KR102500796B1 - Composition for Preventing or Treating Obesity Comprising Surface Layer Protein of Lactic Acid Bacteria from Kefir - Google Patents

Abstract

The present invention relates to an anti-obesity composition and functional food containing surface layer protein (SLP) of lactic acid bacteria derived from kefir. Therefore, it can be used as a functional alternative material that can compensate for the disadvantages of existing processed foods in which daily intake is excessive and it is difficult to maintain probiotics in a viable state during processing and packaging.

Description

Composition for Preventing or Treating Obesity Comprising Surface Layer Protein of Lactic Acid Bacteria from Kefir}

The present invention relates to a composition for preventing or treating obesity, including a cell surface protein (SLP) of kefir-derived lactic acid bacteria. In addition, the present invention relates to a composition for preventing or treating obesity comprising cell surface protein, extracellular polysaccharide, and grape seed powder of kefir-derived lactic acid bacteria.

Probiotics (probiotics) refers to the intestinal flora that is beneficial to health, that is, living microorganisms that provide health benefits to the host, that is, probiotics. Generally, probiotics are consumed as part of fermented foods such as yogurt or as dietary supplements. Microorganisms known as probiotics include lactic acid bacteria (LAB), bifidobacteria, and bacilli. Some of the probiotics are known to have therapeutic effects on obesity, insulin resistance, non-alcoholic fatty liver, and the like.

In addition, recently, the claim that postbiotics, metabolites produced by probiotics, are effective for treatment or disease diagnosis has attracted attention. Although probiotics and postbiotics are claimed to be involved in various activities of the human body, such as immunity, circulation, respiration, and homeostasis, as well as intestinal health, research and development on postbiotics is still insufficient.

For example, Korean Patent Publication No. 10-2017-0033433 discloses prebiotic, probiotic and postbiotic compositions for improving the health and nutrition of commercial livestock and companion animals. And its use method is described, but the culture supernatant used as a postbiotic material is a material presumed to be a metabolic by-product used in the feed composition, and there has been no study on other functions except for the growth promoting function.

On the other hand, kefir (Kefir) is a kind of fermented milk derived from the mountainous region of the Caucasus, and it is a popular drink that Tibetan monks drank for health. Constituent nutrients of kefir include protein and polysaccharide, and include vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin D, vitamin K2, folic acid, nicotinic acid, calcium, iron, and iodine. Such kefir is a yoghurt-like beverage obtained by fermenting milk using a lactic acid bacteria and yeast complex called kefir grain (also referred to as kefir granular bacteria) as an initiator.

Kefir contains a variety of microorganisms, including lactobacilli ( Lactobacillus, Lactococcus, Leuconostoc, Acetobacter , and Streptococcus species), acetobacters (such as Acetobacter ), and yeasts ( Saccharomyces, Kluyveromyces, Torula, and Candida species). Kefir is known to have effects such as anti-mutagenicity, cholesterol lowering, anti-obesity, and fatty liver prevention, but research is conducted to achieve better efficacy by extracting active ingredients such as kefir or kefir-derived lactic acid bacteria, acetic acid bacteria, and yeast. is in short supply.

For example, Korean Patent Registration No. 10-2037897 describes an anti-obesity composition using kefir-derived lactic acid bacteria and grape seed powder, and Korean Patent Registration No. 10-2037898 improves fatty liver using kefir-derived lactic acid bacteria and grape seed powder. Although the composition for use is described, kefir-derived lactic acid bacteria are used as they are, so there remains room for improvement in the utilization of active ingredients. Moreover, in the case of using kefir-derived lactic acid bacteria as they are, it is necessary to consume more than a certain number of bacteria to achieve the anti-obesity effect, and the reduction of bacteria is inevitable during the manufacturing and storage process, which causes a decrease in efficiency. Ticksin grape seed powder had a disadvantage that the daily intake to show anti-obesity effect was excessive.

