KR102011883B1 - Lactobacillus plantarum KC3 having anti-obesity effect and uses thereof - Google Patents

Abstract

Lactobacillus plantarum KC3 strain (Accession No .: KCTC13375BP) having an anti-obesity effect is disclosed. Lactobacillus plantarum KC3 strain according to the present invention has an anti-obesity effect, and thus can be used to suppress the development of metabolic diseases caused by obesity and obesity in modern people.

Description

Lactobacillus plantarum KC3 having anti-obesity effect and uses approximately}

The present invention relates to a Lactobacillus plantarum strain having an anti-obesity effect and its use, and more particularly, to a Lactobacillus plantarum KC3 strain having an anti-obesity effect (Anti-Obesity), a probiotic agent comprising the same, a health function It relates to food compositions and pharmaceutical compositions.

Obesity is a major health problem in most countries. The World Health Organization (WHO) estimates that over 1 billion adults are overweight worldwide, and 300 million of them are clinically obese (Hotamisligil, 2006). Obesity is a major risk factor for cardiovascular disease, stroke, insulin resistance, diabetes, liver disease, neurodegenerative diseases, respiratory and other serious diseases, and is a risk factor for some cancers, including breast and colon cancers. Confirmed. In addition to the effects on physical health, obesity also leads to depression, eating disorders and low self-esteem, which have deleterious effects on quality of life and mental health.

Among these, social changes associated with steady increases in food intake with high energy loads and low exercise are increasing the incidence of metabolic diseases caused by obesity and obesity. However, traditional treatments based on low calorie diets and exercise have little effect on obesity control and only result in temporary weight loss.

Therefore, the development of drugs having an anti-obesity effect is urgently needed.

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Patent Registration No. 10-1500974 (2015.03.13.)

The present invention has been made by the above-mentioned demands, and has an anti-obesity effect, thereby providing a Lactobacillus plantarum KC3 strain which can be used to suppress the development of metabolic diseases caused by obesity and obesity in modern people.

Here, KC3 strain can be isolated from kimchi.

Lactobacillus plantarum KC3 strain (Accession Number: KCTC13375BP) according to an embodiment of the present invention is characterized by having an anti-obesity effect.

The strain is characterized in that it does not produce bio amines from one or more amino acid precursors selected from the group consisting of tyrosine, histidine, ornithine and lysine.

An embodiment of the present invention provides a probiotic formulation comprising at least one selected from the group consisting of the strain, the culture of the strain, the concentrate and the dried product of the culture as an active ingredient.

An embodiment of the present invention provides a health functional food composition comprising at least one selected from the group consisting of the strain, the culture of the strain, the concentrate of the culture and the dried product as an active ingredient.

An embodiment of the present invention provides a pharmaceutical composition comprising at least one selected from the group consisting of the strain, the culture of the strain, the concentrate and the dried product of the culture as an active ingredient.

An embodiment of the present invention provides a beverage comprising at least one of a strain, a culture of the strain, and a concentrate or dried product of the culture as an active ingredient.

An embodiment of the present invention provides a dietary supplement comprising at least one of the strain, the culture of the strain, and a concentrate or dried product of the culture as an active ingredient.

According to embodiments of the present invention, the Lactobacillus plantarum KC3 strain has an anti-obesity effect and can be used to suppress the development of metabolic diseases caused by obesity and obesity in modern people.

1 is a view showing the anti-adipogenic activity results for the Lactobacillus plantarum KC3 strain of the present invention.
Figure 2 is a diagram showing the results of the bile resistance test for Lactobacillus plantarum KC3 strain of the present invention.
Figure 3 is a diagram showing the results of pH resistance test for Lactobacillus plantarum KC3 strain of the present invention.
Figure 4 is a view showing the results of intestinal fixation ability test for Lactobacillus plantarum KC3 strain of the present invention.
5 is a diagram showing the results of the experiment on the weight of 12 weeks in obese mice.
Figure 6 is a diagram showing the weight difference between the testis fat (A), subcutaneous fat (B), and visceral fat (C) extracted after the completion of the mouse experiment.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited or limited by the embodiments, and those skilled in the art can easily change these embodiments. Also, like reference numerals in the drawings denote like elements.

