobility of | - |
| Sporulation | - |
| Catalase Activity | - |
| Growing at 10 DEG C | + |
| Growing at 45 DEG C | - |
| Glucose-derived gas | - |
| Optical activity of lactic acid | L |
TABLE 2
(b) Morphological features
Cell morphology: pediococcus (streptococci)
Colony shape: round (hemispherical) with a diameter of 0.5 to 1.0mm
Colony color: white color
Sporulation: without any means for
(c) Physiological characteristics
Gram staining: positive and negative
Mobility: without any means for
Catalase activity: without any means for
Gas generated from glucose: without any means for
Optical activity of lactic acid: l (L)
Growing at 10 ℃): can survive
Growing at 45 ℃): is not viable
Optimum temperature: 25 to 30 DEG C
Optimum pH: pH 6 to 7
Lactic acid fermentation: homotypic type
(d) Carbon source assimilation character (sugar fermentation character)
The lactococcus lactis subspecies milk fat FC strain of the invention assimilates the following carbon sources:
d-galactose, D-glucose, D-fructose, D-mannose, D-lactose, N-acetylglucosamine and esculin.
(e) Characterization of other characteristics of microorganisms
Based on the above characteristics and the results of homology search using 16S rRNA, the lactococcus lactis subspecies milk fat FC strain of the present invention has 100% homology with existing registered lactococcus lactis subspecies milk FC strains (lactococcus lactis subspecies milk FC, FERM P-20185) and is identified as belonging to the lactococcus lactis subspecies milk, according 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). The nucleotide sequence of the 16S rRNA gene of the lactococcus lactis subspecies milk fat FC strain of the invention is shown in the sequence table as SEQ ID NO: 1.
In addition to the bacteriological characteristics described above, the lactococcus lactis subspecies milk FC strain of the present invention produces a variety of viscous polysaccharides like existing lactococcus lactis subspecies milk FC strains. Thus, the lactococcus lactis subspecies milk FC strain of the present invention is considered to have a variety of useful physiological effects like the existing lactococcus lactis subspecies milk FC strain, such as regulation of intestinal functions, inhibition of blood glucose level elevation, immunostimulation, prevention of influenza virus infection, allergy inhibition, and improvement of motility regulation.
Furthermore, as shown in examples 2 and 3 described later, the lactococcus lactis subspecies milk FC strain of the present invention exhibited higher viability, higher viscosity maintenance and more excellent acid tolerance than the existing lactococcus lactis subspecies milk FC strain under low pH conditions of about pH 4. Furthermore, as shown in example 4 described later, the lactococcus lactis subspecies milk FC strain of the present invention exhibited higher viability under dry conditions and aerobic conditions than the existing lactococcus lactis subspecies milk FC strain, and was more tolerant to the dry conditions and aerobic conditions. Thus, the lactococcus lactis subspecies milk fat FC strain of the present invention has excellent stability in oral compositions such as acidic foods and drinks (e.g., yogurt and lactobacillus beverages) and supplements in a dry state, and is capable of maintaining and exhibiting useful physiological effects thereof for a long period of time in the form of oral compositions.
The lactococcus lactis subspecies milk fat FC strain of the present invention includes not only the lactococcus lactis subspecies milk fat FC strain (deposited strain) and its original strain internationally deposited under accession number NITE BP-03310, but also its subcultured strain.
The subculture of the lactococcus lactis subspecies milk fat FC strain of the present invention can be carried out according to a common method commonly used for the subculture of lactic acid bacteria and is not particularly limited. Examples include a method in which the lactococcus lactis subspecies milk fat FC strain of the 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℃for about 12 to 48 hours. Glycerol may be added to the subcultured lactococcus lactis subsp.lactis FC strain to a final concentration of 10w/v% to 30w/v%, and may be stored at-80 ℃ until use.
In addition to the above TY-Glu medium, a common medium comprising a carbon source, a nitrogen source, inorganic ions and, if necessary, additional organic micronutrients may be used as a medium for the lactococcus lactis milk subspecies FC strain of the present invention. The carbon source may be any carbon source containing an assimilable carbon source, and for example, saccharides such as D-glucose, D-galactose, D-fructose, D-mannose, and D-lactose may be preferably used. The nitrogen source may be any nitrogen source which is or contains an assimilable nitrogen compound, and for example, ammonium sulfate, a casamino acid, peptone, or the like may be used. In addition, inorganic salts of phosphoric acid, iron, potassium, magnesium, zinc, manganese, copper, calcium, and the like can also be suitably used. Furthermore, amino acids, vitamins such as biotin, riboflavin, pyridoxine, niacin, pantothenic acid, folic acid, thiamine, and the like, which are necessary for bacterial growth, may be added to the medium as necessary.
The lactococcus lactis subspecies milk fat FC strain of the invention can be cultured at a temperature of 15 ℃ or higher, preferably about 25 to 30 ℃ and at a pH of 5 or higher, preferably about 6 to 7, to enhance proliferation. The lactococcus lactis subspecies milk fat FC strain of the invention may be grown under aerobic conditions, but is preferably grown statically under anaerobic conditions to enhance proliferation.
