KR102157959B1 - NOVEL STRAIN OF Lactobacillus brevis AND USE THEREOF - Google Patents

Abstract

The present invention relates to a strain of Lactobacillus brevis (KCCM 12213P) having antibacterial activity against Salmonella Typhimurium; a probiotic composition comprising the same as an effective component; an antibacterial composition for Salmonella Typhimurium; and a composition for preventing, ameliorating, or treating a bowel disorder.

Description

Novel Lactobacillus brevis strain and its use {NOVEL STRAIN OF Lactobacillus brevis AND USE THEREOF}

The present invention relates to a novel Lactobacillus brevis strain having antimicrobial activity and its use.

Probiotics are known as living bacteria that settle in the intestine when ingested in an appropriate amount and have a variety of functions such as intestinal health, immunity enhancement and cholesterol reduction. However, probiotics must be resistant to acids and bile for survival in the gut and have the ability to settle in the gut. In addition, in terms of safety, resistance to antibiotics, hemolytic properties, and the production of β-glucuronidase, a carcinogen, must be confirmed. Among them, the Lactobacillus brevis strain is one of the lactic acid bacteria belonging to the representative Lactobacillus spp., which is commonly found in the gut of vegetables, plants, and humans, especially in the late fermentation of cheese or kimchi. It is known to be excellent in acid resistance and bile resistance as lactic acid bacteria that proliferate a lot. In addition, these strains ferment arabinose to produce acetic acid and have an excellent effect on intestinal action. In addition, it has anti-inflammatory, antioxidant, and immune-modulating functions.

The present invention is to provide a Lactobacillus brevis ( Lactobacillus brevis ) strain (KCCM 12213P) having antibacterial activity against Salmonella Typhimurium.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a Lactobacillus brevis strain (KCCM 12213P) having antibacterial activity against Salmonella Typhimurium.

The strain is Escherichia coli ), Listeria monocytogenes , and Staphylococcus aureus may further have antimicrobial activity against one or more selected from the group consisting of.

The strain may further produce at least one selected from the group consisting of formic acid, lactic acid, acetic acid, propionic acid, and butyric acid.

The strain may further have one or more characteristics selected from the group consisting of the following i) to v):

I) acid resistance, bile resistance and intestinal adhesion;

Ii) human safety;

Iii) antibiotic sensitivity;

Iv) the ability to inhibit intestinal adhesion to food poisoning causative bacteria; And

V) antioxidant activity.

The strain may include a DNA sequence encoding 16S rRNA of SEQ ID NO: 1.

In one embodiment of the present invention, it provides a probiotic composition comprising the Lactobacillus brevis strain (KCCM 12213P) as an active ingredient.

The strain may be prepared as a live cell, a dead cell, or a culture filtrate.

In another embodiment of the present invention, it provides a composition for antimicrobial against Salmonella Typhimurium containing Lactobacillus brevis strain (KCCM 12213P) as an active ingredient.

In another embodiment of the present invention, it provides a pharmaceutical composition for preventing or treating intestinal diseases comprising the Lactobacillus brevis strain (KCCM 12213P) as an active ingredient.

The bowel disease may be a bowel disease caused by food poisoning causative bacteria.

The food poisoning causative bacteria is Escherichia coli ), Listeria monocytogenes , Salmonella Typhimurium, and Staphylococcus aureus . It may be one or more selected from the group consisting of.

In another embodiment of the present invention, it provides a health functional food for preventing or improving intestinal diseases comprising the Lactobacillus brevis strain (KCCM 12213P) as an active ingredient.

The Lactobacillus brevis strain (KCCM 12213P) according to the present invention has stable and safe probiotic properties, excellent antibacterial activity against Salmonella Typhimurium, etc., and production of organic acids such as short-chain fatty acids Excellent bar, antibacterial composition or intestinal disease (especially, intestinal disease caused by food poisoning causative bacteria) can be usefully used as a composition for preventing, improving or treating.

1 is a novel Lactobacillus brevis This figure shows the schematic diagram of the KU15153 strain (KCCM 12213P).

Among the Lactobacillus brevis strains having probiotic properties, the present inventors have excellent antibacterial activity against Salmonella Typhimurium and the like, and Lactobacillus brevis KU15153 having excellent ability to produce organic acids such as short-chain fatty acids. The present invention was completed by isolating and identifying strains and evaluating their efficacy.

Hereinafter, the present invention will be described in detail.

The present invention provides a Lactobacillus brevis strain (KCCM 12213P) having antibacterial activity against Salmonella Typhimurium.

The strain (KCCM 12213P) can be seen as a subspecies of Lactobacillus brevis. Conventional Lactobacillus brevis is a kind of hetero lactic acid bacteria that produce lactic acid, acetic acid, ethanol, carbon dioxide, etc., and it is known to proliferate a lot in the late fermentation of kimchi or cheese, and is generally known to have excellent acid resistance and dung resistance.

