WO2021251575A1 - Nouvelle souche de pediococcus pentosaceus et composition alimentaire pour la prévention ou l'amélioration de l'obésité ou d'une stéatose hépatique comprenant un produit fermenté à base de lactosérum associé - Google Patents

Nouvelle souche de pediococcus pentosaceus et composition alimentaire pour la prévention ou l'amélioration de l'obésité ou d'une stéatose hépatique comprenant un produit fermenté à base de lactosérum associé Download PDF

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WO2021251575A1
WO2021251575A1 PCT/KR2020/018781 KR2020018781W WO2021251575A1 WO 2021251575 A1 WO2021251575 A1 WO 2021251575A1 KR 2020018781 W KR2020018781 W KR 2020018781W WO 2021251575 A1 WO2021251575 A1 WO 2021251575A1
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strain
pediococcus pentosaceus
obesity
whey
fatty liver
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PCT/KR2020/018781
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English (en)
Korean (ko)
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윤요한
임상동
하지명
김슬기
강주현
최유나
홍상필
진영욱
이명기
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숙명여자대학교산학협력단
한국식품연구원
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Publication of WO2021251575A1 publication Critical patent/WO2021251575A1/fr

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    • 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
    • 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
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/19—Dairy proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/01—Hydrolysed proteins; Derivatives thereof
    • 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
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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

Definitions

  • the present invention is Pediococcus pentosaceus ( Pediococcus pentosaceus ) KI13 strain of accession number KACC 81116BP showing anti-obesity activity during whey fermentation; Food composition, food additive composition, pharmaceutical composition and feed composition for the prevention or improvement of obesity or fatty liver disease comprising a whey fermented product bioconversion to the strain; A method for preventing or treating obesity or fatty liver disease comprising administering the pharmaceutical composition to an individual; and to the use of a composition comprising a whey ferment bioconverted to a Pediococcus pentosaceus strain.
  • Whey is a by-product formed when milk is processed into cheese, and refers to an aqueous solution excluding curd, that is, a coagulation product produced by adding an acid or a curdging enzyme to skim milk. In the past, whey was considered only a by-product in the production of cheese, and a large amount was discarded every year.
  • bioconversion technology also called bioconversion technology, refers to the process of producing useful substances with higher added value from precursors by using the enzymatic function of microorganisms. That is, by inducing structural changes of physiologically active substances through biological methods such as microbial fermentation or enzyme treatment, it has recently been spotlighted as a technology to increase the content of specific active ingredients, improve absorption, or induce the generation of new active ingredients. have. However, in spite of the research on these technologies, the actual situation that leads to the production of the final product was insignificant.
  • the inventor of the present invention reconsidered the possibility of using whey, which has been previously discarded in large quantities, and at the same time, made a diligent effort to produce a functional substance with higher added value from whey, Pediococcus pentosase separated from kimchi with respect to whey powder.
  • the present invention was completed by confirming that the whey fermented product inoculated and cultured with the mouse KI13 strain (Accession No.: KACC 81116BP) can help obesity and fatty liver disease through bioconversion.
  • the bioconverted whey fermented product with Pediococcus pentosaceus KI13 strain reduces the rate of weight gain through inhibition of ⁇ -amylase and ⁇ -glucosidase activity and mast cell differentiation, and reduces obesity and obesity in liver and adipose tissue. It was confirmed that it reduces the expression of inflammation-related genes and proteins, and a composition comprising the same may be usefully used as a food or pharmaceutical composition having an effect of preventing, improving or treating obesity and fatty liver disease.
  • the present invention aims to solve the above problems and other problems related thereto.
  • An exemplary object of the present invention is to provide a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP.
  • Another exemplary object of the present invention is to provide a food composition for the prevention or improvement of obesity or fatty liver disease, comprising the whey fermented product bioconverted to the strain as an active ingredient.
  • Another exemplary object of the present invention is to provide a food additive composition comprising the bioconverted whey fermented product as an active ingredient into the strain.
  • Another exemplary object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity or fatty liver disease, comprising as an active ingredient a whey ferment bioconverted into the strain.