The lactococcus lactis subspecies milk fat FC strain of the invention, which exhibits high viability under the drying and aerobic conditions as described above, can be prepared as a dried product by subjecting the strain to a drying treatment (e.g. vacuum drying or spray drying) after separation from the culture medium, or by subjecting the strain to drying along with other components (e.g. culture medium components and excipients).
(II) primers for fermentation
The lactococcus lactis subspecies milk fat FC strain of the present invention can be used as a primer (a primer for fermentation, which may be referred to as a "primer culture for lactic acid bacteria") for producing fermented foods and drinks by utilizing its fermentability.
Examples of the fermented foods and drinks include fermented milk foods and drinks (yogurt, lactobacillus beverage, cheese, etc.) mainly made of milk, fermented soybean foods or drinks (yogurt, lactobacillus beverage, etc.) mainly made of soybean, fermented cereal foods and drinks (japanese sweet wine (a sweet beverage made of fermented rice), bread, etc.) mainly made of cereal (rice, wheat, etc.), and fermented vegetable and fruit foods and drinks (salted vegetables, etc.).
The starter for fermentation of the present invention is preferably used for fermentation of a raw material mainly composed of milk or soybean milk, and a fermented milk food or drink or a fermented soybean milk food or drink, such as yogurt or lactobacillus 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) may be any material containing proteins derived from milk or beans (preferably soy), and may 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.
In the present specification, "milk" as 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 may be used in any form, including raw milk, concentrated whole milk, skim milk, concentrated whole milk powder, or skim milk powder. "soy milk" as used in the present invention includes, but is not limited to, soy milk obtained by grinding soybeans and water, followed by pressing. "soy milk" is an emulsified liquid (soy emulsion) made from beans and water. The type of beans used as the raw material is not particularly limited, and any beans such as soybean, small bean, pea, broad bean, ordinary bean, and mung bean may be used.
The starter for fermentation of the present invention is preferably inoculated such that the viable count of the lactococcus lactis subspecies milk FC strain of the present invention in the production of fermented milk food or drink or fermented soybean milk food or drink is about 1X 10 5 Up to 1X 10 9 (cfu/g). Thus, for use, it is preferable that the lactococcus lactis subspecies milk FC strain of the present invention is previously cultured using, for example, a milk raw material or a soybean milk raw material and prepared so that the viable count of the culture is about 1X 10 7 Up to 1X 10 9 (cfu/g) (number of primers). In particular, although not limited, for example, the lactococcus lactis subspecies milk fat FC strain of the present invention can be inoculatedSeed into a pre-sterilized raw material for fermentation (milk raw material or soybean milk raw material) or such a raw material for fermentation containing a yeast extract (e.g., 5% to 10% skim milk powder) and cultured at an optimal temperature to achieve the above-mentioned bacterial concentration. The viable count (colony count) of the lactococcus lactis subspecies milk fat FC strain of the present invention can be determined by counting colonies formed by the pour plate method (25 ℃) using TY-Glu medium (pH 7.0). Cell count and turbidity correlate with each other. Thus, if the correlation between the cell count and turbidity (absorbance at a wavelength of 600 nm) is predetermined, the cell count can be calculated by measuring the turbidity.
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 comprise the lactococcus lactis subsp.cremoris FC strain of the present invention in combination with one or more other microorganisms having fermentability, as long as the effect of the present invention is not impaired. Examples of other microorganisms include, but are not limited to, lactic acid bacteria belonging to the genus Lactococcus (Lactococcus), such as Lactococcus lactis subsp.lactis and Lactococcus lactis subsp.diacetyl variant (Lactococcus lactis subsp. Lactis biovar. Diacetylactis); lactic acid bacteria belonging to the genus Streptococcus (Streptococcus), such as Streptococcus thermophilus; and lactic acid bacteria belonging to the genus Lactobacillus, such as Lactobacillus casei subspecies casei (Lactobacillus casei subsp. Casei), lactobacillus delbrueckii subsp. Bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricum), lactobacillus gasseri (Lactobacillus gasseri) and Lactobacillus acidophilus (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.
The microorganism used in combination with the lactococcus lactis subsp.cremoris FC strain of the present invention is preferably Streptococcus thermophilus (hereinafter referred to as "Streptococcus thermophilus"). As shown in example 3 described later, fermentation using the lactococcus lactis subspecies milk FC strain of the present invention with streptococcus thermophilus maintains a high viable count (improves viability) and suppresses a decrease in viscosity after storage, compared to fermentation using the lactococcus lactis subspecies milk FC strain of the present invention alone, under low pH conditions. In the primer for fermentation of the present invention comprising the lactococcus lactis subspecies milk fat FC strain of the present invention and streptococcus thermophilus (the symbiont of the lactococcus lactis subspecies milk fat FC strain of the present invention and streptococcus thermophilus) in combination, the viable count ratio between the two is not limited. For example, the viable count ratio is such that the lactococcus lactis subspecies milk fat FC strain of the invention, streptococcus thermophilus, is 1:0.001 to 1000, preferably 1:0.01 to 100, and more preferably 1:0.1 to 10.
