US20180264054A1 - Lactobacillus plantarum tci378 and its uses in losing fat and improving gastrointestinal functions - Google Patents

Lactobacillus plantarum tci378 and its uses in losing fat and improving gastrointestinal functions Download PDF

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US20180264054A1
US20180264054A1 US15/926,413 US201815926413A US2018264054A1 US 20180264054 A1 US20180264054 A1 US 20180264054A1 US 201815926413 A US201815926413 A US 201815926413A US 2018264054 A1 US2018264054 A1 US 2018264054A1
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lactobacillus plantarum
food
tci378
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Yung-Hsiang Lin
Chu-Han HUANG
Cheng-Yu Ho
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TCI Co Ltd
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TCI Co Ltd
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Assigned to TCI CO., LTD. reassignment TCI CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LIN, YUNG-HSIANG, HO, CHENG-YU, HUANG, CHU-HAN
Publication of US20180264054A1 publication Critical patent/US20180264054A1/en
Priority to US16/854,676 priority patent/US11273189B2/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
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • 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
    • 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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • 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
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • 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
    • C12R1/25—
    • 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
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • A23V2200/3204—Probiotics, living bacteria to be ingested for action in the digestive tract
    • 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
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/332—Promoters of weight control and weight loss
    • 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
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/169—Plantarum
    • A23Y2220/67—
    • 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
    • A61K2035/115—Probiotics
    • 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
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum

Definitions

  • the present invention relates to a novel Lactobacillus plantarum TCI378.
  • the present invention also relates to the uses of Lactobacillus plantarum TCI378 and/or its metabolites, particularly to the uses of Lactobacillus plantarum TCI378 and/or its metabolites in reducing fat formation, enhancing fat metabolism and/or improving gastrointestinal functions.
  • lipophilic probiotics had brought hope to people for using probiotics in losing fat, however, researchers thereafter indicated that none of the probiotics already discovered was able to live by degrading lipid.
  • Some gram-negative pathogens in human gastrointestinal tract could live by degrading lipid, while too much lipid consumption would lead to a dramatic proliferation of gram-negative pathogens and the lipopolysaccharide (LPS) presenting on the surface of pathogens may induce an inflammation in human body, thereby causing obesity, diabetes mellitus, cardiovascular diseases, and even cancers.
  • LPS lipopolysaccharide
  • Lactobacillus which is one of probiotics presenting in human, can be used in the manufacture of various fermentation foods. There are many bacteria of genus lactobacillus are classified as a food additive through the GRAS process (Generally Recognized As Safe) of FDA (Food and Drug Administration).
  • Inventors of the present invention isolated Lactobacillus plantarum TCI378 and found that the strain and its metabolites are effective in reducing fat formation, enhancing fat metabolism, and improving gastrointestinal functions.
  • An objective of the present invention is to provide a Lactobacillus plantarum TCI378, which was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen, DSMZ) under the accession number DSM 32451.
  • Another objective of the present invention is to provide a composition, comprising Lactobacillus plantarum TCI378 and its metabolites.
  • Still another objective of the present invention is to provide a use of Lactobacillus plantarum TCI378 and its metabolites in the manufacture of a composition.
  • the composition provided in accordance with the present invention is used for at least one of reducing fat formation, enhancing fat metabolism, blocking body fat formation, and improving gastrointestinal functions.
  • the composition is a pharmaceutical composition, a food composition, or a feed composition.
  • the pharmaceutical composition is provided as a form for oral administration.
  • the food composition is a health food, a dietary supplement, a functional food, a nutritional supplement or a special nutritional food.
  • Yet another objective of the present invention is to provide a method for losing fat and/or improving gastrointestinal functions, comprising administering to a subject in need an effective amount of the composition as described above.
  • FIGS. 1 to 4 show the results of adipocytes being treated with different media, wherein FIG. 1 are photographs showing the cells being treated and stained (the dark zones pointed by arrows are oil droplets), FIG. 2 shows, in comparison with the control group, the relative content of lipid in each other group (** presents the result is significantly different from that of the control group, p ⁇ 0.01; ## presents the result is significantly different from that of the blank group, p ⁇ 0.01), FIG.
  • FIG. 3 shows, in comparison with the control group, the expression level of PLIN1 gene in the cells of each other group (* presents the result is significantly different from that of the control group, p ⁇ 0.05; ** presents the result is significantly different from that of the control group, p ⁇ 0.01; # presents the result is significantly different from that of the blank group, p ⁇ 0.05), FIG.
  • FIGS. 5 to 9 illustrate the effects of Lactobacillus plantarum TCI378 on losing fat by comparing the indicators of subjects at the 0 th day (i.e., prior to taking capsules containing Lactobacillus plantarum TCI378) and at the 14 th day (i.e., after taking a capsule containing Lactobacillus plantarum TCI378 per day for 14 consecutive days), wherein FIG. 5 shows whole-body fat percentage (* presents the result is significantly different from that of the 0 th day, p ⁇ 0.05), FIG. 6 shows trunk fat percentage (* presents the result is significantly different from that of the 0 th day, p ⁇ 0.05), FIG.
  • FIG. 7 shows body weight (** presents the result is significantly different from that of the 0 th day, p ⁇ 0.01)
  • FIG. 8 shows waist circumference (* presents the result is significantly different from that of the 0 th day, p ⁇ 0.05)
  • FIG. 9 shows BMI value (**presents the result is significantly different from that of the 0 th day, p ⁇ 0.01);
  • FIG. 10 shows the defecation frequency of the subjects at the 0 th day and at the 14 th day, and thus illustrates the effects of Lactobacillus plantarum TCI378 on improving gastrointestinal functions.
  • Inventors of the present invention isolated a novel strain from kimchi, analyzed the strain by 16S rRNA gene sequence analysis, and identified the strain as a Lactobacillus plantarum based on the phylogenetic relationship, and thus, named the strain as Lactobacillus plantarum TCI378.
  • Lactobacillus plantarum TCI378 has been deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen, DSMZ) under the accession number DSM 32451.