The schematic diagram of the strain (KCCM 12213P) is as shown in FIG. 1, and may be isolated from cabbage kimchi. As a result of analyzing the 16S rRNA sequence to identify the isolated strain, it was confirmed that the strain (KCCM 12213P) has a DNA nucleotide sequence encoding the 16S rRNA of SEQ ID NO: 1, and as of January 17, 2018 It was deposited with the Conservation Center and was given the accession number KCCM 12213P.

In addition, the strain (KCCM 12213P) has excellent antibacterial activity against pathogenic causative bacteria among Lactobacillus brevis strains, and is characterized by having antibacterial activity against Salmonella Typhimurium. Specifically, the strain (KCCM 12213P) is characterized in that it has particularly excellent antibacterial effect against Salmonella Typhimurium, compared to the Lactobacillus rhamnosus LGG strain known as a commercial probiotic.

In addition, Escherichia coli ), Listeria monocytogenes ( Listeria monocytogenes ) and Staphylococcus aureus ( Staphylococcus aureus ) may further have antimicrobial activity against at least one selected from the group consisting of.

In addition, the strain (KCCM 12213P) has excellent organic acid production ability, particularly, short-chain fatty acid production ability among Lactobacillus brevis strains, formic acid, lactic acid, acetic acid, propionic acid, and One or more selected from the group consisting of butyric acid may be further produced. Specifically, the strain (KCCM 12213P) is superior to the Lactobacillus rhamnosus LGG strain, which is known as a commercial probiotic, is superior in producing acetic acid and butyric acid, a kind of short-chain fatty acid. By improving the toxin removal (detox), it is expected to be of great help.

In addition, the strain (KCCM 12213P) may further have one or more characteristics selected from the group consisting of the following i) to v):

I) acid resistance, bile resistance and intestinal adhesion;

Ii) human safety;

Iii) antibiotic sensitivity;

Iv) the ability to inhibit intestinal adhesion to food poisoning causative bacteria; And

V) antioxidant activity.

Specifically, i) with respect to acid resistance, bile resistance, and intestinal adhesion, the acid resistance may have a survival rate of 90% or more (preferably, 97% or more) when reacting for 3 hours under pH 2.5 conditions, and The bile may have a survival rate of 100% or more (preferably, 105% or more) when reacting for 24 hours under 0.3% oxgall conditions.

Ii) With respect to human safety, the human stability can be realized by not producing β-Glucuronidase, which is classified as a cancer-causing enzyme.

Iii) Regarding antibiotic sensitivity, the antibiotic sensitivity may be to Kanamycine, Ampicillin, Gentamicin, Teracycline, Chloramphenicol, and Doxycycline. That is, with the exception of Streptomycin and Ciprofloxacin, Kanamycine, Ampicillin, Gentamicin, Tetracycline, Chloramphenicol, and Doxycycline for Chloramphenicol Since none of them are resistant to antibiotics, concerns about the transfer of antibiotic resistance genes may not arise.

Ⅳ) in relation to the mounting section for the inhibition of food poisoning organisms, the causative agent of foodborne illness, E. coli (Escherichia coli), Listeria monocytogenes Zenith (Listeria monocytogenes ), Salmonella Typhimurium, and Staphylococcus aureus may be one or more selected from the group consisting of, and Salmonella Typhimurium is preferred, but is not limited thereto.

V) With respect to the antioxidant activity, the antioxidant activity can be evaluated through calculation of DPPH free radical scavenging ability or β-carotene oxidation inhibitory ability. Therefore, it can be of great help to anti-aging and toxin removal (detox).

In addition, the present invention provides a probiotic composition comprising a Lactobacillus brevis strain (KCCM 12213P) as an active ingredient.

The strain (KCCM 12213P) is as described above, and may be prepared as the live cells, dead cells, or culture filtrate.

In addition, the present invention provides a composition for antimicrobial against Salmonella Typhimurium containing Lactobacillus brevis strain (KCCM 12213P) as an active ingredient.

The strain (KCCM 12213P) is as described above, and may be prepared as the live cells, dead cells, or culture filtrate.

The dose of the strain (KCCM 12213P) is preferably 1 μg/ml to 100 μg/ml, but is not limited thereto. At this time, when the capacity of the strain (KCCM 12213P) is less than the above range, there is a problem that it is difficult to exert an antibacterial effect against Salmonella Typhimurium, etc., and the capacity of the strain (KCCM 12213P) exceeds the above range. If so, there may be toxicity concerns, including cytotoxicity.

The antibacterial composition is preferably selected from the group consisting of a pharmaceutical composition, a health functional food composition, a feed additive, an antiseptic composition, and a quasi-drug composition, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing or treating intestinal diseases comprising the Lactobacillus brevis strain (KCCM 12213P) as an active ingredient.

In the present specification, the term "intestinal disease" refers to a bowel disease caused by a decrease in digestive function or bowel function in terms of symptoms, and the intestinal disease includes food poisoning, enteritis and diarrhea. In particular, the intestinal disease may be a gastrointestinal tract disease (bacterial bowel disease) caused by a food poisoning causative agent in terms of the cause, and the food poisoning causative agent is Escherichia coli ), Listeria monocytogenes ( Listeria monocytogenes ), Salmonella Typhimurium, and Staphylococcus aureus . It may be one or more selected from the group consisting of.

