WO2024038980A1 - Lactobacillus plantarum ku15120 strain or use thereof - Google Patents

Abstract

The present invention relates to Lactobacillus plantarum KU15120 strain and a use thereof. The (Lactobacillus plantarum KU15120 strain was found to have antioxidative and anti-obese effects, which led to the present invention.

Description

Lactobacillus Plantarum KU15120 strain or use thereof

The present invention relates to Lactobacillus plantarum KU15120 strain or its use.

Modern people are in a more complex relationship with society, and due to irregular changes in eating habits, insufficient amount of exercise, and excessive mental stress, toxic reactive oxygen species such as free organic hydroporoxide, hydrogen peroxide, hydroxy radical, superoxide, and nitrogen monoxide are generated in the human body. It is caused by an imbalance between pro-oxidants and antioxidants leading to ROS), which accelerates aging, causes inflammation in the human body, reduces immunity, makes the body vulnerable to various diseases, and even causes cancer (Ahamed et al., 2011).

Biochemical reactions constantly occur to supply energy in the living body, and most of the free radicals generated during this process are basically destroyed by the body's self-defense mechanism, but free radicals and The production of free radicals is the cause of various diseases such as cancer, as well as adult diseases and circulatory disorders.

Substances that prevent damage to the body due to free radicals, which are the cause of various diseases, are called antioxidants. Antioxidants can be taken as drugs, but they can occur if exposed for a long time. You need to be careful about side effects.

It is known that obesity can act as a risk factor that increases the occurrence of complications such as diabetes and cardiovascular disease when body fat accumulates in the body more than necessary (Cui et al., 2015; Li et al., 2008).

In Korea, the prevalence of obesity among adults aged 19 or older is increasing from 31.7% in 2007 to 35.7% in 2018 (Korea Disease Control and Prevention Agency, 2018 data). In addition, the severely obese population with a body mass of 30 or more increased from 3.5% in 2009 to 6.01% in 2018, and is expected to increase to 9.0% in 2030 (Korean Obesity Society, 2019 data).

As a mechanism related to the inhibition of adipocyte differentiation, adipocytes differentiate from preadipocytes into adipocytes, and as fat synthesis increases in adipocytes, they change into mature adipocytes and adipose tissue increases, leading to adipogenesis and adipogenesis. Inhibiting intracellular lipogenesis can reduce body fat accumulation.

Expression of transcription factors (peroxisome proliferator-activated receptor gamma (PPARr), CCAAT/enhancer binding protein alpha (C/EBPa), sterol-regulatory element binding protein (SREBP), etc.) that reduce the differentiation of preadipocytes into adipocytes. Inhibiting differentiation into adipocytes can be inhibited. Additionally, it can inhibit body fat synthesis by inhibiting the activity or expression of enzymes related to body fat synthesis (Korea Food and Drug Safety Evaluation Institute, 2019).

Probiotics are defined as those that benefit the host's health when consumed in appropriate amounts (FAO/WHO, 2006). Probiotics must survive in the stomach to improve the intestinal environment (Franca et al., 2015). Probiotics are reported to have various functional properties such as antioxidant, immunomodulatory, cholesterol-lowering, anti-diabetic, and anti-obesity effects (Cheon et al., 2011; Lee et al., 2015).

The present invention relates to the Lactobacillus plantarum KU15120 strain, which can act as a probiotic and has probiotic activity, antioxidant, and body fat reduction effects. We confirmed the probiotic properties of Lactobacillus plantarum KU15120 strain isolated from radish kimchi, such as acid resistance, rash resistance, and intestinal adhesion ability, and killed it through antioxidant activity and heat treatment, and used this to suppress the differentiation of adipocytes to reduce body fat. By verifying the reduction effect, its potential as a functional food material capable of antioxidant and body fat reduction was confirmed.

As reference prior art documents, Korean Patent No. 1997362 and Korean Patent No. 2344838 were used.

The present invention was derived from the above-mentioned needs, and the present inventors discovered the antioxidant or anti-obesity effect of the Lactobacillus plantarum KU15120 strain, accession number KCCM 12752P, isolated from radish kimchi, and thus the present invention. Completed.

In order to solve the above problem, the present invention provides Lactobacillus plantarum KU15120 strain, accession number KCCM 12752P.

In one example of the present invention, the strain may include the base sequence of SEQ ID NO: 1.

The present invention provides a probiotic composition comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient.

The present invention provides an antioxidant composition comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating obesity comprising Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, a culture medium thereof, a concentrate of the culture medium, or a dried product thereof as an active ingredient.

