WO2021251575A1 - Novel pediococcus pentosaceus strain and food composition for preventing or improving obesity or fatty liver disease comprising whey fermented product thereof - Google Patents

Abstract

The present invention relates to: a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP that exhibits anti-obesity activity in fermentation of whey; a food composition, a food additive composition, a pharmaceutical composition, and a feed composition for preventing or improving obesity or fatty liver disease comprising a whey fermented product bioconverted with the strain; a method for preventing or treating obesity or fatty liver disease comprising a step of administering the pharmaceutical composition to an individual; and a use of a composition comprising a whey fermented product bioconverted with a Pediococcus pentosaceus strain. In the present invention, the whey fermented product bioconverted with a P. pentosaceus KI13 strain reduces a weight gain rate through inhibition of pancreatic lipase activity, inhibition of α-amylase and α-glucosidase activity, and inhibition of mast cell differentiation, and reduces the expression of genes and proteins related to obesity and inflammation in organs, such as the liver, and adipose tissue, thereby exhibiting an effect of preventing, improving, or treating obesity and fatty liver disease.

Description

Food composition for preventing or improving obesity or fatty liver disease, comprising novel Pediococcus pentosaceus strain and its whey fermented product

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.

On the other hand, "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.

Accordingly, 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.

In the present invention, the bioconverted whey fermented product with Pediococcus pentosaceus KI13 strain reduces the rate of weight gain through inhibition of α-amylase and α-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.

SUMMARY OF THE INVENTION 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.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem for solving the above-mentioned problems, and another problem not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.

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. In the present invention, "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.

In addition, another aspect of the present invention for achieving the above object provides a food composition for preventing or improving obesity or fatty liver disease, comprising a bioconverted whey fermented product as an active ingredient. Specifically, 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.

As used herein, the term "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. In one embodiment of the present invention, 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.

In the present invention, the term "fermented product" refers to the result of enzymatic or metabolic degradation using microorganisms. Specifically, in the present invention, 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'.

In the present invention, the term "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. In the present invention, 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

In the present invention, 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. In addition, in the present invention, 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.

In the present invention, the term “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, and the term “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.

In one embodiment of the present invention, as a result of oral administration of a bioconversion product by P. pentosaceus KI13 strain to mice on a normal diet or a high-fat diet, weight gain was suppressed in the group administered with the bioconversion product, and the liver and adipose tissue It was confirmed that the effects of suppressing fat accumulation, reducing blood ALT, AST and LDL-cholesterol levels, and reducing the expression of obesity/inflammation-related genes or proteins in the liver were significantly superior.

In the food composition of the present invention, the food may include health functional (sexual) food. In the present invention, the term "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. In addition, if 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.

In addition, 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. In addition, 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.

In the food composition of the present invention, 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.

In addition, another aspect of the present invention for achieving the above object provides a food additive composition comprising a bioconverted whey fermented product as an active ingredient. Specifically, 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.

In the present invention, 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.

Another aspect of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating obesity or fatty liver disease, comprising a bioconverted whey fermented product as an active ingredient. Specifically, 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. Specific examples of the carrier, excipient and diluent 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. In addition, 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. 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. . In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used, but the present invention is not limited thereto. In addition, as 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. this can be used 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. As the 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. In addition, 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. For example, the pharmaceutical composition of the present invention may be administered intraperitoneally, intravenously, subcutaneously, intradermally, orally, but is not limited thereto.

In the present invention, the term “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.

As described above, 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.

In the method for treating obesity or fatty liver disease of the present invention, 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 terms “whey”, “fermented product”, “bioconversion”, “obesity”, “prevention” and “improvement” of the present invention are as described above.

In the present invention, 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) 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. Examples include, but are not limited to, silage, wild grass, hay cut and dried, straw from breeders, and leaves from legumes. In special feed, mineral feed such as oyster shell and rock salt, urea feed such as urea or its derivative diureide isobutane, and 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. There are 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. For example, 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.

In the present invention, the whey fermented product bioconverted to the P. pentosaceus KI13 strain reduces the weight gain through inhibition of pancreatic lipase activity, inhibition of α-amylase and α-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.