  • the Lactobacillus plantarum TCI378 has a 16S rRNA fragment of SEQ ID NO: 1.
  • the present invention provides a Lactobacillus plantarum TCI378, a composition comprising Lactobacillus plantarum TCI378 and/or its metabolites, and a use of Lactobacillus plantarum TCI378 and/or its metabolites in the manufacture of a composition, wherein the composition is used for at least one of reducing fat formation, enhancing fat metabolism, blocking body fat formation and improving gastrointestinal functions.
  • Lactobacillus plantarum TCI378 adopted in accordance with the present invention could be generated by cultivating Lactobacillus plantarum TCI378 under an environment suitable for its growth.
  • a Lactobacillus plantarum TCI378's metabolites-containing liquid could be obtained by cultivating Lactobacillus plantarum TCI378 in a suitable medium, and then optionally removing the solids, including bacteria, from the medium.
  • any suitable medium could be chosen and used to perform the cultivation of Lactobacillus plantarum TCI378, as long as the medium can provide the desired nutrients (e.g., yeast extract, protein and glucose) and conditions (e.g., pH value) for the growth and metabolism of Lactobacillus plantarum TCI378.
  • the time period for cultivating Lactobacillus plantarum TCI378 is not specifically limited, as long as the time period is sufficient for the Lactobacillus plantarum TCI378 to complete at least one metabolic cycle.
  • Lactobacillus plantarum TCI378 was cultivated in a MRS broth for 18 hours to let the strain metabolize and generate metabolites.
  • the present invention could directly use a medium that has undergone the metabolic cycles of Lactobacillus plantarum TCI378, which contains the Lactobacillus plantarum TCI378 and its metabolites. Otherwise, the present invention could use a Lactobacillus plantarum TCI378's metabolites-containing liquid, from which the solids such as Lactobacillus plantarum TCI378 have been removed. Any suitable procedure could be employed to remove the solids, as long as the procedure does not adversely affect the desired effects of the metabolites generated after cultivation. Generally, physical approaches, such as centrifugal separation, filter filtration, precipitation and decantation, could be employed to remove the solids. Optionally, the above procedures could be repeated or combined to remove solids (including bacteria) from the medium as much as possible.
  • the pharmaceutical composition in accordance with the present invention could be provided in any suitable form without special limitations.
  • the pharmaceutical composition could be administered to a subject in need by an oral administration.
  • suitable carriers could be chosen and used to provide the pharmaceutical composition, wherein the carriers include excipients, diluents, auxiliaries, stabilizers, absorbent retarders, disintegrating agents, hydrotropic agents, emulsifiers, antioxidants, adhesives, binders, tackifiers, dispersants, suspending agents, lubricants, hygroscopic agents, etc.
  • the pharmaceutical composition provided in accordance with the present invention could comprise any pharmaceutically acceptable carrier that will not adversely affect the desired effects of the active ingredients (i.e., Lactobacillus plantarum TCI378 and its metabolites).
  • suitable carriers include, but is not limited to, water, saline, dextrose, glycerol, ethanol or its analogs, cellulose, starch, sugar bentonite, and combinations thereof.
  • the pharmaceutical composition could be provided in any suitable form for oral administration, such as in a form of a tablet (e.g., dragees), a pill, a capsule, granules, a pulvis, a fluidextract, a solution, a syrup, a suspension, a tincture, etc.
  • a tablet e.g., dragees
  • a pill e.g., a pill
  • a capsule granules, a pulvis, a fluidextract
  • a solution e.g., a syrup, a suspension, a tincture, etc.
  • the pharmaceutical composition provided in accordance with the present invention could be administered at various frequencies, such as once a day, multiple times a day, or once every few days, etc.
  • the ratio of amount of Lactobacillus plantarum TCI378 and/or its metabolites in the pharmaceutical composition provided in accordance with the present invention could be adjusted depending on the requirements of practical application.
  • the pharmaceutical composition could optionally further comprise one or more other active ingredient(s), or to be used in combination with a medicament comprising one or more other active ingredient(s), to further enhance the effects of the pharmaceutical composition, or to increase the application flexibility and application adaptability of preparation thus provided, as long as the other active ingredients do not adversely affect the desired effects of the active ingredients of the present invention (i.e., Lactobacillus plantarum TCI378 and its metabolites).
  • the food composition provided in accordance with the present invention could be a health food, a daily supplement, a functional food, a nutritional supplement, or a special nutritional food, and could be manufactured as dairy products, meat products, breads, pasta, cookies, troche, capsule, fruit juices, teas, sport drinks, nutritional drinks, etc., but is not limited thereby.
  • the health food, daily supplement, functional food, nutritional supplement, and special nutritional food provided in accordance with the present invention could be taken at various frequencies, such as once a day, multiple times a day, or once every few days, etc.
  • the amount of Lactobacillus plantarum TCI378 and/or its metabolites in the health food, daily supplement, functional food, nutritional supplement, and special nutritional food provided in accordance with the present invention could be adjusted, preferably to the amount that it should be taken daily, depending on the specific population.
  • the recommended daily dosage, use standards and use conditions for a specific population e.g., patients suffering from hyperlipidemia, pregnant woman, etc.
  • the recommendations for a use in combination with another food product or medicament could be indicated on the exterior package of the health food, daily supplement, functional food, nutritional supplement, and/or special nutritional food provided in accordance with the present invention.
  • it is suitable for the user to take the health food, daily supplement, functional food, nutritional supplement, and/or special nutritional food by him- or herself safely and securely without the instructions of a doctor, pharmacist, or related executive.
  • the types of Lactobacillus plantarum TCI378 and/or its metabolites are in line with the above descriptions.
  • the pharmaceutical composition, food composition, or feed composition provided in accordance with the present invention could further comprise a suitable amount of additives, such as a flavoring agent, a toner, or a coloring agent for enhancing the palatability and the visual perception of the pharmaceutical composition, food composition, or feed composition, and/or a buffer, a conservative, a preservative, an antibacterial agent, or an antifungal agent for improving the stability and storability of the pharmaceutical composition, food composition, or feed composition.