In another example of the present invention, the strain may inhibit adipocyte differentiation.

In addition, the present invention provides a food composition for preventing or improving obesity comprising the Lactobacillus plantarum KU15120 strain, accession number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient.

In addition, the present invention provides a feed composition for preventing or improving obesity comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient.

The present invention provides a method for preventing or improving obesity in non-human animals, comprising the step of administering Lactobacillus plantarum KU15120 strain, a culture medium thereof, a concentrate of the culture medium, or a dried product thereof, with accession number KCCM 12752P. .

Since the Lactobacillus plantarum KU15120 strain, accession number KCCM 12752P of the present invention, showed antioxidant or anti-obesity activity, it can be usefully used as a pharmaceutical, food or feed composition.

Figure 1 shows the base sequence of strain KU15120.

Figure 2 shows the family diagram of the KU15120 strain.

Figure 3 shows the intestinal adhesion ability of Lactobacillus strains. LGG, L. rhamnosus GG; KU15120, L. plantarum KU15120.

Figure 4 shows the antioxidant activity of Lactobacillus strains. (A) DPPH (B) ABTS (C) b-carotene bleaching inhibition activity of LAB strains. LGG, L. rhamnosus GG; KU15120, L. plantarum KU15120.

Figure 5 shows the cytotoxic effect of Lactobacillus strains. ■, 10 ⁸ CFU/mL; □, 10 ⁹ CFU/mL; LGG, L. rhamnosus GG; KU15120, L. plantarum KU15120.

Figure 6 shows the effect of inhibiting fat differentiation through Oil red O staining of Lactobacillus strains. (A) Microscopic observation (i, Negative control; ii, Positive control; iii, LGG 10 ⁸ CFU/mL; iv, KU15120 10 ⁸ CFU/mL; v, LGG 10 ⁹ CFU/mL; vi, KU15120 10 ⁹ CFU/ mL) (B) Measurement using colorimetric method. □, 10 ⁸ CFU/mL; ■, 10 ⁹ CFU/mL; NC, negative control; PC, positive control; LGG, L. rhamnosus GG; KU15120, L. plantarum KU15120.

Figure 7 shows the effect of inhibiting the triglyceride content of Lactobacillus strains. □, 10 ⁸ CFU/mL; ■, 10 ⁹ CFU/mL; NC, negative control; PC, positive control; LGG, L. rhamnosus GG; KU15120, L. plantarum KU15120.

Figure 8 shows the results of confirming the expression of the ability to suppress fat differentiation through RT-PCR and western blot methods of Lactobacillus strains. (A) FAS mRNA (B) C/EBPa mRNA (C) PPARr mRNA (D) Western blot analysis. □, 10 ⁸ CFU/mL; ■, 10 ⁹ CFU/mL; NC, negative control; PC, positive control; LGG, L. rhamnosus GG; KU15120, L. plantarum KU15120.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details, such as specific components, are shown, but this is provided to facilitate a more general understanding of the present invention, and it is known in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge. Additionally, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

In order to achieve the object of the present invention, the present invention provides Lactobacillus plantarum KU15120 strain, accession number KCCM 12752P.

In one example of the present invention, the strain may include the base sequence of SEQ ID NO: 1.

The strain may be derived from kkakdugi kimchi, but is not limited thereto.

The strain may be a live strain or an attenuated strain (dead strain). “Attenuated” means modifying a strain to reduce its pathogenicity. Attenuation is done for the purpose of reducing toxicity and other side effects when the strain is administered to a subject. Attenuated strains can be prepared through various methods known in the art. For example, attenuation can be achieved by deleting or destroying virulence factors that allow the strain to survive in host cells. The deletion and destruction are well known in the art and are carried out, for example, by methods such as homologous recombination, chemical mutagenesis, irradiation mutagenesis, or transposon mutagenesis.

The present invention provides a probiotic composition comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient.

The term "culture medium" in the present invention refers to the entire medium containing the strain, its metabolites, and extra nutrients obtained by culturing the strain for a certain period of time in a medium that can supply nutrients so that the strain can grow and survive in vitro. means, but is not limited to this.

“Probiotics” of the present invention are live bacteria, that is, microorganisms that can survive in the gastrointestinal tract when ingested by humans or animals, and refer to microbial preparations that are effective in preventing or treating specific pathological conditions. In general, probiotics are effective in treating and improving various symptoms caused by abnormal fermentation of intestinal flora. When administered to humans and animals, they crowd and settle on the walls of the digestive tract and prevent harmful microorganisms from settling, causing lactic acid. It inhibits the growth of harmful microorganisms by lowering the intestinal pH.