Also, the terminology used herein is a term used to properly express a preferred embodiment of the present invention, which may vary depending on a viewer, an operator's intention, or customs in the field to which the present invention belongs. Therefore, the definitions of the terms should be made based on the contents throughout the specification.

Brief description of the experiment on the Lactobacillus plantarum KC3 strain of the present invention.

Alpha-amylase Inhibitory Activity Experiment

Recently, many studies for the development of obesity treatments have been conducted, and one of them is the α-amylase inhibition test (α-amylase inhibition test). α-amylase is an enzyme that breaks down α-D- (1,4) -glucan bonds of carbohydrates and is an important enzyme in carbohydrate metabolism. Currently, for the study of hyperglycemia, diabetes and insulin-related metabolic diseases, it is necessary to control the digestion and absorption of sugars, and a lot of studies on the activity inhibition of α-amylase enzyme have been conducted.

α- Glucosidase  Inhibitory Activity Experiment

α-Glucosidase is an enzyme present in small intestinal epithelial cells, and serves to break down monosaccharides for digestion and absorption of polysaccharides and disaccharide carbohydrates. Similar to α-amylase, inhibition of α-glucosidase activity can inhibit blood sugar elevation, and this principle can be used to measure activity against hyperglycemia and diabetes.

Pancreatic lipase Inhibitory Activity Experiment

Pancreatic lipase acts as a key enzyme to break down triglycerides into 2-monoacylglycerol and fatty acids [Bitou, N., M. Nimomiya, T. Tsjita and H. Okuda. 1999. Screening of lipase inhibitors from marine algae. Lipids 34, 441-445. A representative pancreatic lysosomal inhibitor is tetrahydrolipstatin (Orlistat), a derivative of lipstatin derived from Streptomyces toxitricini , which is known to be effective enough to inhibit about 30% of the ingested fat [Drent, ML, et al., 1995. Orlistat ( RO 18-0647), a lipase inhibitor, in the treatment of human obesity: a multiple dose study. Int. J. Obes. 19, 221 -226.], Currently available as a pharmaceutical. However, despite these effects, tetrahydrolipstatin is known to have side effects such as gastrointestinal disorders, hypersensitivity, bile secretion disorders, and fat-soluble vitamin absorption inhibition. Therefore, recently, research for the development of pancreatic lipase inhibitors from foods and natural products having no side effects has been conducted.

Anti- adipogenic  activity experiment

Obesity, caused by imbalances due to high fat diets, is the biggest feature of excess accumulation of fat in the body and results in adipogenesis. Peroxisome proliferator activated receptor gamma (PPARγ), which plays a key role in adipogenesis, is a process of adipogenesis that is expressed in the liver or adipose tissue in response to ingested fat, which is responsible for the synthesis of genes and lipids required for adipocyte differentiation. And genes expressed by the proteins required for storage. In other words, anti-adipogenic activity is a measure of the differentiation of adipocytes to bacteria. This method is used to determine how much fat cells are degraded by Oil Red O relative staining.

Bile Resistance and pH Resistance Experiments

Bile acids and pH secreted by gastric juice and pancreas are important factors influencing the survival of microorganisms. Therefore, it is a measure of how resistant the microorganisms to the environment of survival inhibition. This study examines the potential of probiotics by investigating the resistance to artificial gastric juice and bile, and selects and identifies the most active and resistant strains among them.

Antimicrobial activity

Antimicrobial activity was tested by Escherichia coli coli ), Salmonella typhimurium , Staphylococcus aureus , and Listeria monocytogenes . The better the inhibition against harmful strains, the better.

The microbial isolation and identification of Lactobacillus plantarum KC3 strain of the present invention was carried out by the inventors using kimchi of different regions and types, and diluted with a peptone dilution solution to bromcresol purple. After inoculating 0.1 ml each of the modified MRS medium containing sodium azide and the top planar method, incubated for 37 to 48 hours, and the colonies were separated purely from the modified MRS medium, and the yellowed fungi were selected as potential lactic acid bacteria. . The selected strains were plated in a modified MRS medium, and then separated by pure aerobic culture. As a result of identifying the purely isolated strains, the strains were Gram-positive bacillus, well grown with or without oxygen, and negative for catalase and motility. In addition, it did not grow at 15 and 45, it did not generate ammonia from gas and arginine from glucose (glucose) was confirmed to belong to the genus Lactobacillus ( Lactobacillus ).

