TW201016847A - A strain of Lactobacillus plantarum and its use for inhibiting Helicobacter pylori growth - Google Patents

Abstract

A strain of Lactobacillus plantarum, which is deposited at Bioresource Colletion and Research Center of Taiwan, with deposit number of BCRC910396. The strain or its metabolic product can inhibit Helicobacter pylori growth. The strain can be used for producing food or pharmaceutical combination.

Description

201016847 IX. Description of the Invention: [Technical Field] The present invention relates to a strain of lactic acid bacteria, and more particularly to a strain of Lactobacillus plantarum which inhibits the growth of Helicobacter pylori. [Prior Art] The biochemical characteristics of Helicobacter pylori (H. py 〖orj) can produce a variety of enzymes such as catalase, oxidase and urease; Urease can decompose urea to produce ammonia and carbon dioxide. Ammonia and carbon dioxide can neutralize gastric acid, making the peripheral environment of Helicobacter pylori neutral, and can survive in the stomach of the human body, causing stomach ulcer, duodenal ulcer or gastritis. The current treatment for Helicobacter pylori is the use of antibiotics, which are treated with two peptic ulcers plus two antibiotics to treat the disease caused by infection with Helicobacter pylori. However, antibiotics are prone to side effects in humans. These side effects include drug rash caused by antibiotic allergy, gastrointestinal discomfort, abnormal liver function or abnormal kidney function, and long-term use of antibiotics to inhibit the growth of Helicobacter pylori. It is resistant to drugs, and the therapeutic effect of antibiotics is reduced, and even the effect of inhibiting the growth of Helicobacter pylori is lost; and a more serious disease is caused. Traditional lactic acid bacteria can be divided into cocci and bacilli. Cocci can be divided into Lactobacillus (Indococcws, Iaciococcz/51), Candida albicans ("(3) Shame 5) and Streptococcus (Arbutus (2) cc^); Genus () and Bifidobacterium (brain). Lactic acid bacteria have the ability to inhibit the growth of harmful bacteria, stimulate the immune response of 201016847, remove peroxides and lower cholesterol. Lactobacillus can inhibit the growth of harmful bacteria caused by lactic acid bacteria, which contain organic acids, toxic anions, Hydrogen peroxide and bacteriocin. The organic acid is lactic acid and acetic acid, lactic acid and acetic acid can be used to check the acidity of the environment, thereby preventing harmful pathogens from adhering to the intestinal mucosa; the toxic anion can acidify the cytoplasm of the harmful pathogen; the hydrogen peroxide can be destroyed The protein structure of the pathogen; the bacteriocin is a small cationic protein ('cation protein), which is composed of 30 to 00 amino acid residues, and has a relatively constant isoelectric point and an amphite (amphiphinc). It can effectively inhibit the growth of Gram-positive bacteria. Lactic acid m in the genus Lactobacillus which inhibits the growth of Helicobacter pylori contains L, acidophilus, L. johnsonii, L. salivarius, and B (four); it makes 厶(10)(10) reduce the ability of Helicobacter pylori to adsorb human intestinal mucosa; Zhiliucai (10)" Slow down the symptoms of gastritis caused by Helicobacter pylori; >5α//ν<3Π·(10) can inhibit Helicobacter pylori release il_8 to prevent inflammatory cells from being attracted to Helicobacter pylori infection; z • Surface protein can inhibit the adhesion of Helicobacter pylori to gastric cancer cell. It is also known that lactic acid bacteria can be used in the production of fermented milk, which is produced by microbial fermentation of sugars to produce alcohols and organic acids, which are acetic acid, lactic acid and propionic acid; in addition, lactic acid bacteria can secrete lactase and enhance Lactose digestion, and the growth of lactic acid bacteria in the intestines, and the production of a large amount of lactic acid to inhibit the growth of harmful bacteria in the intestines, can also antagonize this harmful bacteria; moreover, lactic acid bacteria can produce antibiotics, hydrogen peroxide, etc., can also inhibit the large intestine Bacillus or Staphylococcus aureus (Yamano et al 2006). General microorganisms can only survive for 15 minutes in artificial gastric juice with a pH of 2.0, 201016847

