KR102266314B1 - Lactobacillus plantarum HAC03 and uses thereof - Google Patents

Abstract

The present invention relates to a Lactobacillus plantarum HAC03 strain and use thereof and, more specifically, to a Lactobacillus plantarum HAC03 strain having a preventive or therapeutic effect on inflammatory diseases or obesity, and a functional health food composition, probiotic composition, pharmaceutical composition, and feed composition including the same. According to the present invention, the Lactobacillus plantarum HAC03 strain has excellent acid resistance and bile resistance, has no resistance to various types of antibiotics, has no biological amine production ability, and has an effect of inhibiting the expression of various inflammatory cytokines, thereby being used as a health functional food composition, a probiotic composition, a pharmaceutical composition, a feed composition, and the like. The composition comprises a Lactobacillus plantarum HAC03 strain (Accession No.: KCTC 13242BP) as an active ingredient.

Description

Lactobacillus plantarum HAC03 strain and uses thereof {Lactobacillus plantarum HAC03 and uses thereof}

The present invention relates to a Lactobacillus plantarum HAC03 strain and uses thereof, specifically, a Lactobacillus plantarum HAC03 strain having an inflammatory disease or obesity prevention or treatment effect, a health functional food composition comprising the same, a probiotic composition, and a pharmaceutical compositions and feed compositions.

Due to the recent establishment of a new eating culture that increases the intake of fat and sugar and decreases the intake of fiber due to the improvement of living standards according to the modernization of the economy, preference for a high-protein/high-fat diet is prominent. Obesity is rapidly increasing due to modern lifestyle habits. As the intake of high-calorie foods increases and fiber intake decreases, obesity, especially abdominal obesity, is emerging as a serious social problem. Obesity refers to a state in which adipose tissue is excessively increased, and the increase in body weight due to obesity is mostly due to an increase in fat. Obesity-related mechanism is that by ingesting excess carbohydrates and saturated fats, carbohydrates and saturated fats contained in food are digested and absorbed into the body through the small intestine. Monosaccharides and fatty acids are accepted by adipocytes and converted into fat. The harmfulness of obesity is that obesity itself not only causes constipation, indigestion, and gastrointestinal disorders due to the pressure of the abdomen by adipose tissue in many cases, but also has a problem in that it becomes a risk factor for many adult diseases.

The inflammatory response is one of the immune mechanisms that occur in the body, and when it occurs locally, it is an important response that defends the body from invading pathogens or viruses from the outside. However, if such an inflammatory response is excessively activated systemically and chronically due to the imbalance of the immune response in the body, it causes a disturbance in the metabolism occurring in the body. As chronic inflammation in energy metabolism, the number of monocytes (macrophages, lymphocytes, plasma cells) in metabolic-related tissues increases, resulting in an increase in the expression of inflammatory cytokines. directly or indirectly inhibited. In addition, with the deepening of chronic inflammation in the tissue, the expression of tissue regeneration cytokines also increases, leading to proliferation of fibroblasts and capillaries, and the collagen fibers generated from fibroblasts are deposited and become fibrous. At this time, tissue destruction appears more severe than in acute inflammation. In particular, the chronic inflammatory response induced by obesity has been found to be the cause of various metabolic diseases such as inflammatory bowel disease (IBD), diabetes, dyslipidemia, cardiovascular disease, and arteriosclerosis.

On the other hand, beneficial bacteria (probiotics) have shared their history with humans, and their usefulness is increasing day by day as microorganisms that have a beneficial effect on the health of the host when consumed in a live state. In recent years, research on beneficial bacteria has been actively conducted, and the scope of application is expanding, such as being developed not only as general food but also as health food and medicine. These beneficial bacteria live in various parts of the host's gastrointestinal tract, skin, and mucous membranes, decompose nutrients and fiber, etc. to use them as energy sources, produce useful substances such as lactic acid and antibiotics, and inhibit the growth of harmful bacteria through nutrient competition. It plays an important role in maintaining the health of the host. In addition, beneficial bacteria are used to promote animal growth and improve feed utilization, increase resistance to infection, inhibit proliferation of harmful bacteria, reduce mortality, suppress generation of putrefactive toxic substances, and produce various vitamins.

In addition, it has been reported that changes in the composition of intestinal flora and metabolites according to ingestion of beneficial bacteria contribute to the maintenance of host homeostasis and immune tone. In particular, in the case of short-chain fatty acids (SCFA), inhibition of inflammatory response and energy metabolism by promoting differentiation of regulatory T cells and anti-inflammatory macrophages according to host intestinal cell regeneration and stimulation of G protein-coupled receptors It is pointed out as an important factor showing various functions such as improvement of metabolic diseases according to regulation. However, since these effects appear quite different between subspecies even in the same species, the efficacy of beneficial bacteria should be reviewed for each subspecies. In addition, in order to exhibit this effect, a sufficient amount of lactic acid bacteria flowing in from the outside must reach the intestine and exhibit its function. To this end, when administered orally, destruction by gastric acid should be low, and resistance to bile acids should be strong.

The present inventors have completed the present invention by isolating Lactobacillus plantarum HAC03 strain, a novel probiotic strain exhibiting anti-inflammatory and anti-obesity effects, from white kimchi, and experimentally confirming the characteristics of the strain.