The inventors of the present invention found that when cell surface proteins of kefir-derived lactic acid bacteria are isolated and used, an excellent anti-obesity effect can be exerted, and when these cell surface proteins are used in combination with the extracellular polysaccharide of kefir-derived lactic acid bacteria and grape seed powder, their The present invention was completed by discovering that an excellent anti-obesity effect can be exerted even in a small amount by a synergistic effect.

An object of the present invention is to provide an anti-obesity composition containing cell surface protein (SLP) of lactic acid bacteria derived from kefir as an active ingredient.

Another object of the present invention is to provide an anti-obesity composition containing cell surface protein (SLP) of lactic acid bacteria derived from kefir, extracellular polysaccharide (EPS) of lactic acid bacteria derived from kefir, and grape seed powder.

Another object of the present invention is to provide a functional food containing cell surface protein (SLP) of lactic acid bacteria derived from kefir.

In order to achieve the above object, the present invention provides an anti-obesity composition containing a cell surface protein (surface layer protein, SLP) of lactic acid bacteria derived from kefir as an active ingredient.

In the present invention, the lactic acid bacteria are preferably Lactobacillus kefiri , Leuconostoc mesenteroides , or a combination thereof.

In the present invention, the lactic acid bacteria is more preferably at least one selected from the group consisting of Lactobacillus kefirii DH5, Leuconostoc mecenteroides DH1606 and Leuconostoc mecenteroides DH1608.

In the present invention, the lactic acid bacteria is more preferably Lactobacillus kefirii DH5 or Leuconostoc mesenteroides DH1608.

The present invention also provides an anti-obesity composition containing cell surface layer protein (SLP) of lactic acid bacteria derived from kefir, exopolysaccharide (EPS) of lactic acid bacteria derived from kefir, and grape seed powder. .

In the present invention, the cell surface protein of the kefir-derived lactic acid bacteria may be a cell surface protein of Leuconostoc mecenteroides DH1608.

In the present invention, the extracellular polysaccharide of the kefir-derived lactic acid bacteria may be an extracellular polysaccharide of Lactobacillus kefir DH5.

In the present invention, the grape seed powder may be a by-product of wine production.

In the present invention, the grape seed powder may be prepared from grapes of the Chardonnay variety.

In the present invention, the anti-obesity composition may further include keferan extracellular polysaccharide (exopolysaccharide kefiran).

The present invention also provides a functional food containing a surface layer protein (SLP) of lactic acid bacteria derived from kefir.

In the present invention, the functional food may be a fermented food.

The composition containing the cell surface protein of kefir-derived lactic acid bacteria according to the present invention exhibits anti-obesity and anti-inflammatory effects. Since the composition of the present invention isolates and uses only the active ingredients of kefir-derived lactic acid bacteria, it has a functionality that can compensate for the disadvantages of existing processed foods, which have excessive daily intake and were difficult to maintain probiotics in a viable state during processing and packaging. It can be used as an alternative material.

1 is a result of analyzing components of cell surface proteins of kefir-derived lactic acid bacteria through HPLC (High-Performance Liquid Chromatography).
Figure 2 shows the representative protein ID and its name obtained by analyzing the components of the extracellular polysaccharide of kefir-derived lactic acid bacteria through LCMS (Liquid Chromatograph Mass Spectrometry).
Figure 3 is a graph showing the daily energy intake of the experimental rat according to the type of diet.
Figure 4 is a graph showing the weight gain of the experimental rats according to the type of diet.
Figure 5 is a graph showing the change in the weight of the adipose tissue of the experimental rat according to the type of diet.
6 is a graph showing the plasma glucose concentration of experimental mice according to the type of diet.
7 is a graph showing the plasma insulin concentration of mice according to the type of diet.
8 is a graph showing the results of an insulin tolerance test (ITT) of experimental mice according to the type of diet.
9 and 10 are results showing differences in gene expression levels through microarray.
11 and 12 show the results of Ingenuity pathway analysis (IPA) analysis.
13 shows the results of measuring the expression level of 8 genes from epididymal adipose tissue through Real Time RT-PCR.
14 is a result of measuring the expression level of the HP gene from liver tissue through real-time RT-PCR.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

The present invention is to identify the anti-obesity effect of cell surface protein (SLP) among the cell components of lactic acid bacteria newly isolated from kefir fermented milk. It relates to an anti-obesity composition containing as an active ingredient.