The strain (KCCM 12213P) is as described above, and may be prepared as the live cells, dead cells, or culture filtrate.

The dose of the strain (KCCM 12213P) is preferably 1 μg/ml to 100 μg/ml, but is not limited thereto. At this time, when the capacity of the strain (KCCM 12213P) is less than the above range, there is a problem in that the ability to produce organic acids such as short-chain fatty acids decreases, or the ability to inhibit intestinal adhesion to food poisoning causative bacteria decreases, and the capacity of the strain (KCCM 12213P) is If the range is exceeded, there may be toxicity concerns, including cytotoxicity.

In addition, the present invention provides a health functional food for preventing or improving intestinal diseases comprising the Lactobacillus brevis strain (KCCM 12213P) as an active ingredient.

The strain (KCCM 12213P) is as described above, and may be prepared as the live cells, dead cells, or culture filtrate.

The dose of the strain (KCCM 12213P) is preferably 1 μg/ml to 100 μg/ml, but is not limited thereto. At this time, when the capacity of the strain (KCCM 12213P) is less than the above range, there is a problem in that the ability to produce organic acids such as short-chain fatty acids decreases, or the ability to inhibit intestinal adhesion to food poisoning causative bacteria decreases, and the capacity of the strain (KCCM 12213P) is If the range is exceeded, there may be toxicity concerns, including cytotoxicity.

In addition, the present invention may provide a starter for fermentation, a seed composition, or a fermented food comprising a Lactobacillus brevis strain (KCCM 12213P).

The pharmaceutical composition according to the present invention is formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. For formulation, it may contain a suitable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions.

As the carrier or excipient or diluent, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undecided And various compounds or mixtures including vaginal cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In the case of formulation, it can be prepared using diluents or excipients such as fillers, weight agents, binders, wetting agents, disintegrants, and surfactants that are usually used.

Solid preparations for oral administration may be prepared by mixing at least one excipient, for example, starch, calcium bonate, sucrose or lactose, gelatin, and the like with the strain (KCCM 12213P). In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

Liquid preparations for oral use include suspensions, liquid 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, preservatives, etc. may be included. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, Tween 61, cacao butter, laurin paper, glycerol gelatin, and the like can be used.

The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at 0.0001 to 2,000 mg/kg per day, preferably 0.001 to 2,000 mg/kg. Administration may be administered once a day, or may be divided several times. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition according to the present invention can be administered to mammals such as mice, mice, livestock, and humans by various routes. All modes of administration can be administered by, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In the health functional food composition according to the present invention, when the strain (KCCM 12213P) is used as an additive to a health functional food, it can be added as it is or used with other foods or food ingredients, and can be used appropriately according to a conventional method. I can. The mixing amount of the active ingredient can be appropriately determined according to each purpose of use, such as prevention, health or treatment.

Formulations of health functional foods may be in the form of powders, granules, pills, tablets, capsules, as well as general foods or beverages.

The type of food is not particularly limited, and examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, and dairy products including ice cream. , Various soups, beverages, teas, drinks, alcoholic beverages and vitamin complexes, and may include all foods in the usual sense.

In general, in the manufacture of food or beverage, the strain (KCCM 12213P) may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range.

Among the health functional foods according to the present invention, the beverage may contain various flavoring agents or natural carbohydrates as an additional component, like a conventional beverage. The natural carbohydrates described above may be 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. As the sweetener, natural sweeteners such as taumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food composition according to the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, It may contain glycerin, alcohol, and carbonates used in carbonated beverages. In addition, the health functional food composition according to the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These ingredients may be used independently or in combination. The ratio of these additives is not limited, but it is generally selected from 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food composition according to the present invention.

The feed additive according to the present invention has the effect of replacing the existing antibiotics for animals and suppressing the growth of harmful pathogenic microorganisms to improve the health of the animal body, improve the weight gain and the quality of the animal body, and increase the milk production and immunity. . The feed additive may be prepared in the form of fermented feed, blended feed, pellet form, and silage.

The fermented feed can be prepared by fermenting organic matter by adding various microbial groups or enzymes other than the strain (KCCM 12213P), and the blended feed is prepared by mixing various types of general feed and the strain (KCCM 12213P). can do. The pellet-type feed can be prepared by applying heat and pressure to the blended feed in a pellet machine, and the silage can be prepared by fermenting the blueberry feed with microorganisms. Wet fermented feed collects and transports organic matter such as food waste, mixes excipients for sterilization and moisture control at a certain ratio, and then ferments at a temperature suitable for fermentation for more than 24 hours, so that the moisture content is about 70%. It can be manufactured by controlling. The fermented dry feed may be prepared by controlling the wet fermented feed to contain about 30% to 40% of moisture through an additional drying process.

