KR102064134B1 - Novel Strain of Pediococcus acidilactici CACC 537, and feed composition using thereof - Google Patents

Abstract

The present invention relates to a new strain of Pediococcus acidilactici CACC 537, and a feed composition for pets containing the same as an active ingredient. The Pediococcus acidilactici CACC 537 strain of the present invention is excellent in acid resistance, bile resistance, and intestinal adhesion, and thus is suitable for use as a probiotic strain, has excellent antimicrobial activity against livestock pathogens, and has an excellent effect on immune enhancement, proteinuria suppression, cholesterol level normalization and liver function improvement.

Description

Novel Strain of Pediococcus acidilactici CACC 537, and feed composition using according to the new strain Pediococcus ecidyl lactici CACC 537 and the active ingredient

The present invention relates to a new strain pediococcus ecidyl lactic acid CACC 537 and a pet animal feed composition containing the same as an active ingredient, more specifically, has antimicrobial activity against livestock pathogens, immune enhancement, proteinuria suppression, cholesterol level The present invention relates to a new strain Pediococcus ecidyl lactic acid CACC 537 having an excellent effect on normalization and improving liver function and a feed composition for pets using the same.

At present, the number of one- and two-person households is increasing rapidly due to the nuclear family. As a result, a growing population is growing pets to find emotional stability. Dogs account for the largest portion of pets, and dogs generally live on average for 11 to 13 years, but in recent years, their lives have been extended for 15 to 20 years due to improved public health and improved medical standards. It became.

However, most domestic dogs live in homes, so they lack exercise and are stressed so that their digestion and metabolic activity is easily lowered and their immunity tends to be lowered. As a result, metabolic diseases and immune-related diseases occur similarly to humans, and worsen into more fatal internal and external medical diseases, resulting in a problem of poor quality of life of dogs and dogs.

At present, the interest in animal medical care and welfare in Korea is increasing, but since the cost of medical treatment is high due to the high cost of treatment, it is urgent to develop a drug that can easily prevent the development of metabolic or immune-related diseases caused by lack of exercise of dog to be.

On the other hand, probiotics, living microorganisms, promote the health of animals by activating their function in the intestine (Fuller, 1989). Many researchers have reported that probiotics strains such as Lactobacillus and Bifidobacterium reduce or protect against symptoms such as diarrhea (Gilliland, 1990).

These probiotic strains enter the intestine and enter the intestinal epithelial cells to prevent intestinal colonization of harmful microorganisms and secrete antimicrobial substances, thereby inhibiting the growth of harmful microorganisms and improving diarrhea and constipation. Has the effect of action. Probiotics strains are introduced into the intestine and secreted through its own metabolic activity, so many benefits to the host, many studies using the probiotic strains are in progress. Probiotic strains are most normally present in the intestines of animals and balance the intestinal microflora and have a beneficial effect on the health of the host.

Therefore, in order to select useful strains of intestinal probiotics, several important features are identified and selected (Fuller, 1989; Garriga et al., 1998; Martin et al., 2006; Mishra and Prasad, 2005). That is, because the probiotic strain must survive in the digestive tract, it must have acid resistance or bile resistance, and form colonies in the digestive tract until reaching the intestine. In addition, it should be safe for food or feed or clinical use, while at the same time having a beneficial effect on the host. Recently, probiotic strains with many functional properties have been developed. There are probiotic strains that have a function of inhibiting the proliferation of pathogens, and BSH activity (Begley et al., 2006; Pereira et al., 2003) or starch hydrolysis. Probiotic strains having activity (amylolytic activity, Lee et al., 2001; Vizoso Pinto et al., 2006).

However, research on probiotic strains has been focused mainly on improving human health and livestock productivity, and has yet to develop probiotic strains that can effectively suppress the development of dog metabolic and immune related diseases. Is a sluggish situation.

