KR20110112229A - Pediococcus acidilactici wrl-1 and use of the same - Google Patents

Abstract

The present invention relates to Salmonella gallinarum (Salmonella gallinarum) and novel acid-resistant Pediococcus ecidyl lactic acid (Pediococcus acidilactici) WRL-1 having the ability to inhibit the growth of harmful pathogenic microorganisms, and the bioactive agent containing the same, Pedioco Pediococcus acidilactici WRL-1 effectively inhibits the growth of various pathogenic microorganisms and Salmonella gallinarum that exhibit harmful properties in the body and intestine, and has acid and bile acid resistance functions. As an alternative to antibiotics, it can be administered to humans and various livestock, or used as medicines, feed additives, or animal medicines to stabilize the intestinal microflora, and to treat or prevent the symptoms that can be caused by fermentation of harmful microorganisms in the intestines such as diarrhea. That has a very outstanding effect.

Description

Pediococcus Ecidyl Lactyl JR-1 and its use {Pediococcus acidilactici WRL-1 and use of the same}

The present invention relates to Pediococcus acididactici WRL-1 (Pediococcus acidilactici WRL-1) newly identified from kimchi, and more specifically, Kimchi-derived new strain Pediococcus ecidylactidic WRL- having antibacterial activity. 1 (Pediococcus acidilactici WRL-1) and its use.

The antimicrobial effect of fermented microorganisms is due to the metabolites produced as the microorganisms grow during fermentation. Antibiotics, bacteriocin, acetic acid, and lactic acid are the main metabolites of fermented microorganisms. Antimicrobial peptide production strains include Lactobacillus, Streptococcus, Pediococcus, Leuconostoc, Enterococcus, Corynebacterium and Halobacterium ( Halobacterium). The antimicrobial peptides they produce are limited to strains that are systematically close to the antimicrobial producing strain itself or exhibit a broad antimicrobial spectrum. Currently, these antimicrobial peptides have been commercialized in part, and can be used for improving the shelf life of various foods and feeds such as milk products, meat products, feed products, grains, and sanitizing agents.

Bacteriocin is a microbial-derived natural protein or peptide-based antimicrobial substance, which is composed of proteins. Therefore, bacteriocin is generally non-toxic and non-residual to humans, has high stability against pH and heat, and is degraded by digestive enzymes when ingested by animals.

Lactic acid bacteria are known to produce various antibacterial substances such as bacteriocin in addition to organic acids such as lactic acid and acetic acid, which are known to be important biological preservatives in feed. In addition, lactic acid bacteria improve the self-defense ability of plants by producing a variety of bioactive substances, antibacterial substances, anti-cancer substances, and shows the effect of stabilizing the intestinal microorganisms in the livestock, increased feed efficiency, increased disease resistance. The reason why lactic acid bacteria are of particular interest is because they are generally regarded as safe microorganisms (GRAS) because they have been widely used and consumed in foods in both East and West. However, in order to industrially use lactic acid bacteria correctly, it is generally necessary to go through a procedure to clearly understand and evaluate from a scientific standpoint what the use is and what characteristics are suitable for use, rather than judging by a list of positive effects. Only with such a procedure can you ensure the uniformity of the product and the reproducibility of the effects.

In general, diarrhea is caused by a variety of causes, such as diarrhea caused by pathogenic microorganisms, dyspeptic overeating diarrhea, diarrhea due to body temperature decrease. In the livestock industry, diarrhea caused by pathogenic bacteria such as Salmonella is frequently occurring due to large-scale breeding methods. In young livestock, this bacterial diarrhea is a major cause of lower productivity.

However, when diarrhea occurs, there is no special method other than antibiotic administration. Especially, it is forbidden to administer antibiotics to livestock immediately before shipment, but actually, antibiotics are prescribed to prevent massive damage. This raises the issue of antibiotic retention and resistance through livestock products.

Therefore, there is a need to solve the problem and to prevent or treat diarrhea more safely.

An object of the present invention is to provide a kimchi-derived Pediococcus ecidyl lactic acid (Pediococcus acidilactici) microorganism having antibacterial activity against pathogenic bacteria.

Another object of the present invention is to provide an antimicrobial agent comprising the microorganism or its culture.

It is another object of the present invention to provide a feed additive having an infection suppression effect of a pathogenic bacterium comprising pediococcus ecidyl lactixi WRL-1 or a culture thereof.