In the probiotic composition of the present invention, the strain culture medium contains various antibacterial organic acids and non-proteinaceous antibacterial substances produced by the strain, so when included as an active ingredient in a probiotic composition, it can exhibit the same effect as a composition containing the strain.

In addition, the probiotic composition of the present invention is suitable for consumption by livestock along with the Lactobacillus genus lactic acid bacteria, inhibits the growth of harmful microorganisms upon ingestion, and contains other types of known microorganisms that have the activity of improving the balance of intestinal flora. may additionally be included.

The probiotic preparation of the present invention can be prepared and administered in various formulations and methods according to methods known in the art. For example, the Lactobacillus plantarum JDFM LP11 strain of the present invention, its culture, a concentrate of the culture, or a dried product thereof are mixed with a carrier commonly used in the pharmaceutical field to form powders, liquids and solutions. , it can be manufactured and administered in the form of tablets, capsules, syrup, suspension, or granules. The carrier may include, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a coloring agent, and a flavoring agent, but is not limited thereto. Additionally, the administered dose can be appropriately selected depending on the absorption rate of the active ingredient in the body, the inactivation rate, the excretion rate, the age, gender, species, condition, and severity of the disease of the recipient.

The probiotic composition of the present invention contains a strain or a culture medium thereof that is excellent in inhibition of intestinal pathogenic bacteria, acid resistance, bile resistance, etc., and thus has excellent infection inhibition and treatment effects of various intestinal pathogenic bacteria, and normalizes the host's intestinal flora. Not only can it prevent bacterial diseases, but it can also be usefully used in feed, food, and medicine as a probiotic composition such as an intestinal pathogenic bacteria inhibitor, immune enhancer, sterilizer, digestive agent, intestinal stimulant, and antidiarrheal agent.

The present invention provides an antioxidant composition comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating obesity comprising Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, a culture medium thereof, a concentrate of the culture medium, or a dried product thereof as an active ingredient.

In the present invention, the term "prevention" means suppressing or delaying the occurrence of a disease from its cause.

In this specification, “treatment” means stopping the progression of damage by suppressing the progression and/or worsening of symptoms even if not completely cured, or improving some or all of the symptoms and leading toward healing.

The composition of the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of the composition.

The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, drug treatment, and biological response regulators for the prevention and treatment of diseases.

The pharmaceutical composition of the present invention can be formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injectable solutions according to conventional methods. , tablets, capsules, injections and liquid formulations are more preferable. This formulation can be performed by a method commonly performed in the pharmaceutical field, and can be preferably formulated according to each disease or ingredient using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, and cellulose. , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

When formulated, it can be prepared by additionally using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It is prepared by mixing lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.

Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous agents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween, cacao, laurin, glycerogeratin, etc. can be used.

The preferred dosage of the composition of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for desirable effects, the composition of the present invention may be included in an amount of 0.01 to 99.9% by weight, preferably 0.1 to 99% by weight, per day. The daily dosage may be about 0.1 to 1,000 mg/kg, preferably 100 to 300 mg/kg.

The present invention provides a food composition for preventing or improving obesity comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, a culture medium thereof, a concentrate of the culture medium, or a dried product thereof as an active ingredient.

In the present invention, the term “improvement” refers to any action in which obesity is improved or beneficially changed by administration of the composition of the present invention.

When using the composition of the present invention in food, the composition can be added as is or used together with other health functional foods or health functional food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use. In general, when producing a food or beverage, the composition of the present invention can be added in an amount of preferably 15 parts by weight or less, more preferably 10 parts by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health control and hygiene, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

There is no particular limitation on the type of health functional food that can contain the composition of the present invention, and specific examples include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and ice cream. There are dairy products, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, etc., and can include all health functional foods in the conventional sense, and can include foods used as feed for animals.

There are no particular restrictions on other ingredients that can be included in the food composition of the present invention, and like conventional foods, various herbal extracts, food supplements, or natural carbohydrates can be included as additional ingredients.

In addition, as mentioned above, food auxiliary additives may be additionally added. Food auxiliary additives include food auxiliary additives common in the art, such as flavoring agents, flavors, colorants, fillers, stabilizers, etc. .

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the above-mentioned flavoring agents, natural flavoring agents (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.