【Abstract】 J plant #目# species inhibits the growth of bacterium

The invention has the effect of inhibiting the pyloric rod of the present invention by a Lactobacillus plantarum strain (仏(9)(4)-type material. The invention relates to a Lactobacillus plantarum The strain (1 Furuqin:) and its metabolic silk H, the plant milk (4) strain system> is selected from the kimchi, and the metabolite of the Lactobacillus plantarum strain has excellent anti-Helicobacter pylori ability, and can be widely used. The above-mentioned and other objects, features and advantages of the present invention will be more clearly understood from the following description of the present invention. The present invention has been described in detail with reference to the following drawings. The inventors have screened a strain of Lactobacillus plantarum having excellent anti-Helicobacter pylori (like PMmflrWW; accession number: BCRC910396) through extensive and intensive experiments. The screening process of Bacillus sp. 201016847 strain is as follows: 1. Screening the lactic acid bacteria strain from the sample + the first step of screening the lactic acid bacteria strain of the invention: taking the commercial milk H hiding and the newborn money as The (4) source sample port 0 is selected for 37 〇C, 12 hours of strain colony culture (Enrichment rm and screened for the production of curd samples. The second step of the Hansenjie® strain of the present invention: The sample of the curd phenomenon was subjected to e and cultured under anaerobic conditions for 37 hours to select a single colony of the transparent circle of the flute. The second step of screening the lactic acid bacteria strain of the present invention is to display the (four) The single pass was inoculated in 5 mi s medium and cultured at 37 t for 12 hours. (5) Fine = test, Gram pure and miscellaneous, negative for women, and negative for Gram stain And the appearance is rod-shaped or spherical-shaped. The fourth step of screening the lactic acid _ strain of the present L is to take the preliminary f-selected strain into the two-step method using molecular biology (four) species H the molecular organism Φ The method of the strain is to extract the DNA of the strain for the polymerase chain reaction, the PCR, the PCR, and the strain of the lactic acid strain of the present invention, and the lactic acid bacteria strain of 29G strain is isolated and the 290 strain of the lactic acid bacteria is isolated. Numbered sequentially from 丨 to 29〇, and the 290 strains of lactic acid bacteria Sources from the following table:

—8 201016847

49-122 Marinated kimchi 123~145 Soil 146-225 Commercially available lactic acid bacteria fermented milk product 226-268 Baby stool 269-281 Commercially available lactic acid bacteria powder '282-287 Raw milk 288 BCRC16000 (Lactobacillus vhamnosus) 289 Commercial product (AB. Kefir 2, lactic acid bacteria strain is screened out from 290 strains of lactic acid bacteria. The 290 lactic acid bacteria strain is subjected to an acid resistance test to screen acid-resistant lactic acid bacteria strains, and the first step of the acid resistance test is: Each of 290 strains of lactic acid bacteria was inoculated into 5 ml of MRS culture medium, and cultured at 37 ° C for 12 hours to produce a culture solution of each of 290 strains of lactic acid bacteria. The second step of the acid resistance test is: adding 190 strains of each of the 290 strains of the lactic acid bacteria strain to the 9 ml MRS culture solution of Yang 2 〇, pH 2 $ and pH 3.0, respectively, as an experimental group, and The culture solution of each of the 290 strains of lactic acid bacteria was added to the unacidified treatment.