The preservative composition according to the present invention includes food preservatives, cosmetic preservatives or pharmaceutical preservatives. The food preservatives, cosmetic preservatives and pharmaceutical preservatives are additives used to prevent deterioration, spoilage, discoloration, and chemical change of pharmaceuticals. These include fungicides and antioxidants, and inhibit the growth of microorganisms such as bacteria, mold, and yeast. And functional antibiotics, such as inhibiting the growth of decaying microorganisms or sterilizing in medicines, are also included. Ideal conditions for such an antiseptic composition should be non-toxic and should be effective even in trace amounts.

The quasi-drug composition according to the present invention may be used with the above strain (KCCM 12213P) as it is or with other quasi-drug or quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use.

Specifically, the quasi-drug composition may be a disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment, patch, or filter filler.

As described above, the Lactobacillus brevis strain (KCCM 12213P) according to the present invention has stable and safe probiotic properties, excellent antibacterial activity against Salmonella Typhimurium, etc., and short chain Since the ability to produce organic acids such as fatty acids is excellent, it can be usefully used as an antibacterial composition or a composition for preventing, improving or treating intestinal diseases (particularly, intestinal diseases caused by food poisoning causative bacteria).

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

<Example>

Example 1: Lactobacillus brevis Isolation and identification of KU15153 strain

In order to isolate a new strain, the cabbage kimchi broth made at home was sampled and used as a sample. The cabbage kimchi broth was spread on LBS agar medium using a 10-fold dilution method, streaked several times, and cultured, and then the colony shape was observed to isolate a new strain. 16S rRNA sequencing was requested to Bionics (Seoul, Korea) for identification of the isolated new strain. Specifically, 16S rRNA sequencing was performed, and PCR was performed on the 16S rRNA gene using 27F and 1492R primers. Based on the results of nucleotide sequence analysis, the BLAST program was used to compare homology with other standard strains registered in Genbank's database. The new strain is Lactobacillus brevis It was identified as the KU15153 strain, and the similarity was 99%. In addition, homology was analyzed using the Mega 7 program and a schematic diagram was obtained (FIG. 1). At this time, Lactobacillus brevis The KU15153 strain was deposited with the Korea Microbial Conservation Center on January 17, 2018, and received the accession number KCCM 12213P.

Later, Lactobacillus brevis The KU15153 strain was incubated in lactobacilli MRS broth at 37°C for 12 hours, the initial number of bacteria was adjusted to 10 ⁷ CFU/mL, and then centrifuged (5,000×g, 10 minutes) to prepare a precipitate (cell) and a supernatant (culture filtrate). Separated. After the precipitate was lyophilized, a live cell was prepared, and then, the live cell was washed and resuspended three times with 0.1% peptone water and used in the experiment.

Comparative Example 1: Lactobacillus rhamnosus LGG strain preparation

Lactobacillus rhamnosus LGG strain known as commercial probiotics was prepared. Culture conditions were the same as those of Lactobacillus brevis KU15153 strain.

Experimental Example 1: Evaluation of physiological properties of strain

Lactobacillus brevis isolated in Example 1 As the physiological characteristics of the KU15153 strain, acid resistance, bile resistance, and intestinal adhesion were evaluated.

(1) Acid resistance evaluation

Lactobacillus brevis isolated in Example 1 The KU15153 strain was cultured for 24 hours. Lactobacillus brevis isolated in Example 1 in which 3 mg/mL of pepsin was added to the culture solution test tube adjusted to pH 2.5, and cultured in advance 1 mL of the KU15153 strain was inoculated into each test tube. 0 hours was used as a control, and acid resistance was evaluated by checking the total number of bacteria after 0 hours and 3 hours, and the results are shown in Table 1 below.

(2) Bile resistance evaluation

Lactobacillus brevis isolated in Example 1 The KU15153 strain was cultured for 24 hours. Lactobacillus brevis isolated in Example 1 in which Oxgall 3.0 mg/mL was added to the culture solution test tube, and previously cultured 1 mL of the KU15153 strain was inoculated into each test tube. 0 hours was used as a control, and the total number of bacteria after 0 and 24 hours was checked to evaluate bile resistance, and the results are shown in Table 1 below.

(3) Evaluation of carrying capacity

Lactobacillus brevis isolated in Example 1 A sediment (cell) of the KU15153 strain was prepared as a sample, and HT-29 cells (human colon adenocarcinoma cell line, KCLB 30038) were added 1 mL each at a concentration of 1 Х10 ⁵ cells/mL, and cultured for 24 hours. After adding 100 μL of prepared samples and incubating for 2 hours, the attached cells were removed using 1% Triton × 100 solution, and then the number of attached bacteria was checked to evaluate the intestinal adhesion ability as follows.The results are as follows. It is shown in Table 1:

Intestinal adhesion ability = (number of attached bacteria (Log CFU/mL)/number of inoculated bacteria (Log CFU/mL)) × 100.