Accordingly, the present inventors have diligently researched to find a probiotic strain effective for dog metabolism or immune-related diseases. As a result, the new strain Pediococcus esidilactici CACA 537 isolated from the feces of dogs was resistant to acid, bile, and Excellent intestinal adhesion, suitable for use as a probiotic strain and at the same time have antimicrobial activity against livestock pathogens, confirmed that it has an excellent effect on immune enhancement, inhibition of proteinuria, normalization of cholesterol levels and improvement of liver function, and completed the present invention. .

Therefore, the main object of the present invention is to provide a new strain pediococcus ecidyl lactici CACC 537 which is a probiotic strain excellent in acid resistance, bile resistance, and intestinal adhesion.

In addition, another object of the present invention is to provide a feed composition for pets containing the new strain Pediococcus ecidyl lactici CACC 537 as an active ingredient.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a new strain Pediococcus acidilactici CACC 537 (Accession No .: KACC 81098BP).

The term "Pediococcus ecidyl lactici CACC 537" (Pediococcus acidilactici) of the present invention is a novel pediococcus ecidyl lactici strain that has been newly identified in dog feces by the present inventors, "CACC 537 strain" or Can be used interchangeably with "CACC 537".

 The inventors have investigated and analyzed microorganisms useful for dogs for the development of probiotics that can be used in dogs for many years. It has excellent intestinal adhesion and is suitable for use as a probiotic strain, and also has antimicrobial activity against livestock pathogens, and has an excellent effect on immune enhancement, proteinuria suppression, normalization of cholesterol and improvement of liver function, and no safety problems during storage. It was confirmed.

The new strain Pediococcus ecidyl lactyl CACC 537 (Accession Number: KACC 81098BP) of the present invention is characterized by having the 16s rRNA sequence of SEQ ID NO: 1.

The present inventors compared the gene sequence with those of conventionally known strains, and as a result, the strain of the present invention was identified as a new strain belonging to pediococcus ecidyl lactici. It was deposited on July 11, 2019 at the National Institute of Agricultural Science, Agricultural Genetic Resource Information Center, and was assigned accession number KACC 81098BP.

According to another aspect of the present invention, the present invention provides a feed composition for pets comprising the new strain Pediococcus ecidyl lacticy CACC 537, or a culture thereof as an active ingredient.

The pet food composition of the present invention is excellent in acid resistance, bile resistance, and intestinal adhesion activity, suitable for use as a probiotic strain. In a preferred embodiment of the present invention, the CACC 537 strain of the present invention was confirmed to have excellent acid resistance by measuring a 42% survival rate even in a strong acidic condition of pH 2.5, showing a survival rate of more than 99% even in bile conditions very good bile resistance It was confirmed to have (see Fig. 1). In addition, it was confirmed that the CACC 537 strain of the present invention exhibits a high intestinal adhesion activity of 78.6% (see Fig. 2).

The pet food composition of the present invention is characterized by having antimicrobial activity against livestock pathogens. In the present invention, the CACC 537 strain or its culture is characterized in that it exhibits antimicrobial activity by itself. The CACC 537 strain or its culture medium has antimicrobial activity against various livestock pathogens. In one embodiment of the present invention, it was confirmed that the CACC 537 strain culture according to the present invention has growth inhibition effect against various livestock pathogens (see FIG. 3). Specifically, the livestock pathogen has antibacterial activity against Salmonella Derby, Salmonella Enteritidis, Salmonella Typhimurium, and Escherichia coli , but is not limited thereto. .

The pet food composition of the present invention is characterized in that the storage stability. In the embodiment of the present invention, the CACC 537 strain was observed that no significant change in the number of viable cells during the storage period of 4 months, it was confirmed that the strain with excellent storage stability.

In addition, the pet food composition of the present invention is characterized in that it has an immunostimulating activity. In a preferred embodiment of the present invention, the dog was administered the CACC 537 strain of the present invention was observed that the leukocyte level was increased by 16.7%, platelet count was also increased by 8.6%, through these results the CACC 537 strain of the present invention is cellular White blood cells that play a major role in the immune response was confirmed to increase the immune function by increasing the normal range (see Fig. 5).