  • additives such as a flavoring agent, a toner, or a coloring agent for enhancing the palatability and the visual perception of the pharmaceutical composition, food composition, or feed composition
  • a buffer such as a conservative, a preservative, an antibacterial agent, or an antifungal agent for improving the stability and storability of the pharmaceutical composition, food composition, or feed composition.
  • the present invention also provides a method for reducing fat formation, enhancing fat metabolism, blocking body fat formation, and/or improving gastrointestinal functions, comprising administering to a subject in need an effective amount of a composition, wherein the composition comprises Lactobacillus plantarum TCI378 and/or its metabolites.
  • the types of Lactobacillus plantarum TCI378 and/or its metabolites are also in line with the above descriptions.
  • anaerobic cultivation i.e., the oxygen concentration is less than 5%
  • a colony was picked up by using a sterilized inoculating loop and subjected to the quadrant streaking method on a MSR agar, and then, the MSR agar was incubated in an anaerobic incubator at 37° C. until single colony appeared.
  • the strain selected from (A-1) was subjected to a phylogenetic analysis, and it was confirmed that the strain has a 16S rRNA fragment with a sequence as shown in SEQ ID NO: 1.
  • SEQ ID NO: 1 was compared to the online database of National Center for Biotechnology Information (NCBI), and it was noted that SEQ ID NO: 1 has an identity of about 99% to the 16S rRNA fragment of Lactobacillus plantarum strain.
  • NCBI National Center for Biotechnology Information
  • the strain selected from (A-1) was identified as a Lactobacillus plantarum strain according to the phylogenetic relationship and was named as Lactobacillus plantarum TCI378.
  • TCI378 The collection tube containing Lactobacillus plantarum TCI378 was thawed, and then TCI378 strain was inoculated into a MRS broth at an inoculum concentration of 1% (about 1 ⁇ 10 4 CFU/mL) and cultivated under an anaerobic condition at 37° C. for 18 hours, so as to provide a TCI378 cultivation liquid for use in the following experiments.
  • the TCI378 cultivation liquid obtained from (B-1) was inoculated into a MRS broth at an inoculum concentration of 1% (about 1 ⁇ 10 4 CFU/mL) and cultivated under an anaerobic condition at 37° C. for 18 hours. After the aforementioned cultivation, the broth was subjected to a centrifugation at a speed of 5,000 rpm for 5 minutes to remove the bacteria (i.e., TCI378 strain). And then, the supernatant of the centrifuged medium was filtered with a 0.2 ⁇ m filter to further remove the bacteria. The filtrate was collected form the filtration, and a TCI378 sample (containing metabolites of Lactobacillus plantarum TCI378) was thus obtained.
  • DMEM Dulbecco's Modified Eagle's Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • DMEM Dulbecco's Modified Eagle's Medium
  • a 24-well plate was injected with the Pre-adipocyte expansion medium of (C-1) in each well, and then, the mouse 3T3-L1 fibroblasts (purchased from Food Industry Research and Development Institute, BCRC 60071) were seeded into a 24-well plate at a seeding density of 8 ⁇ 10 4 cells/well and cultivated at 37° C. for 48 hours. Thereafter, the medium was replaced by the differentiation medium of (C-1) and the cultivation was continued for 4 days at 37° C. And then, the medium was further replaced by the adipocyte maintenance medium of (C-1) and the cultivation was continued for 8 days at 37° C., so as to provide adipocytes for use in the following experiments.
  • the adipocytes obtained from [Preparation example C-2] were divided into three groups, and separately cultivated with the following media at 37° C. for 48 hours:
  • Control group adipocyte maintenance medium Blank group adipocyte maintenance medium + 1% MRS broth TCI378 group adipocyte maintenance medium + 1% TCI378 sample
  • the cells of each group obtained from the cultivation of (1-1) were washed with phosphate-buffered saline (PBS) twice, and then, 1 mL of 10% methanol was added into each group to provide a mixture, and the mixture was placed at room temperature for 30 minutes to fix cells. Then, the methanol was removed and the cells were washed with PBS twice. 1 mL of 50% isopropanol was added into each group for 1 minute, and then removed. Finally, oil red O solution was added into each group and incubated at room temperature for 1 hour to stain the oil droplet of cells. The oil red O solution was removed, and the cells were washed with PBS twice. Then, the oil droplets of adipocytes were observed under a microscope and pictures were taken. The results are shown in FIG. 1 (the dark zones pointed by arrows are oil droplets).
  • the cells in each group were treated with 100% isopropanol for 10 minutes to dissolve the accumulated oil droplets. And then, cells in each group was subjected to an absorbance measurement at a wavelength of 510 nm to determine the content of lipid in the cells of each group. Finally, the relative content of lipid in the cells of blank group and TCI378 group was respectively calculated by using the result of control group as a basis (i.e., set the content of lipid in the control group as 1-fold). The results are shown in FIG. 2 .
  • Perilipin can protect the oil droplets of adipocytes from destruction. If the expression level of Perilipin can be decreased, the oil droplets of adipocytes will be destroyed more easily, thereby the fat metabolism can be enhanced. It is also known that the increased expression of GLUT4 gene will affect the ability of adipocytes on absorbing glucose, thereby leading to the production of fatty acids and causing obesity. Therefore, if the expression level of GLUT4 gene can be decreased, the fat formation will be reduced.
  • Lactobacillus plantarum TCI378 and its metabolites have effects on enhancing fat metabolism and/or reducing fat formation
  • the following analysis were conducted to investigate the influences of Lactobacillus plantarum TCI378 and its metabolites on the expression levels of PLIN1 (i.e., the gene which expresses Perilipin) and GLUT4 genes.