  • Another exemplary object of the present invention is to provide a method for preventing or treating obesity or fatty liver disease, comprising administering the pharmaceutical composition to a subject.
  • Another exemplary object of the present invention is to provide a use of a composition comprising a whey ferment bioconverted into a Pediococcus pentosaceus strain for the prevention or treatment of obesity or fatty liver disease.
  • Another exemplary object of the present invention is to provide a use of a composition comprising a fermented whey bioconverted into a Pediococcus pentosaceus strain for the manufacture of a medicament for the prevention or treatment of obesity or fatty liver disease. .
  • Another exemplary object of the present invention is to provide a feed composition for preventing or improving obesity or fatty liver disease, comprising, as an active ingredient, a bioconverted whey fermented product into a Pediococcus pentosaceus strain.
  • One aspect of the present invention for achieving the above object provides a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP that exhibits anti-obesity activity during whey fermentation. Specifically, the strain is characterized in that it exhibits anti-obesity activity during whey fermentation.
  • "Pediococcus pentosaceus KI13 strain” is isolated from kimchi and exhibits whey fermentation ability and obesity suppression ability during whey fermentation, and as of May 14, 2020, the National Academy of Agricultural Sciences (KACC) ) and was given accession number KACC 81116BP.
  • a food composition for preventing or improving obesity or fatty liver disease comprising a bioconverted whey fermented product as an active ingredient.
  • the whey fermented product may be bioconverted into a Pediococcus pentosaceus strain, and more specifically, may be bioconverted into a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP.
  • whey refers to an aqueous solution excluding curd, which is a by-product formed when milk is processed into cheese, that is, a coagulation product produced by adding an acid or a curd enzyme to skim milk.
  • the whey powder is dissolved in PBS to a concentration of 10% and sterilized at 80° C. for 1 minute, and then the P. pentosaceus KI13 strain is inoculated and fermented to obtain a bioconversion product, that is, a whey fermented product. prepared.
  • the term "fermented product” refers to the result of enzymatic or metabolic degradation using microorganisms.
  • the product cultured after inoculating the Pediococcus pentosaceus KI13 strain into whey. can mean Also, in the present specification, 'fermented product' or 'fermented whey' may be used interchangeably with 'bioconversion product'.
  • bioconversion is also referred to as bioconversion, and refers to the production of useful substances with higher added value from precursors by using the enzymatic function of microorganisms. That is, by inducing a structural change of a physiologically active material through a biological method such as microbial fermentation or enzyme treatment, it is possible to induce an increase in the content of an active ingredient, an improvement in absorption rate, and the generation of a new active ingredient.
  • the bioconversion product may refer to a whey fermented product cultured after inoculating the Pediococcus pentosaceus KI13 strain into whey, and the prevention, improvement or therapeutic effect of obesity and fatty liver disease through the bioconversion product can be achieved
  • the term “obesity” refers to a state in which adipose tissue is excessive in the body, and furthermore, it may mean to include all of the various complications caused by obesity.
  • the term “fatty liver disease” includes all fatty liver caused by obesity, alcohol, and diseases and complications derived therefrom, and specifically, fatty liver and nonalcoholic steatohepatitis. , but may mean liver cirrhosis, cirrhosis or liver cancer, but is not particularly limited thereto.
  • prevention refers to any action that suppresses or delays symptoms related to obesity or fatty liver disease by administration of the composition of the present invention
  • improvement in the present invention refers to obesity or fatty liver disease by applying the composition of the present invention. It means to show the effect of alleviating the symptoms of the disease.
  • the food may include health functional (sexual) food.
  • health functional food refers to a food manufactured and processed using raw materials or ingredients useful for the human body.
  • the “functionality” means to obtain a useful effect for health purposes such as regulating nutrients or physiological action with respect to the structure and function of the human body.
  • the health functional food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and components commonly added in the art during manufacturing.
  • the formulation of the health functional food is also recognized as a health functional food, it may be manufactured without limitation.
  • the food composition of the present invention can be prepared in various types of dosage forms, and unlike general drugs, it has the advantage of not having side effects that may occur when taking the drug for a long time using food as a raw material.
