US20150139970A1 - Novel lactic acid bacterium - Google Patents

Novel lactic acid bacterium Download PDF

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Publication number
US20150139970A1
US20150139970A1 US14/611,739 US201514611739A US2015139970A1 US 20150139970 A1 US20150139970 A1 US 20150139970A1 US 201514611739 A US201514611739 A US 201514611739A US 2015139970 A1 US2015139970 A1 US 2015139970A1
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lactic acid
acid bacteria
effect
strain
viability
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Airo Tategaki
Taizo Kawabe
Hozumi Tanaka
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Kaneka Corp
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Kaneka Corp
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Assigned to KANEKA CORPORATION reassignment KANEKA CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KAWABE, TAIZO, TANAKA, HOZUMI, TATEGAKI, AIRO
Publication of US20150139970A1 publication Critical patent/US20150139970A1/en
Priority to US15/204,641 priority Critical patent/US10675311B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/045—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with a leaven or a composition containing acidifying bacteria
    • A23K1/009—
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A23L1/3014—
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/065—Microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/02—Nutrients, e.g. vitamins, minerals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K2035/11—Medicinal preparations comprising living procariotic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present invention relates to novel lactic acid bacteria belonging to the Enterococcus faecium species, a composition comprising such lactic acid bacteria, an agent comprising such composition as an active ingredient, which is any one of agent selected from among an agent for ameliorating fatigue, an agent for improving blood circulation, an agent for reducing stool odor, and an agent for promoting growth, and use of such composition for food and other products.
  • the enteric environment of people of modern times has deteriorated because of undue stress, unbalanced diet, and irregular living habits, such as lack of sleep.
  • the enteric environment is closely related to the health condition, and common symptoms such as extreme fatigue, and excessive sensitivity to cold, shoulder stiffness, skin problems, and lower back pain caused by blood circulation disorder are considered to result from a deteriorated enteric environment. While such symptoms may be relieved if the enteric environment is brought back to a normal state by modifying irregular living habits, it is not easy for busy people of modern times to modify their living habits. Accordingly, food products, functional food products, and pharmaceutical products that can alleviate such symptoms and are safe, and thus can be routinely and continuously ingested, have been desired.
  • lactic acid bacteria have drawn attention as components that exert useful biological activity on humans and animals (so-called “functional components”).
  • functional components include regulation of the functions of the intestines, anti-allergic activity, cholesterol-lowering activity, or antihypertensive activity, upon oral administration to humans or animals.
  • lactic acid bacteria the Lactobacillus johnsonii La1 strain, also referred to as “LC1 lactic acid bacteria” regulate the autonomic nervous system through the central histaminergic system or the hypothalamic suprachiasmatic nucleus in which the circadian clock is present, thereby affecting the blood pressure, body temperature, and other properties (Non-Patent Document 2). Accordingly, research on lactic acid bacterial strains with novel biological activity has been in progress.
  • lactic acid bacteria While a wide variety of lactic acid bacterial species are known to exert useful biological activity on humans and animals as described above, lactic acid bacteria that would effectively ameliorating fatigue or blood circulation disorder have not yet been discovered. In addition, effects attained by providing livestock animals or the like with lactic acid bacteria that have effects of ameliorating fatigue or blood circulation disorder, in particular, effects of reducing stool odor, or promoting growth have not yet been examined.
  • the present inventors have conducted concentrated studies in order to attain the above objects. As a result, surprisingly, they discovered that lactic acid bacteria belonging to the Enterococcus faecium species having particular mycological properties and exhibiting viability of 40% or higher when freeze-dried in the absence of a dispersion medium and viability of 80% or higher in a probiotic preparation when stored at 40° C. for 4 months would have one or more effects selected from among excellent fatigue-ameliorating effect, blood circulation-improving effect, stool odor-reducing effect, and growth-promoting effect. This has led to the completion of the present invention.
  • the present invention includes the following.
  • the lactic acid bacteria have the nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence showing 90% or higher sequence identity with SEQ ID NO: 1.
  • [3] The lactic acid bacteria belonging to the Enterococcus faecium species according to [1] or [2], which are Enterococcus faecium R30 strain (NITE BP-01362), Enterococcus faecium R28 strain (NITE BP-01361), or a variant thereof having a DNA mutation.
  • a composition comprising the lactic acid bacteria according to any of [1] to [3] or a processed product or extraction residue of the lactic acid bacteria.
  • the composition according to [4] which further comprises a protective agent.
  • the composition according to [5] wherein the content of the protective agent is 1% by weight or more relative to the dry weight of bacteria.
  • composition according to [5], wherein the protective agent is at least one member selected from the group consisting of sucrose, trehalose, sodium glutamate, histidine, and malic acid.
  • An agent for ameliorating fatigue comprising, as an active ingredient, the composition according to any of [4] to [7].
  • An agent for improving blood circulation comprising, as an active ingredient, the composition according to any of [4] to [7].
  • An agent for reducing stool odor comprising, as an active ingredient, the composition according to any of [4] to [7].
  • An agent for promoting growth comprising, as an active ingredient, the composition according to any of [4] to [7].