  • the cells of each group provided by (1-1) were harvested and subjected to a RNA extraction with an RNA purification kit (purchased from GeneMark). Then, the RNA was retro-transcribed into cDNA by using a SuperScriptTM Reverse Transcriptase kit (purchased from Invitrogen). Thereafter, the cDNA was subjected to a quantitative polymerase chain reaction (qPCR) by using a SYBR Green Master Mix (purchased from KAPA) and ABI StepOne PlusTM System to determine the expression levels of PLIN1 and GLUT4 genes in the cells of each group.
  • qPCR quantitative polymerase chain reaction
  • each subject was again invited to self-evaluate the frequency of feeling incomplete bowel movement, and measured the body weight, waist circumference, BMI value, whole-body fat percentage, and trunk fat percentage. Finally, the whole-body fat percentage, trunk fat percentage, body weight, waist circumference and BMI value of the ten subjects were averaged and shown in FIG. 5 to FIG. 9 respectively.
  • a statistical analysis was conducted on the self-evaluation results of feeling incomplete bowel movement, and the ratio of subjects “never,” “often,” “sometimes,” and “always” feeling incomplete bowel movement was respectively calculated by setting the total number of subjects as “1”. The result shows in FIG. 10 .
  • both whole-body fat percentage and trunk fat percentage were significantly decreased at the 14 th day (i.e., after taking the capsule containing Lactobacillus plantarum TCI378 for 14 consecutive days).
  • the body weight, waist circumference and BMI value were also decreased at the 14 th day.
  • the frequency of feeling incomplete bowel movement was reduced after the subjects took the capsule containing Lactobacillus plantarum TCI378 for 14 consecutive days.
  • the Lactobacillus plantarum TCI378 and its metabolites provided in accordance with the present invention can be used for at least one of reducing fat formation, enhancing fat metabolism, blocking body fat formation, and improving gastrointestinal functions.

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Abstract

Lactobacillus plantarum TCI378 is provided. The Lactobacillus plantarum TCI378 was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen, DSMZ) under the accession number DSM 32451. A method for losing fat and/or improving gastrointestinal functions is also provided. The method comprises administering to a subject in need an effective amount of a composition, wherein the composition comprises Lactobacillus plantarum TCI378 and/or its metabolites.

Description

    CROSS REFERENCE TO RELATED APPLICATIONS
  • This application claims priority to U.S. Provisional Application Ser. No. 62/473,785 filed on Mar. 20, 2017, in the United States Patent and Trademark Office, the disclosures of which are incorporated herein in their entirety by reference.
    • FIELD OF THE INVENTION
  • The present invention relates to a novel Lactobacillus plantarum TCI378. The present invention also relates to the uses of Lactobacillus plantarum TCI378 and/or its metabolites, particularly to the uses of Lactobacillus plantarum TCI378 and/or its metabolites in reducing fat formation, enhancing fat metabolism and/or improving gastrointestinal functions.
    • BACKGROUND OF THE INVENTION
  • People nowadays live in a lifestyle of sleeping and waking up irregularly, seldom eating at home, suffering from high stress, and being lack of exercise, and thus, the incidence rate of metabolic syndromes, hyperglycemia, hypertension, and hyperlipidemia is getting higher and higher. It is believed that the aforementioned course starts from the weakening of gastrointestinal functions and metabolism, leading to the accumulation of daily intake calories and thus the formation of fat, thereby resulting in constipation, obesity, chronic diseases, and other symptoms or diseases which severely threaten the health of people.
  • Although a regular exercise and a routine consumption of vegetables can improve gastrointestinal functions and enhance fat metabolism, for those being busy and seldom eating at home, there is still a need for more convenient approaches. In the recent years, more and more products (e.g., enzymes obtained from vegetables) associated with the gastrointestinal function improvement or the enhancement of fat metabolism have been developed. Also, probiotics have become a popular target of researches.
  • The discussion of lipophilic probiotics had brought hope to people for using probiotics in losing fat, however, researchers thereafter indicated that none of the probiotics already discovered was able to live by degrading lipid. Some gram-negative pathogens in human gastrointestinal tract could live by degrading lipid, while too much lipid consumption would lead to a dramatic proliferation of gram-negative pathogens and the lipopolysaccharide (LPS) presenting on the surface of pathogens may induce an inflammation in human body, thereby causing obesity, diabetes mellitus, cardiovascular diseases, and even cancers.
  • Lactobacillus, which is one of probiotics presenting in human, can be used in the manufacture of various fermentation foods. There are many bacteria of genus lactobacillus are classified as a food additive through the GRAS process (Generally Recognized As Safe) of FDA (Food and Drug Administration).
  • Inventors of the present invention isolated Lactobacillus plantarum TCI378 and found that the strain and its metabolites are effective in reducing fat formation, enhancing fat metabolism, and improving gastrointestinal functions.
    • SUMMARY OF THE INVENTION
  • An objective of the present invention is to provide a Lactobacillus plantarum TCI378, which was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen, DSMZ) under the accession number DSM 32451.
  • Another objective of the present invention is to provide a composition, comprising Lactobacillus plantarum TCI378 and its metabolites.
  • Still another objective of the present invention is to provide a use of Lactobacillus plantarum TCI378 and its metabolites in the manufacture of a composition.
  • The composition provided in accordance with the present invention is used for at least one of reducing fat formation, enhancing fat metabolism, blocking body fat formation, and improving gastrointestinal functions. The composition is a pharmaceutical composition, a food composition, or a feed composition. Preferably, the pharmaceutical composition is provided as a form for oral administration. The food composition is a health food, a dietary supplement, a functional food, a nutritional supplement or a special nutritional food.
  • Yet another objective of the present invention is to provide a method for losing fat and/or improving gastrointestinal functions, comprising administering to a subject in need an effective amount of the composition as described above.