Results of α-amylase, α-glucosidase, and pancreatic lipase inhibition of Lactobacillus plantarum KC3 strains of the present invention are shown in Table 1 below.

The first part of the numerical values shown in Table 1 mean the mean, and the second part means the standard deviation.

1 is a view showing the anti-adipogenic activity results for the Lactobacillus plantarum KC3 strain of the present invention, Table 2 below shows the anti-adipogenic activity results table for the Lactobacillus plantarum KC3 strain.

As shown in Figure 1 and Table 2, it can be seen that the Lactobacillus plantarum KC3 strain inhibits the anti-adipogenic activity of the adipocytes by 37.59%. In other words, it can be seen that Lactobacillus plantarum KC3 strain has excellent adipocyte inhibitory activity.

Adipocyte differentiation inhibitory ability was analyzed by Oil Red O staining. All values or values were marked with * at p <0.05, and marked with ** at p <0.01, and p <0.001 after Fisher's LSD test. Is indicated by ***.

Figure 2 is a diagram showing the results of the bile resistance test for Lactobacillus plantarum KC3 strain of the present invention.

More specifically, Lactobacillus plantarum KC3 strain contains MRS medium containing 0.03% of oxgall and 0.05% of L-cysteine (with oxgall) and 0.05% of L-cysteine (L). -Cysteine) was grown on MRS medium (without oxgall), all values (or values) are the mean ± standard deviation of three iterations, * denotes the case of p <0.05 between with oxgall and without oxgall. .

Figure 3 is a diagram showing the results of pH resistance test for Lactobacillus plantarum KC3 strain of the present invention.

More specifically, it shows the survival rate of the Lactobacillus plantarum KC3 strain after 3 hours in hydrochloric acid services of pH 2.0, 3.0, 4.0 and 6.4, where * is p <0.05 compared to the start time (or start time). Case is indicated, ** denotes a case of p <0.01, and *** denotes p <0.001.

Table 3 below shows the results of the antimicrobial activity test against Lactobacillus plantarum KC3 strain, and the initial bacterial count of the Lactobacillus plantarum KC3 strain is 2.10 ± 0.17 × 10 ⁶ CFU / mL, and the results for after 6 hours at 37 All figures (or values) are mean ± standard deviation of three replicates.

Table 4 below shows the enzyme activity (Enzyme) pattern for the Lactobacillus plantarum KC3 strain, the standard color is assigned a value from 0 to 2, where 0 represents negative and 5 represents the response of maximum intensity. Values from 1 to 4 represent intermediate responses with intensity levels, and approximate activity can be estimated from color intensity. 1 corresponds to 5 nanomoles, 2 to 10 nanomoles or less, 3 to 20 nanomoles or less, 4 to 30 nanomoles or less, and 5 to 40 nanomoles or less liberation.

Table 5 below shows Antibiotics susceptibility to Lactobacillus plantarum KC3 strains, along with other Lactobacillus plantarum strains. In this case, R represents resistance, which means that the inhibition zone size is 0 mm, IS represents intermediate resistance, which means that the inhibition zone size is 1 to 5 mm, and S, which represents resistance, the size of the inhibition zone. Means greater than 5mm.

Biogenic amines are produced by fermentation of foods and may vary depending on the type of microorganism or chemical and physical conditions. Biological amines produced in fermented foods can cause food poisoning or allergic reactions, which is an important criterion for selecting safe strains from food engineering.

In order to determine whether the strain of the present invention bio-amine formation in MRS liquid medium, strain grown for 16 hours in 37 conditions was transferred to a special medium according to Bover-Cid and Holzapfel (1999) and incubated for 48 hours at 37.