In addition to the above, the food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and It may include its salt, organic acid, protective colloidal thickener, pH adjuster, stabilizer, preservative, glycerin, alcohol, carbonating agent used in carbonated beverages, etc. In addition, it may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination.

The above-mentioned health functional food (functional food) is the same term as food for special health use (FoSHU), and is a medicine processed to efficiently exhibit bioregulatory functions in addition to nutritional supply, with high medical effects. It means food. Here, “function” means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes, such as physiological effects. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Additionally, the food formulation can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various types of formulations, and unlike general drugs, it is made from food as a raw material and has the advantage of not having side effects that may occur when taking the drug for a long period of time. It is also excellent in portability and can be used as a pharmaceutical composition according to the present invention. The food composition can be taken as a supplement.

The present invention provides a feed composition for preventing or improving obesity comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient.

In the present invention, the term "feed" may mean any natural or artificial diet, meal, etc., or a component of the meal, for or suitable for eating, ingestion, and digestion by animals. The feed composition may include feed additives. The above feed additives correspond to supplementary feed under the Feed Management Act.

The type of feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the feed include plant feeds such as grains, roots and fruits, food processing by-products, algae, fiber, pharmaceutical by-products, oils and fats, starches, cucurbits or grain by-products; Examples include animal feed such as proteins, inorganic substances, fats and oils, minerals, single-cell proteins, zooplankton, or food. These may be used individually or in combination of two or more types.

The feed composition according to the present invention can be used by adding it to basic feed at a certain ratio. The basic feed may consist of corn, soybean meal, whey, fish meal, molasses, salt, vitamin premix, mineral premix, etc. The vitamin premix may consist of vitamin A, vitamin D, vitamin E, riboflavin, and niacin, and the mineral premix may consist of, but is not limited to, manganese, iron, zinc, calcium, copper, cobalt, and selenium. .

The present invention provides a method for preventing or improving obesity in non-human animals, comprising the step of administering Lactobacillus plantarum KU15120 strain, a culture medium thereof, a concentrate of the culture medium, or a dried product thereof, with accession number KCCM 12752P. .

The term "individual" in the present invention may include mammals, including rats and livestock, without limitation, but refers to mammals other than humans.

The "administration" means introducing the composition of the present invention into an individual by any appropriate method, and the composition may be administered through various routes such as oral or parenteral. Specifically, for parenteral administration, injection methods such as external injection under the skin or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection can be selected. Additionally, the number of administrations of the composition may be single or multiple administrations in amounts effective to the individual, but are not limited thereto.

In the present invention, “administration route” may be administered through any general or special route that can reach the target tissue.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments are merely intended to ensure that the disclosure of the present invention is complete and that common knowledge in the technical field to which the present invention pertains is not limited. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

<Example 1> Strain used

Lactobacillus rhamnosus GG strain, which is widely used as a commercial strain, was used as a standard strain. Lactobacillus Plantarum KU15120 (strain accession number: KCCM12752P) was used. These strains were cultured using MRS medium.

<Example 2> Measurement of acid resistance and bile resistance of Lactobacillus strains

To measure the acid resistance and bile resistance of the strain, the pH was adjusted to 2.5 and the number of bacteria was cultured on TSB medium with 0.3% pepsin and TSB medium with 0.3% oxgall added at 37°C for 3 hours and 24 hours, respectively. was compared with the number of bacteria at 0 hours.

Survival rate (%)={(Number of viable bacteria in treatment group)/(Number of viable bacteria in control group)}×100

<Example 3> Measurement of enzyme production ability of Lactobacillus strains

Using the API ZYM kit, the production capacity of various enzymes was confirmed, and among them, the production of b-glucuronidase, an enzyme that causes cancer, was confirmed. Lactobacillus Frantarum KU15120 was adjusted to a bacterial count of 1×10 ⁵ CFU/mL, washed twice with PBS, and then zym A and zym B reagents were added and cultured at 37°C for 4 hours.

<Example 4> Measurement of antibiotic resistance of Lactobacillus strains

Antibiotic resistance was measured using disk diffusion assay according to CLSI (Clinical and Laboratory Standards Institute) standards. Eight antibiotics (ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, ciprofloxacin, chloramphenicol, and doxycycline) were used. Lactobacillus bacteria were plated on TSA medium at a concentration of 10 ⁶ CFU/mL, then a paper disk was placed and antibiotics were loaded. After culturing at 37°C for 24 hours, the clear zone was measured.