  • The detailed technology and preferred embodiments implemented for the present invention are described in the following paragraphs accompanying the appended drawings for persons skilled in the art to well appreciate the features of the claimed invention.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1 to 4 show the results of adipocytes being treated with different media, wherein FIG. 1 are photographs showing the cells being treated and stained (the dark zones pointed by arrows are oil droplets), FIG. 2 shows, in comparison with the control group, the relative content of lipid in each other group (** presents the result is significantly different from that of the control group, p<0.01; ## presents the result is significantly different from that of the blank group, p<0.01), FIG. 3 shows, in comparison with the control group, the expression level of PLIN1 gene in the cells of each other group (* presents the result is significantly different from that of the control group, p<0.05; ** presents the result is significantly different from that of the control group, p<0.01; # presents the result is significantly different from that of the blank group, p<0.05), FIG. 4 shows, in comparison with the control group, the expression level of GLUT4 gene in the cells of each other group (** presents the result is significantly different from that of the control group, p<0.01; # presents the result is significantly different from that of the blank group, p<0.05), and wherein, the cells in control group were cultivated in an adipocyte maintenance medium, those in the blank group were cultivated in an adipocyte maintenance medium containing 1% MRS broth, and those in the TCI378 group were cultivated in an adipocyte maintenance medium containing 1% TCI378 sample;
  • FIGS. 5 to 9 illustrate the effects of Lactobacillus plantarum TCI378 on losing fat by comparing the indicators of subjects at the 0th day (i.e., prior to taking capsules containing Lactobacillus plantarum TCI378) and at the 14th day (i.e., after taking a capsule containing Lactobacillus plantarum TCI378 per day for 14 consecutive days), wherein FIG. 5 shows whole-body fat percentage (* presents the result is significantly different from that of the 0th day, p<0.05), FIG. 6 shows trunk fat percentage (* presents the result is significantly different from that of the 0th day, p<0.05), FIG. 7 shows body weight (** presents the result is significantly different from that of the 0th day, p<0.01), FIG. 8 shows waist circumference (* presents the result is significantly different from that of the 0th day, p<0.05), and FIG. 9 shows BMI value (**presents the result is significantly different from that of the 0th day, p<0.01);
  • FIG. 10 shows the defecation frequency of the subjects at the 0th day and at the 14th day, and thus illustrates the effects of Lactobacillus plantarum TCI378 on improving gastrointestinal functions.
    •  DESCRIPTION OF THE PREFERRED EMBODIMENT
  • The following paragraphs will describe some of the embodiments of the present invention in detail. However, without departing from the spirit of the present invention, the present invention may be embodied in various embodiments and should not be limited to the embodiments described in the specification. Unless otherwise indicated herein, the expression “a,” “an,” “the,” or the like recited in the specification of the present invention (especially in the claims) are intended to include both the singular and plural forms; the term “subject” recited in this specification refers to an animal, including mammalian (e.g., human), or non-mammalian (e.g., fowl or bird).
  • Inventors of the present invention isolated a novel strain from kimchi, analyzed the strain by 16S rRNA gene sequence analysis, and identified the strain as a Lactobacillus plantarum based on the phylogenetic relationship, and thus, named the strain as Lactobacillus plantarum TCI378. Lactobacillus plantarum TCI378 has been deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen, DSMZ) under the accession number DSM 32451. The Lactobacillus plantarum TCI378 has a 16S rRNA fragment of SEQ ID NO: 1.
  • Inventors of the present invention found that Lactobacillus plantarum TCI378 and its metabolites are effective in reducing fat formation, enhancing fat metabolism, and improving gastrointestinal functions. Therefore, the present invention provides a Lactobacillus plantarum TCI378, a composition comprising Lactobacillus plantarum TCI378 and/or its metabolites, and a use of Lactobacillus plantarum TCI378 and/or its metabolites in the manufacture of a composition, wherein the composition is used for at least one of reducing fat formation, enhancing fat metabolism, blocking body fat formation and improving gastrointestinal functions.
  • The metabolites of Lactobacillus plantarum TCI378 adopted in accordance with the present invention could be generated by cultivating Lactobacillus plantarum TCI378 under an environment suitable for its growth. For example, a Lactobacillus plantarum TCI378's metabolites-containing liquid could be obtained by cultivating Lactobacillus plantarum TCI378 in a suitable medium, and then optionally removing the solids, including bacteria, from the medium.
  • To provide the desired metabolites, any suitable medium could be chosen and used to perform the cultivation of Lactobacillus plantarum TCI378, as long as the medium can provide the desired nutrients (e.g., yeast extract, protein and glucose) and conditions (e.g., pH value) for the growth and metabolism of Lactobacillus plantarum TCI378. Furthermore, the time period for cultivating Lactobacillus plantarum TCI378 is not specifically limited, as long as the time period is sufficient for the Lactobacillus plantarum TCI378 to complete at least one metabolic cycle. For example, in one embodiment of the present invention, Lactobacillus plantarum TCI378 was cultivated in a MRS broth for 18 hours to let the strain metabolize and generate metabolites.
  • The present invention could directly use a medium that has undergone the metabolic cycles of Lactobacillus plantarum TCI378, which contains the Lactobacillus plantarum TCI378 and its metabolites. Otherwise, the present invention could use a Lactobacillus plantarum TCI378's metabolites-containing liquid, from which the solids such as Lactobacillus plantarum TCI378 have been removed. Any suitable procedure could be employed to remove the solids, as long as the procedure does not adversely affect the desired effects of the metabolites generated after cultivation. Generally, physical approaches, such as centrifugal separation, filter filtration, precipitation and decantation, could be employed to remove the solids. Optionally, the above procedures could be repeated or combined to remove solids (including bacteria) from the medium as much as possible.
  • Depending on the desired purpose, the pharmaceutical composition in accordance with the present invention could be provided in any suitable form without special limitations. For example, the pharmaceutical composition could be administered to a subject in need by an oral administration. Depending on the form and purpose, suitable carriers could be chosen and used to provide the pharmaceutical composition, wherein the carriers include excipients, diluents, auxiliaries, stabilizers, absorbent retarders, disintegrating agents, hydrotropic agents, emulsifiers, antioxidants, adhesives, binders, tackifiers, dispersants, suspending agents, lubricants, hygroscopic agents, etc.
