TWI316545B - Novel lactobacillus rhamnosus strain lv108 and uses thereof - Google Patents

Description

1316545 IX. Description of the invention: [Technical field to which the invention pertains] The present invention belongs to the field of microbiology and relates to the use of a new type of refractory rat glycoside product.冈久代代 [Prior Art] Lactic acid bacteria are - fermentable carbohydrates, and mainly produce lactic acid. The lactic acid bacteria have been extensively studied internationally, and lactic acid bacteria should be used, often 'lactic acid _ containing A amount of lysine (4) Official society skin cells ^ and force. Thus, from the external environment, the milk _ is in the body ^ i: falling its wall. And the 'daily side' is in this type of milk (4) genus that exists on epithelial cells, and the survival characteristics of these epithelial cells bring health effects to the injured. Lactic acid bacteria (with 2):: (1) Forming a dominant flora on the cell surface 'Production of pathogenic microorganisms such as secretion of anti-substance; (3) g strain contributes to digestive activity, (4) produces anti-cancer activity, and the research findings The probiotics for humans are preferably derived from the human body. Force, but when they are taken as food, there is adhesion, so they do not reach the intestine through the stomach. They are almost all killed by stomach acid. Bile This is an urgent need to solve the problems of the application and efficacy of lactic acid bacteria. Due to the normal function of the body, delaying the body's health, and promoting the machine [Invention] The lactic acid g from the sputum source has a more powerful function. Strain, repertoire; species" new human Lactobacillus rhamnosus strains are resistant to disease and can resist acid, bile, and radiation (such as Helicobacter pylori and Escherichia coli) It also has anti-1316545 oxidation function. The use of the substance (4) - Mosquito provides a nucleus-containing squid _ and / or its metabolite resources: the use of two · r r r 味 味 的 ' ' ' 提供 提供 提供 提供 提供 提供/ or i metabolite use for the preparation of inhibition of pyloric snails? ι, foods and/or drugs of metabolites.疋 ® 大 大 大 大 大 大 大 大 大 大 大 大 大 大 大 大 大 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷 囷Stomach _, + lij ίt Difficult digestive tract diseases include: gastritis, sputum - colitis, enteritis, colitis or proctitis. The fourth aspect of the invention provides the use of the rhamnoside and/or 1 metabolite for the preparation of antioxidant foods and/or drugs. I, the substitute (such as the ^ ^ ^ ^ ^ provided - the food composition ^ contains an effective amount of this or 1 〇 2 - 1 〇 l2cfu / g, preferably such as i 〇 5 - i 〇 i 〇 cfu /g or Lime milk f-/'*Λ preferably as described in 1〇6-1〇9cfu/g or i〇s-i〇9cfu/mi) of the mouse metabolites, and a small amount of food can be used a. Solution ί ί , the composition is selected from the group consisting of _, dairy, gas, powdered products, or suspension products. (For example, in the six aspects of t2, provide the same as the compound], which contains an effective amount of 1〇5~l〇\f^ U mL or 1〇2—1〇〗 2cfU/g, preferably such as 1〇5-1 In a preferred embodiment of 〇l°cfu/g or bacilli and generations such as 1Q6_1G9cfu/g or (10)-caffeine® rhamnose latex #3, 3, the pharmaceutical composition dosage form is selected from the group consisting of granules and seismographs. Agent, powder, oral solution, suspension 'or emulsion'. The formulation of the drug or food composition described in the preferred embodiment is as follows: 3 to 10 l2 cfu/mL (or g) of Lactobacillus rhamnosus and/or its metabolite; 1316545 and a small amount of food or pharmaceutically acceptable The carrier used. Other aspects of the invention will be apparent to those skilled in the art from this disclosure. [Embodiment] After extensive and intensive research and experiments, the inventors finally found a strain of Lactobacillus rhamnosus having different antibacterial properties (registered number: bcrc face (6). The strain was named "rat" Lactobacillus licheniformis _,,. A variety of tests have shown that the = and: mLvi08 have the ability to resist harmful bacteria such as Helicobacter pylori and Escherichia coli