MRS liquid medium was added to each of the amino acid precursors of tyrosine, histidine, ornithine and lysine, and the bioamines (tyramine, histamine) were determined by strains in each medium. (histamine), putrescine (putrescine) and cadaverine (cadaverine) was confirmed whether the production. Specifically, inoculated with 1% of Lactobacillus plantarum strain isolated in MRS liquid medium to which 0.1% of the amino acid precursor was added, and then cultured 5-10 times to activate decarboxylase. Decarboxylase medium [tryptone 0.5%, yeast extract 0.5%, cocoon extract 0.5%, sodium chloride 0.5%, glucose 0.25%, Tween-80 0.05%, magnesium sulfate 0.02%, manganese sulfate 0.005%, iron sulfate 0.004%, citric acid Salt 0.2%, thiamine 0.001%, K2PO4 0.2%, calcium carbonate 0.01%, pyridoxal-5-phosphate 0.005%, amino acids 1%, bromocresol purple 0.006% and agar After mixing 2% in distilled water and adjusting the pH to 5.3], the enzyme-activated bacteria were smeared and then cultured at 37 to 24 to 48 hours to determine whether the color of the amine was generated.

Bromocresol purple in decarboxylase medium turns yellow at pH 5.2, but turns purple as the pH rises to 6.8. Therefore, it is possible to check the bio amine production by using a purple color change when the pH rises due to bio amine production.

Table 6 below shows the results of analyzing the bio amine generating ability for the Lactobacillus plantarum KC3 strain, as can be seen from Table 6, putrescine (putrescine), tyramine (histamine) and carda All were negative for cadaverine. From this, the strain of the present invention was confirmed that there is no bio amine generating ability that can cause an overactive immune response.

Figure 4 is a view showing the results of intestinal fixation ability test for Lactobacillus plantarum KC3 strain of the present invention.

More specifically, it shows the ability to adhering to HT-29 cells, showing other Lactobacillus plantarum strains and Lactobacillus rhamnosus GG probiotic strains, and all values (or values) were 3 The mean ± standard deviation of the repetition, and the a ~ e mean values with different superscripts are significantly different (P <0.05).

5 is a view showing the results of the experiment for the weight of 12 weeks obesity-induced mice, showing the other lactic acid bacteria strains together.

More specifically, ND is a normal chow fat diet group, HFD is an intermediate fat (IF) group, Xen is an IF + xenical group, LGG is an IF + LGG group, and 299v is an IF + 299v group. Dosage group, KC3 is IF + KC3 dosing group, KC4 is IF + KC4 dosing group, K10 is IF + K10 dosing group, K50 is IF + K50 dosing group, KC28 is IF + KC28 dosing group, K259 Is the IF + K259 administration group, LR86 is the IF + LR86 administration group, and KID7 is the IF + KID7 administration group. Here, the values are expressed as mean and standard deviation, and the general and mixed diet groups were marked with * at p <0.05 after the Fisher's LSD test post test for the IF diet and ** at p <0.01. and *** when p <0.001.

Figure 6 is a diagram showing the weight difference between the testis fat (A), subcutaneous fat (B), and visceral fat (C) extracted after the completion of the mouse experiment, showing the other lactic acid bacteria strains together.

More specifically, ND is a normal chow fat diet group, HFD is an intermediate fat (IF) group, Xen is an IF + xenical group, LGG is an IF + LGG group, and 299v is an IF + 299v group. Dosage group, KC3 is IF + KC3 dosing group, KC4 is IF + KC4 dosing group, K10 is IF + K10 dosing group, K50 is IF + K50 dosing group, KC28 is IF + KC28 dosing group, K259 Is the IF + K259 administration group, LR86 is the IF + LR86 administration group, and KID7 is the IF + KID7 administration group. Here, the values are expressed as mean and standard deviation, and the general and mixed diet groups were marked with * at p <0.05 after the Fisher's LSD test post test for the IF diet and ** at p <0.01. and *** when p <0.001.

In addition, the present invention may provide a probiotic formulation comprising at least one selected from the group consisting of the Lactobacillus plantarum KC3 strain, the culture of the strain, the concentrate and dried product of the culture as an active ingredient.