The viable count of streptococcus thermophilus can be determined by colony counting using a pour plate method (37 ℃) using plate count agar medium (Nissui Pharmaceutical co., ltd.) containing BCP.
Acetic acid bacteria may also be used as microorganisms used in combination with the lactococcus lactis subsp. Examples of the acetic acid bacteria include, but are not limited to, acetic acid bacteria belonging to the genus Acetobacter (Acetobacter), the genus Gluconobacter (Gluconobacter), and the like.
The primer for fermentation of the present invention may be in any form as long as it exhibits fermentability when used for producing fermented foods and drinks. The primer 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, in terms of sanitation, quality maintenance, ease of handling, etc. The primers for fermentation of the present invention may also be prepared as a dried product by subjecting the primers to a drying treatment together with other components (e.g., medium components or excipients).
The fermentation conditions (culture conditions) using the primers for fermentation of the present invention are, for example, the following: the culture temperature is 15℃or higher, preferably about 25 to 30 ℃; the incubation pH is 5 or higher, preferably pH of about 6 to 7; and the incubation time is 3 hours or more, preferably 8 to 24 hours.
(III) oral compositions
The oral composition of the present invention is characterized by comprising the lactococcus lactis subsp. Oral compositions in the form of food or drink products are prepared in the same manner as usual food and drink products by using a suitable edible carrier (food or drink material). The oral compositions are also prepared in pharmaceutical form (supplement form, pharmaceutical form or quasi-pharmaceutical form) by using suitable formulation raw materials (e.g. pharmaceutically acceptable excipients or diluents).
Like the existing lactococcus lactis subspecies milk fat FC strain, the lactococcus lactis subspecies milk fat FC strain of the present invention is expected to exert various physiological effects in vivo (regulation of intestinal functions, inhibition of blood glucose level elevation, immunostimulation, prevention of influenza virus infection, allergy inhibition, improvement of motility regulation, etc.) when orally ingested. This effect is considered to be the effect of the lactococcus lactis subspecies creamer FC strain of the invention per se and/or of the viscous polysaccharide produced by the lactococcus lactis subspecies creamer FC strain of the invention. Thus, the oral composition of the invention preferably comprises the lactococcus lactis subsp. It is desirable that the oral composition of the invention comprises a large amount of the lactococcus lactis subsp. Cremoris FC strain of the invention as viable bacteria, but this does not exclude the inclusion of the lactococcus lactis subsp. Cremoris FC strain of the invention in a dead state.
The oral composition of the present invention may be a culture of the lactococcus lactis subspecies milk fat FC strain of the present invention, a crude or purified product of the culture, or a freeze-dried or spray-dried product thereof, as long as the oral composition comprises the lactococcus lactis subspecies milk FC strain of the present invention. The culture is not limited and may be obtained, for example, by culturing for about 12 to 48 hours at an optimal temperature of about 25 to 30 ℃ using a medium (e.g., TY-Glu medium, pH 7.0) suitable for the lactococcus lactis subsp. Cells of the lactococcus lactis subspecies milk fat FC strain of the invention can be obtained by centrifugation of the culture after cultivation, for example at 3,000rpm, for about 10 minutes to collect the cells. These may also be purified according to a common method. In addition, the cells may be freeze-dried or spray-dried. The dried cells thus obtained can also be used as active ingredient of the oral composition of the present invention. In the present specification, the lactococcus lactis subspecies milk fat FC strain of the present invention, a culture thereof, a crude product or purified product of the culture, and a dried product thereof are collectively referred to as "the lactococcus lactis subspecies milk FC strain of the present invention and a culture thereof".
The oral composition of the invention may further comprise, if necessary, an appropriate amount of a nutritional component suitable for maintenance and proliferation of the lactococcus lactis subspecies milk fat FC strain of the invention. Specific examples of the nutritional components include those used in a medium for culturing microorganisms, such as carbon sources, e.g., glucose, galactose, fructose, lactose, and mannose; nitrogen sources such as yeast extract and peptone; a vitamin; minerals; trace metal elements; as well as other nutritional components.
The oral compositions of the present invention may be formulated according to conventional methods in the form of food or drink products, pharmaceutical products and quasi-drugs. The carrier used in producing these forms may be an edible carrier or a pharmaceutically acceptable carrier, such as excipients and diluents. Details of producing the food or beverage form and details of the edible carrier used in producing the food or beverage form are described later in the "oral composition in food or beverage form" section below.
The amount of the lactococcus lactis subspecies milk fat FC strain of the present invention contained in the oral composition of the present invention can generally be selected such that the viable count of the lactococcus lactis subspecies milk fat FC strain of the present invention in the oral composition of the present invention is about 1 x 10 7 Up to 1X 10 11 (cfu). The amount of the lactococcus lactis subspecies milk fat FC strain of the present invention can be suitably changed depending on the form, desired effect, etc. of the oral composition of the present invention to be prepared, using the above amounts as a guide.