  • a food product comprising the composition according to any of [4] to [7].
  • a feed or veterinary drug comprising the composition according to any of [4] to [7].
  • a pharmaceutical product comprising the composition according to any of [4] to [7].
  • a method for ameliorating fatigue or blood circulation disorder comprising administering the composition according to any of [4] to [7] to a subject who is in need thereof.
  • a method for reducing stool odor comprising administering the composition according to any of [4] to [7] to a subject who is in need thereof.
  • a method for promoting growth comprising administering the composition according to any of [4] to [7] to a subject who is in need thereof
  • the lactic acid bacteria belonging to the Enterococcus faecium species having particular mycological properties according to the present invention were found to ameliorate fatigue or blood circulation disorder. Accordingly, a composition comprising the lactic acid bacteria of the present invention or a processed product or extraction residue of such lactic acid bacteria can be used with high safety for a food or pharmaceutical product having effects of ameliorating fatigue or blood circulation disorder. In addition, such lactic acid bacteria were found to have stool odor-reducing effect, and growth-promoting effect.
  • compositions comprising the lactic acid bacteria of the present invention or a processed product or extraction residue of such lactic acid bacteria can be used with high safety as an alternative to an agent such as an antibiotic agent, and such composition can also be used for a feed or feed additive having stool odor-reducing effect, and growth-promoting effect in the field of animal husbandry and other fields.
  • the lactic acid bacteria of the present invention belong to the Enterococcus faecium species exhibiting viability of 40% or higher when freeze-dried in the absence of a dispersion medium and viability of 80% or higher in a probiotic preparation when stored at 40° C. for 4 months, preferably viability of 40% or higher when freeze-dried in the absence of a dispersion medium and viability of 90% or higher in a probiotic preparation when stored at 40° C. for 4 months, and more preferably viability of 40% or higher when freeze-dried in the absence of a dispersion medium and viability of 95% or higher in a probiotic preparation when stored at 40° C. for 4 months.
  • bacterial viability is lower than 40% when freeze-dried in the absence of a dispersion medium or it is lower than 80% in a probiotic preparation when stored at 40° C. for 4 months, lactic acid bacteria that have one or more effects selected from among fatigue-ameliorating effect, blood circulation-improving effect, stool odor-reducing effect, and growth-promoting effect cannot be obtained. Thus, such low viability is not preferable.
  • freeze-dried in the absence of a dispersion medium in the present invention means that lactic acid bacterial strains in a culture solution obtained by a conventional technique are collected with the use of a centrifuge or the like, the collected strains are dispersed in water without the addition of a dispersion medium, and the resulting dispersion is then freeze-dried.
  • a “dispersion medium” is a substance capable of homogeneously dispersing the collected bacterial strains, and examples thereof include physiological saline, phosphate-buffered saline, and a solution containing a protective agent.
  • the “protective agent” is a substance capable of reducing the damage imposed on a bacterial strain by freezing or dry stress.
  • Examples thereof include trehalose, bovine serum albumin, dried skim milk, sodium glutamate, L-ascorbic acid, histidine, malic acid, whey, glucose, aspartic acid, methionine, starch, dextrin, sucrose, lactose, sodium chloride, and phosphate.
  • the lactic acid bacteria of the present invention can be obtained by the screening method described below.
  • strains to be screened are cultured at 37° C. for 24 hours, bacterial strains are separated from the culture solution via centrifugation, the culture solution is removed, and the bacterial strains are then collected. An adequate amount of water is added to the collected bacterial strains without the addition of a dispersion medium, and a concentrate of dispersed bacterial strains is obtained.
  • the total number of viable bacteria in the bacterial concentrate is determined by a conventional technique, such as colony counting.
  • the bacterial concentrate is freeze-dried, and the total number of viable bacteria in the bacterial concentrate is determined by a conventional technique, such as colony counting.
  • Bacterial viability when freeze-dried in the absence of a dispersion medium is then calculated on the basis of the total number of viable bacteria in the bacterial concentrate and the total number of viable bacteria in the concentrate of freeze-dried bacterial strains. Thus, lactic acid bacteria exhibiting viability of 40% or higher are selected.
  • the lactic acid bacteria that were found to exhibit viability of 40% or higher when freeze-dried in the absence of a dispersion medium as a result of the primary screening are cultured at 37° C. for 24 hours, bacterial strains are separated from the culture solution via centrifugation, the culture solution is removed, and wet bacterial strains are obtained. After a dispersion medium is added to the wet bacterial strains, the resultant is subjected to freeze-drying, so as to obtain freeze-dried bacterial strains. An excipient is added to the freeze-dried bacterial strains so as to adjust the viable bacterial count to 1.0 ⁇ 10 10 cfu/g. Thus, a probiotic preparation is obtained. The resulting probiotic preparation is stored in an incubator at 40° C.
  • the viable bacterial count per g of the probiotic preparation is then determined by a conventional technique, such as colony counting.
  • Bacterial viability in a probiotic preparation when stored at 40° C. for 4 months is then determined on the basis of the viable bacterial count in the probiotic preparation before storage (1.0 ⁇ 10 10 cfu/g) and the viable bacterial count per g of the probiotic preparation.