Accordingly, the present inventors have tried to isolate lactic acid bacteria showing antibacterial activity against Salmonella gallinarum among animal harmful microorganisms from kimchi among traditional fermented foods, and as a result, invented the strain Lactobacillus pediococcus ecidyl lactish WRL-1, the strain And by confirming that the growth of pathogenic bacteria, including Salmonella gallinarum, E. coli and the like by the culture medium derived from the strain was completed the present invention.

The present invention provides a kimchi-derived Pediococcus acidilactici WRL-1 new strain (KACC91525P, deposited on February 18, 2010) having antibacterial activity against pathogenic bacteria.

Strain characteristics are as follows.

MRS broth medium (10 g proteose peptone, 10 g beef extract, yeast extract 5 g, dextrose 20 g, polysorbate 80 1 g, ammonium citrate 2 g, sodium acetate 5 g, magnesium sulfate 0.1 g, manganese sulfate 0.05 g, dicalcium phosphate) (Dipotassium phosphate) in the form of a bacterium exhibited activity when incubated at 37 ° C. for 48 hours at 2 ° C., and the cells are spherical in shape and are Gram-positive cocci. Its physiological properties grow at a growth temperature of 30 to 45 ° C., preferably 37 to 40 ° C. or less, a growth pH condition of pH 4.0 to 6.0, preferably 4.5 to 5, and the influence of oxygen is usually anaerobic.

In addition, the present invention provides a feed additive having an effect of inhibiting infection of a pathogenic bacterium comprising pediococcus ecidyl lactixi WRL-1 or a culture thereof.

The present invention also provides a feed composition comprising pediococcus ecidyl lactixi WRL-1 or a culture thereof.

The feed additive may be 20 to 90% high concentrate or in powder or granule form.

The feed additives include organic acids such as citric acid, fumaric acid, adipic acid, lactic acid and malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechin, alpha-tocopherol, Rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid and the like may further include any one or two or more.

The feed additive of the present invention and the feed containing the same are supplementary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobials, antioxidants, antifungal enzymes, live microbial agents, etc. Dairy products consisting of vegetable protein feed consisting mainly of barley, oats, corn, wheat and rice, rape, soybeans and sunflower, animal protein feed consisting mainly of blood meal, meat meal, bone meal and fish meal, sugar, various powdered milk and whey powder In addition to the main components, such as the liquid component after mixing all the drying additives and the liquid component after heating, it can be used with substances such as nutritional supplements, digestion and absorption enhancers, growth promoters, disease prevention agents and the like.

The feed additive may be administered to the animal alone in combination with other feed additives in an edible carrier.

In addition, the feed additives can be easily administered in a top dressing or directly in animal feed or in oral formulation separately from the feed, by injection or transdermal or in combination with other ingredients. Typically, single or divided daily dosages can be used as is well known in the art.

When the feed additive is administered separately from the animal feed, as is well known in the art, the dosage form of the culture extract or the probiotic may be prepared in an immediate release or sustained release formulation in combination with a nontoxic and pharmaceutically acceptable edible carrier. have. Such edible carriers include, for example, solid or liquids such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used, the dosage form of the extract may be tablets, capsules, powders, troches or sugar-containing tablets or top dressings in microdisperse form. If a liquid carrier is used, it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. In addition, dosage forms may contain adjuvants, such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like.

In addition, the animal feed comprising the feed additive of the present invention may be any protein-containing organic wheat flour conventionally used to meet the dietary needs of the animal. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures.

The feed additive may be used by adding to the feed by dipping, spraying or mixing.

The invention is applicable to a number of animal diets, including mammals, poultry and fish. More specifically, the diet is commercially important mammals such as pigs, cows, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), furry animals (eg minks, foxes). Etc.) and zoo animals (eg monkeys, etc.), and pets (eg cats, dogs, etc.). Commercially important poultry typically include chickens, ducks, geese, pheasants, turkeys and quails. Commercially raised fish, such as trout, may also be included.

In the animal feed blending method according to the present invention, the composition of the present invention is incorporated into the animal feed about 1g ~ 100g per kg of feed on a dry weight basis.