Oral composition in the form of a food or beverage
The oral composition of the present invention in the form of a food or beverage is ingested as a food or beverage. Preferred specific examples include, but are not limited to, fermented milk foods and drinks such as fermented milk (e.g., yogurt) and lactobacillus beverages produced using milk as a raw material; and fermented soybean milk foods and drinks such as soybean yogurt and fermented soybean milk drinks produced using soybean as a raw material. Examples also include food and beverage products in pharmaceutical form, such as solid formulations (e.g. tablets, pills, powders, granules, microcapsules and capsules) and liquid formulations (e.g. fluids, suspensions, syrups and emulsions) comprising the lactococcus lactis subsp. Examples also include those comprising the lactococcus lactis subsp creamer FC strain of the invention, such as confections, e.g. chewing gums, caramels, fondants, nougats and chocolate; fermented foods and drinks such as fermented vegetable drinks and fermented fruit drinks; dairy products other than the above-mentioned fermented foods and drinks, such as pudding and bavac, etc. The terms "fermented milk" and "lactic acid bacteria drink" used in the present specification conform to the definitions in the ministry of the working of the former japanese ministry of thick birth (Japanese Ministry of Health and Welfare) "in terms of the fermented milk of clause 2-40" and the lactic acid bacteria drink of clause 2-41 "concerning the composition standards of milk and dairy products and the like. That is, "fermented milk" refers to a pasty or liquid product prepared by fermenting milk or a dairy product with lactic acid bacteria. Thus, "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 pasty or liquid product as a main raw material, which is prepared by fermenting milk or a dairy product with lactic acid bacteria and diluting with water.
(IV) method for producing a fermented food or beverage, and fermented food or beverage
The method for producing a fermented food or beverage product of the invention is characterized in that the fermented food or beverage product comprises the lactococcus lactis subsp. The fermented food or drink may be produced according to a common method. For example, a fermented food or beverage can be produced by inoculating the lactococcus lactis subspecies milk FC strain of the present invention into a suitable raw material for fermentation comprising a nutrient source of the lactococcus lactis subspecies milk FC strain of the present invention (e.g., a raw material comprising milk, soy milk (soy emulsion), cereal, vegetable or fruit) and culturing it to cause fermentation of the raw material.
A preferred method for fermentation using the lactococcus lactis subspecies creamer FC strain of the invention is to prepare the primer in advance and inoculate itIn the raw material for fermentation, and fermenting the raw material. The primer may be, for example, the primer for fermentation of the present invention described above. The primer preferably comprises the lactococcus lactis subspecies creamer FC strain of the invention such that the viable count of the lactococcus lactis subspecies creamer FC strain of the invention is about 1 x 10 7 Up to 1X 10 9 (cfu/ml)。
The raw materials used for fermentation may optionally be supplemented with fermentation promoting substances (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) for good growth of the lactococcus lactis subsp.
For example, when milk is used as a raw material for fermentation, the amount of the inoculated lactococcus lactis subspecies milk FC strain of the present invention is selected so that the amount of the lactococcus lactis subspecies milk FC strain of the present invention contained in the milk is 1×10 5 (cfu/ml) or more (preferably about 1X 10) 7 Up to 1X 10 9 (cfu/ml)) is suitable. For the culture conditions, the fermentation temperature is generally selected from the range of 15 ℃ or higher, preferably about 25 to 30 ℃ and the fermentation time is generally selected from the range of about 3 to 24 hours.
The fermentation product thus obtained may be in curd form (yogurt-like or pudding-like form), and such a product may be directly ingested as a solid food. The fermentation product in curd form can be further homogenized to prepare the desired beverage form. The homogenization may be performed using a common emulsifying machine (homogenizer). By such homogenization, a beverage having alcohol and feel can be obtained. During this homogenization, if necessary, appropriate dilution may be performed, organic acids may be added to adjust the pH, or various additives commonly used in the production of beverages, such as sugar, juice, thickeners, surfactants and flavoring agents may be added. The beverage thus obtained can be poured aseptically into suitable containers to provide the final product.
The intake (dosage) of the fermented food or drink (oral composition) depends on the age, sex, weight, severity of the disease of the organism taking the compositionThe degree and the like are appropriately determined, and are not particularly limited. The intake (dose) of the oral composition is generally selected from the group consisting of the lactococcus lactis subsp.cremoris FC strain of the invention in an amount of about 1 x 10 7 Up to 1X 10 11 (cfu/ml). The oral composition may be prepared such that the composition is typically ingested in an amount of about 50 to 1,000ml per person per day.
Specific preferred examples of the oral composition of the present invention in the form of a food or drink include food and drink in the form of a supplement, particularly food and drink in the form of a drug, such as solid preparations, for example, tablets, pills, powders, granules, microcapsules and capsules; and liquid preparations such as fluids, suspensions, syrups and emulsions. The food or beverage product in the form of a medicament is preferably a dry solid formulation.