  • the food composition of the present invention may be prepared in any form, and specifically, health functional food preparations such as tablets, capsules, pills, granules, liquids, powders, pieces, pastes, syrups, gels, jellies, bars, etc.; It may be one or more formulations selected from the group consisting of beverages, gums and candies, but is not particularly limited thereto.
  • the food composition of the present invention may contain food additives in addition to the active ingredient.
  • Food additives can be generally understood as substances that are mixed or infiltrated with food in manufacturing, processing or preserving food, and their safety must be ensured since they are consumed daily and for a long period of time with food.
  • the food additives are divided into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, thickeners, etc. in terms of function, and are not particularly limited as long as the food composition of the present invention meets the intended purpose.
  • the food composition of the present invention may contain physiologically active substances or minerals known in the art for the purpose of functional and nutritional supplementation and guaranteed stability as food additives in addition to the food additives.
  • the physiologically active substances or minerals are not particularly limited as long as the food composition of the present invention meets the intended purpose.
  • the food additives as described above may be included in an effective amount to achieve the purpose of the addition according to the type of product, and with respect to other food additives that may be included in the food composition of the present invention, the food additives of each country or the Food Additives Ordinance.
  • a food additive composition comprising a bioconverted whey fermented product as an active ingredient.
  • the whey fermented product may be bioconverted into a Pediococcus pentosaceus strain, and more specifically, may be bioconverted into a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP.
  • the terms Pediococcus pentosaceus KI13 strain, bioconversion, whey and ferment are as described above.
  • the term "food additive” is used for the purpose of improving preservation, quality, nutritional enhancement, flavor and appearance, etc. in manufacturing, processing or preserving food, added to food, mixing, infiltration and other methods. It can mean a substance that becomes The food additive may be added to the active ingredient of the present invention as it is or used together with other foods or food ingredients, and the mixing amount of the active ingredient may be appropriately determined by a person skilled in the art according to the purpose of use.
  • Pediococcus pentosaceus of the present invention P. pentosaceus
  • Food additive composition comprising a bioconverted whey fermented product to KI13 strain, in addition to the food or food ingredient added, the prevention and improvement of obesity and fatty liver disease can do.
  • a pharmaceutical composition for preventing or treating obesity or fatty liver disease comprising a bioconverted whey fermented product as an active ingredient.
  • the whey fermented product may be bioconverted into a Pediococcus pentosaceus strain, and more specifically, may be bioconverted into a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP.
  • the term Pediococcus pentosaceus KI13 strain, bioconversion, whey, fermented product, obesity, fatty liver disease and prevention are the same as described above.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of a pharmaceutical composition in addition to the whey ferment bioconverted to Pediococcus pentosaceus KI13 strain. have.
  • carrier examples include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used, but is not particularly limited thereto.
  • the pharmaceutical composition of the present invention is selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents and freeze-drying agents according to a conventional method, respectively. It may have any one dosage form, and the dosage form may be in various forms, either oral or parenteral. In the case of formulation, it may be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants, but is not limited thereto. Specifically, tablets, pills, powders, granules, capsules, etc.
  • the solid preparation may be used in the solid preparation for oral administration, and the solid preparation may include at least one or more excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. may be used. .
  • excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc may be used, but the present invention is not limited thereto.
  • liquid formulations for oral administration suspensions, internal solutions, emulsions, syrups, etc. may be used, and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin.
  • a sterile aqueous solution, a non-aqueous solution, a suspension, an emulsion, a freeze-dried formulation, or a suppository may be used as a formulation for parenteral administration.
  • a non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used, but is not limited thereto.
  • the optimal amount and dosing interval of individual administration of the pharmaceutical composition of the present invention will be determined by the nature and extent of oral disease or periodontal disease, dosage form, route and site, and the age and health condition of the subject, and the doctor will ultimately use it. It will be appreciated by those skilled in the art that appropriate dosing will be determined. Such dosing may be repeated as often as appropriate, and the dosage and frequency may be altered or reduced in accordance with ordinary clinical practice.
  • the administration route of the composition of the present invention may be administered through any general route as long as it can achieve the object of the present invention.
  • the pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, orally, but is not limited thereto.