MRS culture medium was used as the Lai group; the experimental group and the test were placed in the ^ °C culture 3, and the experimental group and the test culture were taken out and lm was applied to the MRS plate, and the culture was inhibited for 48 hours. After the calculation of the bacteria. Then, the knot of the simple simplification is consumed in the environment of the test value 3, M), and after 3 hours of treatment, the survival rate of 31 strains of lactic acid bacteria is more than 8G%, and the isolates of the 31 strains of lactic acid bacteria are separated by brittle sauerkraut. 9 strains, numbered 4, 19, 23, 32, 34, 4, 42, 43, and 44; 4 strains isolated from the enzyme kimchi, numbered on the side 201016847 82, 84, 94 and 113'· 4 strains were isolated from soil, numbered 124, 125, 126 and 128 respectively; 9 strains were isolated from infant feces, numbered 227, 228, 235, 243, 248, 249, 261 , 266 and Shi; 4 strains isolated from commercially available lactic acid bacteria powder, numbered as (10), 270, 271 and 272; and one BCRC1 surface (―__ coffee). Further, 31 strains of lactic acid bacteria were subjected to a bile salt-tolerant test to select a strain of lactic acid bacteria which is most suitable for growth in gastric acid and bile salts. ❹ ❹ 3, 31 strains of acid-tolerant lactic acid bacteria strains were selected for the strains of the lactic acid bacteria, and the 31 strains of acid-resistant lactic acid bacteria were further added to 5°. The bile salt-tolerant test system of the present invention was used to plant the lactic acid bacteria strains activated by each strain in mL MRS. In the culture medium, after culturing for 12 hours under 3η:, take the following: Add the human containing the bile salt (〇3%) to the 9th position, culture at a remote rate of 80 rpm, and measure the absorbance at _Cong The knife rate P of the obtained result was the gallbladder salt survival rate of the lactic acid bacteria strain, and the MRS culture solution containing no bile salt was used as the control group. The survival rate of the strain resistant strain of lactic acid bacteria was greater than that of listening, (4) strain milk =: '23, 41, 42, 43, 44, 82, = strain, 266, 268 said %. Material-stepping of the 16 strains of porcine strain _仃 inhibited the pyloric rod g test. 4==The plant milk, " One strain of lactic acid bacteria strain was selected to effectively inhibit the lactic acid bacteria strain of the bacterium, and the 16 strains of the lactic acid bacteria strain above the 201016847 supernatant were tested for inhibition of Helicobacter pylori. The step of inhibiting Helicobacter pylori, the preparation of the supernatant of the 16 strains of the acid-resistant and bile-tolerant salts, and the preparation of the supernatant system (four), 16 strains of milk, and then, under the generation, _xg, centrifuged again - step by step The supernatant of the 16 strains of the lactic acid bacteria was obtained without the supernatant culture solution. The second step of the inhibition of Helicobacter pylori test of the present invention is to carry out culture of Helicobacter pylori, and the present invention is selected from

The Center for Bioresource Conservation and Research of the Food Industry Research Institute, BCRC17023, and the numbers R py/on· k6 and pyI〇n· k7 obtained from patients with gastritis from Kaohsiung Medical University, respectively. Helicobacter pylori was inoculated into a culture medium containing 10% calf serum (iv) and cultured for 3 days at 37t. The Helicobacter pylori cultured for 3 days was centrifuged at 6-xg for 10 minutes, and the supernatant was removed to retain the three strains of Helicobacter pylori. The third step of the Helicobacter pylori test of the present invention is that the three strains of Helicobacter pylori are separately added to the supernatant of each lactic acid bacteria strain, and cultured at 37 t for several hours and appropriately diluted with an anaerobic dilution solution, and then taken 1 The sputum was spread on a blood medium containing sheep blood, cultured at 37 ° C for 3 days under microaerobic conditions, and the number of colonies was counted. The result of inhibiting Helicobacter pylori in the supernatant of the lactic acid bacteria strain is shown in the figure. Only a few strains of lactic acid bacteria can maintain high inhibition against Helicobacter pylori, and according to the result, the inhibition of Helicobacter pylori can be maintained. 6 strains of lactic acid bacteria, the numbers of the 6 strains of lactic acid bacteria are 4, 84, 228, 266, 268 and 288, respectively. 201016847 Further, the lactic acid bacteria strain of the sputum strain was subjected to a test for inhibiting Helicobacter pylori by the lactic acid bacteria milk fermentation supernatant, and the results are shown in the following table:

Strain number Original Helicobacter pylori Value Q〇gCFU/mL 6.61 Helicobacter pylori number after inhibition of lactic acid bacteria milk fermentation supernatant (logCFU/mL)* Helicobacter pylori number reduction value log CFU/ mLl 2.26

266 268 288 6.61 6.61 6.61 6.61 4.74 4.78 5.86 4.28 1.87 1.83 0.75 2.33

As apparent from the above table, the fermentation supernatant of the No. 84 lactic acid bacteria strain has the best inhibitory effect on Helicobacter pylori. Further, the sequence analysis and alignment of the No. 84 lactic acid bacteria strain by molecular biology method, the 16S rRNA sequence length of the No. 84 lactic acid bacteria strain of the present invention is 1329 bP ', and the nucleotide sequence thereof is as shown in SEQ ID NO: 1. And confirming the lactic acid bacteria No. 84 of the present invention and the number EU419598.1 (having 100% sequence similarity, confirming that the present invention is a Lactobacillus plantarum strain in the lactic acid bacteria species. Inhibition of Helicobacter pylori by the Lactobacillus plantarum strain of the present invention Animal test This test used male strain BABL/CByj mice, and divided the mice into five groups of A, B, C, D and E '9 mice per group, and group A was the control block of only Helicobacter pylori. Group B is first infected with Helicobacter pylori and then fed with a high dose of the Lactobacillus plantarum strain of the present invention, group C is first infected with Helicobacter pylori and then fed with a medium dose of the Lactobacillus plantarum of the present invention, and group D is infected with a pyloric rod. 201016847 菌再银食 low dose of the Lactobacillus plantarum bacterium of the present invention, Group E, fermented milk of the plant Lactobacillus strain of the present invention, and injected during the test