In order to more fully understand the drawings cited herein, a brief description of each drawing is provided.

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).

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.

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.

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.

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.

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.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples only.

Example 1: Pediococcus pentosaceus Isolation and identification of KI13 strain

1-1. Isolation and collection of strains from kimchi

It was collected from 50 kinds of kimchi and spread on a plate to which Bromocresol purple and sodium azide were added during the MRS medium composition, and then cultured at 37 °C for 48 hours. Colonies obtained by streaking on MRS agar were cultured in TSA at 37°C for 18 hours and stored. A total of 158 strains were isolated from this.

Thereafter, 158 strains were inoculated into skim milk and whey to measure fermentation ability. To sterilize skim milk and whey, 1% lactic acid bacteria were inoculated into 10% skim milk sterilized for 1 minute at a core temperature of 80 ° C. As a result, it was found that 98 strains out of 158 kimchi-derived strains coagulated and had fermentative ability.

1-2. Strain selection and identification

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.

For the DNA sequence of the strain, universal primers 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3') were used, and solvent EF-Taq was used. and PCR was performed. Amplification process was 95 ℃ 15 minutes; 95 °C, 20 seconds; 50° C., 40 seconds; 7 2 ℃, 1 min 30 sec was performed 30 times, 72 ℃, 5 minutes was finished. For sequencing, the PCR product was purified with a solvent PCR purification kit and automatically analyzed with ABI 3730XL DNA sequencer. As a result, 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.

Example 2: Pediococcus pentosaceus Manufacturing of Whey Bioconversion Products Using KI13

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.

Example 3: Evaluation of In Vitro Obesity Inhibiting Ability of Whey Bioconversion Products

3-1. Confirmation of ability to inhibit pancreatic lipase activity

Porcine pancreatic lipase was used to measure the ability of the composition of the present invention to inhibit lipase activity. After dissolving p-Nitrophenylpalmitate (PNP) in acetonitrile at a concentration of 10 mM, the obtained solution was again dissolved in ethanol (1:2 = acetonitrile: ethanol), and as a result, a 3.33 mM PNP solution was used. Lipase was dissolved in distilled water at a concentration of 5 mg/mL and used. A reaction solution was prepared at the following concentrations and reacted at 37 °C for 10 minutes (Table 1).

Buffer Volume Total reaction mixture 200 μl Enzyme (porcine pancreatic lipase) 0.30 mg/ml Sample 0.1 mg/ml PNP 0.167 mM Tris-HCl buffer 0.061M (pH 8.5)

After the reaction, absorbance was measured at 405 nm, and the enzyme was replaced with distilled water for the blank, and the sample was replaced with a solvent for the control. For each concentration sample, the absorbance of the blank minus lipase was measured to correct the color of the sample. The activity inhibiting ability of lipase was calculated using the formula below (A: absorbance of sample, B: absorbance of control). As a result, the whey bioconversion product using P. pentosaceus KI13 of the present invention showed an inhibition rate of 77.33±2.72%. was confirmed.

That is, this means that 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.

Lipase activity inhibition ability (%) = {1-(A/B)} x 100

3-2. Evaluation of inhibitory ability of α-amylase and α-glucosidase activity

1) Measurement of the ability to inhibit α-amylase activity

α-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.

α-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 α-amylase was treated and when the sample was mixed and treated.

The activity inhibiting ability of α-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 α-amylase activity was 18.97±1.37%.

Activity inhibition of α-amylase (%) = {1-(A/B)} x 100

2) Measurement of the ability to inhibit α-glucosidase activity

α -glucosidase is an enzyme that hydrolyzes α-D-glucosidic bonds in polysaccharides, and inhibition of α-glucosidase is known to inhibit the digestion of disaccharides in the small intestine, thereby delaying carbohydrate absorption.