In addition, the pet food composition of the present invention is characterized by having proteinuria inhibitory activity. In a preferred embodiment of the present invention it was confirmed that the administration of the CACC 537 strain of the present invention can effectively reduce the protein content contained in the dog's urine (see Figure 6).

In addition, the pet food composition of the present invention is directly administered to the dog is characterized by having a cholesterol level normalizing effect and liver function improving activity through cholesterol reducing activity. In a preferred embodiment of the present invention, the CACC 537 strain of the present invention was administered to the diarrhea patient dog for one month, and it was confirmed that effectively reducing the levels of high cholesterol and blood factors associated with hepatic function. Animal feed composition was found to be effective in normalizing cholesterol levels and improving liver function by reducing dog cholesterol.

The feed composition of the present invention may mean any natural or artificial diet, meal, snack or ingredient of the meal for dogs to eat, ingest, digest, or suitable for dogs, various known in the art It can be manufactured in the form.

The type of the feed is not particularly limited, and may be used in addition to the feed commonly used in the art, in which case the feed composition of the present invention serves as a feed additive. Non-limiting examples of feeds that can be added include, but are not limited to, vegetable feeds such as cereals, fruits, food processing by-products, algae, fibres, pharmaceutical by-products, oils, starches, gourds or grain by-products; And animal feeds such as proteins, minerals, fats and oils, minerals, fats and oils, single cell proteins, zooplankton or foods. These may be used alone or in combination of two or more thereof.

In another embodiment, the feed composition may be appropriately used depending on the type of dog, age, and other types of feed ingredients to be administered.

The feed composition of the present invention may further include substances exhibiting various effects such as supplementation of nutrients and prevention of weight loss, enhancement of digestive availability of fiber in the feed, improvement of oil quality, prevention of reproductive disorders and improvement in conception, and prevention of high temperature stress in summer. For example, mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, complex minerals, mineral preparations such as trace minerals such as zinc, copper, cobalt, and selenium, keratin, vitamin E, vitamin A, D, E, nicotinic acid , Vitamins such as vitamin B complex, protective amino acids such as methionine, lyric acid, protective fatty acids such as fatty acid calcium salt, yeast culture, fungi such as fungi fermentation, yeasts and the like may be further included.

In still another aspect, the present invention provides a method of improving the dog's immunity, inhibiting proteinuria, lowering cholesterol and improving liver function, comprising administering the feed composition to a dog.

The administration means introducing the composition of the present invention to the animal in any suitable manner, and the route of administration of the composition of the present invention may be administered via various routes orally or parenterally as long as the target tissue can be reached. The inventors do not show acute toxicity even when orally administering the feed composition of the present invention.

The features and advantages of the present invention are summarized as follows:

(1) The present invention provides a feed strain for a new animal strain Pediococcus ecidyl lactic acid CACC 537 derived from the dog's feces and pet food composition containing the same as an active ingredient.

(2) The new strain Pediococcus ecidyl lactyl CACC 537 of the present invention is excellent in acid resistance, bile resistance, and intestinal adhesion, suitable for use as a probiotic strain, has excellent antibacterial activity against livestock pathogens, It has an excellent effect on improving, inhibiting proteinuria, reducing cholesterol levels and improving liver function.

Figure 1 is a graph showing the measurement of acid resistance and bile resistance of pediococcus ecidyl lactici CACC 537 strain of the present invention.
Figure 2 is a graph showing the measurement of intestinal adhesion of the pediococcus ecidyl lactici CACC 537 strain of the present invention.
Figure 3 is a photograph of the results of experiments by the Disk diffusion method to measure the antimicrobial activity against livestock pathogens of the Pediococcus ecidyl lactici CACC 537 strain of the present invention.
Figure 4 is a graph showing the measurement of the change in viable cell count during long-term storage of the Pediococcus ecidyl lactici CACC 537 strain of the present invention.
Figure 5 is a graph showing the measurement of the immunostimulating effect of the pediococcus ecidyl lactici CACC 537 strain of the present invention.
Figure 6 is a graph showing the measurement of the proteinuria inhibitory effect of pediococcus ecidyl lactici CACC 537 strain of the present invention.
Figure 7 is a schematic diagram showing the schematic diagram of the pedeococcus ecidyl lactici CACC 537 strain of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Validation of Acid Resistance, Bile Resistance, and Intestinal Attachment Activity of Dog Feces-Derived Microorganisms

In order to select candidates for probiotics having antimicrobial and immunostimulating activity against livestock pathogens, the probiotic characteristics of microorganisms collected from fecal samples of dogs were verified.