  • treatment refers to any action in which symptoms of obesity or fatty liver disease are improved or beneficially changed by administration of the composition of the present invention.
  • Another aspect of the present invention for achieving the above object provides a method for preventing or treating obesity or fatty liver disease, comprising administering the pharmaceutical composition to an individual in a pharmaceutically effective amount.
  • the bioconverted whey fermented product to the Pediococcus pentosaceus strain provided in the present invention can be used as an active ingredient for the prevention or treatment of obesity or fatty liver disease, so the composition can reduce obesity or fatty liver disease. It can be used to prevent or treat.
  • the term "individual" of the present invention includes, without limitation, mammals including rats, livestock, humans, and the like that are likely to or have developed obesity or fatty liver disease.
  • the administration route of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue.
  • the pharmaceutical composition of the present invention is not particularly limited thereto, and may be administered through routes such as oral administration and rectal administration, and in some cases may be administered by other routes depending on the purpose.
  • Another aspect of the present invention for achieving the above object provides the use of a composition comprising a whey fermented product bioconverted to a Pediococcus pentosaceus strain for the prevention or treatment of obesity or fatty liver disease.
  • Another aspect of the present invention for achieving the above object is a composition comprising a whey fermented product bioconverted to Pediococcus pentosaceus strain for the production of a medicament for the prevention or treatment of obesity or fatty liver disease. provide use.
  • Another aspect of the present invention for achieving the above object provides a feed composition for preventing or improving obesity or fatty liver disease, comprising as an active ingredient a bioconverted whey fermented product into a Pediococcus pentosaceus strain .
  • the term "feed” refers to any natural or artificial diet, one meal, etc., or a component of the one meal meal for animals to eat, ingest and digest, or various types of feed known in the art. It can be manufactured as, and preferably, a concentrated feed, roughage and/or special feed may be included.
  • Concentrated feed includes seed fruits including grains such as wheat, oats, and corn, bran including rice bran, wheat bran, barley bran, etc.
  • Fish-Soluble a by-product obtained from oil extraction
  • Fish-Soluble is concentrated fish meal, fish waste, and fresh liquid obtained from fish, fish meal, fish waste, and residual starch, which is the main component of starch residue, which is the remainder after removing starch from sweet potatoes and potatoes.
  • Animal feed such as dried whey, yeast, chlorella, and seaweeds, which are fish soluble), meat meal, blood meal, feather meal, skim milk powder, cheese from milk, and whey, which is the remainder of the production of casein from skim milk, but limited thereto doesn't happen
  • Forage includes raw grass feed such as wild grasses, grasses and green cuts, turnips for feed, beets for feed, root vegetables such as ruther bearger, a type of turnip, raw grass, green cut crops, grains, etc.
  • raw grass feed such as wild grasses, grasses and green cuts, turnips for feed, beets for feed
  • root vegetables such as ruther bearger, a type of turnip, raw grass, green cut crops, grains, etc.
  • Examples include, but are not limited to, silage, wild grass, hay cut and dried, straw from breeders, and leaves from legumes.
  • mineral feed such as oyster shell and rock salt
  • urea feed such as urea or its derivative diureide isobutane
  • natural feed ingredients are added to the formulated feed to supplement the ingredients that are likely to be lacking when only natural feed ingredients are mixed, or to increase the storage of feed.
  • feed additives and dietary supplements which are substances added in trace amounts, but are not limited thereto.
  • the feed composition for preventing or ameliorating obesity or fatty liver disease according to the present invention is obtained by adding fermented whey bioconverted to Pediococcus pentosaceus strain in an appropriate effective concentration range according to various feed preparation methods known in the art. can be manufactured by
  • the feed composition according to the present invention is not particularly limited as long as it is an individual for the purpose of preventing or improving obesity or fatty liver disease, and any type of feed composition is applicable.
  • non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cattle, sheep, pigs, goats, etc., birds and fish can be applied to any object.
  • the whey fermented product bioconverted to the P. pentosaceus KI13 strain reduces the weight gain through inhibition of pancreatic lipase activity, inhibition of ⁇ -amylase and ⁇ -glucosidase activity, and inhibition of mast cell differentiation, and It is characterized in that it exhibits the prevention, improvement or therapeutic effect of obesity and fatty liver disease by reducing the expression of genes and proteins related to obesity and inflammation in adipose tissue.