In addition, the feed mixture is thoroughly mixed and then a viscous granulated or granular material is obtained depending on the degree of grinding of the components. It is fed as a mash or formed into the desired discrete shape for further processing and packaging. In order to prevent separation during storage, it is desirable to add water to the animal feed, followed by conventional pelletization, expansion, or extrusion processes, and excess water may be dried or removed.

Pediococcus acidilactici WRL-1 having anti-pathogenic activity isolated from kimchi according to the present invention is isolated from kimchi, so stability is ensured even if ingested live bacteria, Salmonella gallina, the causative agent of diarrheal disease It has the effect of inhibiting the growth of the room, it is very useful for the prevention of livestock diarrheal disease by pathogenic microorganisms as an environmentally friendly microbial agent.

Figure 1 shows the antimicrobial effect of Salmonella gallinarum of the supernatant of the present invention strain pediococcus ecidyl lactic WRL-1 culture.
Figure 2 shows the antimicrobial effect of the culture supernatant and heat-treated culture medium of pediococcus ecidyl lactic WRL-1 against the pathogenic bacterium Bacillus cereus.
Figure 3 shows the experimental results confirming the enzyme production of the culture supernatant of pediococcus ecidyl lactishi WRL-1. On the left is a photo showing cellulose degrading enzyme, and on the right is proteolytic production.
FIG. 4 shows the results of comparing the 16S rRNA sequence of the present invention strain Pediococcus ecidyl lacticci WRL-1 and the Pediococcus acidylacti strain DSPV 348T 16S rRNA sequence and the 16S rRNA nucleotide sequences of the strain of the present invention.

The present invention relates to a kimchi-derived new strain Pediococcus ecidyl lactisi WRL-1 having antibacterial activity against pathogenic bacteria. Pediococcus ecidyl lactici is a Gram-positive bacterium belonging to the genus Pediococcus (Pediococcus) and is a lactic acid bacterium commonly found in kimchi, salted pickles, pickles, pickled olives, and plant fermented foods. WRL-1 is a microorganism newly identified and identified by the inventors. Since the new strain of the present invention is known as a bacterium that is normally identified as a lactic acid bacterium isolated from kimchi, it is obvious to those skilled in the art that even when ingested as live bacteria, it is harmless to the human body.

The new strain of the present invention exhibits antimicrobial activity and growth inhibitory activity against pathogenic bacteria. The antimicrobial activity is relatively heat stable and inhibits the growth of Salmonella gallinarum, the causative agent of diarrhea. The cultures derived from the strains as well as the new strain pediococcus ecidyl lactish WRL-1 of the present invention exhibit the same antimicrobial activity.

As used herein, the term 'culture' may be a culture stock solution including the cells, and may also be a culture supernatant or a dilution of the culture.

The composition of the culture may additionally include components necessary for cultivation of lactic acid bacteria, as well as components that synergistically act on the growth of lactic acid bacteria, and thus the composition may be easily selected by those skilled in the art. have.

The strain and / or the culture may be prepared as an antimicrobial composition having a growth inhibitory effect of the pathogenic bacteria, including the composition for inhibiting infection of the pathogenic bacteria, food and animal feed additives. In addition, the composition for inhibiting infection of pathogenic bacteria of the present invention, food and animal feed additives may include only cultures from which microorganisms have been removed, and the cells may be mixed or manufactured to contain only the cells.

In order to prepare the antimicrobial composition of the present invention, the Pediococcus ecidyl lactisi WRL-1 of the present invention can be cultured in large quantities through a conventional liquid or solid phase culture technique. The antimicrobial agent and composition of the present invention may be composed of the feeder cells or spores of the strain, or a mixed state of the feeder cells and the spores, and in order to formulate the microorganisms, the cells are removed under the conditions of reduced pressure freeze drying (Lyophillized), lyophilization, and hydration. can do.

Pediococcus ecidyl lactyl WRL-1 of the present invention exhibits antimicrobial activity against various pathogenic bacteria and enteric pathogenic bacteria of the genus Salmonella, Escherichia coli and Bacillus.

In another aspect of the invention, the present invention relates to a probiotic having an infection inhibitory effect of a pathogenic bacterium comprising Pediococcus ecidyl lactixi WRL-1 or a culture thereof.