Experiment contents Number of bacteria (Log CFU/mL; %) Example 1 Comparative Example 1 Acid and bile resistance Initial bacterial count 8.30±0.02 8.76±0.06 pH 2.5, 0.3% (w/v) pepsin 8.15±0.10; 98.19 8.48±0.03; 96.80 0.3% (w/v) Oxgall 8.95±0.02 ;107.83 9.04±0.03; 103.20 Attachment ability Number of inoculated bacteria 7.94±0.01 8.77±0.04 Number of attached bacteria 6.89±0.06; 91.74% 7.51±0.14; 90.53%

As shown in Table 1, physiological property evaluation results, Lactobacillus brevis isolated in Example 1 The acid resistance of the KU15153 strain was 98.19%, bile resistance was 107.83%, and intestinal adhesion was 91.74%, compared to Lactobacillus rhamnosus LGG strains known as commercial probiotics, all showed higher levels. That is, Lactobacillus brevis isolated in Example 1 Since KU15153 strain has excellent physiological properties, it can be considered as a beneficial lactic acid bacteria for the human body.

Experimental Example 2: Evaluation of the enzyme production ability of the strain

The enzyme-producing ability of the strain Lactobacillus brevis KU15153 isolated in Example 1 was evaluated.

Specifically, Lactobacillus brevis isolated in Example 1 After adjusting the sediment (cell) of the KU15153 strain to 10 ⁶ CFU/mL, the enzyme production ability was confirmed using the API ZYM kit, and the results are shown in Table 2 below.

enzyme Example 1 Control 0 Alkaline phosphatase 0 Esterase One Esterase lipase 0 Lipase 0 Leucine arylamidase 3 Valine arylamidase 2 Cystine arylamidase 0 Trypsin 0 α-Chymotrypsin 0 Acid phosphatase 4 Naphthol-AS-BI-phosphohydrolase 2 α-Galactosidase 0 β-Galactosidase 2 β-Glucuronidase 0 α-Glucosidase One β-Glucosidase 2 N-Acetyl-β-glucosaminidase 0 α-Mannosidase 0 α-Fucosidase 0

* 0, 1, 2, 3, 4 and 5 refer to the enzyme production ability, 0 is 0 nmol; 1 is 5 nmol; 2 is 10 nmol; 3 is 20 nmol; 4 is 30 nmol; And 5 means 40 nmol or more.

As shown in Table 2, Lactobacillus brevis isolated in Example 1 The KU15153 strain does not produce β-Glucuronidase, which is a cancer-causing enzyme, and thus it is confirmed that biosafety is secured.

Experimental Example 3: Evaluation of antibiotic susceptibility of strain

According to CLSI (Clinical and Laboratory Standards Institute) standards, the antibiotic susceptibility of the Lactobacillus brevis KU15153 strain isolated in Example 1 was evaluated.

Specifically, Lactobacillus brevis isolated in Example 1 The following eight types of antibiotics are ampicillin (0.2 mg/mL), gentamicin (0.2 mg/mL), and kanamycin (0.6 mg/mL) on the MRS medium plated with 10 ⁶ CFU/mL of the KU15153 strain. , streptomycin (0.2 mg/mL), tetracycline (0.6 mg/mL), ciprofloxacin (0.1 mg/mL), chloramphenicol (0.6 mg/mL), and doxycycline (0.6 mg/mL) were dispensed with a paper disk. After incubation at 37° C. for 24 hours, antibiotic sensitivity was evaluated through the diameter (mm) of the inhibition zone, and the results are shown in Table 3 below.

Antibiotic Example 1 Ampicillin S Gentamicin S Kanamycin S Streptomycin R Tetracycline S Ciprofloxacin R Chloramphenicol S Doxycycline S

* S: sensitivity; I: medium; R: means tolerance.

As shown in Table 3, Lactobacillus brevis isolated in Example 1 KU15153 strains are Kanamycine, Ampicillin, Gentamicin, Tetracycline, Tetracycline, Chloramphenicol, and Chloramphenicol, except for Ciprofloxacin. However, since all of them do not have antibiotic resistance, it is confirmed that there is no concern about the transfer of the antibiotic resistance gene.

Experimental Example 4: Evaluation of the antibacterial effect of the strain

The antibacterial effect of the strain Lactobacillus brevis KU15153 isolated in Example 1 was evaluated.

Specifically, Lactobacillus brevis isolated in Example 1 After adjusting the viable cells of the KU15153 strain to 10 ⁷ CFU/mL, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 15313, Salmonella Typhimurium P99, and Staphylococcus aureus KCCM 11335 were also prepared at 10 ⁶ CFU/mL, and then the clear zone was measured by performing the Deferred method to evaluate the antibacterial effect. After loading 3 μL of the strain culture medium on the MRS agar medium, it was incubated for 24 hours in a 37° C. After dispensing 0.75% TSA Soft agar pathogenic bacteria and culturing for 24 hours, the clear zone was confirmed. The results are shown in Table 4 below.