<Example 5> Measurement of intestinal fixation ability of Lactobacillus strains

The Lactobacillus strain was inoculated into 1×10 ⁵ cells/well of HT-29 cells and incubated at 37°C for 2 hours. After culturing, the cells were washed three times with PBS to wash away bacteria that were not attached to the HT-29 cells, and then the attached cells were removed with a 1% Triton X-100 solution and the number of bacteria was measured using TSA medium.

<Example 6> Measurement of antioxidant activity of Lactobacillus strains

1) DPPH free radical scavenging ability

The antioxidant effect by DPPH free radical scavenging ability is one of the antioxidant measurement methods that removes DPPH free radicals and observes the color change of the DPPH solution. The free radical scavenging ability was measured as follows. 0.2 mL of the sample and control were mixed with 1 mL of 100 μM DPPH (1,1-diphenyl-2-picrylhydrazyl), left at room temperature for 15 minutes, and the absorbance (optical density, OD) was measured at 517 nm. At this time, the DPPH free radical scavenging ability was calculated as follows.

DPPH free radical scavenging ability (%)={1-(absorbance of sample/absorbance of control)}×100

2) b-Carotene bleaching assay

The b-carotene bleaching assay tests the oxidation inhibitory ability of the active ingredient against b-carotene. To prepare a b-carotene solution, 3 mg of b-carotene, 66 μL of linoleic acid, and 300 μL of Tween 80 were dissolved in 15 mL of chloroform, the solvent was removed using a vacuum concentrator, and 150 mL of distilled water was added. 0.5 mL of Lactobacillus plantarum KU15120 sample was reacted with 4.5 mL of b-carotene solution in a constant temperature water bath at 50°C for 2 hours, then the absorbance was measured at 470 nm, and the oxidation inhibition ability was calculated as follows.

b-carotene oxidation inhibition ability (%) = {(Absorbance of 2h sample - Absorbance of 2h control group)/(Absorbance of 0h control group - Absorbance of 2h control group)} × 100

<Example 7> Measurement of body fat reduction effect of Lactobacillus strain on adipocytes

1) Preparation of dead cells

The lactic acid bacteria strain is inoculated into MRS medium and cultured for 24 hours. After adjusting the number of bacteria to 1 × 10 ⁹ CFU/mL, the cells were washed twice with PBS and then heat treated at 80°C for 30 minutes to prepare dead cells.

2) Measurement of cytotoxicity on adipocytes

MTT (methylthizol-2-yl-2,5-diphenyl tetrazoliumbromide) assay was performed to measure whether Lactobacillus plantarum KU15120 induces cytotoxicity against 3T3-L1 (mouse embryo, ATCC CL-173). Add 0.1 mL of Lactobacillus plantarum KU15120 suspended in DMEM to a 96 well-plate and culture for 44 hours. After removing the medium, add 100 μL of MTT (2.5 mg/mL) to the well and incubate for 4 hours to induce a reduction reaction. Then, remove the culture medium and add 0.1 mL of DMSO (Dimethyl sulfoxide) solution to react for 15 minutes. The formed crystals were melted. Afterwards, the absorbance was measured at 540 nm, and the equation for cell viability was as follows.

Cell survival rate (%)={(cell survival rate for treatment group)/(cell survival rate for control group)}×100

3) Differentiation of adipocytes

3T3-L1 cells (1.5×10 ⁴ cells/dish) are inoculated on a 6 cm dish and cultured until adipocytes form confluence on the dish. After culturing, the strain was dissolved in DMEM medium (MDI medium) containing 0.5 mM 3-Isobutyl-1-methylxanthine (IBMX), 10 μM dexamethasone, and 5 μg/mL insulin, treated with adipocytes, and cultured for 2 days. The strain was dissolved in DMEM medium containing 5 μg/mL insulin, treated with adipocytes, and cultured at 2-day intervals until the 6th day.

4) Measurement of Oil red O staining

To fix the differentiated adipocytes, they are treated with 10% formaldehyde solution and treated for 2 hours. After washing twice with PBS, treat with 0.5% oil red O solution and stain at room temperature for 20 minutes. Then, observe by photographing, wash twice with PBS, treat with isopropanol to elute the stained oil red O, and measure absorbance at 520 nm.

5) Triglyceride content measurement

Inhibition of adipocyte differentiation was confirmed by measuring the triglyceride content in differentiated adipocytes. To collect proteins from differentiated adipocytes, they were centrifuged at 14,000Хg for 25 minutes, and then the proteins were collected and the triglyceride content was measured using a triglyceride quantification kit (BioVision).