Lactobacillus, such as pediococcus ecidyl lactic acid, acts as a beneficial bacterium for the human body by preventing food spoilage by fermenting and inhibiting food poisoning bacteria by secreting antibacterial substances. Foods produced using lactic acid bacteria fermentation include cheese, buttermilk, yoghurt, whey and the like, and recently, functional yoghurt by lactic acid bacteria fermentation is widely marketed. In addition, lactic acid bacteria fermented foods are manufactured as tablets, granules, and the like as commercial preparations and are commercially available as food compositions and pharmaceuticals.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

<Example 1: Selection of strains having antimicrobial activity against pathogenic bacteria>

Pathogenic bacteria causing food poisoning were distributed from KCTC and used to examine the antimicrobial activity of the culture supernatants of 100 species of lactic acid bacteria isolated from Kimchi on the plate of NB light agar (0.8% agar, Salmonella gallinarum). 25 ml of 1% (v / v)) was solidified and prepared by wells on medium with sterile tips. The supernatant was dispensed into the wells of the prepared medium and cultured at the optimum culture temperature of the indicator strain, and strains having antimicrobial activity were selected on the basis of whether or not clear zones were formed on the NB plate. Screened (FIG. 1).

The selected lactic acid bacteria were examined for morphological and biochemical properties according to the method of Bergey's Manual of Determinative bacteriology, and the optimum growth temperature, gram staining, motility, catalase test, CO ₂ production, and genetic The nucleotide sequence of 16S rDNA was analyzed for identification. As a result, it was confirmed that the selected lactic acid bacteria pediococcus ecidyl lacticci WRL-1 strain had more than 99% homology with Pediococcus ecidyl lactic acid (Table 1). Table 1 shows the results of strain identification of Pediococcus ecidyl lacti WRL-1.

                             Strain
 Characteristies Pediococcus acidilactici  WRL-1 Pediococcus acidilactici Shape // Gram stain C / + C / + Spheres - - Motility - - Growth in air + + Growth anaerobically + + Catalase - - Oxidase - - Glucose (acid) + + O / F //- - F Growth at 40 ℃ + + Growth at 45 ℃ + + Growth at pH4.2 + ND Growth at pH8.5 - d Growth in 6.5% Nacl + + Growth in 18% Nacl - - Arginine hydrolysis + + Carbohydrate, acid from:
                              Arabinose + d                               Ribose + +                               xylose - +                               Rhamnos - d                               Lactose + d                               Maltose - -                               Melezition - -                               Sucrose - -                               Trehalose - d                               Dextrin - -                               Starch - -                               Glycerol - -                               Mannitol - -                               Sorbitol - -                               Arbiutin - ND

d-Different reaction in different strains

C-coccus

Example 2: Antimicrobial Activity of Pediococcus Ecidyl Lacticidal WRL-1 Against Pathogenic Bacteria>

In the same manner as in Example 1, the Pediococcus ecidyl lactisi WRL-1 culture supernatant was used as Salmonella gallinarum (KCTC), Escherichia coli, Korea Research Institute of Bioscience and Biotechnology. KCTC) and the like was dispensed into inoculated solid medium to observe the growth inhibitory effect on the pathogenic bacteria. As a result, it was confirmed that there is a growth inhibitory effect (Table 2). Table 2 shows the antimicrobial effect of Pediococcus ecidyl lactic WRL-1 against other pathogenic bacteria besides Bacillus cereus.

Indicatorstrains Growth conditions inhibition zone diameter (mm) Bacillus cerius
(Bacillus cereus) KCTC NB, 37 ℃, Aerobic 17 Escherichia coli
(Escherichia coli) KCTC NB, 37 ℃, Aerobic 20 Salmonella Entry Tides
(Salmonella entritidis) KCTC NB, 37 ℃, Aerobic 19

Example 3 Antimicrobial Activity of Pediococcus Ecidyl Lacticidal WRL-1

In order to confirm the thermal stability of the antimicrobial activity of pediococcus ecidyl lacticidal WRL-1, the culture supernatant was heat-treated at different temperatures and observed for changes in antimicrobial activity. Samples obtained by heat-treating the culture supernatant of Pediococcus ecidyl Lacti WRL-1, inoculated with the pathogenic bacterium Salmonella gallinarum, were aliquoted to confirm whether antimicrobial activity was observed against the pathogenic bacterium. As a control, the culture supernatant without heat treatment was used.