In the present invention, kefir is a type of fermented milk derived from the mountainous region of the Caucasus, and kefir refers to a yogurt-like beverage obtained by fermenting milk using a strain called kefir grain, which is a complex of lactic acid bacteria and yeast, as an initiator. .

The kefir-derived lactic acid bacteria may be in the form of live or dead bacteria, and more preferably dead bacteria.

In addition, the kefir-derived lactic acid bacteria may be Lactobacillus kefiri , Leuconostoc mesenteroides , or a combination thereof.

In the present invention, it was found that cell surface proteins among the cell components of kefir-derived lactic acid bacteria exhibit excellent effects in improving obesity caused by a high-fat diet, and are furthermore effective in terms of immune enhancement and anti-inflammatory activity. In addition, it was confirmed that these effects are related to the promotion of the expression of genes related to lipid metabolism including brown fat formation and the suppression of the expression of genes causing pro-inflammatory reactions.

Preferably, the kefir-derived lactic acid bacteria applicable in the present invention are Lactobacillus kefir DH1, Lactobacillus kefir DH3, Lactobacillus kefir DH5, Leuconostoc mesenteroides DH1604, Leuconostoc mesenteroides DH1606, Leucono It is at least one selected from Stock Mesenteroides DH1607, Leuconostoc Mesenteroides DH1608 and Leuconostoc Mesenteroides DH1609. All of these can be obtained from the Food Safety Research Center of the College of Veterinary Medicine, Konkuk University. Stock Mesenteroides DH1604 has been deposited with the Korea Microbial Conservation Center under accession number KCCM12117P (date of accession: September 21, 2017), and Leuconostoc mecenteroides DH1608 has been deposited with Korea Research Institute of Bioscience and Biotechnology under accession number KCTC18854P (date of accession: September 21, 2017). 16 October 2020).

In the present invention, particularly preferred kefir-derived lactic acid bacteria for their anti-obesity activity are Lactobacillus kefirii DH5 and Leuconostoc mesenteroides DH1608.

In addition, the present invention identifies the synergistic effect of cell surface protein (SLP) of lactic acid bacteria derived from kefir, exopolysaccharide (EPS) of lactic acid bacteria derived from kefir, and anti-obesity of grape seed powder, It relates to an anti-obesity composition containing cell surface protein (SLP) of peer-derived lactic acid bacteria, extracellular polysaccharide (EPS) of kefir-derived lactic acid bacteria, and grape seed powder.

The cell surface protein (SLP) of the kefir-derived lactic acid bacteria may be a cell surface protein of Lactobacillus kefiri , Leuconostoc mesenteroides , or a composite cell thereof, preferably, and It may be at least one selected from Leuconostoc Mesenteroides DH1609. In particular, it may be at least one selected from the group consisting of cell surface proteins of Lactobacillus kefirii DH5 and cell surface proteins of Leuconostoc mecenteroides DH1608, more preferably, cell surface proteins of Leuconostoc mecenteroides DH1608. may be protein.

In the present invention, the extracellular polysaccharide of kefir-derived lactic acid bacteria may be an extracellular polysaccharide of Lactobacillus kefiri , Leuconostoc mesenteroides , or a composite cell thereof, preferably , Lactobacillus kefirii DH1, Lactobacillus kefirii DH3, Lactobacillus kefirii DH5, Leuconostoc mesenteroides DH1604, Leuconostoc mesenteroides DH1606, Leuconostoc mesenteroides DH1607, Leuconostoc mesenteroides DH1608 And it may be at least one selected from Leuconostoc Mesenteroides DH1609. In particular, it may be at least one selected from the group consisting of an extracellular polysaccharide of Lactobacillus kefirii DH5 and an extracellular polysaccharide of Leuconostoc mecenteroides DH1606, more preferably, an extracellular polysaccharide of Lactobacillus kefirii DH5. can

In one embodiment of the present invention, the extracellular polysaccharide of Lactobacillus kefirii DH5 (accession number KCCM11837P, accession date: April 29, 2016) and Leuconostoc mecenteroides DH1604 (accession number KCCM12117P, accession date: 2017 It was confirmed that the combination of cell surface proteins of September 21) exhibited the best synergistic effect and was very effective in reducing weight gain.