  • lactic acid bacteria exhibiting viability of 80% or higher are selected.
  • lactic acid bacteria of the present invention obtained by the screening process; i.e., the Enterococcus faecium R30 strain (hereafter it is also referred to as the “R30 strain”) and the Enterococcus faecium R28 strain (hereafter it is also referred to as the “R28 strain”), were deposited on May 16, 2012 at the Patent Microorganisms Depository (NPMD) of the National Institute of Technology and Evaluation (NITE) (2-5-8, Kazusakamatari, Kisarazu-shi, Chiba, 292-0818, Japan) under Accession Numbers NITE P-1362 and NITE P-1361, respectively. These strains were transferred to the international depositary authority under the provisions of the Budapest Treaty on Apr. 24, 2013, under Accession Numbers NITE BP-01362 and NITE BP-01361, respectively.
  • NPMD Patent Microorganisms Depository
  • NITE National Institute of Technology and Evaluation
  • the Enterococcus faecium R30 and R28 strains have the mycological properties described below:
  • the mycological properties described above were analyzed using the API 20 Strep system (manufactured by Sysmex bioMerieux Co., Ltd.). As a result, the strains mentioned above exhibited mycological properties identical to those of Enterococcus faecium . Also, as a result of analysis of the entire nucleotide sequence of 16S rDNA, the R30 strain showed 99.8% sequence identity and the R28 strain showed 99.9% sequence identity to the sequence of the Enterococcus faecium DSM20477 strain. However, bacterial viability when freeze-dried in the absence of a dispersion medium and bacterial viability in the probiotic preparation when stored at 40° C. for 4 months are apparently different from those of the DSM20477 strain.
  • lactic acid bacteria of the present invention belonging to the Enterococcus faecium species have the chemotaxonomic properties described below:
  • the lactic acid bacteria have the nucleotide sequence as shown in SEQ ID NO: 1 or a nucleotide sequence showing 90% or higher sequence identity with SEQ ID NO: 1.
  • lactic acid bacteria of the present invention belonging to the Enterococcus faecium species it is sufficient for the lactic acid bacteria of the present invention belonging to the Enterococcus faecium species to have 90% or higher sequence identity to the nucleotide sequence of 16S rDNA of the DSM20477 strain as shown in SEQ ID NO: 1.
  • sequence identity is preferably 95% or higher, more preferably 98% or higher, further preferably 99% or higher, and most preferably 99.5% or 99.8% or higher.
  • Sequence identity of 100% is preferable because lactic acid bacteria that have one or more effects selected from among excellent fatigue-ameliorating effect, blood circulation-improving effect, stool odor-reducing effect, and growth-promoting effect can be obtained.
  • sequence identity is lower than 90%, lactic acid bacteria that have one or more effects selected from among excellent fatigue-ameliorating effect, blood circulation-improving effect, stool odor-reducing effect, and growth-promoting effect cannot be obtained.
  • variants thereof, and/or descendants thereof are within the scope of the present invention, provided that such variants and/or descendants have equivalent effects, and such variants and/or descendants can be included in the composition of the present invention instead of the R30 or R28 strain.
  • variant refers to a lactic acid bacterium having a DNA mutation
  • DNA mutation refers to a treatment that would artificially induce mutagenesis by means of a conventional technique, such as radiation application or the use of a mutagen.
  • a spontaneous DNA mutation is within the scope of the “DNA mutation.”
  • the term “descendant” refers to a lactic acid bacterium that inherits its ancestor's genome. In the present invention, the term “descendant” refers to any lactic acid bacterium arising from parent R30 and/or R28 strains and may comprise any natural mutation or DNA recombination due to bacterial cell division or cell prolifieration.
  • the lactic acid bacteria of the present invention can be cultured in any medium as long as such lactic acid bacteria can grow therein. Culture can be carried out in, for example, a test tube, a flask, or a fermenter.
  • a test tube a flask
  • a fermenter a fermenter
  • an MRS medium that is commonly used for lactic acid bacterial culture may be used, and general lactic acid bacterial culture may be performed under general conditions, although culture conditions are not limited thereto.
  • the composition of the present invention comprises the lactic acid bacteria of the present invention or variants thereof.
  • the lactic acid bacteria or variants thereof contained in the composition of the present invention may be bacterial strains that have not been subjected to any treatment or treated products or extraction residues of the lactic acid bacteria or variants thereof.
  • Bacterial strains contained in the composition of the present invention may be either viable bacteria or dead bacteria.
  • viable bacteria used herein refers to lactic acid bacteria remaining alive and the term “dead bacteria” refers to bacterial strains that have been disinfected via heating, pressurization, drug treatment, or another type of treatment.
  • treated product used in the present invention refers to a product that has been subjected to at least one treatment selected from among grinding or fragmentation, liquefaction via extraction, concentration, paste preparation, drying (e.g., spray-drying, freeze-drying, vacuum drying, or drum drying), and attenuation of lactic acid bacteria.