The activity inhibitory ability of α-glucosidase was measured using a method such as Tibbot and Skadsen (1996). The enzyme was α-glucosidase obtained from yeast (sigma, USA), and the substrate was p-nitrophenyl-α-D- glucopyranoside (Sigma, USA) was used. α-glucosidase was dissolved in 100 mM phosphate buffer (pH 7.0) containing 0.2% BSA and 0.02% NaN ₃ to 0.7 μ/ml and used as an enzyme solution, and p-Nitrophenyl-α-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 μl of each sample was added to a microplate, 100 μl of α-glucosidase enzyme was taken, and then incubated at 25° C. for 5 minutes at room temperature. Absorbance at 405 nm with Multi Detection Reader (Infinite 200, TECAN Group Ltd, Switzerland) was measured. Then, 50 μl of the substrate solution was added, and after 2 minutes, absorbance was measured at 405 nm with a multi-detection reader to calculate α-glucosidase inhibitory ability.

The activity inhibiting ability of α-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 α-glucosidase activity was 7.96±1.38%.

Activity inhibitory activity of α-glucosidase (%) = {1-(A/B)} x 100

3-3. Evaluation of mast cell differentiation inhibitory ability

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. When 3T3-L1 cells are treated with dexamethasone, 3-isobutyylmethy-1-xanthine, and insulin to induce cell differentiation, lipogenesis promoting factors such as PPARγ are expressed and ultimately fat is accumulated in the cells. In general, 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).

As a result, it was confirmed that the whey bioconversion product using P. pentosaceus KI13 of the present invention exhibited 78.99±0.62% differentiation inhibitory ability against adipocyte 3T3-L1 cells.

Example 4: Evaluation of in vivo obesity suppression ability using an animal model

4-1. Manufacture of high-fat diet animal models

A 5-week-old male C57BL/6 mouse was purchased from Orient Bio and used in the experiment. 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.

army substance to be administered N NF-PBS regular diet + PBS 5 HF-PBS high fat diet + PBS 8 HF-KI 35W High fat diet + whey + P. pentosaceus KI 13* 8 HF-KI 35B High-fat diet + P. pentosaceus KI 13 bioconversion product** 8

*KI 13 strain: used after being killed

*Bioconversion product: 1,500 mg/kg

4-2. Preparation of test bacterial solution

1) Preparation of test bacteria solution of HF-KI 13W group

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 ℃), washed twice with the same amount of PBS, and then subjected to a bacteriostatic process at 98 ℃ 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.

2) Preparation of test bacteria solution of HF-KI 13B group

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) in sterile distilled water to a concentration of 10%, sterilize it for 1 minute at a central temperature of 80 ℃, inoculate the prepared bacterial solution to 0.1%, incubate at 37 ℃ for 24 hours, and then 0.1 to pH 6.5 Titrated with N NaOH. Then, the 35% concentrated solution using a vacuum concentrator was powdered using a spray dryer. Finally, the bioconversion product of the present invention was dissolved in PBS to 1,500 mg/kg and used.

3) Administration period, cycle and dose

After a one-week acclimatization period and a four-week obesity induction period, 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.

4-3. Weight gain analysis result

As a result of measuring mouse body weight once a week at a certain time, the weight change rate in the group administered with the bioconversion product of the present invention (HF-KI 13B) was higher than in the group administered with the P. pentosaceus KI13 strain (HF-KI 13W). It was confirmed that it appeared significantly less (FIG. 2).

4-4. Organ and tissue observations

As a result of visual observation of liver, epididymal fat, and lateral abdominal adipose tissue in the normal diet group (NF-PBS) and the high-fat diet group (HF-PBS, HF-KI 13W, HF-KI 13B), bioconversion products It was confirmed that there was a significant difference between the groups administered with HF-KI 13B. Specifically, in the case of the group administered with the bioconversion product of the present invention (HF-KI 13B), the size of the mesenchymal was relatively small and the ratio of triglyceride particles was small, so it had a bright red color, while in the other high-fat diet group (HF) -PBS, HF-KI 13W) showed a high accumulation rate of triglyceride particles in the liver tissue, resulting in a white to yellow color (FIG. 3). That is, this means that the accumulation of triglycerides in the liver tissue can be significantly reduced when the bioconversion product of the present invention is administered.