1-1. Acid and Bile Resistance

To study the acid resistance and bile characteristics of the strains isolated from dog feces, the isolated strains were used for the experiment by incubating for 24 hours at 37 ° C. using MRS broth ((BD Difco ™, USA). In MRS broth adjusted to pH 2.5, bile resistance was inoculated in MRS medium containing 0.3%, 1% (w / v) Oxgall (BD Difco ™, USA) and incubated for 2 hours, followed by 0 and 2 hours. The viable cell count after time was measured and the survival rate (%) was measured.

TABLE 1

As a result, the CACC 537 strain of the isolated candidate group was confirmed to have excellent acid resistance by measuring 42% of the survival rate even in the strong acidic condition of pH 2.5, and showed very good bile resistance by showing the survival rate of 99% or more in the bile condition. I could confirm it.

1-2. Intestinal adhesion

Intestinal epithelial HT-29 cell line with 10% fecal calf serum (Gibdo-BRL, USA) and 1% penicillin / streptomycin (10,000 U / ml, Gibdo-BRL, USA) added DMEM (Gibdo-BRL, USA) The medium was incubated at 37 ° C., 5% CO ₂ . Candidate strains of 1.0 × 10 ⁸ CFU / mL or more were inoculated into a 24-well plate on which a HT-29 cell monolayer was formed, and after 2 hours of incubation, HT-29 cells and the strain were used using 0.1% triton X 100. The number of viable cells was measured after removing. Lactobacillus rhamnosus GG strain was used as a control strain, and is shown in Table 2 below.

TABLE 2

As a result, the intestinal adhesion capacity of the CACC 537 strain was determined to be 78.6% among the isolated candidate groups, and the intestinal adhesion effect of the CACC 537 strain was Lactobacillus rhamnosus GG strain, which is known to have excellent intestinal adhesion ability among probiotic strains. It was measured to be better even when compared with.

Example 2. Validation of antimicrobial activity against livestock pathogens

In order to verify the antimicrobial activity against livestock pathogens, the antimicrobial activity was verified by the Disk diffusion method. Pathogens include Salmonella Derby NCCP 12238, Salmonella Enteritidis NCCP 14546, Salmonella Typhimurium NCCP 10328, and Escherichia Four strains of coli KCTC 2617 were used. The four pathogens were incubated at 37 ° C. for 24 hours using BHI medium (Becton, Dickinson and Company).

Strains isolated from dog feces were inoculated on MRS broth, incubated for 24 hours at 37 ° C, and 80 µl of the culture supernatant sterilized with 0.22㎛ pore size membrane filter was inoculated on 8mm paper disc to measure the antimicrobial activity. Evaluated.

TABLE 3

As a result, among the isolated candidates, the CACC 537 strain was observed to form a clear zone of 11-12 mm against the livestock pathogens Salmonella Derby NCCP 12238 and Salmonella Enteritidis NCCP 14546 strains, Salmonella Typhimurium NCCP 10328, and Escherichia Forming a clear zone of 9-10mm for the coli KCTC 2617 strain was confirmed to have a very good antibacterial activity (see Figure 3).

Example 3. Storage Stability Assessment

The CACC 537 strain was incubated at 37 ° C. for 48 hours using MRS broth, centrifuged at 13,000 rpm for 5 minutes, and only cells were collected. The collected cells were lyophilized by the addition of lactic acid bacteria probiotic powder lyophilization protector (10% skim milk, 15% trehalose). After freeze-dried cells were pulverized and powdered, 0.2g (5 x 10 ⁹ CFU / g) was packaged in one packet and stored for 4 months at 5 ℃. The number of viable cells during the storage period was measured to confirm formulation stability.