Dexamenthasone, to reduce the mouse immune system, let Helicobacter pylori infection mice and cause gastritis in mice, and after the sacrifice of each mouse, take the stomach and = digestive bacteria for bacterial number analysis 'to explore the health food The Lactobacillus plantarum strain of the invention and the fermentation thereof are produced to inhibit the growth of the pyloric rod g. 1. Analysis of the gg-number of the stomach in the mouse. The result of the analysis of the weight of the stomach in the stomach (4) g-number analysis is shown in Figure 2, 'By the second ®, the average value of the bacilli in group A is 45〇-CFU/mL, The average number of Helicobacter pylori bacteria in groups B, C, D, and E were 1.34, 2.04, 2.89, and 3.34 Log CFU/mL, respectively, indicating that the mice were left with the lactic acid bacteria or fermented milk of the Lactobacillus plantarum strain of the present invention. Helps to reduce the number of Helicobacter pylori in the stomach; and, in Figure 2, it is also found that the lactic acid bacteria content in the stomach of the B, C, D and E groups is greater than that of the group a, and the plant medicinal rod of the present invention can be fed sideways. The fermented milk of the gg strain or the Lactobacillus plantarum φ strain of the present invention survives gastric acid and survives in the stomach of the mouse. 2. Results of analysis of Helicobacter pylori bacteria in mice

The results of the analysis of the number of Helicobacter pylori bacteria in the mouse are shown in Fig. 3 - the ’ image shows that the average number of Helicobacter species in the A grade is higher than b and C. The average number of sputum sputum bacteria in the group' indicates that the lactic acid bacteria or fermented milk of the plant-derived plant milk strain of the present month helps to reduce the effect of the bacterium of the duodenum; and, in the third figure, the small gas is also found. The lactic acid bacteria content of the duodenum, the average number of bacteria in the sputum, C, D and sputum groups are all greater than A Μ 'can be bribed. The plant buds of the invention are 植 或 本 本 本 本 或 或 或 或 — — — — gg gg gg gg gg gg gg gg gg gg gg It can resist the bile salt domain and live in the stomach of mice. - + As described above, compared with the conventional use of antibiotics for the treatment of infection of the pyloric rod S, it is easy to cause side effects and the disadvantages of the antibiotic resistance of the antibiotics; the present invention is a plant beneficial to the living body. Lactobacillus strain (lactic acid bacteria) inhibits the growth of Helicobacter pylori or treats a condition caused by infection with Helicobacter pylori, which includes duodenal ulcer, gastric ulceration, and stomach (4) 'The vegetable milk (4) (milk _) metabolite not only stimulates The biological immune response, the elimination of peroxides and the reduction of cholesterol do not cause side effects of organisms or resistance to Helicobacter pylori; therefore, the metabolites of the Lactobacillus strains of the present invention not only have excellent anti-Helicobacter pylori months. b force 'can also be widely used in the processing of food compositions or pharmaceutical compositions' to produce a health food or medicine that can inhibit the pyloric rod g, which can be a dairy product such as fermented milk. Although the present invention has been made in accordance with the present invention, any skilled person skilled in the art can make various changes and modifications without departing from the scope of the present invention. The money _ is attached to the ΐ 专 专 专 专 专 。 。 。 。 。. —14 — 201016847 [Simple description of the diagram] Figure 1: Lactobacillus plantarum strains inhibit the inhibition range of the sand/ow BCRC17023, //. pj/orz· k6 and E py/on· k7, respectively. Fig. 2 is a histogram of the number of Lactobacillus plantarum and the number of Helicobacter pylori in the stomach of mice. Figure 3: Bar graph of the number of Lactobacillus plantarum and the number of Helicobacter pylori in the duodenum of mice. ❹ [Main component symbol description] (none) —15 —