Domestic Registered Patent No. 10-2011883

The problem to be solved by the present invention is to provide a Lactobacillus plantarum HAC03 strain having an inflammatory disease or obesity prevention or treatment effect, a health functional food composition comprising the strain, a probiotic composition, a pharmaceutical composition and a feed composition.

In order to solve the above problems, the present invention provides a Lactobacillus plantarum HAC03 strain having a 16s rDNA sequence represented by SEQ ID NO: 1, deposited as accession number KCTC 13242BP.

The strain may not produce biogenic amines from one or more amino acid precursors selected from the group consisting of tyrosine, histidine, ornithine and lysine.

The strain may not be resistant to one or more antibiotics selected from the group consisting of erythromycin, gentamicin, ampicillin, tetracycline, chloramphenicol, streptomycin, ciprofloxacin and penicillin.

The strain may have anti-inflammatory and anti-obesity effects by producing short chain fatty acids.

In addition, the present invention is an inflammatory disease or obesity prevention or improvement health containing at least one selected from the group consisting of the strain, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution as an active ingredient A functional food composition is provided.

The inflammatory disease is from the group consisting of enteritis, gastritis, hepatitis, bronchitis, pleurisy, arthritis, rhinitis, keratitis, nephritis, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis, degenerative neuritis and inflammatory pain. It may be one or more selected.

In addition, the present invention provides a probiotic composition containing as an active ingredient at least one selected from the group consisting of the strain, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution.

In addition, the present invention is a pharmaceutical for preventing or treating inflammatory diseases or obesity containing as an active ingredient at least one selected from the group consisting of the strain, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution. A composition is provided.

The inflammatory disease is from the group consisting of enteritis, gastritis, hepatitis, bronchitis, pleurisy, arthritis, rhinitis, keratitis, nephritis, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis, degenerative neuritis and inflammatory pain. It may be one or more selected.

In addition, the present invention provides a feed composition containing as an active ingredient at least one selected from the group consisting of the strain, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution.

The Lactobacillus plantarum HAC03 strain of the present invention has excellent acid resistance and bile resistance, has no resistance to various types of antibiotics, has no biological amine production ability, and has the effect of inhibiting the expression of various inflammatory cytokines. , health functional food, probiotic preparation, feed, etc. In addition, the strain can be used as a functional starter for fermented food for producing various lactic acid bacteria fermented milk and fermented products.

In addition, the Lactobacillus plantarum HAC03 strain of the present invention promotes the production of short-chain fatty acids in the intestine, thereby exhibiting an anti-inflammatory effect through maintenance of immune tone homeostasis, and preventing/treating obesity by alleviating blood glucose, cholesterol and triglyceride levels It is also available as a pharmaceutical composition for

1 shows the 16SrRNA sequence (A) of the Lactobacillus plantarum HAC03 strain and the result (B) matching the blast to the NCBI database (database). The highest coincidence rate is 99% seen with Lactobacillus plantarum subspecies (strain) JCM1149 (1,402/1,404), confirming that this strain is a Lactobacillus plantarum subspecies (strain), and at the same time it is a new subspecies that does not match the existing strain can
Figure 2 is a result of analyzing the amount of short-chain fatty acids after inoculation of lactic acid bacteria in the mouse cecum. A is the result of analyzing acetic acid, propionic acid, and butric acid, and B is the result of analyzing the total amount of short-chain fatty acids.
Figure 3 confirms the change in immune cytokine expression by the Lactobacillus plantarum HAC03 strain inoculation. A shows the analysis of the expression of inflammatory cytokines under infection conditions, and B shows the analysis of the expression of anti-inflammatory cytokines under normal conditions.
Figure 4 confirms the effect of improving enteritis of the Lactobacillus plantarum HAC03 strain. A is a weight loss analysis, B is a result of a decrease in colon length analysis, and C is a confirmation of cytokine changes in the extracted colon tissue.
5 and 6 show the anti-obesity effect of the Lactobacillus plantarum HAC03 strain. Fig. 5A is a change in body weight, Fig. 5B is a measurement result of tissue weight related to energy metabolism after a 12-week breeding period, Fig. 6A is an H&E staining photograph of testicular fat, Fig. 6B is an average size of adipocytes, and Fig. 6C is an adipocyte size. The distribution according to

The present inventors isolated Lactobacillus plantarum, a lactic acid bacterium from white kimchi, and analyzed the acid resistance and bile resistance, antibiotic resistance, and biological amine production ability of the isolated lactic acid bacteria. As a result, the isolated lactic acid bacteria showed excellent acid resistance and bile resistance. , it was confirmed that there is no antibiotic resistance and no biological amine production ability, and the isolated strain was named Lactobacillus plantarum HAC03 and deposited with the Korea Research Institute of Bioscience and Biotechnology on December 28, 2020 (accession number: KCTC 132424BP).

Lactobacillus plantarum of the present invention ( Lactobacillus plantarum ) may be used synonymously with Lactobacillus plantarum ( Lactoplantibacillus plantarum ).

Therefore, in one aspect, the present invention relates to a novel probiotic Lactobacillus plantarum HAC03 (Accession No.: KCTC 13242BP) strain, wherein the strain comprises a 16s rDNA sequence represented by SEQ ID NO: 1 do it with

<SEQ ID NO: 1>

The Lactobacillus plantarum HAC03 strain of the present invention is not resistant to erythromycin, gentamicin, ampicillin, tetracycline, chloramphenicol, streptomycin, ciprofloxacin or penicillin, but is not limited to the antibiotics listed above.