Lactobacillus licheniformis, "L. rhamnosus strain surface. The rhamnophoid larvae of Lactobacillus rhamnosus is the rhamnosperm gg strain (4) 8 of the present invention, which is passed through Xinjiang and:: specific medium and (10) long-term, initial; === acid bacteria selection force τ species Advantages of lactobacilli; (4) from preliminary confirmation = comparison of our processing and growth characteristics, (d) superior sorrel, bile acid salt capacity, excellent resistance to sputum, sputum characteristics, acid resistance and processing, functional foods Production and promotion of ά 攸 攸 can be widely used in dairy products. The present invention uses the following:; = drug development. Biochemical characteristics are analyzed; (2) Let: (1) Analyze the present invention using physiological and over-resolved sequence analysis: (3) Compare and confirm that the fine-dry strain is subjected to (TM) to obtain the result. The iterative AADD polymerase-bonded reaction with a standard strain is Lactobacillus rhamnosus. ° Fruit, sweet out of the lactobacillus Specifically, using ΑΡί κ arc vinegar fermentation, using a specific program analysis, the 1316545 real accuracy belongs to rhamnose lactate®. By using the 16S ship sequence BLAST analysis, the strain was confirmed to be rhamnose = bacteria with the above accuracy. Furthermore, when PCR was performed using a primer specific for Lactobacillus rhamnosus, about 300b segments were found in both the strain of the present invention and the proven Lactobacillus rhamnosus], which proves that the strain of the present invention belongs to Lactobacillus rhamnosus. On the other hand, when comparing the results of desensitization sequencing, there are some differences in the Lactobacillus rhamnosus MCRF-412 and MCRF-271 strains of the present invention. ^When using "Ganggang special L73R and (10) to find the enzyme-bonded reaction", the fragment at 448 bp is common in the test strain and other (10) milk rods, while the other bands are not. However, when PCR was performed on the (R) Lactobacillus rhamnosus-specific Rhal and Rha2 primers, it was found in the strain that about = primer was found to have a monthly difference from other strains at _. Therefore, using Rhal and Rha2, as well as the 〇pG4 primer, the present strain = strain can be identified, thereby demonstrating that the strain has its own specific characteristics. ', you are the safety of the fine-grained strain, choose to use ICM, mouse as an experimental animal. In the first implementation of the present invention, _ _ _ 丨, take healthy small (four) only, male and female, weight; ^,: 砚 * day # move normal, eating condition is good, fasting 12 hours after the experiment, weighing , points, according to 0. 4mL / l 〇g filling the sample, 2 times / day, after feeding, that is, the animal's mental separation, Mao Chang H, spoon, Hongtian Guyang 7 days '2 months after the detection of its safety Sexuality, the organ was found to maintain positive H, and the digestive organs including the gastric side and the small intestine were similar to those of the control group. The invention (4) milking (4) also has acid resistance and _ ability. For the purpose of bis, bile ability, the present inventors used a mixed acid to adjust the MRS medium to AW medium, PH 3. ° MRS medium, pH 4.0 MRS medium, at pH 2 · 0 2 〇% ' 3 · 〇 % ' 4 ·〇%', _ This callus strain can be placed in ρΗ2, 3.0% bile acid for 5 hours, indicating that it can have strong resistance to acid and bile salts. 1316545 has the ability to detect strains, _ ability, and the strength of the invention. In vitro drilling test for cells. The competitive adhesion inhibition of the strains of ===-0-2 bacteria was measured, and compared with the pathogenic large intestine rods. It was confirmed by experiments that the strain has intestinal deprivation ability. The strain of the bacterium was put on the surface of the 3rci vol.

Come, with the Meek standard turbidity tube for the turbid surface to elute 10 CfymL. Pipette 0. 5mL in the corresponding solid culture to make the K degree control in the aseptic operating table through the sterile air, soil, with a sterile applicator stick, after punching, the diameter of the hole 4 coffee 'fixed on the surface hole of the plate , can not overflow the hole, in the appropriate difference, δ. The positive/next degree of the acid bacteria bacteria solution is fully used. Single layer nutrition

The isolated strain of acid bacteria inhibited Escherichia coli, and it was found that ICR mice, male and female, and Bacillus strains have antioxidant capacity. Using the positive and positive control group VE, for 8 consecutive days, the culture of the gavage strain (about lxlD9Cfu/g) was tested for SOD activity and 1 hour after the MDA containing the red blood, the eyeball was taken for blood, the blood oxidation capacity was separated, and the lipid culture in the body was reduced. The substance has significantly improved mouse resistance to the present invention and also provides for the preparation of the composition. The metabolite or the antibacterial component of the Lactobacillus rhamnosus or the composition thereof of the present invention has a stomach inflammation, gastric ulcer caused by E. coli and sputum And the role of twelve proctitis. Enteritis caused by pathogenic bacteria, colitis and