❹ 201016847 Sequence Listing <110〉National Pingtung University of Science &Technology<120〉Use of a plant Lactobacillus strain and a p-type Helicobacter pylori growth <160〉 1 <170> Patentln version 3.5

<210> 1 <211> 1329 <212> DNA <213> Lactobacillus plantarum <221> rRNA <222>0)..(1329) <400> 1 gcagtcgaac gaactctggt attgatt^t gcttgcatca tgatttacat ttgagtgagt ^ cgaact ^ tgagtaacac gtgggaaacc tgcccagaag cgg ^ gataa cacct ^ aaa cagatgctaa taccgcataa caactt ^ ac cgcatggtcc gagtttgaaa gat ^ cttcg gctatcactt ttggat ^ tc ccgcggcgta ttagctagat ggtgg ^ taa cggctcacca t ^ caatgat acgtagccga cctgagags taatc ^ cca catt ^ act gagacacggc ccaaactcct acgggaggca gcagta ^ a atcttccaca at ^ acgaaa gtctgatgga gcaacgccgc gtgagtgaag aa ^ gtttcg gctcgtaaaa ctctgttgtt aaagaagaac atatctgaga gtaactgttc a ^ tattgac ^ tatttaac cagaaagcca c ^ ctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgttg tcc ^ attta tt ^ cgtaa agcgagcgca ggcggttttt taagtctgat gtgaaagcct tcggctcaac cgaagaagtg 60 120 180 240 300 360 420 480 540 600 201016847 Catc^aaac t^gaaactt gagtgcagaa gaggacagtg gaactccatg tgtagc^tg 660 aaatgcgtag atatat^aa gaacaccagt ggcgaa^cg gctgtctggt ctgtaactga 720 cgctgaggct cgaaagtatg ggtagcaaac aggattagat acc ctggtag tccataccgt 780 aaacgatgaa tgctaagtgt t ^ agggttt ccgcccttca gtgctgcagc taacgcatta 840 agcattccgc ctg ^ agta c ^ ccgcaag gctgaaactc aaa ^ aattg ac ^ ggccc 900 gcacaagcgg t ^ agcatgt ^ tttaattc gaagctacgc gaagaacctt acca ^ tctt 960 ❹ gacatactat gcaaatctaa gagattagac gttcccttcg gggacatgga tacaggtggt 1020 gcatggttgt cgtcagctcg tgtcgtgaga tgtt ^ gtta agtcccgcaa cgagcgcaac 1080 ccttattatc agttgccagc attaagttgg gcactctggt gagactgccg gtgacaaacc 1140 ^ aggaaggt ^^ atgacg tcaaatcatc atgcccctta tgacct ^ c tacacacgtg 1200 ctacaat ^ a tggtacaacg agttgcgaac tcgcgagagt aagctaatct cttaaagcca 1260 ttctcagttc ^ attgtagg ctgcaactcg cctacatgaa gtcggaatc ^ ctagtaatcg 1320 c ^ atcagc 1329 -17 -

Claims (1)

Lu Wei 201016847 X. Applying for a patented version · 1 , 7 A strain of isolated Lactobacillus plantarum [- ‘ is deposited in the Republic of China Food Industry Development Research Institute'. The registration number is BCRC910396. A composition for the growth of Helicobacter pylori comprising the Lactobacillus plantarum strain or the metabolite thereof as described in claim 1 of the patent application (4). And a composition for treating a Helicobacter pylori infection, which comprises the plant milk bud (four) strain or the metabolite thereof according to the above-mentioned patent scope. A composition for treating duodenal ulcer, gastric ulcer and gastritis, such as the Lactobacillus plantarum strain or its metabolite as described in claim j. 5. A composition according to claim 2, wherein the composition is a food composition or a pharmaceutical composition. 6. The composition of claim 3, wherein the composition is a composition or a pharmaceutical composition. 7. The composition according to claim 4, wherein the composition is a food composition or a pharmaceutical composition, <8, the composition according to claim 5, 6 or 7, wherein The food composition is fermented milk. X 9. The Lactobacillus plantarum strain according to the scope of the patent application, which is an acid-resistant strain having an acid resistance of pH 3.0. 10. The plant mast g_, a strain resistant to bile salts, according to item i of the patent application scope. —18 —