The food or beverage product in the form of a supplement may be produced by using diluents or excipients commonly used in the art, such as fillers, compatibilizers, binders, wetting agents, disintegrants, surfactants and lubricants, as a pharmaceutical carrier.
The amount of lactic acid bacteria of the present invention contained in an oral composition in the form of a food or drink may be appropriately selected from a wide range as long as it is sufficient to exert the action of lactic acid bacteria. Preferably, the lactic acid bacteria are generally included in an amount of about 1X 10 7 Up to 1X 10 11 (cfu)/units per intake (dose).
The method for administering the composition is not particularly limited as long as the composition is orally ingested, and the method is determined according to the form, age, sex and other conditions of the patient, severity of disease, and the like.
The intake amount of the composition is appropriately selected according to the method of use, age, sex, and other conditions of the patient, and the like. As described above, the lactococcus lactis subspecies creamer FC strain of the invention is preferably used at about 1X 10 7 Up to 1X 10 11 (cfu)/amount per intake (dose). The lactococcus lactis subspecies milk fat FC strain of the invention preferably has an intake of about 1X 10 as active ingredient 7 (cfu) or more per day, and can be taken in multiple portions per day. Preferably connected withThe oral composition of the present invention is continuously taken to effectively obtain its effect. For example, the oral compositions of the present invention may be ingested continuously or intermittently over a period of 2 weeks or more.
As with the existing lactococcus lactis subsp.lactis FC strain, it is expected that it exerts a useful physiological effect in vivo when an oral composition comprising the lactococcus lactis subsp.lactis FC strain of the present invention in a living state is ingested (administered). Thus, the oral composition of the present invention can be effectively used as a health enhancer based on its physiological effects. Thus, the oral composition of the present invention comprises foods and drinks which have a specific function and are ingested for health maintenance and the like. In particular, the oral compositions of the present invention comprise foods and drinks having, for example, an effect of regulating intestinal functions, an effect of inhibiting an increase in blood glucose levels, an immunostimulatory effect, an effect of preventing influenza virus infection, an allergic reaction inhibiting effect, and/or an effect of improving motility regulation. Such foods and drinks include, for example, foods and drinks produced and sold together with the above-described action or purpose tags or statements, such as foods with health statements (foods with nutritional function statements, foods with function statements, and foods for specific health uses), foods for special dietary uses (foods for specific health uses), and similar health foods.
In this specification, the terms "comprising" and "including" encompass the meaning "consisting essentially of.
Examples
In order to facilitate 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 indicated, the following experiments were conducted at room temperature (25.+ -. 5 ℃) and atmospheric pressure. In the following, "%" means "% by mass" and "parts" means "parts by mass" unless otherwise specified.
Example 1: isolation and identification of novel lactococcus lactis strain FC of the milk subspecies
The lactococcus lactis subspecies milk FC strain (FERM P-20185) was inoculated (1%) into a sterilized 8% skim milk powder liquid and left at its optimal temperature (25 ℃) for about 10 hours to activate the lactococcus lactis subspecies milk FC strain (primer 1). Streptococcus thermophilus (hereinafter referred to as "Streptococcus thermophilus") was inoculated (1%) into the sterilized 8% skim milk powder liquid and left at its optimum temperature (37 ℃) for about 10 hours to activate Streptococcus thermophilus (primer 2). Subsequently, the above prepared primers 1 and 2 were inoculated into separately prepared sterilized milk, and fermentation was performed at 30 ℃. When curd formation was observed, cooling (4 ℃) was started.
The resulting fermented milk was stored in the dark at 4℃for 46 days, inoculated in a proportion of 4% into 3L 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), and then cultured at 26℃for 72 hours. The culture broth was streaked onto TY-Gal agar medium (pH 7.0) and incubated at 25℃for 48 hours, and the grown colonies were each cultured in sterilized 8% skim milk powder liquid and stored.
Among them, a strain having 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 a general method. 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 biomerieux Japan ltd.). Table 2 shows the results. Table 1 is as described in paragraph [0015], and Table 2 is as described in paragraph [0016 ].
These results demonstrate that the obtained strain has the same bacteriological characteristics and sugar assimilation characteristics as the lactococcus lactis subspecies milk FC strain used as the parent strain (hereinafter simply referred to as "parent FC strain"). Therefore, this strain was identified as a strain belonging to the lactococcus lactis subspecies creamer and named lactococcus lactis subspecies creamer FC46 strain (hereinafter referred to as "lactococcus lactis subspecies creamer FC46 strain" or simply "FC46 strain"). The lactococcus lactis subspecies milk FC46 strain was identified by the identification "lactococcus lactis subspecies milk FC46" on month 11 and 6 of 2020 (date of issuance of the accession number: month 11 and 30 of 2020) as deposited internationally with the national institute of technical evaluation's patent microbiological deposit (122 Chamber, 2-5-8Kazusakamatari, kisarazu-shi, chiba, japan).
