KR20120060252A - Novel Lactobacillus salivarius FC113 and probiotics containing the same - Google Patents

Abstract

PURPOSE: A novel Lactobacillus salivarius FC113 with immunity and phytase is provided to ensure excellent resistance and immunity against diseases. CONSTITUTION: A Lactobacillus salivarius FC113(KCCM 11087P) is gram-positive bacillus and has a sequence of sequence number 1. The strain is isolated from chicken feces. The probiotic contains the strain. A composition for feeding contains the strain. The probiotics is manufactured in a solid and liquid phase.

Description

New Lactobacillus salivarius FC113 and probiotics using the same

The present invention is a novel Lactobacillus salivarius ( Lactobacillus) salivarius ) FC113 strain and probiotics using the same, more specifically, Lactobacillus excellent in immune activity and phytate production from chicken feces It is about salivarius FC113.

The livestock industry has continued to use antibiotics as growth promoters to prevent livestock disease and increase livestock productivity. However, due to the abuse of these antibiotics, antibiotics remain in the livestock products and transfer to the human body, resulting in diseases such as allergies, and also increase the body's resistance to reduce the therapeutic effect of the drug when the disease occurs. There is a problem. In Korea, the range has been greatly reduced to 25 items for antibiotics in 2005, and the European Union (EU) has taken even more intense measures. The situation is forbidding the addition. Therefore, as a substitute for these, many studies are being conducted on natural products, and probiotics are a representative example.

Probiotics are considered to be microorganisms that can promote growth, increase feed utilization, prevent intestinal fermentation or diarrhea, and eliminate nutrient inhibitors. The bacteria used for probiotics are resident bacteria commonly found in the intestines of healthy people, mainly composed of Lactobacillus and Bifidus. The necessary properties as probiotic probiotics include safety, functional aspects (survivability, fixation, formatting, antimicrobial production ability, immune stimulating ability, antigenetic activity, inhibitory of pathogenic bacteria) and technical aspects (functional properties, stability, bacteriophage) Resistance, viability during manufacture), and GRAS (Genearlly Recognized As Safe) microorganisms. Probiotics have the opposite characteristics of antibiotics. If antibiotics are metabolites produced by microorganisms and they inhibit or kill the development of other microorganisms in small amounts, probiotics promote immune function by utilizing the symbiosis and coexistence of bacteria. It has been shown to have the effect of inhibiting the growth of harmful microorganisms, and thus functional substances and probiotics having such immune enhancing functions can be used as substitutes for antibiotics, which are currently becoming social problems due to misuse and abuse. There are also cases where lactic acid bacteria-related probiotics have been used directly in chickens [ http://www.chosun.com/national/news/200503/200503070014.html ]. The reported case relates to a method of treating Lactobacillus kimchii by feeding a culture medium filtered only lactic acid bacteria to chickens infected with viral respiratory disease. Korean Patent Registration No. 10-0691413 describes antimicrobial probiotics for swine using the new strain Lactobacillus Permanent IB 261 (KCTC 18107P) isolated from the human body, and Korean Patent Publication No. 10-2010-0081778 describes Bacillus. Lactobacillus sp. KU4 (KCCM10975P) has been described for lactic acid bacteria probiotics for food and feed that inhibit the intestinal epithelial cell adhesion of Escherichia coli O157: H7 bacteria. As such, many studies have been made to utilize probiotics in the livestock industry to prevent infectious diseases of animals and to boost the immune system without the use of antibiotics.

