KR102269256B1 - Probiotic yeast Kluyveromyces marxianus A5 from kefir and uses thereof - Google Patents

Abstract

The present invention is a Kluyveromyces marxianus A5 strain entrusted with accession number KCCM 12598P and uses thereof, and the strain and its culture medium have antibacterial activity against Salmonella spp. It can be used as a feed additive and a pharmaceutical composition for preventing or treating food poisoning.

Description

Probiotic yeast Kluyveromyces marxianus A5 from kefir and uses thereof

The present invention relates to kefir-derived Kluyveromyces marxianus A5 strain and uses thereof.

Salmonella is a microorganism of Gram-negative bacillus belonging to the Enterobacteriaceae family, and is a representative bacterium that causes bacterial food poisoning in humans as well as causing gastrointestinal tract infections in animals. Salmonella exists in the intestines of animals and remains in human food due to incomplete cooking, causing food poisoning. Animals that can carry Salmonella are very diverse, including mammals such as cattle and pigs, birds such as chickens, and reptiles and fish.

Accordingly, it can be said that it is important to reduce Salmonella infection in carrier animals in order to prevent food poisoning accidents in humans and minimize the possibility of potential infectious diseases in animals. it is not the case. In the past, various enterobacteriaceae including Salmonella were reduced due to the regulation of the intestinal microflora through the addition of antibiotics to the feed, but even this has been prohibited today due to the occurrence of resistant bacteria due to the chronic use of antibiotics. Therefore, there is a need for an alternative material that can interfere with the colonization of Salmonella in the intestinal tract of chickens.

Korean Patent Publication No. 10-2017-0024967

It is an object of the present invention to provide a Kluyveromyces maxianus A5 strain deposited with accession number KCCM 12598P.

Another object of the present invention is to provide an antibacterial composition comprising the Kluyveromyces maxianus A5 strain entrusted with accession number KCCM 12598P or a culture solution thereof as an active ingredient.

Another object of the present invention is to provide a probiotic comprising the Kluyveromyces maxianus A5 strain deposited with accession number KCCM 12598P or a culture solution thereof as an active ingredient.

Another object of the present invention is to provide a feed additive comprising the Kluyveromyces maxianus A5 strain entrusted with accession number KCCM 12598P or a culture solution thereof as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of food poisoning comprising the Kluyveromyces maxianus A5 strain entrusted with accession number KCCM 12598P or a culture solution thereof as an active ingredient.

Another object of the present invention is to provide a method for reducing Salmonella spp. comprising the step of treating the sample with the Kluyveromyces maxianus A5 strain or its culture medium deposited with accession number KCCM 12598P.

In order to achieve the above object, the present invention provides a Kluyveromyces maxianus A5 strain deposited with accession number KCCM 12598P.

In one embodiment of the present invention, the strain may be isolated from kefir, but is not limited thereto.

In one embodiment of the present invention, the strain may have antibacterial activity, and preferably, may have antibacterial activity against the Salmonella spp. strain.

In addition, the present invention provides an antibacterial composition comprising the Kluyveromyces maxianus A5 strain entrusted with accession number KCCM 12598P or a culture solution thereof as an active ingredient.

In one embodiment of the present invention, the composition may have antibacterial activity against the Salmonella spp. strain, and the Salmonella spp. strain is Salmonella enterica (Salmonella enterica), Salmonella bongori (Salmonella bongori) , but may be selected from the group consisting of Salmonella Enteritidis and Salmonella typhymurium, but is not limited thereto.

In addition, the present invention provides a probiotic comprising the Kluyveromyces maxianus A5 strain deposited under Accession No. KCCM 12598P or a culture solution thereof as an active ingredient.

In addition, the present invention provides a feed additive comprising the Kluyveromyces maxianus A5 strain entrusted with accession number KCCM 12598P or a culture solution thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating food poisoning comprising the Kluyveromyces maxianus A5 strain entrusted with accession number KCCM 12598P or a culture solution thereof as an active ingredient.

In an embodiment of the present invention, the food poisoning may be caused by a Salmonella sp. strain, and the strain may have antibacterial activity against the Salmonella sp. strain.