Example 2: evaluation of acid tolerance
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℃for 15 hours to prepare a preculture solution. The resulting preculture solution was inoculated into 40mL of TY-Glu medium adjusted to pH 4.0 with lactic acid to reach a final concentration of 1%, followed by stationary culture at 26℃for 27 hours. The viable count (cfu/ml) of the FC46 strain in the culture broth was measured over time by a pour plate culture method using plate count agar medium (Nissui Pharmaceutical co., ltd.) containing BCP, and the survival rate after 27 hours was calculated. As a comparative test, the FC parent strain was used in place of the FC46 strain and cultured in the same manner, the viable count (cfu/ml) of the parent FC strain in the culture broth was measured over time, and the survival rate (%) after 27 hours was calculated.
FIG. 1 shows the results of measurements of viable count (cfu/ml) of lactococcus lactis subspecies milk fat strains (FC 46 strain and parent FC strain) over time. As shown in FIG. 1, the parent FC strain was rapidly killed at a low pH of 4.0 and the viable count was reduced to 6.6X10 after 27 hours of cultivation 3 (cfu/ml) (survival rate: 0.2%). In contrast, the FC46 strain exhibited high viability even at a low pH condition of pH 4.0, and maintained 1.9×10 even after 27 hours of culture at that pH condition 6 High viable count (survival rate: 33.6%) of (cfu/ml). This result confirmed that the FC46 strain was a lactococcus lactis subsp.
Example 3: evaluation of stability in fermented milk
Fermented milk was prepared using the FC46 strain, and its stability in fermented milk was evaluated. As a control, fermented milk was prepared in the same manner using the parent FC strain, and the stability in the fermented milk was evaluated.
1. Preparation of fermented milk
Four fermented milks (fermented milks 1 to 4) were prepared using the following strains of activated bacteria as primers:
(1) Fermented milk 1: parent FC strain (primer 1)
(2) Fermented milk 2: FC46 strain (primer 3)
(3) Fermented milk 3: parent FC strain (primer 1) +Streptococcus thermophilus (ST strain) (primer 2)
(4) Fermented milk 4: FC46 strain (primer 3) +streptococcus thermophilus (primer 2).
Primer 1 was prepared by inoculating the parent FC strain (1%) into a sterilized 8% skim milk powder liquid and allowing to stand at 30 ℃ for about 10 hours for activation. Primer 3 was prepared by inoculating FC46 strain (1%) into sterilized 8% skim milk powder liquid and allowing them to stand at 30 ℃ for about 10 hours for activation. Primer 2 was prepared by inoculating streptococcus thermophilus (1%) into a sterilized 8% skim milk powder liquid and allowing them to stand at an optimal temperature (37 ℃) for about 10 hours for activation.
Each of the above prepared primers was inoculated into sterilized milk and fermented at 30 ℃. When curd formation was observed, cooling (4 ℃) was started to inhibit fermentation, thereby obtaining fermented milk.
2. Evaluation of bacterial stability in fermented milk
After preparing fermented milk 1 to 4, it was stored in the dark at 10 ℃ for 30 days. The number of viable bacteria (cfu/ml) in the fermented milk (the number of viable bacteria of lactococcus lactis subspecies milk in fermented milk 1 and fermented milk 2, and the number of viable bacteria of lactococcus lactis subspecies milk and the number of viable bacteria of Streptococcus thermophilus in fermented milk 3 and fermented milk 4) was determined after storage. The viable count of lactococcus lactis subspecies creamed was determined by the pour plate method (25 ℃) using TY-Gal medium (pH 7.0), and the viable count of Streptococcus thermophilus was determined by the pour plate method (37 ℃) using plate count agar medium (pH 7.0) containing BCP.
The pH, acidity, viscosity of each fermented milk were measured by the following methods.
Measurement of pH
After the fermentation products were adjusted to 10 ℃, the pH of each fermentation product was measured using an F-52pH meter (produced by Horiba, ltd.).
Measurement of acidity
10g of each fermentation product was weighed precisely and diluted with the same amount of distilled water. 0.5ml of phenolphthalein solution was added as an indicator and titration was performed with 0.1mol/L aqueous sodium hydroxide until a pale pink color lasted 30 seconds. The percentage of lactic acid per 100g of sample was determined as acidity. Note that 1mL of a 0.1mol/L aqueous sodium hydroxide solution corresponds to 9mg of lactic acid.
Measurement of viscosity
After the fermentation products were adjusted to 10 ℃, 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.
Table 3 shows the results on day 1 after the start of storage, and table 4 shows the results on day 30 after the start of storage.
TABLE 3 Table 3
TABLE 4 Table 4
As shown in tables 3 and 4, the parent FC strain was rapidly killed during storage and only a few viable bacteria per mL (fermented milk 1) remained after 30 days. In contrast, the FC46 strain remained 1.1×10 even after 30 days 4 High viable count (survival rate: 0.0026%) of (cfu/ml) (fermented milk 2). When fermented with Streptococcus thermophilus, both the parent FC strain and the FC46 strain remain more parent than when used aloneThe present FC strain or FC46 strain has a higher viable count (fermented milk 3 and fermented milk 4) when subjected to fermentation, and particularly, the FC46 strain shows a very high viable count of 5.8X10 6 (cfu/ml) (survival rate: 2.8%) (fermented milk 4).