Phytase is an enzyme that breaks down phytic acid to produce phosphate, phosphate inositol and inositol. Phitsic acid accounts for 50-70% of the grains used for livestock feed, and they are indigestible, reducing the efficiency of feed, and discharging them as feces, acting as a serious environmental pollutant. It is a factor causing. Therefore, in order to solve this problem, a method of directly feeding phytase to livestock has been developed, and many studies have been made to reduce environmental pollution by reducing economic benefits and phosphorus emission by increasing feed efficiency. To date, studies of phytates in Europe [Ehrlich, KC, Montalbano, BG, Mullaney, EJ, Dischinger, HC Jr., and Ullah, AH (1993) Identification and cloning of a second phytase gene (phyB) from Aspergillus niger ( ficuum ). Microbiology 148, 2097-2109; Piddington, CS, Houston, CS, Paloheimo, M., Cantrell, M., Miettinen-Oinonen, A., Nevalainen, H., and Rambosek, J. (1993) The cloning and sequencing of the genes encoding phytase (phy) and pH 2.5-optimum acid phosphatase (aph) from Aspergillus niger var. awamori . Gene 133, 55-62; Ullah, AHJ and Phillippy, BQ (1994) Substrate selectivity in Aspergillus ficuum phytase and acid phosphatases using myo-inositol phosphates. J. Agri. Food Chem. 43, 423-425; Urbano, G., Lopez-Jurado, M., Aranda, P., Vidal-Valverde1, C., Tenorio, E., and Porres, J. (2000) The role of phytic acid in legumes: Antinutrient or beneficial function? J. Physiol. Biochem. 56, 1138-7548; Zamudio, M., Gonza´lez, A., and Medina, JA (2001) Lactobacillus plantarum phytase activity is due to non-specific acid phosphatase. Lett. Appl. Microbiol. 32, 181-184], and studies on the effect of phytase on animals [Young, LG, Leunissen, M., and Atkinson, JL (1993) Addition of microbial phytase to diets of young pigs. J. Anim. Sci. 71, 2147-150; Lei, XG, Ku, PK, Miller, ER, Yokoyama, MT, and Ullrey, DE (1994) Calcium level affects the efficacy of supplemental microbial phytase in corn-soybean meal diets of weanling pigs. J. Anim. Sci. 72, 139-143; Mroz, Z., Jongbloed, AW, and Kemme, PA (1994) Apparent digestibility and retention of nutrients bound to phytate complexes as influenced by microbial phytase and feeding regimen in pigs. J. Anim. Sci. 72, 126-132, and the like. However, phytates developed to date show limitations due to economic problems such as enzyme titer and production cost.

The present invention has been made in view of the above points, and an object of the present invention is to provide a lactic acid bacterium excellent in immune function and phytase enzyme activity isolated from chicken feces and probiotics using the lactic acid bacteria.

The object of the present invention is to maximize the feed efficiency by screening lactic acid bacteria with excellent immunity and phytase enzyme activity from chicken feces and verifying their probiotic properties, and through the development of disease-resistant probiotic strains of livestock farms Achieved by demonstrating disease resistance and economics.

The present invention maximizes feed efficiency by providing a microorganism having excellent immune function and phytase enzyme activity, and exhibits excellent resistance and immunity to diseases, thereby providing a cost saving effect and disease prevention effect. In addition, environmentally, there is an excellent effect to significantly remove the environmental pollution factors of livestock manure.

1 shows L. salivarius isolated from chicken feces in artificial gastric juice Survival of FC113 is shown. ○ is the control group without artificial gastric juice and ● is the gastric juice treatment (pH 2.5).
Figure 2 L. salivarius isolated from chicken feces treated with artificial bile acid after artificial gastric juice (pH 2.5) treatment Survival of FC113 is shown. ○ is the control group without artificial bile acid after artificial gastric juice treatment, and ● is the one with artificial gastric juice (pH 2.5) and artificial bile acid.

The present invention is a novel Lactobacillus excellent in immune activity and phytase production ability isolated from chicken feces salivarius FC113 and probiotics using the same. In addition, the present invention is a novel Lactobacillus Provided is a feed composition comprising salivarius FC113.