  • FIG. 1 shows a phylogenetic diagram for Pediococcus pentosaceus KI13 strain according to the present invention.
  • Figure 2 shows the results of comparison/analysis of weight gain after oral administration of a whey bioconversion product by P. pentosaceus KI13 to mice fed a general diet or a high-fat diet (NF-PBS: general diet + PBS, HF-PBS: high-fat diet+PBS, HF-KI 13W: high-fat+whey+ P. pentosaceus KI13, HF-KI 13B: high-fat+ P. pentosaceus KI13 bioconversion products).
  • NF-PBS general diet + PBS
  • HF-PBS high-fat diet+PBS
  • HF-KI 13W high-fat+whey+ P. pentosaceus KI13
  • HF-KI 13B high-fat+ P. pentosaceus KI13 bioconversion products
  • Figure 3 shows the liver, epididymal fat, and lateral abdominal adipose tissue of mice when a whey bioconversion product by P. pentosaceus KI13 or the like is orally administered to a mouse fed a general diet or a high-fat diet.
  • Figure 4 shows the results of checking the liver level and LDL-cholesterol level in the blood when the whey bioconversion product by P. pentosaceus KI13 or the like was orally administered to mice fed a general diet or a high-fat diet.
  • FIG. 5 shows the results of analyzing the expression level of obesity-related genes in the liver when a whey bioconversion product by P. pentosaceus KI13 or the like was orally administered to mice fed a general diet or a high-fat diet.
  • FIG. 6 shows the results of analysis of the expression level of inflammation-related proteins in the liver when a whey bioconversion product by P. pentosaceus KI13 or the like was orally administered to mice fed a general diet or a high-fat diet.
  • FIG. 7 shows the results of analyzing the expression level of obesity-related genes in adipose tissue when a whey bioconversion product by P. pentosaceus KI13, etc., was orally administered to mice fed a general diet or a high-fat diet.
  • FIG. 8 shows a microscopic photograph of hepatocytes in the case of oral administration of a whey bioconversion product by P. pentosaceus KI13 to mice fed a general diet or a high-fat diet.
  • 9 is a microscopic view of epididymal fat when orally administered with a whey bioconversion product by P. pentosaceus KI13 to mice fed a general diet or a high-fat diet.
  • Example 1 Pediococcus pentosaceus Isolation and identification of KI13 strain
  • the strain was finally selected through the evaluation of the obesity suppression ability (Examples 3 and 4) for the whey fermented product (bioconversion product) using 98 strains.
  • the isolated and selected strains were subcultured twice or more in MRS liquid medium to increase their activity, and then used in the experiment. Identification of the strain was carried out by the method of Hammes et al. (1992).
  • the purely isolated strain was subjected to Gram staining, sporulation, aerobic and anaerobic growth, catalase production, growth at 15 ° C and 45 ° C, gas production from glucose, ammonia production from arginine, microscopic observation, and the like.
  • the strain of the present invention shows 99% (1549/1554) homology to the Pediococcus pentosaceus species, and the novel microorganism of the present invention belongs to the Pediococcus pentosaceus species.
  • the strain was confirmed. Therefore, the finally selected strain was named Pediococcus pentosaceus KI13, and was deposited in the Microbial Bank of the National Academy of Agricultural Sciences (KACC) on May 14, 2020 (accession number: KACC 81116BP).
  • the 16S rRNA base sequence of the strain of the present invention is shown in SEQ ID NO: 1, and the phylogenetic tree for the Pediococcus pentosaceus KI13 strain was analyzed, and the results are shown in FIG.
  • the whey powder was dissolved in PBS to a concentration of 10%, sterilized at 80 ° C. for 1 minute , inoculated with the P. pentosaceus KI13 strain of Example 1, and then fermented to prepare the bioconversion product of the present invention.
  • PNP p-Nitrophenylpalmitate
  • the bioconversion product of the present invention inhibits pancreatic lipase to inhibit the digestion and absorption of lipids, thereby achieving prevention, improvement and therapeutic effects of obesity or fatty liver disease.