The anti-obesity composition of the present invention may include cell surface protein (SLP) of kefir-derived lactic acid bacteria and extracellular polysaccharide (EPS) of kefir-derived lactic acid bacteria in a weight ratio of 1:1 to 1:10, preferably It may be included in a weight ratio of 1:1 to 1:3.

The present inventors confirmed that grape seed powder acts as a prebiotic that has a beneficial effect on the activity of cell surface protein (SLP) and extracellular polysaccharide (EPS) of kefir-derived lactic acid bacteria, which is a postbiotic derived from probiotics.

Although there is no particular limitation on the variety, production area or harvesting time of the grapes used in the present invention, it is most preferable to use Chardonnay grapes.

The grape seed powder of the present invention may be produced from by-products generated during the manufacturing process of beverages or alcoholic beverages made from grapes, and preferably from by-products generated during the manufacturing process of wine or wine. Here, "by-product" is a generic term for products other than wine or wine distillate produced in the distillation process after crushing and fermentation of grapes as raw materials.

Typically, wine or by-products generated during winemaking are discarded. However, such waste amounts to about 40% of raw grapes, which is a great loss economically. According to the present invention, since by-products generated during wine production can be used as prebiotics, there is a great environmental and economical advantage.

"Grape seed flour" (GSF) according to an embodiment of the present invention is separated from grape seeds from by-products left after producing wine, dried, and then extracted from grape seeds using "cold press" or "heat press" techniques. It can be produced by extracting the oil and then grinding it into a powder. Separation, drying, compression, and grinding of grape seed powder may be performed in various forms by conventionally known techniques.

The particle size of the grapeseed meal can be varied depending on the grinding environment, and sieving can be added to produce a powder with smaller particles.

The particle size of grape seed powder can be adjusted according to the use and purpose of food, medicine, etc.

In the present invention, when the cell surface protein (SLP) of kefir-derived lactic acid bacteria, the extracellular polysaccharide (EPS) of kefir-derived lactic acid bacteria, and grape seed powder are combined, an excellent effect on improving obesity due to a high-fat diet is obtained even in small amounts It was further found to be effective in terms of immune enhancement and anti-inflammatory activity. In particular, it was confirmed that they exhibit a synergistic effect when ingested together, and exhibit remarkable effects that are difficult to predict from the case of ingesting them individually.

In the anti-obesity composition of the present invention, the total content of the cell surface protein (SLP) of kefir-derived lactic acid bacteria and the extracellular polysaccharide (EPS) of kefir-derived lactic acid bacteria may be 0.1 to 10% by weight relative to the content of grape seed powder. And, preferably, it may be 0.5 to 5% by weight.

The composition containing the cell surface protein (SLP) of kefir-derived lactic acid bacteria according to the present invention can be applied as a functional food for anti-obesity.

The functional food is preferably a fermented food, but is not limited thereto. For example, the functional food may be beverages, gum, tea, vitamin complexes, health supplements, and more specifically, dairy products, confectionery products, seasonings, beverages and drinks, snacks, candies, jellies, ice cream and frozen Desserts, breakfast cereals, nutrition bars, snack bars Chocolate products, processed foods, grain products and pastas, soups, sauces and dressings, confectionery products, oil and fat products, dairy drinks and milk drinks, tea, soy milk and soybeans It may be any one selected from the group consisting of soy dairy-like products, frozen foods, prepared foods and alternative foods, meat products, cheese, yogurt, bread and rolls, cakes, cookies and crackers.