4-5. blood analysis results

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).

4-6. Results of analysis of the expression level of obesity-related genes in the liver

Mouse liver was immersed in 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 Epoch™ Microplate Spectrophotometer (BioTek Instruments, Inc., USA) and stored at -20 °C. For transcript analysis, 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. The qRT-PCR conditions and the sequences of the primers used are shown in Tables 3 and 4 below, respectively, and β- 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 ΔΔCt} method.

Step Time Temperature PCR initial activation step 5 min 95℃ Number of cycles as below 35 cycles Denaturation 5 s 95℃ Combined annealing/extension 10 s 60℃

Gene Sequence (5' to 3') Reference β- actin Forward TGTCCACCTTCCAGCAGATGT Fan et al., 2014 Reverse AGCTCAGTAACAGTCCGCCTAGA SREBP Forward AAGCAAATCACTGAAGGACCTGG Oliveira et al., 2015 Reverse AAAGACAAGGGGCTACTCTGGGAG PPAR-γ Forward GCTTCCACTATGGAGTTCATGCT Lu et al., 2013 Reverse CCGGCAGTAAGATCACACCTAT CPT-2 Forward GCCTGCTGTTGCGTGACTG Yoo et al., 2013 Reverse TGGTGGGTACGATGCTGTGC

As a result of analyzing the expression level of obesity-related genes in the liver, SREBP , a transcriptional activator that regulates fatty acid and cholesterol synthesis, was low expressed in the bioconversion product-administered group (HF-KI 13B), and was PPAR-γ, which is involved in growth inhibition, was highly expressed in the bioconversion product-administered group (HF-KI 13B). In addition, CPT-2 , which plays a role in helping fatty acid oxidation, was expressed low in the bioconversion product-administered group (HF-KI 13B) (FIG. 5). That is, this indicates that the bioconversion product of the present invention has a preventive, ameliorating or therapeutic effect on obesity by increasing or decreasing the expression of obesity-related genes in the liver.

4-7. Results of analysis of the expression level of inflammation-related proteins in the liver

To extract total protein from the liver, 30 mg of frozen liver tissue was incubated with one Stainless still bead and 600 ul of PRO-PREP™ 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.

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 β-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. Antigen/antibody complexes were detected at room temperature for 1 min using ECL Select™ 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).

Antibody Concentration Incubation condition Size (kDa) Cat No. (Manufacturer) Clonal type β-actin 1:1,000 3 h, RT ¹⁾ 43 sc-81178 (Santa Cruz) Mouse monoclonal IL-6 1:500 12h, 4℃ 21 sc-57315 (Santa Cruz) Mouse monoclonal

¹⁾ RT, room temperature

As a result of analysis of the expression level of inflammation-related protein in the liver, it was confirmed that IL-6, an inflammatory index, was significantly lower in the group administered with the bioconversion product of the present invention (HF-KI 13B) than in HF-PBS, a control group on a high-fat diet, This indicates that the bioconversion product of the present invention suppresses the liver inflammatory response caused by obesity (FIG. 6).

4-8. Results of analysis of gene expression levels related to inflammation and obesity in adipose tissue

Fat was immersed in 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 Epoch™ Microplate Spectrophotometer (BioTek Instruments, Inc., USA) and stored at -20 °C. For transcript analysis, 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 β- 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 ΔΔCt method.}

Step Time Temperature PCR initial activation step 5 min 95℃ Number of cycles as below 35 cycles Denaturation 5 s 95℃ Combined annealing/extension 10 s 60℃

Gene Sequence (5' to 3') Reference β- actin Forward TGTCCACCTTCCAGCAGATGT Fan et al., 2014 Reverse AGCTCAGTAACAGTCCGCCTAGA IL-6 Forward ACAAGTCGGAGGCTTAATTACACAT Rube et al., 2004 Reverse AATCAGAATTGCCATTGCACAA MCP1 Forward GACCCGTAAATCTGAAGCTAA Chow et al., 2006 Reverse CACACTGGTCACTCCTACAGAA