In addition, the Lactobacillus plantarum HAC03 strain of the present invention is characterized in that it does not produce biogenic amines such as tyramine, histamine, putrescine or cadaverine from tyrosine, histidine, ornithine, or lysine amino acid precursors. It is not limited to biogenic amines.

In addition, the Lactobacillus plantarum HAC03 strain of the present invention is characterized in that it has the effect of preventing or improving anti-inflammatory and metabolic diseases by producing short chain fatty acids. The short chain fatty acid may be acetic acid, propionic acid or butric acid, but is not limited thereto.

The term "acid resistance" of the present invention refers to the property of resisting high acidity. When probiotics have acid resistance, they can be ingested through various routes of administration, including oral, to prevent degradation or damage even when exposed to strong acidic conditions in the stomach.

As used herein, the term "biliary tolerance" refers to resistance to digestive enzymes in the bile. Bile is a slightly alkaline, greenish-brown liquid that is made in the liver, stored in the gallbladder, and aids in the digestion of fats in the small intestine. It emulsifies fats to aid digestion and absorption. As such bile also acts on probiotics ingested through various routes, including oral, it corresponds to one of the main causes of reducing the effect of probiotic administration.

It was confirmed that the Lactobacillus plantarum HAC03 strain of the present invention has excellent acid resistance and bile resistance. In one embodiment of the present invention, as a result of simultaneously comparing Lactobacillus rhamnosus GG, a well-known probiotic strain, and Lactobacillus plantarum ATCC8014, a subspecies within the same species, the survival rate of the Lactobacillus plantarum HAC03 strain of the present invention is about 18.6 %, it was confirmed to exhibit superior acid resistance and bile resistance than the comparative strain (Example 5).

In addition, in another aspect, the present invention contains at least one selected from the group consisting of the Lactobacillus plantarum HAC03 strain, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution as an active ingredient. It relates to a functional health food composition for preventing or improving inflammatory diseases or obesity.

The inflammatory disease is from the group consisting of enteritis, gastritis, hepatitis, bronchitis, pleurisy, arthritis, rhinitis, keratitis, nephritis, peritonitis, spondylitis, pancreatitis, urethritis, cystitis, burn inflammation, dermatitis, periodontitis, gingivitis, degenerative neuritis and inflammatory pain. It may be one or more selected, but is not limited thereto. Preferably, the inflammatory disease may be enteritis, and in one embodiment of the present invention, the effect of improving enteritis of the Lactobacillus plantarum HAC03 strain was confirmed (Example 7).

The food composition means a natural product or processed product containing one or more nutrients, and preferably means a state that can be eaten directly through some processing process, and in a conventional sense, food, It refers to all food additives, health functional foods and functional beverages.

The food composition of the present invention may include the form of pills, powders, granules, needles, tablets, capsules or liquids, etc., and the type of food is not particularly limited, for example, various beverages, gum, tea, vitamin complexes, There are health supplements, etc.

Other ingredients may be added to the food composition, and the type thereof is not particularly limited. For example, it may contain, as an additional component, various herbal extracts, food-logically acceptable food supplements or natural carbohydrates, such as conventional food, but is not limited thereto.

As used herein, the term "food supplement additive" refers to a component that can be supplementally added to food, and is added to manufacture health functional food of each formulation, and those skilled in the art can appropriately select and use it. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc., but the above examples are not limited to the types of food supplement additives of the present invention.

Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Hygin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used.

The food composition of the present invention may include a health functional food. The term "health functional food" used in the present invention refers to food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients useful in the human body. Here, the term "functionality" refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects with respect to the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. In addition, unlike general drugs, food is used as a raw material, so there are no side effects that may occur when taking the drug for a long time, and it can be excellent in portability.

In general, the amount of Lactobacillus plantarum HAC03 strain included in the food composition may be added to 0.1 to 90% by weight of the total food weight. However, in the case of long-term ingestion for health and hygiene or health control, the amount may be less than or equal to the above range. In addition, the food composition according to the present invention preferably contains other ingredients that can give a synergistic effect to the main effect within the range that does not impair the main effect of the present invention in addition to the Lactobacillus plantarum HAC03 strain. free of charge

In addition, in another aspect, the present invention is one or more selected from the group consisting of the Lactobacillus plantarum HAC03 strain, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution as an active ingredient. It relates to a probiotic composition containing.

As used herein, the term “probiotics” refers to live bacteria that enter the body and give good health effects. Most of the probiotics known to date have been consumed as fermented milk products made using lactic acid bacteria such as Lactobacillus, but recently, in addition to Lactobacillus, some strains such as Bifidobacterium and Entyrococcus are included in fermented milk, granules, and powders is sold as The Lactobacillus plantarum HAC03 strain of the present invention may also be used in the form of fermented milk, granules, powder, etc., but is not limited thereto.

The probiotic formulation may be prepared and administered in various formulations and methods according to methods known in the art. For example, the Lactobacillus plantarum HAC03 strain of the present invention, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution are mixed with a carrier commonly used in the pharmaceutical field and mixed with a powder (powder) ), liquids and solutions, tablets, capsules, syrups, suspensions or granules may be prepared and administered. The carrier may be, for example, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a fragrance, and the like, but is not limited thereto. In addition, the administration dose can be appropriately selected according to the absorption of the active ingredient in the body, the inactivation rate, the excretion rate, the age, sex, breed, condition and severity of the disease of the recipient.