Pathogenic bacteria Clear zone(nm) Example 1 Comparative Example 1 Escherichia coli ATCC 25922 13.00 ± 2.00 18.67 ± 4.73 Listeria monocytogenes ATCC 15313 14.67 ± 1.15 22.33 ± 0.58 Salmonella Typhimurium P99 16.67 ± 2.89 3.00 ± 0.00 Staphylococcus aureus KCCM 11335 18.00 ± 3.00 22.33 ± 8.69

As shown in Table 4, the antibacterial effect evaluation result, Lactobacillus brevis isolated in Example 1 The KU15153 strain is Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 15313, Salmonella Typhimurium P99, and Staphylococcus aureus KCCM 11335 are all confirmed to have large clear zones, which can be considered to have excellent antibacterial effects. Specifically, Lactobacillus brevis isolated in Example 1 The KU15153 strain was found to have a particularly large clear zone for Salmonella Typhimurium P99 compared to the Lactobacillus rhamnosus LGG strain, which is known as a commercial probiotic, and it can be seen that the antibacterial effect is very excellent.

Experimental Example 5: Evaluation of intestinal adhesion inhibitory ability of strains against food poisoning causative bacteria

Lactobacillus brevis isolated in Example 1 The ability of KU15153 strain to inhibit intestinal adhesion to food poisoning causative bacteria was evaluated.

Specifically, in order to evaluate the intestinal adhesion inhibitory ability against food poisoning causative bacteria, HT-29 (human colon adenocarcinoma cell line) cell line inoculated at 1×10 ⁵ cells/well was cultured for 24 hours and then mixed bacteria (food poisoning causative bacteria and Lactobacillus brevis Mixed bacteria of KU15153 strain) (10 ⁶ cells/well) were treated and incubated for an additional 2 hours at 37°C. Washing three times with Phosphate buffered saline to remove non-adherent bacteria. After removing the cell line using 1% Triton X-100 solution, the number of bacteria attached to the cell line was measured using a plate smear method, and the results are shown in Table 5. Indicated.

Food poisoning causative bacteria Number of bacteria (Log CFU/mL) Food poisoning causative bacteria Food poisoning causative bacteria and
Lactobacillus brevis KU15153 strain Escherichia coli ATCC 25922 7.49 ± 0.84 6.90 ± 0.09 Listeria monocytogenes ATCC 15313 5.17 ± 0.17 5.05 ± 0.14 Salmonella Typhimurium P99 5.70 ± 0.11 4.75 ± 0.26 Staphylococcus aureus KCCM 11335 6.99 ± 0.02 6.50 ± 0.01

As shown in Table 5, Lactobacillus brevis isolated in Example 1 The KU15153 strain is Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 15313, Salmonella Typhimurium P99, and Staphylococcus aureus KCCM 11335 are all confirmed to have intestinal adhesion inhibition. In particular, Salmonella Typhimurium P99 and Lactobacillus brevis isolated in Example 1 When KU15153 strains are cultured at the same time, the number of bacteria decreased the most to 0.95 Log CFU/mL, and it can be seen that it is particularly effective in the prevention, improvement, or treatment of intestinal diseases that may be caused by Salmonella.

Experimental Example 6: Evaluation of antioxidant effect on strain

The antioxidant effect on the strain Lactobacillus brevis KU15153 isolated in Example 1 was evaluated.

(1) DPPH free radical scavenging ability

DPPH free radical scavenging activity is one of the antioxidant measurement methods to observe the color change of DPPH solution by removing DPPH free radical. The scavenging activity of free radicals was measured as follows. Lactobacillus brevis isolated in Example 1 150 μL of live cells and control group of KU15153 strain were mixed with 150 μL of 40 μM DPPH (1,1-diphenyl-2-picrylhydrazyl), left for 30 minutes in a dark room at 37° C., and absorbance (optical density, OD) was measured at 517 nm. Measured. At this time, the DPPH free radical scavenging ability was evaluated as follows, and the results are shown in Table 4 below:

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

(2) Oxidation inhibitory ability against β-carotene

In order to confirm the antioxidant ability for β-carotene, β-carotene assay was performed. To prepare a β-carotene solution, 2 mg of β-carotene, 44 μL of linoleic acid, and 200 μL of Tween 80 were dissolved in 10 mL of chloroform, and then the solvent was removed with a vacuum concentrator and 150 mL of distilled water was added. Lactobacillus brevis isolated in Example 1 4.5 mL of β-carotene solution was reacted to 0.5 mL of live cells of the KU15153 strain for 2 hours in a 50°C constant temperature water bath, and then the absorbance was measured at 470 nm, and the oxidation inhibitory ability was evaluated as follows, and the results are shown in Table 4 below. Shown in:

β-carotene oxidation inhibitory ability (%) = (absorbance after reaction/absorbance before reaction) × 100.

Example 1 Comparative Example 1 DPPH free radical scavenging activity (%) 64.25 ± 6.27 ^a 19.21 ± 2.92 ^a β-carotene oxidation inhibitory ability (%) 71.62 ± 6.87 ^a 44.14 ± 0.23 ^b

As shown in Table 6, antioxidant effect evaluation results, Lactobacillus brevis isolated in Example 1 As the KU15153 strain is confirmed to have a high antioxidant effect, it is expected to be of great help in anti-aging and removal of toxins (detox). On the other hand, it was confirmed that Lactobacillus rhamnosus LGG strain, known as a commercial probiotic, significantly reduced the antioxidant effect.