As a result of analyzing the gene expression of the proinflammatory cytokine IL-6 in adipose tissue, it was confirmed that IL-6 was expressed significantly lower in the bioconversion product-administered group (HF-KI 13B) than in the high-fat diet, HF-PBS. In addition, as a result of analyzing the expression of obesity-related genes in adipose tissue, MCP-1, which affects macrophage accumulation and increases glucose uptake by stimulating insulin, was found to be high-fat diet in the bioconversion product-administered group (HF-KI 13B). It was confirmed that the expression was significantly lower than that of the control group HF-PBS (FIG. 7). That is, this indicates that the bioconversion product of the present invention has a preventive, ameliorating or therapeutic effect on obesity by increasing or decreasing the expression of obesity-related genes in adipose tissue.

4-9. Histopathological analysis results

After the extracted mouse liver tissue was fixed with 10% formalin, a paraffin block was made to make a section, and then, hematoxylin and eosin (H&E) staining was performed and observed using an optical microscope. As a result of confirming a tissue slide made of liver tissue collected from the left leteral lobe of HF-PBS, a high-fat control group, and HF-KI 13B, a group administered with a bioconversion product, macrovesicular fatty changes in hepatocytes were observed in the group administered with the bioconversion product of the present invention. It was confirmed that there is a small effect of reducing steatosis (FIG. 8).

4-10. Observation of fat sphere size in adipose tissue

As a result of observing the size of fat globules in the epididymal adipose tissue of mice at 20 magnification using an optical microscope, the size of fat globules was small and relatively regular in the group administered with the bioconversion product of the present invention (HF-KI 13B), despite the high fat diet. was confirmed (FIG. 9).

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

[Accession number]

Name of depositary: National Academy of Agricultural Sciences Microbial Bank (KACC)

Accession number: KACC 81116BP

Deposit date: 20200514

Claims (15)

Pediococcus pentosaceus ( Pediococcus pentosaceus ) A food composition for preventing or improving obesity or fatty liver disease, comprising as an active ingredient a whey fermented product bioconverted to a strain. The food composition according to claim 1, wherein the Pediococcus pentosaceus strain is a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP. The food composition according to claim 1, wherein the fatty liver disease is selected from the group consisting of fatty liver, nonalcoholic steatohepatitis, liver cirrhosis, cirrhosis and liver cancer. The food composition according to claim 1, wherein the food is a health functional food. According to claim 4, wherein the health functional food is a tablet, capsule, pill, granule, liquid, powder, flaky, paste, syrup, gel, jelly, bar health functional food preparations such as beverages, gums and candies. One or more formulations selected from the group, the food composition. Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP showing anti-obesity activity during whey fermentation. A food additive composition comprising, as an active ingredient, a whey fermented product that is bioconverted to a Pediococcus pentosaceus strain. The food additive composition of claim 7, wherein the Pediococcus pentosaceus strain is a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP. A pharmaceutical composition for the prevention or treatment of obesity or fatty liver disease, comprising, as an active ingredient, a whey fermented product bioconverted to a Pediococcus pentosaceus strain. The pharmaceutical composition of claim 9, wherein the Pediococcus pentosaceus strain is a Pediococcus pentosaceus KI13 strain of accession number KACC 81116BP. The pharmaceutical composition of claim 9, wherein the fatty liver disease is selected from the group consisting of fatty liver, nonalcoholic steatohepatitis, liver cirrhosis, cirrhosis and liver cancer. . A method for preventing or treating obesity or fatty liver disease, comprising administering the composition of any one of claims 9 to 11 to a subject. Use of a composition comprising a fermented whey bioconverted into a Pediococcus pentosaceus strain for the prevention or treatment of obesity or fatty liver disease. Use of a composition comprising a whey ferment bioconverted into a Pediococcus pentosaceus strain for the manufacture of a medicament for the prevention or treatment of obesity or fatty liver disease. A feed composition for preventing or improving obesity or fatty liver disease, comprising, as an active ingredient, a whey fermented product bioconverted to a Pediococcus pentosaceus strain.

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