  • ⁇ -amylase is an enzyme that catalyzes the hydrolysis of 1,4-alpha-glucosidic bonds in polysaccharides, and is known as a biomarker related to obesity by participating in glycogen metabolism.
  • ⁇ -amylase (amylase) was diluted with distilled water to a concentration of 0.1 g / 10 mL, and soluble starch as a substrate was prepared at 0.5% with distilled water, mixed with the sample, and reacted at 25 ° C. for 10 minutes. After stopping the reaction with 0.1N HCl solution, the color was developed using iodine solution for 30 minutes, and absorbance was measured at 660 nm. The results were shown by comparing the absorbance when only ⁇ -amylase was treated and when the sample was mixed and treated.
  • the activity inhibiting ability of ⁇ -amylase was calculated through the following formula (A: absorbance of sample, B: absorbance of control), and a culture cultured for 18 hours at 37°C by inoculating 1% of the strain in MRS broth as a control. As a result of the measurement, it was confirmed that the ability to inhibit ⁇ -amylase activity was 18.97 ⁇ 1.37%.
  • ⁇ -glucosidase is an enzyme that hydrolyzes ⁇ -D-glucosidic bonds in polysaccharides, and inhibition of ⁇ -glucosidase is known to inhibit the digestion of disaccharides in the small intestine, thereby delaying carbohydrate absorption.
  • the activity inhibitory ability of ⁇ -glucosidase was measured using a method such as Tibbot and Skadsen (1996).
  • the enzyme was ⁇ -glucosidase obtained from yeast (sigma, USA), and the substrate was p-nitrophenyl- ⁇ -D- glucopyranoside (Sigma, USA) was used.
  • ⁇ -glucosidase was dissolved in 100 mM phosphate buffer (pH 7.0) containing 0.2% BSA and 0.02% NaN 3 to 0.7 ⁇ /ml and used as an enzyme solution, and p-Nitrophenyl- ⁇ -D -glucopyranoside was 100 It was dissolved in mM phosphate buffer (pH 7.0) to 10 mM and used as a substrate solution. After that, 50 ⁇ l of each sample was added to a microplate, 100 ⁇ l of ⁇ -glucosidase enzyme was taken, and then incubated at 25° C. for 5 minutes at room temperature.
  • the activity inhibiting ability of ⁇ -glucosidase was calculated through the following formula (A: absorbance of sample, B: absorbance of control), and a culture cultured at 37 ° C. for 18 hours by inoculating 1% of the strain in MRS broth as a control group. As a result of the measurement, it was confirmed that the ability to inhibit ⁇ -glucosidase activity was 7.96 ⁇ 1.38%.
  • the 3T3-L1 cell culture method was used by modifying the method of Hemati et al. (1997).
  • Adipogenesis measurement is a method of differentiating 3T3-L1 cell line and inducing adipogenesis to measure the effect of the addition of a specific material on fat accumulation.
  • 3T3-L1 cells are treated with dexamethasone, 3-isobutyylmethy-1-xanthine, and insulin to induce cell differentiation, lipogenesis promoting factors such as PPAR ⁇ are expressed and ultimately fat is accumulated in the cells.
  • fat accumulation occurs within 9 days, including the period of adipocyte progenitor cells in 3T3-L1 cells, and the degree of intracellular fat accumulation is measured by extracting these accumulated adipocytes with Oil Red O solution and measuring the absorbance at 520 nm. (Ramirez-Zacarias et al., 1992).
  • Example 4 Evaluation of in vivo obesity suppression ability using an animal model
  • mice were separated to minimize the difference in body weight between groups, and then 3-4 mice per cage were bred in the laboratory animal room of Sookmyung Women's University. Contrast was controlled in the experimental animal room every 12 hours, and the temperature was maintained at 20-23°C and humidity 40-60%. After acclimatization to the animal room environment for 1 week, they were bred by dividing them into a normal diet (normal fat diet, NF) and a high fat diet (HF). All diets were supplied with 10% fat and 60% fat experimental animal feed produced by DK Bio, and the bedding was replaced once a week.