The probiotic formulations can be prepared and administered in a variety of formulations and methods according to methods known in the art. For example, the Lactobacillus plantarum KC3 strain of the present invention, cultures thereof, concentrates of the cultures or dried products thereof may be mixed with carriers commonly used in the pharmaceutical field to provide powders, liquids and solutions, It may be prepared and administered in the form of tablets, capsules, syrups, suspensions or granules. The carrier may be, for example, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, a perfume, and the like. In addition, the dosage may be appropriately selected depending on the degree of absorption, inactivation rate, excretion rate, age, sex, breeder, condition, and severity of disease of the active ingredient in the body.

The present invention also provides a dietary supplement for preventing or improving obesity containing Lactobacillus plantarum KC3 strain, at least one selected from the group consisting of a culture of the strain, a concentrate of the culture and a dried product as an active ingredient. to provide.

The dietary supplement composition of the present invention may be prepared in any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions and rings. The health functional food composition according to the present invention includes the Lactobacillus plantarum KC3 strain, etc. as an active ingredient to be formulated in the form of powder, liquid, tablets, soft capsules, granules, tea bags, instant tea or drink. Can be. The content of Lactobacillus plantarum KC3 strain as an active ingredient can be suitably determined depending on the purpose of use (prevention or improvement). In addition, the food composition according to the present invention, in addition to the Lactobacillus plantarum KC3 strain, also containing other components that can give a synergistic effect to the main effect, preferably within a range that does not impair the main effect of the present invention. It's okay.

The food composition formulated in such a form may be added to the food as it is, or used with other foods or food ingredients, and may be appropriately used according to conventional methods. Examples of foods include drinks, meat, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, confectionery, pizza, ramen, dairy products including other noodles, gums, ice creams, various soups, beverages, alcoholic beverages and vitamin complexes. , Dairy products and dairy products, and all functional foods in the conventional sense.

When the food composition of the present invention is a beverage, it contains Lactobacillus plantarum KC3 strain as an essential ingredient in the indicated ratio, and there are no particular limitations on other ingredients used for preparing other beverages, Eggplant flavor or natural carbohydrate and the like may be included as additional ingredients. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent other than the above-mentioned, a natural flavor, a synthetic flavor, etc. can be used. The proportion of such natural carbohydrates is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition, the food composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, colorants and enhancers (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and salts thereof , Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain a pulp for producing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the Lactobacillus plantarum KC3 strain.

In addition, the present invention provides a pharmaceutical composition for preventing or treating obesity, containing one or more selected from the group consisting of the Lactobacillus plantarum KC3 strain, the culture of the strain, the concentrate and dried product of the culture as an active ingredient. do.

The throughput of the pharmaceutical composition for preventing or treating obesity used in the present invention should be a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level may refer to an individual type and severity, age, sex, It can be determined according to the type of virus infected, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the drug used concurrently and other factors well known in the medical arts. Effective amounts may vary depending on the route of treatment, the use of excipients, and the possibility of use with other agents, as will be appreciated by those skilled in the art.

Pharmaceutical compositions for the prevention or treatment of obesity of the present invention can be prepared in pharmaceutical formulations using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. In the preparation of the formulation, it is preferred that the active ingredient is mixed or diluted with the carrier or enclosed in a carrier in the form of a container.

Accordingly, the pharmaceutical composition for preventing or treating obesity of the present invention may be formulated in the form of powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and the like, external preparations and patches according to a conventional method. It may be used, and may further comprise a suitable carrier, excipient or diluent conventionally used in the preparation of the composition.

For example, carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used.

In addition, the Lactobacillus Lactobacillus plantarum KC3 strains described herein may be contained in various types of products, such as dietary supplements, beverages, fermented foods including fermented dairy products, and the like.

Claims (7)

Lactobacillus plantarum KC3 strain having an anti-obesity effect (Accession No .: KCTC13375BP), and tested by Oil Red O relative staining technique, the fat cell differentiation activity (Anti-adipogenic activity) of fat precursor cells 37.59% inhibition, 76.80% inhibition and 13-15% intestinal fixation ability when tested for antibacterial activity of Salmonella typhimurium using KC3 strain of 2.10 ± 0.17 Х 10 ⁶ CFU / mL Lactobacillus plantarum KC3 strain to reduce the weight of the mice of the IF (intermediate fat) administration group, and to reduce the visceral fat weight of the mice of the IF diet administration group. delete delete delete delete delete delete

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