The titer of the antimicrobial activity is expressed in AU (activity unit) / mL, and the supernatant is serially diluted twice, taking the inverse of the maximum dilution factor that forms the inhibitory ring, and multiplied by the conversion factor converted to 1 ml. It was. As a result, antimicrobial activity was observed in all (FIG. 4). On the basis of the above results, it was confirmed that pediococcus ecidyl lactisi WRL-1 culture medium had a heat-stable secretory component that inhibited the growth of pathogenic bacteria Salmonella gallinarum.

Example 4 Confirmation of Enzyme Production of Pediococcus Ecidyl Lacticidal WRL-1

To identify the enzymes present in the culture supernatant of Pediococcus ecidyl lactish WRL-1, 2.0% (w / v) skimmed milk and 1.5% (w / v) with protease identification medium. v) Agar was mixed to make a medium. In order to identify cellulose degrading enzyme (cellulase), a medium was prepared by mixing 1.0% (w / v) CMC (carboxy methly cellulose) and 1.5% (w / v) agar. All medium was sterilized (121 ° C., 15 minutes) and dispensed 25 ml each time in a disposable petri dish. The wells were made with sterile tips on the plate, and then dried by adding supernatant of Pediococcus ecidyl Lacticidal WRL-1 culture, followed by incubation at 37 ° C. for 24 hours to confirm whether the enzyme was produced.

Proteolytic enzyme productivity was confirmed by the clear ring on the scheme milk plate, cellulose enzyme productivity was confirmed by staining with Congo red 0.5% (w / v) buffer for 30 minutes in CMC plate with 1M NaCl buffer solution After washing, a yellow (orange) ring was identified on the well (FIG. 5).

As mentioned above, specific parts of the present invention have been described in detail, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. . Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Institute of Agricultural Biotechnology KACC91525 20100129

<110> WOORI LIFESCIENCE CO., LTD <120> Pediococcus acidilactici WRL-1 and use of the same <130> WOORI-PEDIOCOCCUS <150> KR1020100031454 <151> 2010-04-06 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1072 <212> DNA <213> Pediococcus acidilactici <400> 1 gctgctatct gcagtcgaac gaacttccgt taattgatta tgacgtgctt gcactgaatg 60 agattttaac acgaagtgag tggcggacgg gtgagtaaca cgtgggtaac ctgcccagaa 120 gcaggggata acacctggaa acagatgcta ataccgtata acagagaaaa ccgcctggtt 180 ttcttttaaa agatggctct gctatcactt ctggatggac ccgcggcgca ttagctagtt 240 ggtgaggtaa cggctcacca aggcgatgat gcgtagccga cctgagaggg taatcggcca 300 cattgggact gagacacggc ccagactcct acgggaggca gcagtaggga atcttccaca 360 atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag aagggtttcg gctcgtaaag 420 ctctgttgtt aaagaagaac gtgggtgaga gtaactgttc acccagtgac ggtatttaac 480 cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta 540 tccggattta ttgggcgtaa agcgagcgca ggcggtcttt taagtctaat gtgaaagcct 600 tcggctcaac cgaagaagtg cattggaaac tgggagactt gagtgcagaa gaggatagtg 660 gaactccatg tgtagcggtg aaatgcgtag atatatggaa gaacaccagt ggcgaaggcg 720 gctgtctggt ctgtaactga cgctgaggct cgaaagcatg ggtagcgaac aggattagat 780 accctggtag tccatgccgt aaacgatgat tactaagtgt tggagggttt ccgcccttca 840 gtgctgcagc taacgcatta agtaatccgc ctggggagta cgaccgcaag gttgaaactc 900 aaaagaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc ggagctacgc 960 gaagacctta ccagtcttga catcttctgc cactagagat tagcgttccc ttcggggaca 1020 aatgacagtg gtgcatggtt gtcgtcagct cgtgcgtgga aatgttgggt ta 1072

Claims (3)

Kimchi-derived Pediococcus ecidyl lactisi WRL-1 (KACC91525P) exhibiting antimicrobial activity against Bacillus cereus
Feed additive containing pediococcus ecidyl lacticidal WRL-1 or its culture medium which exhibits antimicrobial activity against Bacillus cereus of claim 1
Feed composition containing Pediococcus ecidyl lacticidal WRL-1 or its culture solution showing antibacterial activity against Bacillus cereus of claim 1

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