The probiotics of the present invention can be prepared into the final product by adding and blending various ingredients and flavorings in order to improve the taste or to make the required shape according to conventional methods. As a component which can be added and mix | blended with the probiotics of this invention, various sugars, an emulsifier, a sweetener, an acidulant, fruit juice, a fragrance, etc. are mentioned. Specifically, glucose, sucrose, fructose, fructose, sugar such as beeswax, sucrose fatty acid ester, glycerin fatty acid ester, emulsifier such as lecithin, sorbitol, xylitol, lecithin ( lecithin), sweeteners such as lactitol, citric acid, acetic acid, tartaric acid, lactic acid, fumaric acid, malic acid, succinic acid, acidulants such as gluconodelta-lactone, yogurt, berry, orange, quince tree, tea plant, Spices such as citrus, tortilla, mint, grape, fair, custard cream, peach, melon, banana, tropical fruit juice, tea, coffee, herbal extract, plant extract, grains, vegetable ingredients, milk ingredients , Vitamins A, B vitamins, vitamin C, vitamin E and the like, etc. There may be used one or two or more combination components selected from the group.

In addition, the probiotics of this invention can be a product of any form, such as a solid and a liquid.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the invention in more detail, the embodiments according to the invention can be modified in many different forms, the scope of the invention is construed as limited to the embodiments described below It should not be lost.

Example 1. Isolation and Identification of Strains

To isolate acid-resistant strains from chicken feces, aseptically add chicken feces to a dilution adjusted to pH 3.5 and shake for 2 hours, then smear using LBS Agar (BBL, Cockeysville, USA) as a selective medium. Then, the cultures were selected after incubating at 24-48 hours at 37 ° C. Acid-resistant Lactobacillus spp. Was isolated from chicken feces, and their phytate activity was confirmed.

The phytase enzyme activity was measured by Shimizu (1992) (Shimizu, M., 1992, Purification and characterization of phytase from Bacillus). subtilis . Biosci. Biotech. Biochem. 56, 1266-1269) was determined by measuring the inorganic phosphate dissociated from sodium phytate (Sigma, USA). 100 μL cell suspension and 900 μL of 2 mM substrate (lysed in acetate buffer (pH 4.5)) were mixed and reacted at 50 ° C. for 10 minutes. 1 mL of 10% trichloroacetic acid was added to stop the reaction and developed for 10 minutes. The coloring agent was used by mixing 1.5% ammonium molybdate (soluble in 5.5% sulfuric acid) and 2.7% ferrous sulfate in a ratio of 4: 1. The amount of dissociated phosphoric acid was determined by measuring the absorbance at 700 nm. As a standard curve, monopotassium phosphate (monopotassium phosphate) was obtained using Samchun Chemical, Seoul, Korea. One unit of phytase was defined as the amount of enzyme required to dissociate 1 μmol at 50 ° C.

Among the isolates, FC113 showed the highest activity with 403.6 U. Pediococcus pentosaceus CFR R38 and CFR R35 produced 89 and 213 U of phytase, respectively, when incubated at 50 ° C (Ragharvendra and Halami, 2009). Lactobacillus rhamnosus GG ATCC 53510 and Lactobacillus amylovorus B4552 have been reported to produce 15 and 8 U at lower values, respectively. Therefore, it was confirmed that the FC113 strain produced significantly higher phytate than the existing strains.

Example  2. Identification of Strains

For the identification of the strain, FC113 lactic acid bacteria were identified through microscopy, Gram staining and carbon source availability (API 50CHL kit (Biomeriuex, Lyon, France), 16S rRNA sequencing (Macrogen Inc., Seoul, Korea), a molecular biological method.

Lactobacillus FC113 strain was Gram-positive, bacillus. Lactobacillus as a result of checking carbon availability salivarius and 98.7%. In addition, 16S rRNA sequencing results (SEQ ID NO: 1), Lactobacillus with a significance of 99.0% It was identified as salivarius . Based on these results, the FC113 strain Lactobacillus salivarius It was named FC113. FC 113 strain was deposited with the Korea Microorganism Conservation Center (KCCM) (Accession Number: KCCM 11087P).