Furthermore, the parent FC strain showed a decrease in viscosity after 30 days of storage, whereas the decrease in viscosity in the FC46 strain was suppressed compared to the parent FC strain. The same trend was observed when fermentation was carried out with streptococcus thermophilus.
The fermented milks 1 to 4 produced in the present example all have good appearance (color, presence or absence of syneresis), good smell and good taste, and have no difference in these respects.
Example 4: preparation of compositions comprising lactic acid bacteria and evaluation of stability
The FC46 strain was used to prepare a composition comprising lactic acid bacteria and its storage stability was evaluated in a dry state. For comparison, a composition comprising lactic acid bacteria was prepared using a parent FC strain instead of the FC46 strain, and its storage stability in the dry state was also evaluated.
1. Preparation of lactic acid bacteria liquid
The FC46 strain or the parent FC strain was inoculated into Difco M17 medium (BD) supplemented with glucose at a ratio of 4% to reach a final concentration of 0.5%, and cultured at 25 ℃ for 16 hours to prepare a preculture solution. 4% of the preculture solution was inoculated into the same kind of medium as that described above and cultured at 25℃for 16 hours to obtain 10L of the culture solution. The culture broth was centrifuged (3,000 rpm,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.
To each of the produced lactic acid bacteria liquids was added skim milk powder in a proportion of 10%, followed by freeze-drying treatment according to a usual method using a freeze-dryer (FD-81,Tokyo Rikakikai Co, ltd.) to obtain freeze-dried lactic acid bacteria (FC 46 strain and parent FC strain, about 50g each).
The obtained freeze-dried lactic acid bacteria (FC 46 strain and parent FC strain, about 0.5g each) were placed in sterilized laminate film zipper bags (LAMIZIP (registered trademark); manufactured by Seisannipponsha Ltd.) and stored under dark conditions at 37 ℃. The number of viable bacteria 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 (%).
Table 5 shows the results.
TABLE 5 survival after storage at 37 ℃ (%)
| First day | After 3 weeks | After 4 weeks | |
| FC46 strain | 100.0 | 14.67 | 6.03 |
| Parent FC strain | 100.0 | 1.58 | 0.07 |
The parent FC strain was rapidly killed during storage at a temperature of 37℃and the viable count of the parent FC strain after 3 weeks was 3.2X10 9 (cfu/g) (survival rate: about 1.6%).In contrast, the FC46 strain showed a very high viable count of 1.1X10) 10 (cfu/g) (survival: about 14.7%). These results confirm that the FC46 strain in dry powder form has excellent storage stability even under aerobic conditions compared to the parent FC strain.
Free text of sequence Listing
SEQ ID NO. 1 shows the base sequence of 16S rRNA of the lactococcus lactis subspecies milk fat FC strain of the present invention.
Sequence listing
<110> FUJICCO CO., LTD.
<120> novel lactic acid bacteria strain and composition comprising the same
<130> P21-046WO
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1507
<212> DNA
<213> lactococcus lactis milk fat subspecies (Lactococcus lactis subsp. Cremoris)
<220>
<221>
<222>
<223> 16S rRNA Gene
<400> 1
gagtttgatc ctggctcagg acgaacgctg gcggcgtgcc taatacatgc aagttgagcg 60
atgaagattg gtgcttgcac caatttgaag agcagcgaac gggtgagtaa cgcgtgggga 120
atctgccttt gagcggggga caacatttgg aaacgaatgc taataccgca taacaacttt 180
aaacataagt tttaagtttg aaagatgcaa ttgcatcact caaagatgat cccgcgttgt 240
attagctagt tggtgaggta aaggctcacc aaggcgatga tacatagccg acctgagagg 300
gtgatcggcc acattgggac tgagacacgg cccaaactcc tacgggaggc agcagtaggg 360
aatcttcggc aatggacgaa agtctgaccg agcaacgccg cgtgagtgaa gaaggttttc 420
ggatcgtaaa actctgttgg tagagaagaa cgttggtgag agtggaaagc tcatcaagtg 480
acggtaacta cccagaaagg gacggctaac tacgtgccag cagccgcggt aatacgtagg 540
tcccgagcgt tgtccggatt tattgggcgt aaagcgagcg caggtggttt attaagtctg 600
gtgtaaaagg cagtggctca accattgtat gcattggaaa ctggtagact tgagtgcagg 660
agaggagagt ggaattccat gtgtagcggt gaaatgcgta gatatatgga ggaacaccgg 720
tggcgaaagc ggctctctgg cctgtaactg acactgaggc tcgaaagcgt ggggagcaaa 780
caggattaga taccctggta gtccacgccg taaacgatga gtgctagatg tagggagcta 840
taagttctct gtatcgcagc taacgcaata agcactccgc ctggggagta cgaccgcaag 900
gttgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960
gaagcaacgc gaagaacctt accaggtctt gacatactcg tgctattcct agagatagga 1020
agttccttcg ggacacggga tacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga 1080
tgttgggtta agtcccgcaa cgagcgcaac ccctattgtt agttgccatc attaagttgg 1140