  • extraction residue refers to, for example, a precipitate obtained by treating bacterial strains with boiled water or hot water, obtaining the extract from bacterial strains, and collecting the residue via centrifugation. It should be noted that the “treated product” and the “extraction residue” are not limited thereto.
  • composition comprising viable lactic acid bacteria of the present invention can be obtained by, for example, collecting bacterial strains in the culture solution that have been cultured in accordance with a conventional technique, adding a solution of a protective agent to the collected bacterial strains, drying the mixture, and mixing the resultant with an adequate excipient.
  • any substance can be used as the protective agent, provided that such substance can reduce damage imposed by freezing or dry stress on bacterial strains.
  • examples thereof include trehalose, bovine serum albumin, dried skim milk, sodium glutamate, L-ascorbic acid, histidine, malic acid, whey, glucose, aspartic acid, methionine, starch, dextrin, sucrose, lactose, sodium chloride, and phosphate.
  • Such protective agents can be used alone or in combination.
  • bacterial strains in the culture solution that have been cultured in accordance with a conventional technique may be collected via centrifugation, and a solution of a protective agent, such as trehalose, sodium glutamate, histidine, malic acid, or sucrose, may be added to the bacterial strains.
  • a protective agent such as trehalose, sodium glutamate, histidine, malic acid, or sucrose
  • the mixing ratio of protective agents is not particularly limited; however, the lower limit for the total amount of the protective agent is 1% by weight or more, preferably 10% by weight or more, and further preferably 100% by weight or more, relative to the dry weight of bacterial strains. If the final concentration of the protective agent is less than 1% by weight relative to the dry weight of bacterial strains, damage imposed by freezing or dry stress on bacterial strains cannot be sufficiently reduced. Accordingly, such low concentration is not preferable.
  • the composition comprising dead lactic acid bacteria of the present invention can be obtained by, for example, collecting bacterial strains in a culture solution that have been cultured in accordance with a conventional technique, disinfecting the collected bacterial strains, drying the resulting bacterial strains, and mixing the resultant with an adequate excipient.
  • the composition of the present invention has excellent fatigue-ameliorating effect and it can thus be used as an agent for ameliorating fatigue.
  • the agent for ameliorating fatigue of the present invention has effects of preventing or ameliorating a sense of fatigue caused by undue stress, unbalanced diet, or irregular living habits such as lack of sleep, improving motility function, or increasing endurance.
  • composition of the present invention has excellent blood circulation-improving effect, and it can thus be used as an agent for improving blood circulation.
  • the agent for improving blood circulation of the present invention has an action of preventing or ameliorating symptoms such as excessive sensitivity to cold, shoulder stiffness, skin problems, or lower back pain caused by undue stress, unbalanced diet, or irregular living habits such as lack of sleep.
  • the composition of the present invention has excellent stool odor-reducing effect, and it can thus be used as an agent for reducing stool odor.
  • the agent for reducing stool odor of the present invention has an action of reducing stool odor of infants who often suffer from enteric imbalance, elderly people, or people who have lost strength.
  • the agent has an action of reducing stool odor of livestock animals that suffer from deteriorated enteric environments because of stress caused by group breeding or drug administration such as vaccination.
  • composition of the present invention has excellent growth-promoting effect, and it can thus be used as an agent for promoting growth.
  • the agent for promoting growth of the present invention has an action of promoting growth of infants or suppressing weight loss of elderly people or sick people.
  • the agent has an action of promoting growth of livestock animals that suffer from deteriorated enteric environments because of stress caused by group breeding or drug administration such as vaccination.
  • fatigue-ameliorating effect can be evaluated by, for example, having a rat to run on a treadmill at 10 m/min, gradually increasing the rate by 5 m/min every 3 minutes, and determining the duration of time until the rat can no longer run (i.e., the maximal running time), as described in the Examples below. If the maximal running time of the rat is prolonged after the test substance has been administered in comparison with the maximal running time of the rat before the test substance has been administered, the test substance is evaluated as having fatigue-ameliorating effect.
  • blood circulation-improving effect can be evaluated by, for example, having a volunteer to immerse his left hand in cold water at 15° C. for 10 seconds and measuring the skin temperature immediately before the application of cold water stress and 10 minutes after the application of cold water stress with the use of thermography, as described in the Examples below.
  • the skin temperature of the subject measured immediately after the application of cold water stress is compared with that measured 10 minutes after the application of cold water stress. If the difference in the subject's skin temperature therebetween becomes greater as a result of ingestion of the test substance, such test substance is evaluated as having blood circulation-improving effect.
  • stool odor-reducing effect can be evaluated by, for example, examining the stool odor of a baby chicken, as described in the Examples below.
  • a reduction in stool odor is determined based on the stool odor before administration of the test substance and the stool odor after administration of the test substance, and the test substance is evaluated as having stool odor-reducing effect if the reduction in stool odor is significant.
  • Stool odor may be evaluated by a sensory evaluation, or it may be evaluating by measuring, for example, the concentration of acetic acid gas in a stool sample.