  • As a dosage form for oral administration, the pharmaceutical composition provided in accordance with the present invention could comprise any pharmaceutically acceptable carrier that will not adversely affect the desired effects of the active ingredients (i.e., Lactobacillus plantarum TCI378 and its metabolites). Examples of suitable carriers include, but is not limited to, water, saline, dextrose, glycerol, ethanol or its analogs, cellulose, starch, sugar bentonite, and combinations thereof. The pharmaceutical composition could be provided in any suitable form for oral administration, such as in a form of a tablet (e.g., dragees), a pill, a capsule, granules, a pulvis, a fluidextract, a solution, a syrup, a suspension, a tincture, etc.
  • Depending on the need, age, body weight, and health conditions of the subject, the pharmaceutical composition provided in accordance with the present invention could be administered at various frequencies, such as once a day, multiple times a day, or once every few days, etc. The ratio of amount of Lactobacillus plantarum TCI378 and/or its metabolites in the pharmaceutical composition provided in accordance with the present invention could be adjusted depending on the requirements of practical application. In addition, the pharmaceutical composition could optionally further comprise one or more other active ingredient(s), or to be used in combination with a medicament comprising one or more other active ingredient(s), to further enhance the effects of the pharmaceutical composition, or to increase the application flexibility and application adaptability of preparation thus provided, as long as the other active ingredients do not adversely affect the desired effects of the active ingredients of the present invention (i.e., Lactobacillus plantarum TCI378 and its metabolites).
  • The food composition provided in accordance with the present invention could be a health food, a daily supplement, a functional food, a nutritional supplement, or a special nutritional food, and could be manufactured as dairy products, meat products, breads, pasta, cookies, troche, capsule, fruit juices, teas, sport drinks, nutritional drinks, etc., but is not limited thereby.
  • Depending on the need, age, body weight and health conditions of the subject, the health food, daily supplement, functional food, nutritional supplement, and special nutritional food provided in accordance with the present invention could be taken at various frequencies, such as once a day, multiple times a day, or once every few days, etc. The amount of Lactobacillus plantarum TCI378 and/or its metabolites in the health food, daily supplement, functional food, nutritional supplement, and special nutritional food provided in accordance with the present invention could be adjusted, preferably to the amount that it should be taken daily, depending on the specific population.
  • The recommended daily dosage, use standards and use conditions for a specific population (e.g., patients suffering from hyperlipidemia, pregnant woman, etc.), or the recommendations for a use in combination with another food product or medicament could be indicated on the exterior package of the health food, daily supplement, functional food, nutritional supplement, and/or special nutritional food provided in accordance with the present invention. Thus, it is suitable for the user to take the health food, daily supplement, functional food, nutritional supplement, and/or special nutritional food by him- or herself safely and securely without the instructions of a doctor, pharmacist, or related executive. In the food composition provided in accordance with the present invention, the types of Lactobacillus plantarum TCI378 and/or its metabolites are in line with the above descriptions.
  • Optionally, the pharmaceutical composition, food composition, or feed composition provided in accordance with the present invention could further comprise a suitable amount of additives, such as a flavoring agent, a toner, or a coloring agent for enhancing the palatability and the visual perception of the pharmaceutical composition, food composition, or feed composition, and/or a buffer, a conservative, a preservative, an antibacterial agent, or an antifungal agent for improving the stability and storability of the pharmaceutical composition, food composition, or feed composition.
  • The present invention also provides a method for reducing fat formation, enhancing fat metabolism, blocking body fat formation, and/or improving gastrointestinal functions, comprising administering to a subject in need an effective amount of a composition, wherein the composition comprises Lactobacillus plantarum TCI378 and/or its metabolites. The types of Lactobacillus plantarum TCI378 and/or its metabolites are also in line with the above descriptions.
  • The present invention will be further illustrated in detail with specific examples as follows. However, the following examples are provided only for illustrating the present invention and the scope of the present invention is not limited thereby. The scope of the present invention will be indicated in the appended claims.
    • EXAMPLES Preparation Examples
  • A. Selection and Identification of Lactobacillus plantarum TCI378
  • (A-1) Selection
  • The homogenate of kimchi (including kimchi and souse) was mixed with a MRS broth at a volume ratio of kimchi:MRS broth=1:100, and then the mixture thus provided was subjected to an anaerobic cultivation (i.e., the oxygen concentration is less than 5%) at a temperature of 37° C. for 18 hours. Thereafter, the cultivated mixture was spread onto a MSR agar, and then the agar was subjected to an anaerobic cultivation at 37° C., to form different colonies on the agar. Then, to confirm the uniqueness of the strain, a colony was picked up by using a sterilized inoculating loop and subjected to the quadrant streaking method on a MSR agar, and then, the MSR agar was incubated in an anaerobic incubator at 37° C. until single colony appeared.
  • (A-2) Identification
  • The strain selected from (A-1) was subjected to a phylogenetic analysis, and it was confirmed that the strain has a 16S rRNA fragment with a sequence as shown in SEQ ID NO: 1. SEQ ID NO: 1 was compared to the online database of National Center for Biotechnology Information (NCBI), and it was noted that SEQ ID NO: 1 has an identity of about 99% to the 16S rRNA fragment of Lactobacillus plantarum strain. Hence, depending on the phylogenetic identification, the strain selected from (A-1) was identified as a Lactobacillus plantarum strain according to the phylogenetic relationship and was named as Lactobacillus plantarum TCI378.
  • (A-3) Storage
  • The unique strain obtained from (A-1) was subjected to a liquid cultivation with a MRS broth to provide a bacteria cultivation liquid. Thereafter, glycerol was added into the bacteria cultivation liquid at a volume ratio of glycerol:bacteria cultivation liquid=1:3 to provide a mixture, and then, the mixture was placed into collection tubes and stored at a temperature of −80° C.