In addition, the functional food may be prepared in formulations such as capsules, tablets, powders, liquid suspensions, pills and granules containing at least one cell surface protein of kefir-derived lactic acid bacteria.

In addition, the functional food may further include ingredients commonly added during food preparation as active ingredients in addition to cell surface proteins of kefir-derived lactic acid bacteria.

The functional food of the present invention is various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts , organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like, and may contain fruit flesh for producing fruit juice or vegetable beverages.

The cell surface protein of the kefir-derived lactic acid bacteria of the present invention can also be applied to a pharmaceutical composition for preventing or treating obesity.

The pharmaceutical composition contains the cell surface protein of kefir-derived lactic acid bacteria as an active ingredient, and may further include an appropriate pharmaceutically acceptable carrier, excipient or diluent according to conventional methods. The pharmaceutically acceptable carrier is one commonly used in formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components. Regarding suitable pharmaceutically acceptable carriers and formulations, it can be preferably formulated according to each component using the method disclosed in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered either orally or parenterally, and parenteral administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

Formulations for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. chlorose, mannitol, sorbitol, cellulose and/or glycine), lubricants such as silica, talc, stearic acid and magnesium or calcium salts thereof and/or polyethylene glycol. In addition, the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid or a disintegrant or effervescent mixture, such as its sodium salt, and/or absorbents, colorants, flavors, and sweeteners. The formulation may be prepared by conventional mixing, granulating or coating methods.

In addition, a typical formulation for parenteral administration is an injection formulation, and water, Ringer's solution, isotonic physiological saline or suspension may be mentioned as a solvent for the injection formulation. Sterile fixed oils of the above injectable preparations may be used as a solvent or suspension medium, and any bland fixed oil may be used for this purpose, including mono- and di-glycerides. In addition, the formulation for injection may use a fatty acid such as oleic acid.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type, severity, and activity of the drug of the patient's disease , sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and weight, and is generally 0.001 to 150 mg per 1 kg of body weight, preferably 0.01 to 100 mg daily or every other day, or 1 to 1 per day. It can be divided into 3 doses. However, since it may increase or decrease depending on the route of administration, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

Example

The present invention will be described in more detail through the following examples. However, these examples show some experimental methods and compositions to illustratively explain the present invention, and the scope of the present invention is not limited to these examples.

Experimental Example 1: Mass culture of kefir-derived lactic acid bacteria

The three strains of kefir-derived lactic acid bacteria listed in Table 1 below were spread on MRS (man Rogosa Sharpe) agar and cultured for 48 hours. Then, it was transferred to 10 ml of MRS broth and cultured by shaking at 120 rpm for 48 hours. Then, it was transferred to 500 ml of MRS broth and cultured in large quantities by shaking at 120 rpm for 48 hours. At this time, Lactobacillus kefir was cultured at a temperature of 37 ° C, and Leuconostoc mecenteroides was cultured at a temperature of 30 ° C.

strain short name source Lactobacillus Kefir DH5 DH5 Konkuk University Veterinary Medicine Food Safety Research Center Leuconostoc mecenteroides DH1606 LCM6 Leuconostoc mesenteroides DH1608 LCM8

Experimental Example 2: Extracellular polysaccharide (EPS) extraction and component analysis from kefir-derived lactic acid bacteria

Among the lactic acid bacteria mass-cultivated in Experimental Example 1, 80% of trichloroacetic acid (TCA) was added to each of DH5 and LCM6 to obtain a final concentration of 17.5%. Thereafter, the mixture was reacted for 30 minutes in a shaking incubator and then centrifuged at 4° C. for 20 minutes at a speed of 8,000×g. The supernatant was separated, mixed with 2-fold volume of ethanol (99.9%), and placed in a refrigerator to stand for 24 hours. This process was repeated twice, and after discarding the upper layer of ethanol, a precipitate was obtained.