  • growth-promoting effect can be evaluated by, for example, measuring body weights of mice, as described in the Examples below. If the body weight of a mouse to which the test substance has been administered is greater than that of a mouse to which the test substance has not been administered, the test substance is evaluated as having growth-promoting effect.
  • composition of the present invention can be used for a food product, functional food product or supplement, feed (including pet food), veterinary drug, or pharmaceutical product.
  • the content of lactic acid bacteria or a treated product or extraction residue thereof is not particularly limited.
  • the lower limit of such content is 0.00001% by weight, preferably 0.001% by weight, and further preferably 0.1% by weight in a food product
  • the upper limit thereof is 100% by weight, preferably 50% by weight, and further preferably 30% by weight in a food product.
  • composition of the present invention can also be prepared in the form of a supplement, such as a capsule or tablet, and it can be used for Food With Health Claims, such as Food for Specified Health Uses or Food with Nutrient Function Claims, or functional food products, such as health food products or nutritional supplements.
  • Food With Health Claims such as Food for Specified Health Uses or Food with Nutrient Function Claims
  • functional food products such as health food products or nutritional supplements.
  • the use thereof can be indicated as use for prevention or amelioration of fatigue, improvement of motility function, improvement of endurance, and/or prevention or amelioration of excessive sensitivity to cold, shoulder stiffness, skin problems, or lower back pain, and/or reduction or moderation of stool odor, and/or promotion of growth.
  • composition of the present invention is administered to a subject once or in several separate instances per day, so that an adult subject can ingest at least 0.01 mg and preferably at least 1 mg of lactic acid bacteria or a treated product or extraction residue thereof per kg of the body weight of the subject per day.
  • the maximal amount thereof that can be administered is 1,000 mg and preferably 300 mg thereof per kg of the body weight of an adult per day.
  • the dosage form thereof is not particularly limited. Examples thereof include capsules, syrups, tablets, pills, powders, granules, beverages, injection preparations, infusion solutions, nasal drops, eye drops, suppositories, adhesive skin patches, and sprays, although the dosage form is not limited thereto.
  • another pharmaceutically acceptable agent such as an excipient, disintegrator, lubricant, antioxidant, coloring agent, anti-coagulant, absorption accelerator, solubilizer, or stabilizer, can be adequately added.
  • composition of the present invention When the composition of the present invention is used for a feed (including pet food) or a veterinary drug, the lactic acid bacteria or a treated product or extraction residue thereof are used as main raw materials.
  • Raw materials used for a common feed mixture may be adequately added in accordance with animal species, growth stage, or breeding environment, such as the area of breeding.
  • raw materials include cereals and processed cereals (e.g., maize, milo, barley, wheat, rye, oat, millet, flour, and wheat germ powder), cereal grain by-products (e.g., bran, rice bran, and corn gluten feed), vegetable oil meals (e.g., soybean oil meal, sesame oil meal, cottonseed oil meal, peanut meal, sunflower seed oil, and safflower oil meal), animal-derived raw materials (e.g., dried skim milk, fish meal, and bone-meal feed), minerals (e.g., calcium carbonate, calcium phosphate, salt, and anhydrous silicic acid), vitamins (e.g., vitamin A, vitamin D, vitamin E, vitamin K, vitamin B1, vitamin B2, vitamin B6, vitamin B12, calcium pantothenate, nicotinic-acid amide, and folic acid), amino acids (e.g., glycine and methionine), yeast such as brewer's yeast, and fine powders
  • the feed of the present invention may comprise, in addition to the raw materials of feed described above, a feed additive that is commonly used for a feed mixture, such as an excipient, filler, binder, thickener, emulsifier, coloring agent, aroma chemical, food additive, or seasoning agent, and other components, such as an antibiotic, disinfectant, anthelmintic, or antiseptic agent, according to need.
  • a feed additive that is commonly used for a feed mixture, such as an excipient, filler, binder, thickener, emulsifier, coloring agent, aroma chemical, food additive, or seasoning agent, and other components, such as an antibiotic, disinfectant, anthelmintic, or antiseptic agent, according to need.
  • composition of the present invention is administered in a single instance or several separate instances per day, so that at least 0.001 mg, preferably 0.01 mg, and further preferably 0.1 mg of lactic acid bacteria or a treated product or extraction residue thereof can be administered to a target animal per kg of the body weight thereof, regardless of the animal species.
  • the maximal amount thereof that can be administered is 1,000 mg and preferably 500 mg per kg of the body weight of an animal per day.
  • the dosage form thereof is not particularly limited. Examples thereof include capsules, syrups, tablets, pills, powders, granules, drinkable preparations, injection preparations, infusion solutions, nasal drops, eye drops, suppositories, adhesive skin patches, and sprays.
  • another pharmaceutically acceptable agent such as an excipient, disintegrator, lubricant, binder, antioxidant, coloring agent, anti-coagulant, absorption accelerator, solubilizer, or stabilizer, can be adequately added.
  • the amount of the composition of the present invention administered in the form of a pharmaceutical product may be determined in accordance with the amount of the functional food product or supplement to be administered.
  • lactic acid bacteria was selected through the methods of primary screening and secondary screening described below, and biological activity was evaluated by evaluating fatigue-ameliorating effect, blood circulation-improving effect, stool odor-reducing effect, and growth-promoting effect.