In addition, in another aspect, the present invention is one or more selected from the group consisting of the Lactobacillus plantarum HAC03 strain, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution as an active ingredient. It relates to a pharmaceutical composition for preventing or treating an inflammatory disease or obesity containing.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried agents and suppositories It may have a dosage form, and may be oral or parenteral various dosage forms. In the case of formulation, it is prepared 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 include at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin. It can be prepared by mixing and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical composition of the present invention may be administered in 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 the effective dose level includes the subject type and severity, age, sex, and disease type. , drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of treating an inflammatory disease or obesity, and any pharmaceutical composition is applicable. For example, any of non-human animals such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, human, bird and fish can be used, and the pharmaceutical composition is parenterally, subcutaneously , intraperitoneally, intrapulmonary and intranasally, and for local treatment, if necessary, by any suitable method including intralesional administration. The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the individual, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. For example, it may be administered by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection, but is not limited thereto.

A suitable total daily amount can be determined by treatment within the scope of sound medical judgment, and is generally in an amount of 0.001 to 1000 mg/kg, preferably 0.05 to 200 mg/kg, more preferably 0.1 to 100 mg/kg. It can be administered in divided doses from once to several times a day.

In addition, in another aspect, the present invention is one or more selected from the group consisting of the Lactobacillus plantarum HAC03 strain, the culture solution of the strain, the concentrate of the culture solution, the dried product of the culture solution, and the extract of the culture solution as an active ingredient. It relates to a feed composition containing.

The composition of the present invention may also be provided in the form of an additive for animal feed containing the strain or an animal feed containing the same.

In the present invention, the term "feed" may refer to any natural or artificial diet, one-meal, etc., or a component of the one-meal, which is suitable for or suitable for the animal to eat, ingest, and digest. The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton or food. These may be used alone or in mixture of two or more.

The additive for animal feed of the present invention may be in the form of a dry or liquid formulation, and may further include other non-pathogenic microorganisms in addition to the Lactobacillus plantarum HAC03 strain. Microorganisms that can be added include, for example, Bacillus subtilis capable of producing proteolytic enzymes, lipolytic enzymes and sugar converting enzymes, lactobacilli having physiological activity and organic matter decomposition ability under anaerobic conditions such as the stomach of cattle. Bacillus strains, filamentous fungi such as Aspergillus oryzae and yeasts such as Saccharomyces cerevisiae , which increase the body weight of livestock, increase milk production, and increase the digestibility and absorption rate of feed etc. may be used.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Example 1] Isolation and identification of strains

After purchasing white kimchi that is fermenting 10 to 15 days after production, the pH was measured to confirm the decrease in pH due to lactic acid production, and then lactic acid bacteria were isolated.

Put 90 mL physiological saline (0.85% NaCl/) in an autoclaved plastic bag, add 10 g of white kimchi sample, and mix evenly at 200 rpm for 5 minutes (Stomacher® 400 Circulator, Seward, UK). Dispense 1 ml to 9 ml of physiological saline (0.85% NaCl/L) was serially diluted 10 times and spread on MRS solid medium (5.5% Lactobacillus MRS broth, BD Difco, USA and 1.5% Bacteriological Agar, Affymetrix, USA). was grown for 24 to 48 hours, isolated and identified.

First, in order to estimate the type of strain on the plate, the colony morphology was compared by observation under a microscope (Imager.A2, ZEISS, Germany), and the short rod-shaped bacteria with rounded ends were Lactobacillus brevis (Lactobacillus). brevis ), the convex rod shape of short or medium length was Lactobacillus plantarum ( Lactobacillus plantarum ), and the spherical rod shape was estimated as Lactobacillus sakei ( Lactobacillus sakei ).

Next, the grown bacteria were cultured at 37° C. for 24 to 48 hours using 3% hydrogen peroxide to analyze catalase activity and Gram staining. Catalase-negative strains were identified as taxonomic species through bidirectional 16S rDNA gene sequencing. The sequencing of the strain was commissioned by Solgent (Korea).

As a result, the strain of the present invention isolated from white kimchi was catalase-negative, gram-positive, and identified as Lactobacillus plantarum as a rod-shaped strain, and was named Lactobacillus plantarum HAC03. The strain was deposited with the Korea Research Institute of Bioscience and Biotechnology on December 28, 2020 (accession number: KCTC 13242BP).

[Example 2] Confirmation of sugar utilization characteristics of Lactobacillus plantarum HAC03 strain

The sugar utilization characteristics of the Lactobacillus plantarum HAC03 strain isolated in the present invention were tested by using the API 50 CH kit (Biomerieux) according to the method of the manual. The sugar utilization characteristics of each strain are shown in Table 2 below.

[Example 3] Analysis of biogenic amine production of Lactobacillus plantarum HAC03 strain

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

In order to check whether the Lactobacillus plantarum HAC03 strain forms biological amines, MRS liquid medium, strains grown for 16 hours at 37°C were transferred to a special medium according to Bover Cid and Holzapfel (1999), and at 37°C, 48 hours. incubated during

Prepare an MRS liquid medium to which amino acid precursors of tyrosine, histidine, ornithine and lysine are added, and biological amines, specifically tyramine, by the strain in each medium , histamine (histamine), putrescine (putrescine) and cadaverine (cadaverine) was confirmed whether the production. Specifically, 1% of each Lactobacillus plantarum strain isolated in the MRS liquid medium to which 0.1% of the amino acid precursor was added was inoculated, and subcultured 5 to 10 times to activate decarboxylase. Decarboxylase medium [Trypton 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 acid 1%, bromocresol purple 0.006% and agar 2% in distilled water After mixing, adjust the pH to 5.3, smear the enzyme-activated bacteria for use, and incubate at 37° C. for 24 to 48 hours to determine whether the color changes to purple.