6) Investigation of gene expression related to body fat reduction and measurement of protein content in adipocytes

3T3-L1 cells, which are preadipocytes, were differentiated and treated with Lactobacillus strains. The inhibition of adipocyte differentiation and the increase or decrease of factors related to adipocyte differentiation, such as FAS, C/EBPa, and PPARr, were measured using real-time PCR and western blot.

Gene expression was confirmed using RNA primers (Table 1) and real-time PCR.

RNA purity was determined using Multiscan GO (Thermo Fisher Scientific, Waltham, MA, USA), and cDNA was converted using the Revertaid first strand cDNA synthesis kit (Thermo Fisher Scientific). Semi-quantitative real-time PCR used SYBR Green PCR Master Mix, and PCR conditions were 20 μL total volume, 95°C for 2 min as initial denaturation; 40 cycle 95℃ for 5 s as denaturation/60℃ for 15 s as annealing and extension; It was set at 60℃ for 10 min extension.

To collect proteins from segmented adipocytes, they were centrifuged at 14,000Хg for 25 minutes, then the proteins were collected, loaded on a 10% SDS-Page gel, electrophoresed at 100 V for 2 hours, and separated according to molecular weight. Electrophoresis was performed for 2 hours under conditions below 400A and transferred to a Bio-trace PVDF membrane (PALL Life Sciences, USA). The membrane to which the protein was transferred was treated with Tris-buffered saline (10 mM Tris-HCl, pH 7.6, 150 mM NaCl) containing 0.1% Tween-20 and 5% skim milk to remove the portion where the protein was not bound to the membrane. After blocking, primary antibodies [anti-PPARr antibody or anti-FAS antibody and C/EBPa antibody (Santa Cruz Biotechnology, Inc., USA)] diluted according to the manufacturer's protocol were bound to the blocking solution, and then washed. did. Afterwards, it was reacted with a secondary antibody (anti-rabbit-IgG-HRP; Amersham Bioscience, UK) diluted 1:5,000 in blocking solution for 2 hours, and then washed again. A luminescent reaction was induced using an ECL detection kit (Thermo Fisher Scientific, USA), and the binding reaction was observed by exposure to a film.

Gene Primer sequence b-Actin
Sense 5'-TGT CCA CCT TCC AGC AGA TGT-3' Antisense 5'-AGC TCA GTA ACA GTC CGC CTA GA-3' FAS
Sense 5'-AGG GGT CGA CCT GGT CCT CA-3' Antisense 5'-GCC ATG CCC AGA GGG TGG TT-3' C/EBPa Sense 5'-GGA ACT TGA AGC ACA ATC GAT C-3' Antisense 5'-TGG TTT AGC ATA GAC GTG CAC A-3' PPARr
Sense 5'-TTG ATT TCT CCA GCA TTT CT-3′' Antisense 5'-RTG TTG TAG AGC TGG GTC TTT-3′'

<Test Example 1> Isolation and identification of Lactobacillus strains

As a result of partial purified 16S rRNA gene analysis of the KU15120 strain isolated from radish kimchi, the nucleotide sequence shown in Figure 1 was obtained, and a family tree was obtained accordingly (Figure 2). It was confirmed that the KU15120 strain was Lactobacillus plantarum.

<Test Example 2> Acid resistance and bile resistance effects of Lactobacillus strains

Acid resistance and bile resistance were measured under the conditions of artificial gastric fluid (pepsin 3 mg/mL, pH 2.5, 3 hours) and artificial bile fluid (oxgall 3 mg/mL, pH 7.0, 24 hours). As a result, Lactobacillus plantarum KU15120 was confirmed at 93.18% and 83.09%, respectively (Table 2). The comparative strain, Lactobacillus rhamnosus GG, was 96.80% and 103.20%, respectively ( p <0.05).

Therefore, Lactobacillus plantarum The KU15120 strain was confirmed to have excellent acid resistance and bile resistance.

LAB strains L. rhamnosus GG L. plantarum KU15120 Initial cell number (Log CFU/mL) 8.31±0.09 8.80±0.06 0.3% Pepsin, pH 2.5, 3 h (Log CFU/mL) 8.25±0.10 8.21±0.04 Survival rate (%) 99.27±0.73 93.18±0.09 Initial cell number (Log CFU/mL) 8.31±0.09 8.80±0.06 0.3% Oxgall, 24 h (Log CFU/mL) 8.55±0.02 7.33±0.08 Survival rate (%) 102.88±0.17 83.09±0.09

All values are expressed as the mean±standard deviation. Values with different letters in the same row indicate significant differences for each characteristic ( p <0.05).