  • Lactic acid bacteria were cultured in 10 ml of MRS liquid medium (a solution of 0.52 g of MRS broth (manufactured by Kanto Chemical Co., Inc.) dissolved in 10 ml of water, sterilized in an autoclave at 121° C. for 15 minutes) at 37° C. for 24 hours to obtain a preculture solution.
  • the preculture solution (1 ml) was added to 100 ml of MRS liquid medium, and culture was conducted at 37° C. for 24 hours. After the completion of culture, the culture product was centrifuged at 8,000 rpm for 10 minutes to separate bacterial strains from the culture solution.
  • the bacterial strains were washed with 100 ml of sterile water and centrifuged again to separate the bacterial strains from the sterile water. Fresh sterile water (2 ml) was added to the bacterial strains to obtain a bacterial concentrate.
  • the bacterial concentrate (0.1 ml) was suspended in 0.9 ml of physiological saline, an MRS agar medium (this MRS agar medium was prepared by dissolving 1.24 g of an MRS agar medium (manufactured by Kanto Chemical Co., Inc.) in 20 ml of water, sterilizing the resultant in an autoclave at 121° C.
  • the bacterial concentrate was freeze-dried in a freeze-dryer, the VirTis AdVantage Plus (freezing conditions: ⁇ 30° C. for 12 hours; drying conditions: 10 ⁇ 10 ⁇ 1 torr at a shelf temperature of 20° C. for 48 hours) to obtain freeze-dried bacterial strains.
  • the freeze-dried bacterial strains (20 mg) were dispersed in 1 ml of physiological saline to obtain a bacterial suspension, and the resulting bacterial suspension was diluted 10 6 -fold with physiological saline.
  • An MRS agar medium was coated with 0.1 ml of the resulting diluent, culture was conducted at 37° C. for 2 days, and the developed colonies were counted to determine the total viable bacterial count in the freeze-dried bacterial strains.
  • Bacterial viability when freeze-dried in the absence of a dispersion medium was calculated using the equation shown below, and lactic acid bacteria exhibiting viability of 40% or higher were selected.
  • the lactic acid bacteria selected via primary screening method were cultured in 10 ml of MRS liquid medium at 37° C. for 24 hours to obtain a preculture solution.
  • the preculture solution (10 ml) was added to 1 liter of MRS liquid medium and culture was conducted at 37° C. for 24 hours.
  • the culture product was centrifuged at 8,000 rpm for 10 minutes to separate bacterial strains from the culture solution.
  • the bacterial strains were washed with 1 liter of sterile water and centrifuged again to separate the bacterial strains from sterile water. Sterile water was added to the bacterial strains so as to adjust the volume of the bacterial solution to 20 ml. Thus, a bacterial concentrate was obtained.
  • a solution (2 ml) of a protective agent comprising 0.4% by weight of sucrose, 0.2% by weight of trehalose, 0.2% by weight of sodium glutamate, 0.2% by weight of histidine, and 0.2% by weight of malic acid was added to the bacterial concentrate, and the resultant was freeze-dried under the same conditions as described above to obtain freeze-dried bacterial strains.
  • the freeze-dried bacterial strains were mixed with dextrin to adjust the viable bacterial count to 1.0 ⁇ 10 10 cfu/g, and a probiotic preparation was thus obtained.
  • the probiotic preparation was introduced into separate polyethylene bags with zippers (Unipac B-8, manufactured by Seisannipponsha Ltd.) in amounts of 10 g each, packed into aluminum pouches (Lamizip, manufactured by Seisannipponsha Ltd.) each containing 1 g of silica gel (manufactured by Fuji Silysia Chemical Ltd.), and heat-sealed.
  • the resultant was designated as a storage sample.
  • the storage sample was stored in an incubator at 40° C.
  • a probiotic preparation 100 mg was dispersed in 1 ml of physiological saline to prepare a bacterial suspension, the suspension was diluted 10 7 -fold with physiological saline, and an MRS agar medium was coated with 0.1 ml of the resulting diluent.
  • Culture was conducted at 37° C. for 2 days, the developed colonies were counted, and bacterial viability in the probiotic preparation when stored at 40° C. for 4 months was calculated using the equation below. Thus, lactic acid bacteria exhibiting viability of 80% or higher were selected.
  • the maximal running time was measured before administration of the test substance and 2 hours after administration of the test substance, and the maximal running time measured before administration of the test substance was subtracted from the maximal running time measured 2 hours after administration of the test substance to determine the difference in the maximal running time before and after the administration of the test substance. Fatigue-ameliorating effect was evaluated on the basis of whether or not the difference between the maximal running time measured before administration of the test substance and that measured after administration of the test substance had increased as a result of the administration of the test substance.
  • the cold water stress test was performed by having subjects to immerse their left hands in cold water at 15° C. for 10 seconds and measuring the skin temperature immediately thereafter and 10 minutes thereafter with the use of thermography (TVS 600, manufactured by Nippon Avionics Co., Ltd.).