Bromocresol purple in the decarboxylase medium is yellow at pH 5.2, but turns purple as the pH increases to 6.8. Therefore, it is possible to confirm the production of biological amines by using the purple color when the pH is increased due to the production of biological amines.

Whether biogenic amine is produced by amino acid precursor metabolism strain tyramine histamine Putrescine cadaverine Lactobacillus plantarum HAC03 - - - -

As shown in Table 3, as a result of analyzing the biological amine production ability of the Lactobacillus plantarum HAC03 strain, it was confirmed that all of tyramine, histamine, putrescine, and cadaverine were negative. From this, it was confirmed that the strain of the present invention has no biological amine-producing ability that can induce an overactive immune response.

[Example 4] Antibiotic resistance of Lactobacillus plantarum HAC03 strain

Erythromycin, gentamycin, ampicillin, tetracycline, chloramphenicol, streptomycin, ciprofloxacin and penicillin G against penicillin G Antibiotic resistance of Lantarum HAC03 strain was confirmed according to Danielsen and Wind (Susceptibility of Lactobacillus spp. to antimicrobial agents. International Journal of Food Microbiology, 82, 1e11, 2013).

Specifically, each antibiotic was serially diluted (1:2) and mixed in MRS solid medium at a concentration of 0.025 to 64 μg/ml. The Lactobacillus plantarum strain cultured for 18 hours ^{was inoculated into a solid medium mixed with the antibiotic to 1 x 10 5} CFU, and then cultured at 37° C. for 24 hours to measure the minimum inhibitory concentration (MIC).

strain Minimum inhibitory concentration (㎍/mL) Em Gm Am Te Ch Sm Ci Pe Lactobacillus plantarum HAC03 < 0.25 32 2 64 4 128< 32 2 4 128 4 64 16 256< 32< 4 * Em: erythromycin; Gm: gentamicin; Am: ampicillin; Te: tetracycline; Ch: chloramphenicol; Sm: streptomycin; Ci: ciprofloxacin; Pe: penicillin G
* Standard value: Lactobacillus plantarum antibiotic resistance discrimination standard value according to Danielsen and Wind (2003)

As a result, as shown in Table 4, erythromycin, gentamicin, ampicillin, tetracycline, chloramphenicol, streptomycin, ciprofloxacin and penicillin were confirmed to have no resistance to a total of eight antibiotics based on the minimum inhibitory concentration.

[Example 5] Acid resistance and bile resistance analysis of Lactobacillus plantarum HAC03 strain

When ingested with food, lactic acid bacteria stay for about 1 hour in the stomach that secretes strong gastric acid, and then move to the intestine after staying in the high-concentration bile of the duodenum for about 2 hours. When lactic acid bacteria survive in such an extreme environment and can enter the intestine, they attach to the intestine and provide good nutrition to the host, and can act as a probiotic. Therefore, in order to confirm the acid resistance and bile resistance of the lactic acid bacteria isolated in the present invention, a simulated stomach duodenum passage (SSDP) method set under conditions similar to those of the human body (stomach and duodenum) was performed.

Specifically, after culturing the Lactobacillus plantarum HAC03 strain at 37 ° C. for 16 hours in MRS liquid medium, 1 ml of the strain solution is placed in a sterile disposable tube and centrifuged at 12,000 x g for 5 minutes at 4 ° C. The supernatant was discarded and the process of washing the cell pellet with physiological saline was repeated twice. Thereafter, physiological saline was removed, and the precipitated bacteria were mixed with 10 ml of MRS liquid medium adjusted to pH 3, and then thoroughly mixed. 1 ml of the suspension was serially diluted 10-fold, plated on MRS solid medium, and the initial cell number (CFU/ml) was counted. The remaining 9 ml is incubated at 37° C. for one hour, and then 4 ml of bile acid (10 g of oxgall mixed with 100 ml of distilled water and autoclaved) and 17 ml of duodenum juice (6.4 g/L NaHCO3, 0.239 g/L KCl, and 1.28 g/L NaCl were mixed well in distilled water, and then the pH was adjusted to 7.4 and autoclaved) were continuously mixed. After incubation at 37° C. for 2 hours or more, serially diluted 10-fold and smeared on MRS solid medium to count the number of surviving bacteria. And the number of cells (CFU/ml) of the first bacteria counted above and the number of cells remaining alive (CFU/ml) after 1 hour and 3 hours of incubation were compared and expressed as the viability (Table 5).

subordination initial cell count
(log CFU/mL) After 1 hour at pH 3.0
(log CFU/mL) 2 in 1.3% Oxgal
After more than an hour (log CFU/mL) Lactobacillus plantarum HAC03 100 85.11 ± 0.67 18.62 ± 0.39 Lactobacillus plantarum ATCC8014 100 10.61 ± 0.15 5.87 ± 0.32 Lactobacillus rhamnosus GG 100 26.30 ± 0.26 0.50 ± 0.18

Referring to Table 5 above, when analyzing the acid resistance and bile resistance of the strain, Lactobacillus rhamnosus GG, a well-known probiotic strain, and Lactobacillus plantarum ATCC8014, a subspecies within the same species, were simultaneously compared, and Lactobacillus planta of the present invention It was confirmed that the survival rate of the Rum HAC03 strain was about 18.6%, indicating superior acid resistance and bile resistance than the comparative strain (Table 5).