In addition, the present invention provides a method for reducing Salmonella spp. comprising the step of treating the sample with the Kluyveromyces maxianus A5 strain or its culture medium deposited with the accession number KCCM 12598P.

Kluyveromyces maxianus A5 strain of accession number KCCM 12598P according to the present invention is Salmonella enterica ( Salmonella enterica ), Salmonella bongori ( Salmonella bongori ), Salmonella enteritidis ( Salmonella Enteritidis ) and Salmonella typhymurium ( Salmonella typhymurium ) ) and has antibacterial activity against Salmonella spp., it can be used as an antibacterial composition, probiotic or feed additive.

The present invention provides a Kluyveromyces marxianus A5 strain deposited with accession number KCCM 12598P.

The present inventors have identified a strain isolated from Kefir, and identified the Kluyveromyces maxianus A5 strain as a strain with significant antibacterial activity against Salmonella, and reported it to the Korea Microbial Conservation Center as of October 02, 2019. It was deposited and was given an accession number KCCM 12598P.

In addition, the present invention provides an antibacterial composition comprising the Kluyveromyces maxianus A5 strain or a culture solution thereof as an active ingredient.

The composition may have antibacterial activity against the Salmonella spp. strain, and the Salmonella spp. strain is Salmonella enterica , Salmonella bongori , Salmonella Enteritidis. And Salmonella typhymurium may be selected from the group consisting of, but is not limited thereto.

The term "antibacterial" or "antibacterial activity" refers to a property that resists microorganisms such as bacteria or fungi, and more particularly, refers to a property of inhibiting the growth or proliferation of bacteria or killing bacteria itself. In the present invention, the antibacterial or antibacterial activity may be used as a property of inhibiting or killing the growth or proliferation of a Salmonella sp.

In addition, the present invention provides a probiotic comprising the Kluyveromyces maxianus A5 strain or a culture solution thereof as an active ingredient.

As used herein, the term "probiotic" refers to a microorganism that can survive in the gastrointestinal tract when ingested by an animal, and is effective in preventing or treating a specific pathological condition. In general, probiotics are effective in treating and improving symptoms caused by abnormal fermentation of the intestinal flora, and when administered into the body, they densely settle on the walls of the digestive tract in the intestine, preventing harmful microorganisms from settling, and preventing miscarriage. Inhibits the growth of harmful microorganisms by lowering the intestinal pH. In addition, the administered probiotic serves to help the activity of the intestinal villi responsible for the absorption of nutrients.

In addition, the present invention provides a feed additive comprising the Kluyveromyces maxianus A5 strain or a culture solution thereof as an active ingredient.

The feed additive has antibacterial activity, and by continuously ingesting poultry or livestock, it is possible to prevent bacterial diseases, and to improve bacterial diseases that have already occurred. Feed can be classified into various types according to nutritional value, main ingredient, distribution, moisture content, blending state and processing type, etc. The feed can be roughage, concentrated feed, supplementary feed, protein feed, starch feed, fat feed or fiber feed. .

The feed additive may additionally contain an acceptable carrier, for example, the feed additive may be added as it is or a known carrier, stabilizer, or the like. In addition, if necessary, various nutrients such as vitamins, amino acids, minerals, antioxidants, antibiotics, antibacterial agents and other additives may be added, and the shape thereof may be in a suitable state such as powder, granule, pellet, suspension, etc. . The feed additive of the present invention may be supplied alone or mixed with feed.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of food poisoning comprising the Kluyveromyces maxianus A5 strain or a culture solution thereof as an active ingredient.

The food poisoning may be caused by the Salmonella sp. strain, and the Kluyveromyces maxianus A5 strain or culture solution thereof of the present invention may have antibacterial activity against the Salmonella sp. strain.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” refers to exhibiting non-toxic properties to cells or humans exposed to the composition. The carrier may be used without limitation as long as it is known in the art, such as buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, and the like.

In addition, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. can Furthermore, it may be used in the form of an ointment, lotion, spray, patch, cream, powder, suspension, gel, or external preparation for skin in the form of a gel. Carriers, excipients and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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 in the Cycotria rubra extract, for example, starch, calcium carbonate, It is prepared by mixing sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, 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, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents 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 is administered in a pharmaceutically effective amount. The term "administration" of the present invention means introducing a predetermined substance to an individual by an appropriate method, and the administration route of the composition may be administered through any general route as long as it can reach a target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, may be administered intrarectally, but is not limited thereto.