  • the average skin temperature measured immediately after cold water stress application was subtracted from the average skin temperature measured 10 minutes after cold water stress application (i.e., the difference in average skin temperature), and blood circulation-improving effect was evaluated on the basis of whether or not the difference in average skin temperature had significantly increased as a result of ingestion of the test substance.
  • the average skin temperature was determined by measuring the skin temperature at 3 sites; i.e., the tip of the middle finger on the opisthenar side, the midpoint of the proximal phalanx of the middle finger, and the midpoint of the third metacarpal bone.
  • the test feed containing the test substance i.e., a feed mixture containing the test substance
  • Stool odor was evaluated in terms of an odor index, hydrogen sulfide gas concentration, and acetic acid gas concentration of stool samples. Reductions in the odor index, hydrogen sulfide gas concentration, and acetic acid gas concentration of stool samples after the test were determined.
  • the odor index values were determined by suspending 1 g of a stool sample in 100 ml of odorless distilled water, allowing the sample to stand at 25° C. for 30 minutes, diluting the sample to 1/10th initial concentration, and analyzing the resulting test solution using a three-point comparison-type flask method.
  • Hydrogen sulfide gas concentration and acetic acid gas concentration were determined by mounting 100 g of stool sample on a glass petri dish, introducing the petri dish into a 10-liter polystyrene bag, creating a vacuum in the bag, filling the bag with odorless air purified with the aid of active carbon to hermetically seal the bag, allowing the resultant to stand at room temperature (about 22° C.) for 1 hour, and determining hydrogen sulfide gas concentration and acetic acid gas concentration in the bag with the use of a gas-detecting tube.
  • the collected stool samples were hermetically sealed and stored at ⁇ 80° C. before the test.
  • Roden CAFE Oriental Yeast Co., Ltd.
  • the Enterococcus faecium DSM20477 strain which is a reference strain of Enterococcus faecium , is referred to as the “reference strain.”
  • the reference strain is freeze-dried in the absence of a dispersion medium, the bacterial viability is 25%.
  • the bacterial viability in a probiotic preparation is 40% when stored at 40° C. for 4 months.
  • Lactic acid bacterial strains (125 strains: Isolate 1 to Isolate 125) separated from food materials and the like were subjected to the process of primary screening described above, and the viability of each isolate when freeze-dried in the absence of a dispersion medium was determined.
  • Isolate 17 and Isolate 82 exhibited viability of 40% or higher when freeze-dried in the absence of a dispersion medium.
  • Isolate 17 and Isolate 82 were subjected to the process of secondary screening described above, and the viability of each thereof in a probiotic preparation when stored at 40° C. for 4 months was determined. The results are shown in Table 1.
  • Isolate 82 was found to exhibit viability of 80% or higher in a probiotic preparation when stored at 40° C. for 4 months.
  • Isolate 82 Mycological properties of Isolate 82 were examined using the API 20 Strep system (manufactured by Sysmex bioMerieux Co., Ltd.), and Isolate 82 was found to exhibit mycological properties identical to those of Enterococcus faecium . Further, the nucleotide sequence of 16S rDNA of Isolate 82 was found to exhibit sequence identity of 99.8% to the sequence as shown in SEQ ID NO: 1. Thus, Isolate 82 was found to belong to the Enterococcus faecium species. In the following examples, Isolate 82 is referred to as the R30 strain.
  • freeze-dried bacterial strains of the R30 strain and the reference strain were prepared, and the freeze-dried bacterial strains were mixed with dextrin to adjust the viable bacterial count to 5.0 ⁇ 10 10 cfu/g.
  • the R30 strain administration group to which a probiotic preparation of the R30 strain was administered in an amount of 1 g/kg, the reference strain administration group to which a probiotic preparation of the reference strain was administered in an amount of 1 g/kg, and the dextrin administration group to which dextrin was administered in an amount of 1 g/kg were subjected to evaluation regarding fatigue-ameliorating effect as described above. The results thereof are shown in Table 2.
  • the R30 strain administration group exhibited a difference in the maximal running time before and after the administration of the test substance significantly greater than that of the dextrin administration group and the reference strain administration group. Thus, the R30 strain was found to have fatigue-ameliorating effect.
  • the R30 strain ingestion group that was to ingest 1 g of a probiotic preparation of the R30 strain prepared in Example 2 every day, the reference strain ingestion group that was to ingest 1 g of a probiotic preparation of the reference strain prepared in Example 2 every day, and the dextrin ingestion group that was to ingest 1 g of dextrin every day were subjected to evaluation of blood circulation-improving effect as described above. The results thereof are shown in Table 3.
  • the R30 strain ingestion group exhibited significantly higher average skin temperature after the ingestion of the test substance for a week than the dextrin ingestion group and the reference strain ingestion group. Thus, the R30 strain was found to have blood circulation-improving effect.
  • freeze-dried strains of R30 strain were prepared and a probiotic preparation thereof was prepared by mixing the freeze-dried bacterial strains with dextrin to adjust the viable bacterial count to 1.0 ⁇ 10 10 cfu/g.