Thereafter, the precipitate was subdivided into conical tubes and centrifuged for 20 minutes at a temperature of 4° C. at a speed of 8,000×g. After discarding the supernatant, the precipitate was mixed with tertiary distilled water and placed in a Spectra/Por 4 RC dialysis membrane, followed by dialysis in tertiary distilled water for 48 hours. After the dialysis, the extracellular polysaccharide was transferred to a 50ml conical tube and stored for 24 hours at -80°C in a deep freezer. Then, it was lyophilized and powdered to obtain extracellular polysaccharide (EPS) of DH5 and LCM6.

Then, the components of the EPS of DH5 and LCM6 were analyzed through HPLC (High-Performance Liquid Chromatography), and the results are shown in (a) and (b) of FIG. 1, respectively.

1, it can be seen that the main components of the sugar constituting the EPS of DH5 and LCM6 are mannose, glucose, galactose, arabinose and fucose. .

Experimental Example 3: Cell surface protein (SLP) extraction and component analysis from kefir-derived lactic acid bacteria

Each of DH5 and LCM8 among the lactic acid bacteria mass-cultivated in Experimental Example 1 was subdivided into 250ml each in a 500ml centrifuge bottle and centrifuged at a speed of 10,000×g at a temperature of 4° C. for 10 minutes. After discarding the supernatant, the precipitate was suspended in 1X PBS (pH 7.2). Then, centrifugation was performed again at 10,000×g at a temperature of 4° C. for 10 minutes.

Thereafter, 150 ml of 5M LiCl per bottle was added to the precipitate after discarding the supernatant, and vortexing was performed, followed by reaction at a speed of 200 rpm in a shaker for 1 hour.

Then, it was subdivided into a 50ml conical tube and centrifuged at a speed of 16,000×g at 4° C. for 30 minutes. After collecting only the supernatant, it was filtered under reduced pressure using a 0.45 μm cellulose ester membrane and collected in a sterilized flask.

The filtrate was placed on a Spectra/Por 4 RC dialysis membrane, and dialysis was performed in a mixture of 1X PBS and 0.05% tween-20 for 24 hours. After dialysis, it was subdivided into 50 ml conical tubes and stored for 24 hours at -80 ° C in a cryogenic refrigerator. Then, it was lyophilized and powdered to obtain cell surface proteins (SLPs) of DH5 and LCM8.

Thereafter, the components of SLP of LCM8 were analyzed through LCMS (Liquid Chromatograph Mass Spectrometry), and the representative protein ID and its name obtained as a result are shown in FIG. 2.

2, it can be seen that the most abundant protein in the SLP of LCM8 is a transcriptional regulator with CBS domains closely related to anti-inflammatory metabolism.

Experimental Example 4: Analysis of body weight change of mice according to diet

C57BL/6 mice were divided into 11 groups and provided with the diet shown in Table 2 below for 7 weeks.

In Table 2, BW represents body weight, and the content of grape seed powder is the ratio (wt%) to the total weight of the high-fat diet. As the grape seed powder, Chardonnay grape seed powder was used.

Group No. type of diet One normal diet 2 High-fat diet (HF) 3 High-fat diet containing EPS (250mg/Kg BW) of DH5 4 High-fat diet containing EPS (250 mg/Kg BW) of LCM6 5 High-fat diet containing DH5's SLP (120mg/Kg BW) 6 High-fat diet containing SLP (120mg/Kg BW) of LCM8 7 High-fat diet containing EPS from DH5 (62.5mg/Kg BW), SLP from DH5 (30mg/Kg BW) and grape seed powder (0.5%) 8 High-fat diet containing EPS (62.5mg/Kg BW) from LCM6, SLP (30mg/Kg BW) from LCM8 and grape seed powder (0.5%) 9 High-fat diet containing EPS (62.5mg/Kg BW) from DH5, SLP (30mg/Kg BW) from LCM8 and grape seed powder (0.5%) 10 High-fat diet containing EPS (62.5mg/Kg BW) from LCM8, SLP (30mg/Kg BW) from DH5 and grape seed powder (0.5%) 11 High-fat diet containing grape seed powder (2%)

Figure 3 shows the daily energy intake (kcal) of the experimental rat according to the type of diet, Figure 4 shows the weight gain (g) of the experimental rat according to the type of diet.