Experimental Example 7: Evaluation of organic acid production of strain

Organic acid production of the strain Lactobacillus brevis KU15153 isolated in Example 1 was evaluated. At this time, the organic acid is a substance essential to improve the intestinal environment by adjusting the pH of the intestine, and among them, those having less than 6 carbons in molecular structure are collectively referred to as short-chain fatty acids. Short-chain fatty acids are metabolites produced by decomposition of indigestible oligosaccharides by intestinal bacteria. It has been reported that it inhibits the accumulation of fat, aids in immunity, and also aids in anticancer activity.

Specifically, in the case of organic acid production evaluation, the strain was inoculated with MRS broth and cultured at 37°C for 12 hours. After incubation, the supernatant was separated using a centrifuge, filtered through a 0.45 μm membrane filter, and used as an HPLC sample. In the case of the evaluation of short-chain fatty acid production, it was carried out in the same manner as in the evaluation of organic acid production, except that 2% arabinose was added during cultivation. At this time, the used column was separated with a Hypersil GOLD aQ column (4.6 mm×150 mm, 5 μm), and the mobile phase was flowed at 1.25 mL/min using a 50 mM potassium phosphate buffer (pH 2.8). The sample injection amount was 20 μL and was detected at 210 nm using a UV detector, and the results are shown in Table 7.

Organic acid (mg/kg) formic acid lactic acid acetic acid propionic acid butyric acid Example 1 1,846.78±118.74 11,077.75±259.62 960.18±79.04 143.01±11.44 576.48±84.77 Comparative Example 1 1,319.29±75.06 10,026.64±264.66 743.66±98.88 2,001.57±201.61 126.73±98.92

As shown in Table 7, the Lactobacillus brevis KU15153 strain isolated in Example 1 was compared to the Lactobacillus rhamnosus LGG strain known as a commercial probiotic, excluding propionic acid, formic acid, lactic acid, and acetic acid. (acetic acid) and butyric acid (butyric acid) production is confirmed to be excellent. In particular, the Lactobacillus brevis KU15153 strain isolated in Example 1 was significantly superior to the Lactobacillus rhamnosus LGG strain known as a commercial probiotic, acetic acid and butyric acid, which are a kind of short-chain fatty acids. ), it is confirmed to be about 4 times excellent), and it is expected to be of great help in removing toxins (detox) by improving the intestinal environment by increasing the intestinal function.

Hereinafter, an example of the preparation of a composition comprising the strain of the present invention as an active ingredient will be described, but the present invention is not intended to limit it, but is intended to be described in detail.

Formulation Example 1: Preparation of powder

Lactobacillus brevis KU15153 strain 20 mg

100 mg lactose hydrate

10 mg of talc

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2: Preparation of tablets

Lactobacillus brevis KU15153 Strain 10 mg

100 mg corn starch

100 mg lactose hydrate

2 mg of magnesium stearate

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet preparation method.

Formulation Example 3: Preparation of capsules

Lactobacillus brevis KU15153 strain 10 mg

Microcrystalline cellulose 3 mg

Lactose Hydrate 14.8 mg

Magnesium stearate 0.2 mg

After mixing the above ingredients, the capsules were prepared by filling them into gelatin capsules according to a conventional capsule preparation method.

Formulation Example 4: Preparation of injection

Lactobacillus brevis KU15153 Strain 10 mg

Mannitol 180 mg

2974 mg of sterile distilled water for injection

Sodium monohydrogen phosphate 26 mg

After mixing the above ingredients, it was prepared in the amount of the above ingredients per ampoule (2 mL) according to a conventional method for preparing injections.

Formulation Example 5: Preparation of liquid formulation

Lactobacillus brevis KU15153 Strain 10 mg

10 g of isomerized sugar

5 g of mannitol

Purified water appropriate amount

Lemon flavor appropriate amount

The above ingredients are dissolved by adding each ingredient to purified water according to a conventional manufacturing method, added an appropriate amount of lemon flavor, adjusted to 100 mL by adding purified water, sterilized and filled in a brown bottle to prepare a liquid formulation.

Formulation Example 6: Preparation of health functional food

Lactobacillus brevis KU15153 strain 10 mg

Vitamin mixture right amount

Vitamin A acetate 70 ㎍

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 ㎍

Vitamin C 10 mg

Biotin 10 ㎍

Nicotinic acid amide 1.7 mg

Folic acid 50 ㎍

0.5 mg of calcium pantothenate

Suitable amount of inorganic mixture

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dicalcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride 24.8 mg

The composition ratio of the vitamin and mineral mixture is relatively suitable for the health functional food, but it is possible to arbitrarily modify the composition, and the above ingredients are mixed according to the general health functional food manufacturing method. Then, granules are prepared, and can be used in the manufacture of health functional foods according to a conventional method.