Example 3. Strains and Medium

FC113 lactic acid bacteria used in the present invention was activated by passage 3-3 times, preserved at -70 ℃ by glycerol stock method, working culture was used by subculture once a month. Culture medium of FC113 lactic acid bacteria was used lactobacilli MRS medium (MRS; Difco Laboratories, Detroit, USA). FC113 lactobacillus was inoculated in 10 mL of MRS culture and pre-cultured at 37 ° C. for 12 hours, followed by inoculation into MRS culture in a 500 mL flask (working volume: 300 mL, 1% inoculation ratio) for 12 hours. Incubated.

Example 4. Cell line culture

Cell lines Raw 264.7 (KCLB 40071) and Caco-2 (KCLB 30037.1) were purchased from Korea Cell Line Bank (Seoul, Korea). Raw 264.7 and Caco-2 cells were cultured in 10% fetal calf serum and 1% streptomycin in DMEM (Dulbecco's Modified Eagle Medium; Gibco Laboratories, Grand Island, NY, USA) and MEM (Minimum Essential Medium; Gibco Laboratories), respectively. Penicillin was added and used. These were incubated at 37 ° C. with a composition of 5% CO ₂ /95% air. Raw 264.7 cells, macrophages, were used to confirm nitrous acid (NO), IL-1α and TNF-α production, and Caco-2 cells, colon cancer cells, were used to confirm bowel fixation.

Example 5. Resistance to artificial gastric juice and artificial bile

Artificial gastric fluid was used by adding 1% pepsin to the culture medium adjusted to pH 2.5 and pH 4.0 using 5 N HCl according to the method of Kobayashi et al. (Kobayashi, Y .; K.Tohyama; T.Terashima, 1974 Tolerance of the multiple antibiotic resistant strains, L. casei PSR 3002, to artificial digestive fluids.Jpn. J. Microbiol. 29, 691-697). To test the resistance of L. salivarius FC113 to artificial gastric juice, 1 mL of 12 hours culture was added to 9 mL of artificial gastric juice in a test tube and cultured at intervals of 0, 1 and 2 hours using MRS agar plates. The total bacterial count was measured. The control was experimented using the same method as above for cultures without pH adjustment and without the addition of pepsin. The number of bacteria was continuously diluted 10 times with 0.1% peptone water, and then dispensed by dispensing 0.1 mL in plate medium and incubated at optimal temperature, and the total number of bacteria was calculated by measuring colony forming units. .

Artificial bile acids were used by adding sterile 0.3% bull's bile (oxgall) (Difco, USA) to the sterilized culture. In order to test the resistance of artificial bile acid, the culture medium subjected to artificial gastric juice was added to a test tube containing artificial bile acid, and cultured at 37 ° C. for 24 hours, and then the total bacterial count was measured to examine viability. The control was tested using the same method as above for the culture medium without oxgall. The number of bacteria was continuously diluted 10 times with 0.1% peptone water, and then dispensed by dispensing 0.1 mL in plate medium and incubated at optimal temperature, and the total colony count was calculated by measuring colony forming units.

As shown in FIGS. 1 and 2, L. salivarius FC113 was found to be very stable (96.4%) for artificial gastric juice (pH 2.5), but stable for 8 hours for artificial bile acids, but decreased 2 Log CFU / mL at 24 hours.

Example 6. Enzyme Productivity

In order to measure the production of various enzymes, the bacterial culture was centrifuged to recover only the cells, washed twice with sterile water, and then suspended in sterile water to have a concentration of about 10 ⁶ CFU / mL. The suspension was inoculated in API ZYM kit (bioMerieux Co., France) and incubated for 4 hours at 37 ° C. in the dark. ZYM A and B reagents were dropped one drop into each chapel to increase expression activity and dissolution, and the enzyme activity was measured by observing the change of color after reacting for about 5 minutes in a bright place.