In FIG. 3, although there was no significant difference in daily energy intake between the experimental groups (groups 2 to 11) consuming a high-fat diet, the group consuming kefir-derived lactic acid bacteria cell surface protein (SLP) in FIG. 4 5 and 6 had significantly lower weight gain (over 20% weight gain compared to control group 2) decrease) was observed.

In addition, comparing the results of groups 7 to 10 who ingested the cell surface protein (SLP) of kefir-derived lactic acid bacteria, the extracellular polysaccharide (EPS) of kefir-derived lactic acid bacteria, and grape seed powder in combination, the EPS of DH5 and the SLP of DH5 Group 7, which consumed grape seed powder together, showed less weight loss than group 5, which consumed only SLP of DH5, whereas group 9, which consumed EPS of DH5 and SLP of LCM8 together with grape seed powder, showed the greatest weight loss among all groups. It was confirmed that there was a synergistic effect on obesity by showing weight loss (about 28% decrease in weight gain compared to the control group 2).

Experimental Example 5: Analysis of weight change in adipose tissue of experimental mice according to diet

Figure 5 shows the weight change (g) of the adipose tissue of the experimental rat according to the type of diet.

Although there was no significant difference in total energy intake between the experimental groups (groups 2 to 11) consuming a high-fat diet, groups 5 and 6 consuming kefir-derived lactic acid bacteria cell surface protein (SLP) showed significant differences. showed a low adipose tissue weight change, and the groups 8 to 10 who ingested kefir-derived lactic acid bacteria cell surface protein (SLP), kefir-derived lactic acid bacteria extracellular polysaccharide (EPS), and grape seed powder in combination also had significantly lower Adipose tissue weight change was shown.

Experimental Example 6: Biochemical test

Tests for plasma glucose concentration (mg / dL) and plasma insulin concentration (mg / dL) were performed using an automated biochemical analyzer (7020, HITACHI, JAPAN) from the blood of experimental mice fed the diet for 7 weeks in Experimental Example 4 And the results are shown in Figures 6 and 7, respectively.

In FIG. 6, the plasma glucose concentration was lower in groups 5 to 10 who consumed kefir-derived lactic acid bacteria cell surface protein (SLP), and in particular, group 9 who ingested EPS of DH5 and SLP of LCM8 in combination showed the lowest value.

The result of plasma insulin concentration in FIG. 7 also showed a similar trend to FIG. 6 .

Experimental Example 7: Insulin tolerance test (ITT)

Before the end of the experiment in Experimental Example 4, 5 to 6 rats were selected for each group, fasted for 5 to 7 hours, and insulin (0.5 U/kg) was intraperitoneally injected. Blood glucose levels (mg/dL) were measured by collecting blood from the tail vein at 0, 15, 30, 60, and 90 minutes after insulin administration, and the results are shown in FIG. 8 , respectively.

In FIG. 8, the AUC (Area Under Curve) value of ITT was significantly decreased compared to the control group in groups 8, 9, and 10 ingesting LCM8 with SLP alone (group 6) or EPS and SLP with grape seed powder. In the case of obesity, since insulin resistance increases and the response to insulin decreases, the overall blood glucose level remains high after insulin administration, and the AUC value is higher than normal. In FIG. 8 , the group in which the AUC value decreased indicates that blood glucose control by insulin was well achieved, and thus, the response to insulin was improved in groups 6, 8, 9, and 10.

Experimental Example 8: Gene profiling by microarray analysis in epididymal adipose tissue

After the experiment in Experimental Example 4 was completed, RNA was isolated from the epididymal tissue of the rats of Groups 2, 6, and 9, and microarray analysis was performed using an Agilent microarray chip. The difference in gene expression level between Group 6 and the control group 2 is shown in FIG. 9 , and the difference between the gene expression level between Group 9 and the control group 2 is shown in FIG. 10 .