As used herein, the term “individual” refers to all animals including humans, rats, mice, and livestock. Preferably, it may be a mammal including a human.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is dependent on the patient's sex, age, body weight, health condition, type, severity, drug activity, sensitivity to drug, administration method, administration time, administration route, and excretion rate, treatment duration, factors including drugs used in combination or concomitantly, and other fields of medicine It can be readily determined by one of ordinary skill in the art according to well-known factors. For administration, the recommended dosage may be administered once a day, or divided into several administrations.

Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Isolation and identification of yeast strains

The yeast strain used in the present invention was isolated from kefir, a natural complex fermented milk. Briefly, the colonies obtained by diluting kefir with sterile physiological saline and spreading it on potato dextrose agar were confirmed and identified through ITS sequencing.

As a result, we disconnect the 10 weeks yeast strain, that the separated yeast as follows: Kluyveromyces marxianus A1, Kluyveromyces marxianus A2, Kluyveromyces marxianus A5, Kluyveromyces marxianus AGB, Kluyveromyces marxianus K5, Kluyveromyces marxianus D2, Saccharomyces unisporus 26S1, Saccharomyces unisporus 2603 , Saccharomyces unisporus 2604 and Saccharomyces unisporus 3701.

Example 2. Screening results based on anti-salmonella ability of the isolated yeast strain

The present inventors performed an anti-Salmonella ability test for 10 strains of yeast isolated from kefir. The anti-salmonella test was performed using a growth curve analysis technique. Yeast cultures and Salmonella enteritidis After mixing 100 μL of each of RR-SE (rifampicin resistant Salmonella Enteritidis) strain into a 96-well microplate to make a total of 200 μL, incubated at 37° C. for 24 hours to observe the growth of Salmonella. If the yeast strain completely inhibits the growth of Salmonella and induces death, it is indicated by (+++), when the growth is completely inhibited, it is indicated by (++), and when the growth is partially inhibited, it is indicated by (+), If growth was not inhibited, the result was judged by displaying (-).

As a result, as shown in Table 1, Kluyveromyces marxianus A5 (Kluyveromyces marxianus A5) out of 10 yeast strains isolated from kefir completely inhibits the growth of Salmonella and induces death, resulting in the best anti-Salmonella ability. was confirmed to indicate In the remaining yeast, it was confirmed that it was possible to inhibit the growth of Salmonella, but did not kill it, or did not affect the growth of Salmonella.

yeast name anti-salmonella Kluyveromyces marxianus A1 ++ Kluyveromyces marxianus A2 + Kluyveromyces marxianus A5 +++ Kluyveromyces marxianus AGB + Kluyveromyces marxianus K5 ++ Kluyveromyces marxianus D2 ++ Saccharomyces unisporus 26S1 + Saccharomyces unisporus 2603 + Saccharomyces unisporus 2604 + Saccharomyces unisporus 3701 -

Example 3. In vivo using the selected yeast strain ( in vivo ) Salmonella reduction effect verification

The present inventors performed two experiments to determine whether or not the Salmonella reducing effect in chickens using Kluyveromyces maxianus A5, which has the best anti-Salmonella activity. Briefly, the first experiment was conducted in 1-day-old chicks to confirm the protective effect of Salmonella immediately after hatching. The experiment was performed with a total of three experimental groups, a negative control group, a positive control group, and a Kluyveromyces maxianus A5 administration group, and a total of 6 chicks in each group. The Kluyveromyces maxianus A5 administration group was ^{orally administered with 1.23x10 6} cfu of yeast strain to 1-day-old chicks, and then attacked Salmonella the next day (2 days old), and the negative control group was inoculated with only saline, and the positive control group was 3.2x10 ⁷ cfu of Salmonella bacteria (rifampicin resistant Salmonella Enteritidis) was challenged. After that, all experimental groups were challenged with Salmonella and counted Salmonella in the cecum of chicks through autopsy the day after.