  • the probiotic preparation of the R30 strain was mixed with the reference feed (i.e., the reference feed for early-stage broiler fattening, manufactured by Nippon Formula Feed Manufacturing Company Limited) in an amount of 0.3% by weight thereof, and the resulting test feed (i.e., a feed mixture containing R30 strain) was evaluated in terms of stool odor-reducing effect as described above. The results thereof are shown in Table 4.
  • the probiotic preparation of the R30 strain prepared in Example 4 was mixed with the reference feed (powder feed CE-2, manufactured by CLEA Japan, Inc.) in an amount of 0.1% by weight thereof to prepare a test feed (i.e., a feed mixture containing R30 strain), and the resulting test feed was evaluated in terms of growth-promoting effect as described above.
  • a test feed i.e., a feed mixture containing R30 strain
  • body weights of the group of mice to which the reference feed had been administered were measured and designated as 100%, and the proportion of the body weight of the group to which the feed mixture containing R30 strain had been administered to that of the group to which the reference feed had been administered was determined. The results thereof are shown in Table 5.
  • the body weight of the group to which the feed mixture containing R30 strain had been administered was 107% relative to the body weight of the group to which the reference feed had been administered. That is, the R30 strain was found to have growth-promoting effect.
  • the average amounts of feed ingested by the reference feed group and the group to which a feed mixture containing R30 strain had been administered during the test were 200 g and 199 g, respectively. That is, there were no significant differences. Thus, the R30 strain was found to have effects of improving feed efficiency.
  • the R30 strain has fatigue-ameliorating effect, blood circulation-improving effect, stool odor-reducing effect, and growth-promoting effect.
  • the present inventors decided to further screen for lactic acid bacteria of the Enterococcus faecium species having mycological properties identical to those of the R30 strain, exhibiting viability of 40% or higher when freeze-dried in the absence of a dispersion medium, and exhibiting 80% or higher bacterial viability in a probiotic preparation when stored at 40° C. for 4 months.
  • Lactic acid bacterial strains separated from food materials and the like were examined with the use of the API 20 Strep system (manufactured by Sysmex bioMerieux Co., Ltd.) to select 10 strains exhibiting mycological properties identical to those of the Enterococcus faecium species (the Enterococcus faecium strains 1 to 10).
  • the selected strains were subjected to the process of primary screening described above, three strains exhibiting 40% or higher viability when freeze-dried in the absence of a dispersion medium (i.e., Enterococcus faecium 1, Enterococcus faecium 2, and Enterococcus faecium 3) were subjected to secondary screening, and bacterial viability in a probiotic preparation when stored at 40° C. for 4 months was determined. The results thereof are shown in Table 6.
  • Enterococcus faecium 1 exhibits viability of 40% or higher when freeze-dried in the absence of a dispersion medium and viability of 80% or higher in a probiotic preparation when stored at 40° C. for 4 months.
  • the entire nucleotide sequence of 16S rDNA of Enterococcus faecium 1 was analyzed and, as a result, it was found to exhibit 99.9% sequence identity to the sequence as shown in SEQ ID NO: 1.
  • Enterococcus faecium 1 is referred to as the R28 strain.
  • the R28 strain has significant fatigue-ameliorating effect and blood circulation improvement, compared with Enterococcus faecium 2 and Enterococcus faecium 4.
  • lactic acid bacteria belonging to the Enterococcus faecium species were found to have particular mycological properties and excellent effects; that is, such lactic acid bacteria would exhibit viability of 40% or higher when freeze-dried in the absence of a dispersion medium and viability of 80% or higher in a probiotic preparation when stored at 40° C. for 4 months.
  • the R30 strain was cultured in 1 liter of MRS liquid medium at 37° C. for 24 hours. After the completion of culture, the bacterial strains were centrifuged, washed with 1 liter of sterile water, and then centrifuged again to separate the bacterial strains from sterile water.
  • a solution of a protective agent comprising sucrose, trehalose, sodium glutamate, histidine, and malic acid dissolved in amounts of 0, 1, 3, 10, 100, 1,000, and 10,000% by weight, respectively, relative to the dry weight of bacteria in the bacterial concentrate was added to the bacterial concentrate, the volume of which had been adjusted to 20 ml with sterile water, and the resultant was then freeze-dried.
  • the freeze-dried bacterial strains were mixed with dextrin to adjust the viable bacterial count to 1.0 ⁇ 10 10 cfu/g, a probiotic preparation was prepared, and bacterial viability in a probiotic preparation when stored at 40° C. for 4 months was determined.
  • the R28 strain was subjected to a similar experiment. The results thereof are shown in Table 9.
  • bacterial viability in a probiotic preparation when stored at 40° C. for 4 months was improved with the addition of 1% by weight or more protective agent relative to the dry weight of bacteria. Also, by increasing the amount of the protective agent, from 3% by weight to 10% by weight and then to 100% by weight, relative to the dry weight of bacteria, bacterial viability in a probiotic preparation when stored at 40° C. for 4 months was improved.
  • the present invention is applicable in the field of manufacture of food products such as functional food products and supplements, feeds, and pharmaceutical products utilizing lactic acid bacteria.

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