L. salivarius FC113 did not produce β-glucuronidase, an enzyme that causes cancer, and α-galactosidase and β-galactosidase (β- galactosidase) production was confirmed. This may also help with lactose intolerance.

L. salivarius isolated from chicken feces measured by API Zym kit Enzyme Activity of FC113 enzyme Enzyme activity enzyme Enzyme activity contrast 0 Acid phospatase 2 Alkaline phosphatase 0 Naphtol-AS-BI-phosphohydrolase 3 Esterase One α-Galactosidase 2 Esterase Lipase One β-Galactosidase One Lipase 0 β-Glucuronidase 0 Leucine arylamidase 3 α-Glucosidase 0 Valine arylamidase One β-Glucosidase 0 Crystine arylamidase One N-Acetyl-β-glucosaminidase One Trypsin One α-Mannosidase 0 α-Chymotrypsin 0 α-Fucosidase One

0, 0 nmol; 1, 5 nmol; 2, 10 nmol; 3, 20 nmol; 4, 30 nmol; 5, ≧ 40 nmol.

Example 7. Resistance to antibiotics

The paper disc method was used to measure antibiotic resistance. That is, incubated for 12 hours at the optimum temperature of the bacteria, inoculated with 100 μL of the culture solution in MRS soft agar (0.75% agar) was overlayed on agar plate. The paper plate was placed on the overlay plate with sterile tweezers, and antibiotic resistance was measured by dropping 10 μL of antibiotics at each concentration onto the paper disc and incubating for 12 hours. The results are shown in Table 2.

L. salivarius Resistance to antibiotics of FC113 Antibiotic (µg / mL) FC113 Antibiotic (µg / mL) FC113 Nisin 10
20
100
200 + ¹⁾
+
+
+ Gentamycin 10
20
100
200 +
+
+
+ Streptomycin 10
20
100
200 +
+
+
+ Rifampicin 10
20
100
200 -
-
-
- Neomycin 10
20
100
200 +
+
+
+ Erythromycin 10
20
100
200 +
-
-
- Roxithromycin 10
20
100
200 +
+
-
- Ciprofloxacin 10
20
100
200 +
+
+
- Chloramphenicol 10
20
100
200 -
-
-
- Ampicillin 10
20
100
200 +
-
-
-

¹⁾ +, growth; -Not growing.

Example 8. Immune Boosting Activity

In order to measure the immune enhancing activity of L. salivarius FC113, production of NO, IL-1α, TNF-α was confirmed. Raw 264.7 cells were dispensed in 96 × well plates at 2 × 10 ⁴ concentrations and incubated for 24 hours at 37 ° C. and 5% CO ₂ /95% air. L. salivarius FC113 strain was heat-treated to obtain the dead bacteria, the bacterial concentration was dissolved in PBS at a concentration of 1 × 10 ⁶ , 1 × 10 ⁷ , 1 × 10 ⁸ CFU / mL inoculated in the wells and incubated for 48 hours. The supernatant of the culture solution was collected to confirm the production of NO, IL-1α and TNF-α.

NO production was confirmed by adding Griess reagent (Fluka, Germany), reacting for 15 minutes, and measuring absorbance at 540 nm. To analyze IL-1α and TNF-α, Koma Biotech Inc. ELISA kit sold by (Korea) was used, and the production capacity was examined according to the manual.

When 10 ⁷ CFU / mL was added, production of 12.51 ± 0.90 μM NO was confirmed, and production of 175.5 ± 36.40 pg / mL of IL-1α and 353.5 ± 62.479 pg / mL of TNF-α was confirmed.