<Test Example 3> Enzyme production ability of Lactobacillus strain

The enzyme production ability of the Lactobacillus strain was verified using the API ZYM kit (Table 3). It was confirmed that all Lactobacillus strains do not produce b-glucuronidase, an enzyme that induces the production of carcinogenic substances. Meanwhile, Lactobacillus plantarum KU15120 showed excellent b-Galactosidase production ability and showed an enzyme production pattern similar to Lactobacillus rhamnosus GG strain.

Enzyme L. rhamnosus GG L. plantarum KU15120 Control 0 ^a 0 Alkaline phosphatase 0 0 Esterase 2 0 Esterase Lipase One 0 Lipase 0 0 Leucine arylamidase 3 4 Valine arylamidase 3 3 Cystine arylamidase 0 0 Trypsin 0 0 a-Chymotrypsin 0 0 Acid phosphatase One One Naphthol-AS-BI-phosphohydrolase 2 2 a-Galactosidase 0 0 b-Galactosidase One 3 b-Glucuronidase 0 0 a-Glucosidase 0 One b-Glucosidase One 5 N-Acetyl-b-glucosaminidase 0 2 a-Mannosidase 0 0 a-Fucosidase 0 0

<Test Example 4> Antibiotic resistance of Lactobacillus strains

Lactobacillus Plantarum The antibiotic resistance of KU15120 was tested according to CLSI standards, and antibiotic resistance can cause problems in terms of transfer of resistance genes. Lactobacillus Plantarum When compared to the industrial strain Lactobacillus rhamnosus GG, KU15120 was found to be resistant to streptomycin and chloramphenicol, and showed the same resistance to the others, so there was no concern about transfer of resistance genes (Table 4).

Antibiotics L. rhamnosus GG L. plantarum KU15120 Ampicillin (10 mg) S S Gentamicin (10 mg) R R Kanamycin (30 mg) R R Streptomycin (10 mg) R R Tetracycline (30 mg) S R Ciprofloxacin (30 mg) R R Chloramphenicol (30 mg) S I Doxycycline (30 mg) S S

<Test Example 5> Intestinal attachment ability of Lactobacillus strains

The intestinal adhesion ability of probiotics is one of the most important factors. Lactobacillus Plantarum of KU15120 It was confirmed that the intestinal attachment ability was 5.66% (Figure 3), and considering that the standard for intestinal attachment ability of existing useful lactic acid bacteria is usually 1%, Lactobacillus plantarum KU15120 was confirmed to be a strain with excellent intestinal adhesion ability.

<Test Example 6> Antioxidant effect of Lactobacillus strain

The antioxidant effect of Lactobacillus strains was evaluated using the following three methods (Figure 4). In the case of DPPH radical scavenging ability, the industrial strain Lactobacillus rhamnosus GG showed a radical scavenging ability of 36.60%, but Lactobacillus plantarum showed a radical scavenging ability of 36.60%. The KU15120 strain showed excellent radical scavenging ability of 44.68%. For ABTS radical scavenging activity, Lactobacillus rhamnosus GG and Lactobacillus plantarum The KU15120 strain showed similar radical scavenging abilities of 18.33% and 18.11%, respectively. When evaluating the degree of inhibition of lipid oxidation using b-Carotene, Lactobacillus plantarum Strain KU15120 showed a higher lipid oxidation inhibition ability at 37.61% than Lactobacillus rhamnosus GG (35.08%). When evaluated comprehensively, Lactobacillus Plantarum It can be seen that the KU15120 strain showed the most excellent antioxidant ability in the previous three experiments.

<Test Example 7> Cytotoxic effect of Lactobacillus strain on 3T3-L1 cells

MTT assay was used to confirm the cytotoxic effect of the Lactobacillus strain on 3T3-L1 cells, which are preadipocytes (Figure 5). Lactobacillus Plantarum Both KU15120 and Lactobacillus rhamnosus GG strains showed no cytotoxicity against 3T3-L1 cells, preadipocytes, at two concentrations of 10 ⁸ CFU/mL and 10 ⁹ CFU/mL, and the lipolysis inhibitory effect was tested.

<Test Example 8> Inhibition effect on fat differentiation through Oil red O staining of Lactobacillus strains

3T3-L1 cells, which are adipocytes, were treated with lactic acid bacteria and the inhibitory effect on differentiation of adipocytes was confirmed through Oil red O staining (Figure 6).