As a result, as shown in Table 2, when Kluyveromyces maxianus A5 was orally administered to 1-day-old chicks immediately after hatching, it was confirmed that the number of Salmonella bacteria in the cecum was significantly reduced compared to the positive control group 1 day after the Salmonella attack inoculation. did.

autopsy time Average number of Salmonella in the cecum (cfu/g) negative control positive control Kluyveromyces marxianus A5 group Salmonella attack vaccination
1 day later not detected 94.10 X 10 ⁵ 4.00 X 10 ⁵ Salmonella attack vaccination
after 6 days not detected 8.00 X 10 ⁵ 6.09 X 10 ⁵ Salmonella attack vaccination
after 10 days not detected 4.06 X 10 ⁵ 1.88 X 10 ⁵

In addition, the second experiment was conducted to confirm the protective effect of Salmonella 10 days after hatching. The experiment was performed with a total of three experimental groups, a negative control group, a positive control group, and a Kluyveromyces maxianus A5 administration group, and a total of 6 chicks in each group. The Kluyveromyces maxianus A5 administration group was ^{orally administered with 1.23x10 6} cfu of yeast strain to 10-day-old chicks, and then attacked Salmonella the next day (11 days old), the negative control group was inoculated with only saline, and the positive control group was 3.2x10 ⁷ cfu of Salmonella bacteria (rifampicin resistant Salmonella Enteritidis) was challenged. After that, all experimental groups were challenged with Salmonella and counted Salmonella in the cecum of chicks through autopsy the day after.

As a result, as shown in Table 3, when Kluyveromyces maxianus A5 was orally administered to chicks 10 days after hatching, it was confirmed that the number of Salmonella bacteria in the cecum was significantly reduced compared to the positive control group.

autopsy time Average number of Salmonella in the cecum (cfu/g) negative control positive control Kluyveromyces marxianus A5 group Salmonella attack vaccination
1 day later not detected 95.50 X 10 ⁴ 25.70 X 10 ⁴

From the above results, the present inventors found that the Kluyveromyces maxianus A5 strain of accession number KCCM 12598P isolated from kefir has antibacterial activity against the Salmonella spp. strain, and by the activity, the antibacterial It was confirmed that it can be used in compositions, probiotics, and animal feed additives, and can be used as a composition for preventing or treating food poisoning through reduction of Salmonella spp.

Korea Microorganism Conservation Center (Overseas) KCCM12598P 20191002

Claims (11)

As a Kluyveromyces marxianus A5 strain or a culture solution thereof isolated from Kefir deposited with accession number KCCM 12598P,
The Cluj Vero MRS film cyano Salmonella Entebbe Rica (Salmonella enterica), Salmonella rod ring (Salmonella bongori), Salmonella Entebbe utility disk (Salmonella enteritidis) and Salmonella typhimurium bunch antimicrobial activity of the Titanium (Salmonella typhimurium) strain Taunus A5 is from poultry Kluyveromyces marxianus , characterized in that it has a ( Kluyveromyces marxianus ) A5 strain or a culture solution thereof. delete delete As a poultry antibacterial composition comprising the strain of claim 1 or a culture solution thereof as an active ingredient,
The strain is Salmonella enterica in poultry ( Salmonella enterica ), Salmonella bongori ( Salmonella bongori ), Salmonella enteritidis ( Salmonella enteritidis ) and Salmonella typhimurium ( Salmonella typhimurium ) Antibacterial activity, characterized in that it has antibacterial activity against the strain for composition. delete delete A probiotic for poultry comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A feed additive for poultry comprising the strain of claim 1 or a culture solution thereof as an active ingredient. A pharmaceutical composition for preventing or treating poultry food poisoning comprising the strain of claim 1 or a culture solution thereof as an active ingredient. 10. The method of claim 9,
The food poisoning is Salmonella enterica ( Salmonella enterica ), Salmonella bongori ( Salmonella bongori ), Salmonella enteritidis ( Salmonella enteritidis ) and Salmonella typhimurium ( Salmonella typhimurium ) Composition, characterized in that caused by the strain. Claim 1 strain or step Salmonella Entebbe Rica (Salmonella enterica), Salmonella rod ring (Salmonella bongori) in poultry comprising a handling the culture thereof in a sample, of Salmonella Entebbe utility disk (Salmonella enteritidis) and Salmonella typhimurium (Salmonella typhimurium ) A method for reducing strains.