As a result, the CACC 537 strain was observed that no significant change in the number of viable cells during the storage period, it was confirmed that the strain with excellent storage stability.

Example 4 Clinical Trials on Immunostimulating Effects

Five dogs were subjected to clinical trials on the immune-promoting effects of CACC 537 strains whose probiotic activity was verified in Examples 1 and 2 with the consent of the guardian.

In the experiment, the CACC 537 strain was formulated in powder form as in Example 3, and fed 0.2 g (5 x 10 ⁹ CFU / g) once a day for 1 month, and then blood was collected before and after the microbial supplement. Was carried out.

TABLE 4

As a result, the dog's leukocyte level increased by 16.7% and platelet level increased by 8.6% through the administration of the CACC 537 strain. In addition, erythrocyte and hemoglobin levels were also increased to 4%.

Through the above results, it was confirmed that the CACC 537 strain has an effect of increasing the immune function by increasing the white blood cells that play a major role in the cellular immune response in the normal range.

Example 5 Clinical Trials on Proteinuria Inhibitory Effects

Five dogs were subjected to clinical trials on the proteinuria inhibitory effects of CACC 537 strains whose probiotic activity was verified in Examples 1 and 2 with the consent of the guardian.

First, the CACC 537 strain was formulated in powder form and fed for 1 month, and then the dog's urine was collected before and after the microbial supplement and the protein content contained in the urine was examined.

TABLE 5

As a result, it was observed that the protein content in the dog's urine decreased by 70.5% through the administration of the CACC 537 strain.

Through the above results, it was confirmed that the CACC 537 strain has an effect of effectively reducing the proteinuria of the dog.

Example 6 Liver Function Improvement Effect

Liver function may decrease due to various causes such as liver disease and autoimmune diseases. The following experiment was performed to evaluate the effect of the CACC 537 strain of the present invention on cholesterol normalization and liver function improvement.

First of all, 5 dogs with diarrhea patients were examined with blood immunochemical changes before and after CACC 537 strains whose immunostimulatory activity was verified under the parental consent.

In the experiment, the CACC 537 strain was formulated in powder form and fed to diarrhea patients for 1 month, blood was collected before and after microbial supplementation, and blood tests were performed.

TABLE 6

As a result, the cholesterol (cholesterol, CHOL) levels significantly higher before feeding was significantly reduced to a normal range due to the feeding of the CACC 537 strain, it was confirmed that the CACC 537 strain of the present invention has a high cholesterol level reducing effect. In addition, alkaline phosphatase (ALP), alanine amino-trasferase (ALT), gamma glutamyl transferase (GGT), and blood urea nitrogen / creatinine ratio (B / C) were slightly higher than normal after feeding, but the CACC 537 strain was 1 It was found to be effective in improving liver function after a month of drastic reduction. Through the above results, it was confirmed that the CACC 537 strain of the present invention has an excellent effect on improving liver function through improving the intestinal environment of the dog.

Example  7. Strain Identification

16S rRNA gene sequencing analysis was performed to identify CACC 537 strains whose probiotic activity was verified in Examples 1 to 5.

As primers, 27F and 1492R primers were used and 16S rRNA genes were amplified using a PCR instrument, and nucleotides of about 1,400bp or more were obtained using 785F and 907R primers, which are inter-primers. The obtained sequence was analyzed by 16S rRNA using 16S-based ID of Ezbiocloud (https://www.ezbiocloud.net), and showed a new 99.8% homology with P. acidilactici DSM 20284T (accession number, GL397069). It was identified as P. acidilactici strain.

The phylogenetic tree is shown in FIG. 7 based on the neighbor joining method using a Mega 7 program.

 The present inventors named the strain of the present invention having excellent probiotic activity as pediococcus ecidyl lactyl CACC 537, and deposited it on July 11, 2019, under the deposit number (KACC 81098BP) at the National Institute of Agricultural Science. .