gcactctaac gagactgccg gtgataaacc ggaggaaggt ggggatgacg tcaaatcatc 1200
atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg agtcgcgaga 1260
cagtgatgtt tagctaatct cttaaaacca ttctcagttc ggattgtagg ctgcaactcg 1320
cctacatgaa gtcggaatcg ctagtaatcg cggatcagca cgccgcggtg aatacgttcc 1380
cgggccttgt acacaccgcc cgtcacacca cgggagttgg gagtacccga agtaggttgc 1440
ctaaccgcaa ggagggcgct tcctaaggta agaccgatga ctggggtgaa gtcgtaacaa 1500
ggtagcc 1507
Claim (modification according to treaty 19)
1. Lactic acid bacteria, which are lactococcus lactis subsp. Cremoris (Lactococcus lactis subsp. Cremoris) FC46 (accession number: NITE BP-03310) or a subcultured strain thereof,
the subcultured strain is a lactic acid bacterium: it belongs to the lactococcus lactis subspecies of milk fat and has the same bacteriological, morphological, physiological, carbon source assimilation, viscous polysaccharide production, acid tolerance and viability under dry and aerobic conditions as those of the lactic acid bacteria of the lactococcus lactis subspecies of FC 46.
2. The lactic acid bacterium according to claim 1, which is in a dry state.
3. A primer for fermentation comprising the lactic acid bacterium according to claim 1 or 2.
4. A primer for fermentation according to claim 3, further comprising streptococcus thermophilus (Streptococcus thermophilus).
5. A primer for fermentation according to claim 4, which is lactococcus lactis subspecies milk fat FC46 (accession number: NITE BP-03310) or a symbiont of a subcultured strain thereof with Streptococcus thermophilus.
6. The primer 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.
8. The oral composition of claim 7, which is a fermented food or beverage product.
9. A method for producing a fermented food or beverage, comprising inoculating the lactic acid bacterium 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 beverage, and fermenting the raw material.
Claims (9)
1. Lactic acid bacteria, which are lactococcus lactis subsp. Cremoris (Lactococcus lactis subsp. Cremoris) FC46 (accession number: NITE BP-03310) or a subcultured strain thereof.
2. The lactic acid bacterium according to claim 1, which is in a dry state.
3. A primer for fermentation comprising the lactic acid bacterium according to claim 1 or 2.
4. A primer for fermentation according to claim 3, further comprising streptococcus thermophilus (Streptococcus thermophilus).
5. A primer for fermentation according to claim 4, which is lactococcus lactis subspecies milk fat FC46 (accession number: NITE BP-03310) or a symbiont of a subcultured strain thereof with Streptococcus thermophilus.
6. The primer 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.
8. The oral composition of claim 7, which is a fermented food or beverage product.
9. A method for producing a fermented food or beverage, comprising inoculating the lactic acid bacterium 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 beverage, and fermenting the raw material.
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| JP2008000102A (en) * | 2006-06-26 | 2008-01-10 | Bio Ken:Kk | Spawn for producing yoghurt |
| CN102399730A (en) * | 2011-12-07 | 2012-04-04 | 天津科技大学 | High-extracellular-polysaccharide-yield Lactobacillus, application and extracellular polysaccharide thereof |
| CN106047773A (en) * | 2016-08-10 | 2016-10-26 | 江南大学 | Lactococcal lactis and application thereof |
| CN106434441A (en) * | 2016-09-21 | 2017-02-22 | 光明乳业股份有限公司 | Lactococcus lactis subsp.cremoris and application thereof in cheese production |
| KR20200067299A (en) * | 2018-12-03 | 2020-06-12 | 김상태 | Lactococcus lactis strain for treating degenerative brain disease and uses thereof |
| CN111295096A (en) * | 2017-08-30 | 2020-06-16 | 科·汉森有限公司 | Method for producing medium-temperature fermented dairy product |
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2021
- 2021-03-02 CN CN202180095075.0A patent/CN117062905A/en active Pending
- 2021-03-02 JP JP2021559615A patent/JP7035283B1/en active Active
- 2021-03-02 WO PCT/JP2021/008002 patent/WO2022185419A1/en active Application Filing
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| CN111295096A (en) * | 2017-08-30 | 2020-06-16 | 科·汉森有限公司 | Method for producing medium-temperature fermented dairy product |
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| TW202242092A (en) | 2022-11-01 |
| JPWO2022185419A1 (en) | 2022-09-09 |
| WO2022185419A1 (en) | 2022-09-09 |
| JP7035283B1 (en) | 2022-03-14 |
| TWI799153B (en) | 2023-04-11 |
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