  • B. Lactobacillus plantarum TCI378 Activation and Sample Preparation
  • (B-1) Activation
  • The collection tube containing Lactobacillus plantarum TCI378 was thawed, and then TCI378 strain was inoculated into a MRS broth at an inoculum concentration of 1% (about 1×104 CFU/mL) and cultivated under an anaerobic condition at 37° C. for 18 hours, so as to provide a TCI378 cultivation liquid for use in the following experiments.
  • (B-2) Sample Preparation
  • The TCI378 cultivation liquid obtained from (B-1) was inoculated into a MRS broth at an inoculum concentration of 1% (about 1×104 CFU/mL) and cultivated under an anaerobic condition at 37° C. for 18 hours. After the aforementioned cultivation, the broth was subjected to a centrifugation at a speed of 5,000 rpm for 5 minutes to remove the bacteria (i.e., TCI378 strain). And then, the supernatant of the centrifuged medium was filtered with a 0.2 μm filter to further remove the bacteria. The filtrate was collected form the filtration, and a TCI378 sample (containing metabolites of Lactobacillus plantarum TCI378) was thus obtained.
  • C. Cultivation of Adipocytes
  • (C-1) Medium
    • (1) Pre-Adipocyte Expansion Medium:
  • 90% Dulbecco's Modified Eagle's Medium (DMEM; purchased from Gibco);
  • 10% Bovine Serum (purchased from Gibco); and
  • 1% Penicillin-streptomycin (purchased from Gibco).
    • (2) Differentiation Medium:
  • 90% Dulbecco's Modified Eagle's Medium (DMEM; purchased from Gibco);
  • 10% Fetal Bovine Serum (purchased from Gibco);
  • 1% Penicillin-streptomycin (purchased from Gibco);
  • 1.0 μmole/mL Dexamethasone (purchased from Sigma);
  • 0.5 mmole/mL Methylisobutylxanthine (purchased from Sigma); and
  • 1.0 μg/mL insulin (purchased from Sigma).
    • (3) Adipocyte Maintenance Medium:
  • 90% Dulbecco's Modified Eagle's Medium (DMEM; purchased from Gibco);
  • 10% Fetal Bovine Serum (purchased from Gibco);
  • 1% Penicillin-streptomycin (purchased from Gibco); and
  • 1.0 μg/mL insulin (purchased from Sigma).
  • (C-2) Differentiation of Adipocytes
  • A 24-well plate was injected with the Pre-adipocyte expansion medium of (C-1) in each well, and then, the mouse 3T3-L1 fibroblasts (purchased from Food Industry Research and Development Institute, BCRC 60071) were seeded into a 24-well plate at a seeding density of 8×104 cells/well and cultivated at 37° C. for 48 hours. Thereafter, the medium was replaced by the differentiation medium of (C-1) and the cultivation was continued for 4 days at 37° C. And then, the medium was further replaced by the adipocyte maintenance medium of (C-1) and the cultivation was continued for 8 days at 37° C., so as to provide adipocytes for use in the following experiments.
    • Example 1: Effects of Lactobacillus plantarum TCI378 and its Metabolites on Losing Fat
  • (1-1) Cell Treatment
  • The adipocytes obtained from [Preparation example C-2] were divided into three groups, and separately cultivated with the following media at 37° C. for 48 hours:
  • Group Condition of the medium
    Control group adipocyte maintenance medium
    Blank group adipocyte maintenance medium + 1% MRS broth
    TCI378 group adipocyte maintenance medium + 1% TCI378 sample
  • (1-2) Lipid (Oil Red 0) Staining
  • The cells of each group obtained from the cultivation of (1-1) were washed with phosphate-buffered saline (PBS) twice, and then, 1 mL of 10% methanol was added into each group to provide a mixture, and the mixture was placed at room temperature for 30 minutes to fix cells. Then, the methanol was removed and the cells were washed with PBS twice. 1 mL of 50% isopropanol was added into each group for 1 minute, and then removed. Finally, oil red O solution was added into each group and incubated at room temperature for 1 hour to stain the oil droplet of cells. The oil red O solution was removed, and the cells were washed with PBS twice. Then, the oil droplets of adipocytes were observed under a microscope and pictures were taken. The results are shown in FIG. 1 (the dark zones pointed by arrows are oil droplets).
  • After the above observation and recording of oil droplets were completed, the cells in each group were treated with 100% isopropanol for 10 minutes to dissolve the accumulated oil droplets. And then, cells in each group was subjected to an absorbance measurement at a wavelength of 510 nm to determine the content of lipid in the cells of each group. Finally, the relative content of lipid in the cells of blank group and TCI378 group was respectively calculated by using the result of control group as a basis (i.e., set the content of lipid in the control group as 1-fold). The results are shown in FIG. 2.
  • As shown in FIG. 1, in comparison with the control group, the oil droplets in the TCI378 group were smaller, while the oil droplets in the blank group did not change significantly. As shown in FIG. 2, no matter in comparison with the control group and blank group, the relative content of lipid in the cells of TCI378 group was decreased about 15%. These results indicate that Lactobacillus plantarum TCI378 and its metabolites are effective in decreasing the content of lipid in adipocytes.
  • (1-3) Analysis of Gene Expression
  • It is known that the protein, Perilipin, can protect the oil droplets of adipocytes from destruction. If the expression level of Perilipin can be decreased, the oil droplets of adipocytes will be destroyed more easily, thereby the fat metabolism can be enhanced. It is also known that the increased expression of GLUT4 gene will affect the ability of adipocytes on absorbing glucose, thereby leading to the production of fatty acids and causing obesity. Therefore, if the expression level of GLUT4 gene can be decreased, the fat formation will be reduced. To confirm whether Lactobacillus plantarum TCI378 and its metabolites have effects on enhancing fat metabolism and/or reducing fat formation, the following analysis were conducted to investigate the influences of Lactobacillus plantarum TCI378 and its metabolites on the expression levels of PLIN1 (i.e., the gene which expresses Perilipin) and GLUT4 genes.