Lactobacillus plantarum compared to PC (positive control) It was confirmed that there was less staining in the fat globules in KU15120 and Lactobacillus rhamnosus GG (Figure 6A). In particular, it was confirmed that fat globules were reduced at 10 ⁹ CFU/mL rather than 10 ⁸ CFU/mL, and the comparative strains Lactobacillus rhamnosus GG and Lactobacillus plantarum It was confirmed that the size of the fat globules of KU15120 was similarly reduced. At a concentration of 10 ⁹ CFU/mL, Lactobacillus rhamnosus GG and Lactobacillus plantarum KU15120 reduced it by 54.98% and 59.88%, respectively (Figure 6B).

<Test Example 9> Effect of Inhibiting Triglyceride Content of Lactobacillus Strains

It was confirmed that treating fat cells with kimchi lactic acid bacteria reduced the content of triglyceride, a substance accumulated in fat cells (Figure 7).

It was confirmed that the triglyceride content decreased overall compared to PC, and that 10 ⁹ CFU/mL decreased the triglyceride content by about 2mM more than 10 ⁸ CFU/mL.

Lactobacillus Plantarum KU15120 appears to have reduced triglyceride content by about 70.71% compared to PC, and the triglyceride content is about 0.46% lower than the comparative strain Lactobacillus rhamnosus GG, which is similar to Lactobacillus plantarum. KU15120 appears to have excellent ability to inhibit differentiation of adipocytes.

<Test Example 10> Confirmation of expression of fat differentiation inhibition ability of Lactobacillus strain through RT-PCR and western blot method

In the process of differentiation from preadipocytes to adipocytes, transcription factors such as peroxisome proliferator-activated receptor gamma (PPARr) and CCAAT/enhancer-binding protein alpha (C/EBPa) are transferred to adipocyte-specific proteins such as glucose transporter (GLUT4) and adipocyte-specific proteins. It is related to the continuous expression of specific fatty acid binding protein 2 (aP2), and the expression of these proteins causes fat cells to absorb glucose or fatty acids and synthesize or accumulate fat, increasing the size of fat cells and fatty acid synthase (FAS). ) is a protein that promotes the synthesis of fatty acids.

Proteins related to fat accumulation in the above adipocytes were confirmed through genetic analysis and protein expression (Figure 8).

As shown in Figure 8 (A, B, C), PPARr, C/EBPa, and FAS all showed RT-PCR results of Lactobacillus plantarum. The overall expression content of KU15120 was decreased compared to PC, and it was confirmed that the expression amount was lower at a concentration of 10 ⁹ CFU/mL than at 10 ⁸ CFU/mL, and the expression amount was higher than that of the comparative strain Lactobacillus rhamnosus GG. Lactobacillus plantarum was confirmed to be less than Lactobacillus rhamnosus GG. It was confirmed that strain KU15120 had good differentiation inhibition ability.

It was confirmed that protein expression through the Western blot method (Figure 8D) also showed the same results as the genetic analysis.

<References>

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[Accession number]

Name of depository organization: Korea Microbial Conservation Center

Accession number: KCCM12752P

Trust date: 20200619

Claims (12)

Lactobacillus plantarum KU15120 strain with accession number KCCM 12752P. The strain according to claim 1, wherein the strain contains the base sequence of SEQ ID NO: 1. A probiotic composition comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient. Antioxidant composition comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient. Pharmaceutical composition for preventing or treating obesity comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient. The pharmaceutical composition for preventing or treating obesity according to claim 5, wherein the strain comprises the base sequence of SEQ ID NO: 1. The pharmaceutical composition for preventing or treating obesity according to claim 5, wherein the strain inhibits adipocyte differentiation. A food composition for preventing or improving obesity comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient. The food composition for preventing or improving obesity according to claim 8, wherein the strain comprises the base sequence of SEQ ID NO: 1. The food composition for preventing or improving obesity according to claim 8, wherein the strain inhibits adipocyte differentiation. A feed composition for preventing or improving obesity comprising the Lactobacillus plantarum KU15120 strain with deposit number KCCM 12752P, its culture, a concentrate of the culture, or its dried product as an active ingredient. Method for preventing or improving obesity in non-human animals comprising administering the Lactobacillus plantarum KU15120 strain with accession number KCCM 12752P, a culture medium thereof, a concentrate of the culture medium, or a dried product thereof.

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