[Example 6] Short-chain fatty acid production effect of Lactobacillus plantarum HAC03 strain

Intestinal lactic acid bacteria use intestinal substances to produce metabolites, thereby having a two-way effect with the host human body. Intestinal bacteria ingest and break down carbohydrates to produce short chain fatty acids (SCFAs), which have several beneficial functions in the intestine. Short-chain fatty acids, including acetate, propionate, and butyrate, increase intestinal motility, interfere with the colonization of pathogens, and act as an energy source for intestinal mucosal epithelial cells to metabolize It can help prevent obesity by activating fat and preventing fat accumulation. In addition, when short-chain fatty acids are absorbed in the intestine and enter the liver, it reduces cholesterol synthesis, lowers blood cholesterol levels, and inhibits glucose absorption, thereby helping to prevent and treat metabolic diseases such as hyperlipidemia and diabetes.

By measuring the amount of change in short-chain fatty acids by co-culture with each lactic acid strain in a mouse cecum sample, it is possible to investigate the intestinal production capacity of the strains relatively simply. In the present invention, an animal experiment was performed to confirm the short-chain fatty acids produced in the intestine by the isolated Lactobacillus plantarum strain.

Specifically, 7-week-old male C57BL/6J mice (specific pathogen free) were purchased and raised in an environment of 23°C, 55±10% humidity, and a light/dark cycle for 12 hours. After separating the mouse cecum, and inoculating each of the Lactobacillus plantarum HAC03 strain or the comparative strain ( L. plantraum 299v) of the present invention, it was cultured for 6 hours in an anaerobic chamber. After it was fermented in an anaerobic environment, the amount of short-chain fatty acids in each sample was analyzed through gas chromatography (Shimadzu GC2010). As a control, the same amount of sterile PBS was inoculated instead of the strain, and the results were compared. By comparing the amount of short-chain fatty acids in the culture medium, it was possible to confirm the change in the short-chain fatty acid production ability of the intestinal flora caused by probiotics.

Gas chromatography was analyzed using a Shimadzu GC2010 instrument. After analyzing a standard curve and a peak detection time (retention time) using a volatile fatty acid mixture (ultrapure) manufactured by Sigma (Supelco), short-chain fatty acids were extracted from feces and contrasted with the standard curve. .

Short-chain fatty acid extraction was described by Schwiertz et. The method described in Al., (2009) was used. Briefly, an extract containing oxalic acid (0.1 mole/L) and sodium azide (40 mmole/L) is mixed with the sample (up to 80 mg), followed by shaking incubation at room temperature for 1 hour. ) and centrifuged at 16,000 x g, 24° C. for 5 minutes, and the supernatant obtained was analyzed using a FID instrument. The column was analyzed using a HP INNO-WAS 30 m x 32 mm at a splitter temperature of 260 °C, FID 260 °C, and the column was analyzed from 100 °C to 180 °C at a rate of 25 °C/m at a pressure of 27.1 psi.

As a result, as shown in FIG. 2, the Lactobacillus plantarum HAC03 of the present invention significantly increased the production of propionic acid compared to the control group not inoculated with the strain (FIG. 2A), and Lactobacillus in the total production of short-chain fatty acids Unlike the Plantarum 299V strain, it was confirmed that it was significantly higher than that of the control group (FIG. 2B).

[Example 7] Anti-inflammatory effect of Lactobacillus plantarum HAC03 strain

1. Immune function activation effect

Animal cell experiments were performed to confirm the degree of immune function activation by the Lactobacillus plantarum HAC03 strain under normal and infected conditions.

Specifically, the mouse macrophage cell line (Raw 264.7) was cultured in a 36.5° C. 5% CO ₂ incubator using a medium supplemented with 10% FBS in RPMI1640 medium. After 2 hours of subculture at 1X106 /well in a 12 well tissue culture plate, Lactobacillus plantarum 299V, Lactobacillus rhamnosus GG and Lactobacillus plantarum HAC03 strains were each 1:1 (1X10 ⁶ CFU/well) ratio. was treated for 16 hours. In addition, in order to simulate a severe inflammatory state, lipopolysaccharide (LPS) was simultaneously treated with each strain in a unit of 100 ng/ml.