Formulation Example 7: Preparation of health drink

Lactobacillus brevis KU15153 strain 10 mg

15 g of vitamin C

100 g of vitamin E (powder)

19.75 g of iron lactate

Zinc oxide 3.5 g

3.5 g of nicotinic acid amide

0.2 g of vitamin A

0.25 g of vitamin B1

0.3 g vitamin B2

Purified water quantification

After mixing the above ingredients according to the general health drink manufacturing method, stirring and heating the mixture at 85°C for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized, and stored in a refrigerator. It is used in the manufacture of the health beverage composition of the invention.

The composition ratio is a mixture of ingredients suitable for a relatively preferred beverage in a preferred embodiment, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as the demand class, the country of demand, and the purpose of use.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

Korea Microorganism Conservation Center (overseas) KCCM12213P 20180117

<110> Konkuk University Industrial Cooperation Corp <120> NOVEL STRAIN OF Lactobacillus brevis AND USE THEREOF <130> 1065594 <160> 1 <170> KopatentIn 2.0 <210> 1 <211> 1091 <212> DNA <213> Lactobacillus brevis <400> 1 gagctgacga caaccatgca ccacctgtca ttctgtcccc gaagggaacg tcttatctct 60 aagattggca gaagatgtca agacctggta aggttcttcg cgtagcttcg aattaaacca 120 catgctccac cgcttgtgcg ggcccccgtc aattcctttg agtttcaacc ttgcggtcgt 180 actccccagg cggagtgctt aatgcgttag ctgcagcact gaagggcgga aaccctccaa 240 cacttagcac tcatcgttta cggcatggac taccagggta tctaatcctg ttcgctaccc 300 atgctttcga gcctcagcgt cagttacaga ctagacagcc gccttcgcca ctggtgttct 360 tccatatatc tacgcattcc accgctacac atggagttcc actgtcctct tctgcactca 420 agtctcccag tttccgatgc acttctccgg ttaagccgaa ggctttcaca tcagacttaa 480 aaaaccgcct gcgctcgctt tacgcccaat aaatccggac aacgcttgcc acctacgtat 540 taccgcggct gctggcacgt agttagccgt ggctttctgg ttaaataccg tcaacccttg 600 aacagttact ctcaaaggtg ttcttcttta acaacagagt tttacgagcc gaaacccttc 660 ttcactcacg cggcattgct ccatcagact ttcgtccatt gtggaagatt ccctactgct 720 gcctcccgta ggagtttggg ccgtgtctca gtcccaatgt ggccgattac cctctcaggt 780 cggctacgta tcatcgtctt ggtgggcctt tacctcacca actaactaat acgccgcggg 840 atcatccaga agtgatagcc gaagccacct ttcaaacaaa atccatgcgg attttgttgt 900 tatacggtat tagcacctgt ttccaagtgt tatcccctgc ttctgggcag atttcccacg 960 tgttactcac cagttcgcca ctcgcttcat tgttgaaatc agtgcaagca cgtcattcaa 1020 cggaagctcg ttcgacttgc atgtattagg catgccgcca gcgttcgtcc tgagccataa 1080 tccaaactct a 1091

Claims (12)

Has antibacterial activity against Salmonella Typhimurium and Staphylococcus aureus , formic acid, lactic acid, acetic acid, propionic acid, and Butyric acid (butyric acid) producing, Lactobacillus brevis ( Lactobacillus brevis ) strain (KCCM 12213P).
The method of claim 1,
The strain is E. coli ( Escherichia coli ) or Listeria monocytogenes ( Listeria monocytogenes ) further has an antibacterial activity, Lactobacillus brevis ( Lactobacillus brevis ) strain (KCCM 12213P).
delete The method of claim 1,
The strain is Lactobacillus brevis ( Lactobacillus brevis ) strain (KCCM 12213P), further having at least one characteristic selected from the group consisting of the following i) to v):
I) acid resistance, bile resistance and intestinal adhesion;
Ii) human safety;
Iii) Antibiotic susceptibility to Kanamycine, Ampicillin, Gentamicin, Teracycline, Chloramphenicol and Doxycycline;
Iv) the ability to inhibit intestinal adhesion to food poisoning causative bacteria; And
V) antioxidant activity.
The method of claim 1,
The strain comprises a DNA sequence encoding the 16S rRNA of SEQ ID NO: 1, Lactobacillus brevis ( Lactobacillus brevis ) strain (KCCM 12213P).
Lactobacillus brevis ( Lactobacillus brevis ) containing the strain (KCCM 12213P) as an active ingredient,
The strain has antibacterial activity against Salmonella Typhimurium and Staphylococcus aureus , and formic acid, lactic acid, acetic acid, propionic acid acid) and butyric acid (butyric acid) to produce, probiotic composition.
The method of claim 6,
The strain is a probiotic composition that is produced as a live cell or a dead cell.
Lactobacillus brevis ( Lactobacillus brevis ) containing the strain (KCCM 12213P) as an active ingredient,
The strain has antibacterial activity against Salmonella Typhimurium and Staphylococcus aureus , and formic acid, lactic acid, acetic acid, propionic acid acid) and butyric acid (butyric acid) to produce, Salmonella Typhimurium ( Salmonella Typhimurium) and Staphylococcus aureus ( Staphylococcus aureus ) for antibacterial composition.
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