L. salivarius isolated from chicken feces Immunostimulatory Activity of FC113 Cell concentration NO (μM) IL-1α (pg / mL) TNF-α (pg / mL) 10 ⁶ 0.35 ± 1.56 136.4 ± 7.66 153.3 ± 17.64 10 ⁷ 12.51 ± 0.90 175.5 ± 36.40 353.5 ± 61.79 10 ⁸ 33.88 ± 15.99 113.4 ± 24.84 308.4 ± 57.91

Korea Microbial Conservation Center KCCM11087 20100730

<110> Konkuk University Industrial Cooperation Corp <120> Novel Lactobacillus salivarius FC113 and probiotics containing          the same <130> YEIL0001 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1652 <212> DNA <213> Lactobacillus salivarius FC113 <400> 1 caatcatctg tcccacttag acggctggct ccttgcggtt accccaccgg ctttgggtgt 60 tacaaactct catggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcga 120 catgctgatt cgcgattact agcgattccg acttcatgta ggcgagttgc agcctacaat 180 ccgaactgag aacggcttta agagattagc taaacctcgc ggtcttgcga ctcgttgtac 240 cgtccattgt agcacgtgtg tagcccaggt cataaggggc atgatgactt gacgtcgtcc 300 ccaccttcct ccggtttgtc accggcagtc tcgccagagt gcccaactta atgctggcaa 360 ctgacaacaa gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga 420 cgacagccat gcaccacctg tcactttgtc cccgaaggga aagcctaatc tcttaggtgg 480 tcaaaggatg tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc 540 caccgcttgt gcgggccccc gtcaattcct ttgagtttca accttgcggt cgtactcccc 600 aggcggaatg cttattgcgt tagctgcggc actgaagggc ggaaaccctc caacacctag 660 cattcatcgt ttacggcgtg gactaccagg gtatctaatc ctgtttgcta cccacgcttt 720 cgaacctcag cgtcagttac agaccagaga gccgctttcg ccactggtgt tcttccatat 780 atctacgcat ttcaccgcta cacatggagt tccactctcc tcttctgcac tcaagtcttc 840 cagtttccaa tgcactactc cggttaagcc gaaggctttc acatcagact taaaagaccg 900 cctgcgttcc ctttacgccc aataaatccg gacaacgctt gccacctacg tattaccgcg 960 gctgctggca cgtagttagc cgtgacttgc tggttagata ccgtcatcga atgaacagtt 1020 actctcactc gtgttcttct ctaacaacag agttttacga tccgaagacc ttcttcactc 1080 acgcggcgtt gctccatcag acttgcgtcc attgtggaag attccctact gctgcctccc 1140 gtaggagttt gggccgtgtc tcagtcccaa tgtggccgat caacctctca gttcggctac 1200 gtatcatcac cttggtaggc cgttacccca ccaactagtt aatacgccgc gggtccatct 1260 aaaagcgata gcagaaccat ctttcatcta aggatcatgc gatccttaga gatatacggt 1320 attagcacct gtttccaagt gttatcccct tcttttaggc aggttaccca cgtgttactc 1380 acccgtccgc cactcaactt cttacggtga atgcaagcat tcggtgtaag aaagtttcgt 1440 tcgacttgca tgtattaggc acgccgccag cgttcgtcct gagcagtaaa aaaaattcta 1500 taanannccc gaaaaacatg aaaggtcgga tcttcttagc ggtttcctca cccgattttt 1560 gcgggtctta aaacagaaaa gcctgtcacc cgggggagtc tacctcacgg ttataaaaat 1620 acaacgaact gtgccggtgt ccgcacaaaa aa 1652

Claims (3)

The rods represent the positive Gram stain when raised Lactobacillus having the sequence of SEQ ID NO: 1 bariwooseu (Lactobacillus salivarius ) FC113 (KCCM 11087P). Lactobacillus of claim 1 salivarius ) probiotics including FC113 (KCCM 11087P). Lactobacillus of claim 1 salivarius ) Feed composition comprising FC113 (KCCM 11087P).

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