Ct 로 계산하였다.In order to confirm the expression level of cytokines, RNA was isolated from the cells of each experimental group and real time RT PCR was performed. After material treatment, the cells were washed twice with sterile PBS and dissolved in Trizol® (RNA extraction buffer, Thermo Fisher), and RNA was extracted according to the manufacturer's manual. The extracted RNA was quantified using nano drops (BMG LabTech). and cDNA was synthesized using RTase (Promega) and Oligo_dT (20) Real-time PCR was performed using the synthesized cDNA and the primers in Table 6 below specific to each gene, SYBR Green premix (GoTaq®, Promega) and the relative gene expression level

Calculated as Ct.

target Forward
(5'→ 3') Reverse
(5'→3') Annealing Temp(℃) IL-1β AGACAGGTCGCTCAGGGTCA
(SEQ ID NO: 2) AAGTGGTTGCCCATCAGAGG
(SEQ ID NO: 3) 60℃ IL-6 TCCAGTTGCCTTCTTGGGAC
(SEQ ID NO: 4) AGTCTCCTCTCCGGACTTGT
(SEQ ID NO: 5) IL-10 TGCCTGCTCTTACTGACTGG (SEQ ID NO: 6) CTGGGAAGTGGGTGCAGTTA
(SEQ ID NO: 7) MCP-1 GTGCTGACCCCAAGAAGGAA (SEQ ID NO: 8) GTGCTGAAGACCTTAGGGCA
(SEQ ID NO: 9) TGF-β CCACCTGCAAGACCATCGAC (SEQ ID NO: 10) CTGGCGAGCCTTAGTTTGGAC
(SEQ ID NO: 11) TNF-α CACAAGATGCTGGGACAGTGA (SEQ ID NO: 12) GAGGCTCCAGTGAATTCGGA
(SEQ ID NO: 13)

As a result, as shown in FIG. 3A, the Lactobacillus plantarum HAC03 strain significantly promotes the expression of inflammatory cytokines compared to the Lactobacillus plantarum 299V, a subspecies within the same species, or the Lactobacillus rhamnosus GG treatment group, which is another probiotic strain. By doing so, it was confirmed that the removal of the infectious agent can be induced through a rapid inflammatory response in the inflammatory situation caused by an external stimulus.

On the other hand, the expression of anti-inflammatory cytokines by inoculation of each strain under normal conditions without external stimuli was observed in the Lactobacillus plantarum 299V, a subspecies within the same species, or Lactobacillus rhamnosus GG, a subspecies of other probiotic strains. In comparison, it was significantly increased in the Lactobacillus plantarum HAC03 strain inoculated group. Through this, the inoculation of Lactobacillus plantarum HAC03 confirmed the possibility of maintaining immune tone homeostasis and suppressing chronic inflammatory diseases (FIG. 3B).

2. Colitis improvement effect

In order to confirm the effect of preventing and inhibiting enteritis caused by the Lactobacillus plantarum HAC03 strain, an animal experiment was performed. As the enteritis model, a colitis model induced by DSS (dextran sulfate sodium) feeding was selected.

After orally administering PBS or Lactobacillus plantarum HAC03 strain suspension to 8-week-old C57BL/6J female mice at a concentration of 1X109 CFU/mouse for 2 weeks, dextran sulfate sodium (DSS) was turbid 3% in drinking water and treated for 7 days. There was a daily wash out period. And in order to check the expression level of cytokines, RNA was isolated from the cells of each experimental group and real-time RT PCR was performed. After material treatment, cells were washed twice with sterile PBS and dissolved in Trizol® (RNA extraction buffer, Thermo Fisher), and RNA was extracted according to the manufacturer's manual. The extracted RNA was quantified using a nano drop (BMG LabTech). And cDNA was synthesized using RTase Promea and Oligo_dT (20) Real-time PCR was performed using the synthesized cDNA and each gene-specific primer in Table 6, SYBR Green premix (GoTaq®, Promega), The relative gene expression level was calculated as ΔΔCt.

As a result, as can be seen in FIG. 4 , it was confirmed that the decrease in body weight (A) and colon length (B) caused by DSS-induced enteritis was alleviated in the Lactobacillus plantarum HAC03 strain administered group. In addition, as a result of confirming the cytokine changes in the excised colon tissue, it was found that the expression of inflammation-related cytokines induced by DSS decreased and the expression of IL-10, an anti-inflammatory cytokine, was significantly increased (C).

[Example 8] Anti-obesity effect of Lactobacillus plantarum HAC03 strain

In order to confirm the effect of improving energy metabolism by administration of the Lactobacillus plantarum HAC03 strain, the Lactobacillus plantarum HAC03 strain was orally administered to a diet-induced metabolic disease model, and changes in body weight and energy metabolism-related tissues were confirmed.

Specifically, after acclimatizing 5-week-old C57BL/6J male mice to the breeding facility for 1 week, high-fat diet [AIN 93G 100g, Sucrose 99g, Maltodextrin 99g, Casein 80g, Lard 128g; Carbohydrate 40%kcal, Fat 45%kcal, Protein 15%kcal; 5.95 kcal/g] or a normal diet (AIN 93G) for 2 weeks. After 2 weeks of induction of a high-fat diet, Lactobacillus plantarum HAC03, Lactobacillus rhamnosus GG, Lactobacillus plantarum 299V (1X10 ⁹ CFU/mouse/day) or Orlistat (30mpk) was added orally administered for 10 weeks, respectively. .

Body weight, drinking water, and dietary intake were measured periodically once a week, and after the end of the 12-week experimental period, the weights of liver, testicular fat, subcutaneous fat, and mesenteric fat, which are involved in energy metabolism, were measured to suppress obesity at the tissue level. The effect was confirmed. In addition, according to the report that hypoxia and inflammatory reaction of adipocytes caused by hypertrophy of adipose tissue cause various metabolic diseases accompanying obesity, testicular adipose tissue was fixed in 3.8% formalin aqueous solution, paraffin block production and slide production , the average size and distribution of adipocytes were confirmed through H&E staining.