  • NF normal fat diet
  • HF high fat diet
  • the isolated strain P. pentosaceus KI13 received from the Korea Food Research Institute was aerobically cultured in 10 ml of MRS broth (MRSB, BD, USA) at 37 °C for 24 hours, and then the same in 10 ml of new MRSB (BD, USA). cultured under conditions.
  • the culture solution was transferred to a conical tube, centrifuged (1912 ⁇ g, 15 min, 4 °C), washed twice with the same amount of PBS, and then subjected to a bacteriostatic process at 98 °C for 10 min. Finally, a 10-fold dilution in 10% whey (prepared by dissolving whey powder in PBS to a concentration of 10% and sterilizing at 80°C for 1 minute) was used as the test bacterial solution.
  • glycerol strain stock solution 100 ul of the glycerol strain stock solution was activated in 10 ml of MRSB (BD, USA) autoclaved at 121° C. for 15 minutes, and cultured at 37° C. for 18 hours to prepare a bacterial solution.
  • Dissolve whey powder processed ginseng milk
  • sterile distilled water to a concentration of 10%, sterilize it for 1 minute at a central temperature of 80 °C
  • inoculate the prepared bacterial solution to 0.1% incubate at 37 °C for 24 hours, and then 0.1 to pH 6.5 Titrated with N NaOH.
  • the 35% concentrated solution using a vacuum concentrator was powdered using a spray dryer.
  • the bioconversion product of the present invention was dissolved in PBS to 1,500 mg/kg and used.
  • lactic acid bacteria and the bioconversion product were orally administered to 6-week-old mice at 2-day intervals for a total of 10 weeks.
  • the size of the mesenchymal was relatively small and the ratio of triglyceride particles was small, so it had a bright red color
  • HF-KI 13W high-fat diet group
  • mice After anesthetizing the mice in each group, the abdomen was incised and blood was collected from the aorta. Blood collected from mice was placed in a serum separation tube (Microtaner; BD, USA) and left at room temperature for about 30 minutes, then centrifuged at 4 °C at 5,000 rpm for 10 minutes to obtain serum from the supernatant, and then at -70 °C. It was stored frozen. As a result of blood analysis, it was confirmed that ALT and AST levels were significantly lower in the bioconversion product administration group (HF-KI 13B) among the high-fat diet group (HF), and liver damage did not occur in the bioconversion product administration group. It was confirmed that the level of cholesterol was significantly reduced (FIG. 4).
  • RNA later (Qiagen, Germany), which is an RNA stabilization reagent, and stored at 2-8 °C, and total mRNA was extracted using RNeasy Mini Kit (Qiagen, Germany). Total mRNA was quantified using an EpochTM Microplate Spectrophotometer (BioTek Instruments, Inc., USA) and stored at -20 °C.
  • cDNA was synthesized from total mRNA using the QuantiTech Reverse Transcription Kit (Qiagen, Germany).
  • qRT-PCR was performed using cDNA as a template and Rotor-Gene SYBR Green PCR Kit (Qiagen, Germany) according to the manufacturer's manual under the conditions shown in the table below.
  • qRT-PCR conditions and the sequences of the primers used are shown in Tables 3 and 4 below, respectively, and ⁇ - actin was used as a housekeeping gene to normalize the expression level. PCR was performed in two replicates, and the relative gene expression level was calculated by the -2 ⁇ Ct method.
  • Step Time Temperature PCR initial activation step 5 min 95°C Number of cycles as below 35 cycles Denaturation 5 s 95°C Combined annealing/extension 10 s 60°C
  • liver tissue was incubated with one Stainless still bead and 600 ul of PRO-PREPTM protein extraction solution (iNtRON Biotechnology Inc., Korea), followed by a tissue lyzer (Qiagen, Germany). ) was used to completely homogenize the tissue under 50 Hz conditions. After sonication, the beads were removed, stored on ice for 30 minutes, centrifuged (13,000 rpm, 4 °C, 10 minutes), and the supernatant was stored at -20 °C.