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

[Accession number]

Depositary Name: Institute of Agricultural Biotechnology

Accession number: KACC81098BP

Deposit Date: 20190711

<110> Center for Industrialization of Agricultural and Livestock Microorganisms <120> Novel Strain of Pediococcus acidilactici CACC 537, and feed          composition using <130> PN190061AN <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1499 <212> DNA <213> Pediococcus acidilactici <400> 1 gatgaacgct ggcggcgtgc ctaatacatg caagtcgaac gaacttccgt taattgatca 60 ggacgtgctt gcactgaatg agattttaac acgaagtgag tggcggacgg gtgagtaaca 120 cgtgggtaac ctgcccagaa gcaggggata acacctggaa acagatgcta ataccgtata 180 acagagaaaa ccgcctggtt ttcttttaaa agatggctct gctatcactt ctggatggac 240 ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcgatgat gcgtagccga 300 cctgagaggg taatcggcca cattgggact gagacacggc ccagactcct acgggaggca 360 gcagtaggga atcttccaca atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag 420 aagggtttcg gctcgtaaag ctctgttgtt aaagaagaac gtgggtgaga gtaactgttc 480 acccagtgac ggtatttaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa 540 tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa agcgagcgca ggcggtcttt 600 taagtctaat gtgaaagcct tcggctcaac cgaagaagtg cattggaaac tgggagactt 660 gagtgcagaa gaggacagtg gaactccatg tgtagcggtg aaatgcgtag atatatggaa 720 gaacaccagt ggcgaaggcg gctgtctggt ctgtaactga cgctgaggct cgaaagcatg 780 ggtagcgaac aggattagat accctggtag tccatgccgt aaacgatgat tactaagtgt 840 tggagggttt ccgcccttca gtgctgcagc taacgcatta agtaatccgc ctggggagta 900 cgaccgcaag gttgaaactc aaaagaattg acgggggccc gcacaagcgg tggagcatgt 960 ggtttaattc gaagctacgc gaagaacctt accaggtctt gacatcttct gccaacctaa 1020 gagattaggc gttcccttcg gggacagaat gacaggtggt gcatggttgt cgtcagctcg 1080 tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttattact agttgccagc 1140 attcagttgg gcactctagt gagactgccg gtgacaaacc ggaggaaggt ggggacgacg 1200 tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga tggtacaacg 1260 agtcgcgaaa ccgcgaggtt tagctaatct cttaaaacca ttctcagttc ggactgtagg 1320 ctgcaactcg cctacacgaa gtcggaatcg ctagtaatcg cggatcagca tgccgcggtg 1380 aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgagagtttg taacacccaa 1440 agccggtggg gtaacctttt aggagctagc cgtctaaggt gggacagatg attagggtg 1499

Claims (10)

New strain Pediococcus ecidyl lacti CACC 537 (Accession No .: KACC 81098BP). The method of claim 1,
The strain is a new strain pediococcus ecidyl lactici CACC 537 (Accession No .: KACC 81098BP), characterized in that it has a 16s rRNA sequence of SEQ ID NO: 1. A pet food composition comprising the strain of claim 1 or a culture thereof as an active ingredient. The method of claim 3,
The composition is a pet food composition, characterized in that the acid resistance, bile resistance, and intestinal adhesion activity. The method of claim 3,
The composition is characterized in that it has antimicrobial activity to at least one livestock pathogen selected from the group consisting of Salmonella Derby, Salmonella Enteritidis, Salmonella Typhimurium, and Escherichia coli . Feed composition for pets. delete The method of claim 3,
The composition is a pet food composition, characterized in that it has an immunostimulating activity. The method of claim 3,
The composition is a pet food composition, characterized in that it has a proteinuria inhibitory activity. The method of claim 3,
The composition is a pet food composition, characterized in that it has a cholesterol reducing activity. The method of claim 3,
The composition is a pet food composition, characterized in that it has a liver function improving activity.

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