In addition, significant pathways through Ingenuity pathway analysis (IPA) are shown in FIGS. 11 and 12, respectively. Specifically, FIG. 11 shows the five major biochemical functions, standards, and related metabolic pathways in epididymal adipose tissue of group 6 compared to group 2, the control group, and FIG. 12 shows the epididymal adipose tissue of group 9 compared to group 2, the control group. It shows the five main biochemical functions and canonical and related metabolic pathways shown in .

9 and 10, it can be seen that groups 6 and 9 showed increased expression of Ebf2 and Cfd genes and decreased expression of genes such as Dock8 and Ubd , compared to group 2, which is a control group. Ebf2 is a gene that causes brown fat to be formed from white fat, and Cfd , a gene related to inflammation and immune response, tends to decrease in obesity. In addition, Dock8 is a gene associated with B cell immune synapse formation, and Ubd is a gene that promotes an inflammatory response and inhibits fat metabolism. These results suggest that the intake of kefir-derived lactobacillus cell surface protein (SLP) or the combined intake of kefir-derived lactobacillus cell surface protein (SLP), kefir-derived lactobacillus extracellular polysaccharide (EPS), and grape seed powder can reduce obesity. It means that it is effective in suppressing and improving immune and inflammatory responses.

As a result of Ingenuity pathway analysis (IPA) analysis of FIGS. 11 and 12, intake of cell surface protein (SLP) of kefir-derived lactic acid bacteria, or cell surface protein (SLP) of kefir-derived lactic acid bacteria, or extracellular polysaccharide of kefir-derived lactic acid bacteria (EPS) and grape seed powder are significantly related to the improvement of immune and inflammatory responses and the prevention of metabolic diseases.

Experimental Example 9: Measurement of gene expression in adipose tissue and liver tissue through real-time RT-PCR

In order to synthesize the cRNA required for real-time RT-PCR, epididymal adipose tissue and group 2, group 3, group 5, group 6, and group 8 of rats of groups 2, 6, and 9 after the experiment of Example 4 were completed. , RNA was isolated from liver tissues of mice of groups 9 and 10, and the concentration was adjusted to 250 ng/μl. Mix 2 μl of 5X Prime script buffer, 0.5 μl of PrimeScript RT enzyme mix, 0.5 μl of Oligo dT Primer (50 μM), and 0.5 μl of Random 6 mers (100 μM) from the Takara Prime Script reagent kit, and add RNA and RNase free water to each sample. A final 10 μl of mixture was made.

Subsequently, the expression levels of the following 8 genes ( Acsm3, Atp6v0d2, Cfd, Dock8, Ebf2, Mrc2, Trem2, Ubd ) among the genes that were significant as a result of microarray analysis of Experimental Example 8 were measured using adipose tissue in Real-Time RT-PCR. The results are shown in FIG. 13, and the expression level of the HP gene was measured using liver tissue, and the results are shown in FIG. 14.

It can be seen from FIG. 13 that the expression levels of all genes tested in adipose tissue tend to coincide with the results of Microarray analysis in Experimental Example 8.

In addition, it can be seen from FIG. 14 that the expression level of the HP gene related to intestinal permeability in liver tissue was significantly decreased, especially in group 9.

As above, specific parts of the content of the present invention have been described in detail, and for those skilled in the art, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. It will be clear. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (5)

Kefir-derived lactic acid bacterium Leuconostoc mesenteroides LCM8 (Accession Number: KCTC18854P) cell surface protein (surface layer protein, SLP), Lactobacillus kefiri DH5 (Accession Number: A functional food composition for improving obesity, comprising exopolysaccharide (EPS) of KCCM11837P) and grape seed powder as active ingredients. delete delete delete Kefir-derived lactic acid bacterium Leuconostoc mesenteroides LCM8 (Accession Number: KCTC18854P) cell surface protein (surface layer protein, SLP), Lactobacillus kefiri DH5 (Lactobacillus kefiri DH5, Accession Number: KCCM11837P) extracellular polysaccharide (exopolysaccharide, EPS) and a pharmaceutical composition for preventing or treating obesity, including grape seed powder.

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