  • PRO-PREPTM protein extraction solution iNtRON Biotechnology Inc., Korea
  • tissue lyzer Qiagen, Germany
  • Protein expression was quantified by western blot. A total of 30 ug of total protein from each sample was separated by 12% SDS-PAGE at 120 V for 1 hour. After moving the protein to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Life Science, USA) at 60 V for 2.5 hours, the membrane was blocked with 5% skimmed milk (Sigma-Aldrich Corp., USA) for 1 hour at room temperature. did Immunoblotting was performed using the primary antibodies listed in Table 5 below together with ⁇ -actin used as a reference protein, and anti-mouse IgG HRP (sc-2005, 1:5,000, Santa Cruz Biotechnologies, Inc., USA) was used. It was used as a secondary antibody.
  • PVDF polyvinylidene difluoride
  • Antigen/antibody complexes were detected at room temperature for 1 min using ECL SelectTM Western blottng Detection Reagent (GE Healthcare Life Science, USA). Immune response bands were visualized using a LAS-3000 Imager (Fujifilm, Japan) and band intensities were analyzed using GelQuant software v.2.7. (DNR Imaging System Ltd., Israel).
  • RNA later (Qiagen, Germany), which is an RNA stabilizing reagent, and stored at 2-8 °C, and total mRNA was extracted using RNeasy Lipid Tissue Mini Kit (Qiagen, Germany). Total mRNA was quantified using an EpochTM Microplate Spectrophotometer (BioTek Instruments, Inc., USA) and stored at -20 °C.
  • cDNA was synthesized from total mRNA using the QuantiTech Reverse Transcription Kit (Qiagen, Germany).
  • qRT-PCR was performed using cDNA as a template and the Rotor-Gene SYBR Green PCR Kit (Qiagen, Germany) according to the manufacturer's manual under the conditions shown in Table 6 below.
  • the sequences of the primers used are shown in Table 7 below, and ⁇ - actin was used as a housekeeping gene to normalize the expression level. PCR was performed in two replicates, and the relative gene expression level was calculated using the -2 ⁇ Ct method.
  • Step Time Temperature PCR initial activation step 5 min 95°C Number of cycles as below 35 cycles Denaturation 5 s 95°C Combined annealing/extension 10 s 60°C

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Abstract

La présente invention concerne : une souche de Pediococcus Pentosaceus KI13 de numéro d'accès KACC 81116BP qui présente un effet de prévention de l'obésité dans une fermentation à base de lactosérum ; une composition alimentaire, une composition d'additif alimentaire, une composition pharmaceutique et une composition d'aliment visant à prévenir ou à soigner l'obésité ou la stéatose hépatique comprenant un produit fermenté à base de lactosérum bioconverti contenant la souche ; un procédé de prévention ou de traitement de l'obésité ou de la stéatose hépatique comprenant une étape d'administration de la composition pharmaceutique à un individu ; et l'utilisation d'une composition comprenant un produit fermenté à base de lactosérum bioconverti contenant une souche de Pediococcus pentosaceus. Dans la présente invention, le produit fermenté à base de lactosérum bioconverti contenant une souche de P. pentosaceus KI13 réduit un taux de gain de poids par l'inhibition de l'activité de la lipase pancréatique, l'inhibition de l'activité d'α-amylase et d'α-glucosidase ainsi que l'inhibition de la différenciation de mastocytes, et réduit l'expression de gènes et de protéines associés à l'obésité et à l'inflammation d'organes, tels que le foie, et de tissu adipeux, ce qui a pour effet de prévenir, de soigner ou de traiter l'obésité et la stéatose hépatique.
PCT/KR2020/018781 2020-06-12 2020-12-21 Nouvelle souche de pediococcus pentosaceus et composition alimentaire pour la prévention ou l'amélioration de l'obésité ou d'une stéatose hépatique comprenant un produit fermenté à base de lactosérum associé WO2021251575A1 (fr)

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CN117143785B (zh) * 2023-11-01 2024-02-27 中国农业科学院饲料研究所 一株片球菌及其应用
CN117625478A (zh) * 2023-11-29 2024-03-01 广西爱生生命科技有限公司 一种具有治疗非酒精性脂肪肝和/或减脂功能的戊糖片球菌菌株a21243及其应用

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