  • The cells of each group provided by (1-1) were harvested and subjected to a RNA extraction with an RNA purification kit (purchased from GeneMark). Then, the RNA was retro-transcribed into cDNA by using a SuperScript™ Reverse Transcriptase kit (purchased from Invitrogen). Thereafter, the cDNA was subjected to a quantitative polymerase chain reaction (qPCR) by using a SYBR Green Master Mix (purchased from KAPA) and ABI StepOne Plus™ System to determine the expression levels of PLIN1 and GLUT4 genes in the cells of each group. Then, the relative expression level of PLIN1 and GLUT4 genes of the TCI378 group were calculated by using the result of the control group as a basis (i.e., set the expression level of the control group as 1-fold). The results are shown in FIG. 3 and FIG. 4.
  • As shown in FIG. 3 and FIG. 4, no matter in comparison with the control group and blank group, the expression levels of PLIN1 and GLUT4 genes in the TCI378 group were significantly lower.
  • The results of (1-2) and (1-3) indicate that Lactobacillus plantarum TCI378 and its metabolites in accordance with the present invention are effective in reducing fat formation and enhancing fat metabolism, and thus can be used for decreasing the content of lipid in adipocytes, thereby blocking body fat formation.
    • Example 2: Effects of Lactobacillus plantarum TCI378 on Losing Fat and Improving Gastrointestinal Functions
  • Ten volunteered subjects, who have a body fat percentage more than 27% and did not take any diet foods or drugs, were recruited. Prior to conducting the experiment (i.e., before taking the capsule containing Lactobacillus plantarum TCI378), each subject was invited to self-evaluate the frequency of feeling incomplete bowel movement (selecting one form the group consisting of “never,” “often,” “sometimes,” and “always”), and measure the body weight, waist circumference, BMI value, whole-body fat percentage, and trunk fat percentage. Then, each subject took a capsule containing Lactobacillus plantarum TCI378 (5×109 CFU/capsule) a day for 14 consecutive days (i.e., each subject took the capsule 14 times). Thereafter, each subject was again invited to self-evaluate the frequency of feeling incomplete bowel movement, and measured the body weight, waist circumference, BMI value, whole-body fat percentage, and trunk fat percentage. Finally, the whole-body fat percentage, trunk fat percentage, body weight, waist circumference and BMI value of the ten subjects were averaged and shown in FIG. 5 to FIG. 9 respectively. In addition, a statistical analysis was conducted on the self-evaluation results of feeling incomplete bowel movement, and the ratio of subjects “never,” “often,” “sometimes,” and “always” feeling incomplete bowel movement was respectively calculated by setting the total number of subjects as “1”. The result shows in FIG. 10.
  • As shown in FIG. 5 to FIG. 9, in comparison with those at the 0th day (i.e., prior to taking the capsule containing Lactobacillus plantarum TCI378), both whole-body fat percentage and trunk fat percentage were significantly decreased at the 14th day (i.e., after taking the capsule containing Lactobacillus plantarum TCI378 for 14 consecutive days). In comparison with those at the 0th day, the body weight, waist circumference and BMI value were also decreased at the 14th day. In addition, as shown in FIG. 10, the frequency of feeling incomplete bowel movement was reduced after the subjects took the capsule containing Lactobacillus plantarum TCI378 for 14 consecutive days.
  • The above results show that, after the subjects took the capsule containing Lactobacillus plantarum TCI378, their fat accumulation and gastrointestinal functions are both improved, and their body weight, BMI value and waist circumference were decreased thereby. Therefore, the Lactobacillus plantarum TCI378 and its metabolites provided in accordance with the present invention can be used for at least one of reducing fat formation, enhancing fat metabolism, blocking body fat formation, and improving gastrointestinal functions.
    • Deposit of Biological Material
    • Depository institute: German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen, DSMZ)
    • Address: Inhoffenstraße 7 B, 38124 Braunschweig, GERMANY
    • Date: 2017 Mar. 13
    • Deposited biological material: Lactobacillus plantarum TCI378
    • Accession number: DSM32451

Claims (14)

What is claimed is:
1. A Lactobacillus plantarum TCI378, which was deposited at German Collection of Microorganisms and Cell Cultures (Deutsche Sammlung von Mikroorganismen and Zellkulturen, DSMZ) under the accession number DSM 32451.
2. A composition, comprising the Lactobacillus plantarum TCI378 as claimed in claim 1 and/or its metabolites.
3. The composition as claimed in claim 2, which is for at least one of reducing fat formation, enhancing fat metabolism, blocking body fat formation, and improving gastrointestinal functions.
4. The composition as claimed in claim 3, which is a pharmaceutical composition, a food composition, or a feed composition.
5. The composition as claimed in claim 4, which is a pharmaceutical composition, and the pharmaceutical composition is for oral administration.
6. The composition as claimed in claim 4, which is a food composition, and the food composition is a health food, a daily supplement, a functional food, a nutritional supplement, or a special nutritional food.
7. A method for losing fat, comprising administering to a subject in need an effective amount of the composition as claimed in claim 2.
8. The method as claimed in claim 7, wherein the composition is administered to the subject as a pharmaceutical composition, a food composition, or a feed composition, thereby achieving at least one of reducing fat formation, enhancing fat metabolism, and blocking body fat formation.
9. The method as claimed in claim 8, wherein the composition is administered to the subject as a pharmaceutical composition.
10. The method as claimed in claim 9, wherein the pharmaceutical composition is administered to the subject by oral administration.
11. A method for improving gastrointestinal functions, comprising administering to a subject in need an effective amount of the composition as claimed in claim 2.
12. The method as claimed in claim 11, wherein the composition is administered to the subject as a pharmaceutical composition, a food composition, or a feed composition, thereby relieving incomplete bowel movement.
13. The method as claimed in claim 12, wherein the composition is administered to the subject as a pharmaceutical composition.
14. The method as claimed in claim 13, wherein the pharmaceutical composition is administered to the subject by oral administration.
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CN110684701A (en) * 2019-11-19 2020-01-14 西南大学 Lactobacillus plantarum S58 and application thereof in preparation of products for relieving obesity
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