As a result, as can be seen in FIG. 5 , unlike Lactobacillus plantarum 299V, a significant weight gain inhibitory effect was exhibited by administration of the Lactobacillus plantarum HAC03 strain (A). In addition, as a result of measuring tissue weight related to energy metabolism after 12 weeks of breeding, the Lactobacillus plantarum HAC03 strain administered group had liver and testicular fat (epididymal adipose tissue, EAT), subcutaneous adipose tissue (SAT), mesenteric fat (mesenteric) It was confirmed that the weight of adipose tissue (MAT) was significantly reduced.

In addition, by checking the H&E staining photograph of testicular fat (A), the average size of adipocytes (B), and the distribution according to adipocyte size (C), hypertrophy of adipocytes by administration of Lactobacillus plantarum HAC03 strain was confirmed. It was confirmed that there is an inhibitory effect (FIG. 6).

Korea Research Institute of Bioscience and Biotechnology Center KCTC13242BP 20201228

<110> Handong global university Industry-Academic Cooperation Foundation <120> Lactobacillus plantarum HAC03 and uses thereof <130> P-1 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 1404 <212> DNA <213> Unknown <220> <223> Lactobacillus plantarum HAC03 16s rDNA <400> 1 ctctggtatt gattggtgct tgcatcatga tttacatttg agtgagtggc gaactggtga 60 gtaacacgtg ggaaacctgc ccagaagcgg gggataacac ctggaaacag atgctaatac 120 cgcataacaa cttggaccgc atggtccgag cttgaaagat ggcttcggct atcacttttg 180 gatggtcccg cggcgtatta gctagatggt ggggtaacgg ctcaccatgg caatgatacg 240 tagccgacct gagagggtaa tcggccacat tgggactgag acacggccca aactcctacg 300 ggaggcagca gtagggaatc ttccacaatg gacgaaagtc tgatggagca acgccgcgtg 360 agtgaagaag ggtttcggct cgtaaaactc tgttgttaaa gaagaacata tctgagagta 420 actgttcagg tattgacggt atttaaccag aaagccacgg ctaactacgt gccagcagcc 480 gcggtaatac gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc 540 ggttttttaa gtctgatgtg aaagccttcg gctcaaccga agaagtgcat cggaaactgg 600 gaaacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata 660 tatggaagaa caccagtggc gaaggcggct gtctggtctg taactgacgc tgaggctcga 720 aagtatgggt agcaaacagg attagatacc ctggtagtcc ataccgtaaa cgatgaatgc 780 taagtgttgg agggtttccg cccttcagtg ctgcagctaa cgcattaagc attccgcctg 840 gggagtacgg ccgcaaggct gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 900 agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc aggtcttgac atactatgca 960 aatctaagag attagacgtt cccttcgggg acatggatac aggtggtgca tggttgtcgt 1020 cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tattatcagt 1080 tgccagcatt aagttgggca ctctggtgag actgccggtg acaaaccgga ggaaggtggg 1140 gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg 1200 tacaacgagt tgcgaactcg cgagagtaag ctaatctctt aaagccattc tcagttcgga 1260 ttgtaggctg caactcgcct acatgaagtc ggaatcgcta gtaatcgcgg atcagcatgc 1320 cgcggtgaat acgttcccgg gccttgtaca caccgtccgt cacaaccatg agagtttgta 1380 acacccaaag tcggtggggt aacc 1404 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1b_F <400> 2 agacaggtcg ctcagggtca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-1b_R <400> 3 aagtggttgc ccatcagagg 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6_F <400> 4 tccagttgcc ttcttgggac 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-6_R <400> 5 agtctcctct ccggacttgt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10_F <400> 6 tgcctgctct tactgactgg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> IL-10_R <400> 7 ctgggaagtg ggtgcagtta 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MCP-1_F <400> 8 gtgctgaccc caagaaggaa 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> MCP-1_R <400> 9 gtgctgaaga ccttagggca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TGF-b_F <400> 10 ccacctgcaa gaccatcgac 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TGF-b_R <400> 11 ctggcgagcc ttagtttgga c 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TNF-a_F <400> 12 cacaagatgc tgggacagtg a 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TNF-a_R <400> 13 gaggctccag tgaattcgga 20

Claims (10)

As a health functional food composition for preventing or improving colitis containing Lactobacillus plantarum HAC03 strain (Accession No.: KCTC 13242BP) as an active ingredient,
The strain is i) has the 16s rDNA sequence shown in SEQ ID NO: 1, ii) does not produce biogenic amines from tyrosine, histidine, ornithine and lysine, iii) erythromycin, gentamicin, ampicillin, tetracycline, chloramphenicol A food composition, characterized in that it is not resistant to streptomycin, ciprofloxacin and penicillin, and iv) produces short chain fatty acids.
delete delete delete delete delete delete As a pharmaceutical composition for preventing or treating colitis containing Lactobacillus plantarum HAC03 strain (Accession No.: KCTC 13242BP) as an active ingredient,
The strain is i) has the 16s rDNA sequence shown in SEQ ID NO: 1, ii) does not produce biogenic amines from tyrosine, histidine, ornithine and lysine, iii) erythromycin, gentamicin, ampicillin, tetracycline, chloramphenicol , which is not resistant to streptomycin, ciprofloxacin and penicillin, and iv) produces short chain fatty acids.

delete delete

Images