10 1316545 The product or the anti-(four) substance f is an active ingredient of Lactobacillus rhamnosus or its metabolism caused by E. coli bacterium gastritis, gastric ulcer and duodenitis, and more (4) disease caused by enteritis, colitis and straightness The role of enteritis and the like. Other foods, such as various powders prepared from fermented milk or products containing the spirulina®, are obtained from the culture of the rat __, plus 嶋峨 ^ = Various cultures of bacteria = Lactobacillus rhamnosus or inactivated extract of Lactobacillus syringae and purified body, solid powder such as and containing the milk, in the invention, surgery! A variety of edible forms of preparation. Composition or composition towel. ^ Table transfer component can be - the domain used in the present invention is mixed in the term "=,, = 'term" is mainly composed of ·.., the spear "consisting of, the package applies; ί:; Gamma: a pharmaceutically acceptable ''or food-usable' ingredient, ie, ==f:r should be (eg, toxic, irritating, and "used" or "food usable" Or the medicinal product which can be used in foods or transferred to an animal or a human is preferably _ 杂 转 转 ( 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四 四A carrier of product activity, or a food-effective amount of sputum, or a sputum, for administering a medicament to a mammal for prevention and/or treatment by a substance. The composition of the present invention can be used by a large intestine® and other intestines, f' , j"Mental μ, gastric ulcer and duodenitis, can be used for anti-oxidation. Intestinal rickets (4) from edema, colitis and straight-line, etc.···田本发明的_糖乳杆_代代上_ On the way, they can be mixed with 1316545 = === accepted 峨 excipients, such as solvents, ί;: suspended fines such as (four) 纳娜糊, 峨二二, ::: or 10, f two U / m 1, more particularly 'can contain about cf9cfU / e / e U m W although the active ingredients of the gas and the fermenting drum. And can be "mi妓4'' green denomination / _ filament or activity The amount accepted by the 1〇$ character. For example, in this fortune, u 105-l〇C'' or 1〇10 CfU/ml can be prepared (special ' can contain 1〇5一1〇1°cfu /g or the effective dose of the drug to be treated and the severity of the disease to be treated. Applicable to about 10' containing 1〇51〇 intimately mixed with _ or liquid pharmaceutically acceptable carrier 1. fg or 1〇-1〇12 cfu/ml (in particular, may contain 105-10lfl Cfu/g or activity: more specifically, may contain 1〇6-1〇9cfu/g or 1〇6-coffee) The active ingredient produced by the fermentation or fermentation can be adjusted. This dosage regimen can be adjusted to provide the most appropriate response. For example, 'the urgent need for a therapeutic condition can be administered several times per day, in a dose, or proportionally. The L. lactis or its metabolites can be administered by oral or other means. Solid: powder, lactose, broken m曰曰 cellulose, and white clay II H' package Including: medium, polyethylene glycol, non-ionic surfactants, and edible oils (eg, peanut oil and sesame oil) as long as it is suitable for the specific mode of administration of the mutagenic mast g or its metabolite property σ. The composition towel usually allows the (iv) adjuvant to also be 'included' such as flavors, colors, preservatives and antioxidants such as vitamins C, BHI^dBHA. , , , , = = easy to prepare and administer the position of ' Preferred pharmaceutical compositions are solid compositions, especially/, sputum tablets and solid filled or liquid filled capsules. Oral administration is a major advantage of the present invention in that: ' 1316545 (a) this & month, syrup Milk (4) has excellent resistance to fineness, excellent acid and bile acid resistance, and excellent oxidation resistance. (b) This 33(d)(9) Lactobacillus has lions and treats gastritis caused by pyloric helix (iv), with /S and 十日彳, and enteritis caused by Escherichia coli and other intestinal pathogenic bacteria, colitis and The function of proctitis.

below',,. The present invention is described in the following section. It is to be understood that these examples are only used to limit the scope of the invention. The following women do not specify the specific conditions of the * * test loose, it often touch the f-conditions such as sambIOQk et al., laboratory manuals such as: (4) Spring HarbOT LatoratQry Press, (10), or ^ "Microorganism: Experiment Manual (James Cappucc according to the manufacturer's recommended conditions - cattle or Example 1: sample source and collection of the invention _ strain from China's two I 44 * L · β apricot village Guangxi # f ' is also the world's 4 and the 5th long poor village Guangxi Bamaru Autonomous County and Xinjiang Hetian area Long Bama County is located in the northwest of Guangxi, 400-600 meters, with more negative oxygen ions, air, and main 斩~ also P 夂 f The average elevation is in the new. Although the local people are scattered and live in mountainous areas, river valleys, and protect eyesight. Plant species up to 2_ species = four seasons Shenyue' are beneficial to a certain degree of hard water, containing a large number of drops and water resources. Rich. Drinking water has no heat in summer, long warmth and strictness. It has a few days in the Tmall Factory. The rain is hot in the same season, the spring is cool and cool, and the good self-sufficiency is thick. (4) The longevity of the Bama people provides the Hetian in Xinjiang. The southernmost point, the ancient name is in the I sound ^ two-way shirt. #=Products of the second production, (10) million. The Uighurs accounted for the total population, and the skill was 4,000 square kilometers. The population and the original material exchanges. 68 ° BC = in the Shang and Zhou dynasties, the ancient Yuge on the country', Yuge officially accepted the people of the Central Government of the Han Dynasty under the "fighting guards of the west." The ancient Yuge is the earliest 1316545 Buddhist center in the Western Region, with rich Buddhist cultural heritage; many of them have been involved in Hetian. Unique folk customs? Two Fa Xian, Tang Shixuan exotic style. And tons of waiting warmth, special products rich in the grain and cotton warehouse, Silk Road, the fruit of the country is known as the world of gold and jade, traditional products are well-known at home and abroad. Here There are the world's second largest '„silk and other samples collected by the invention three times, including Guangxi 2-large, a typical longevity family of different generations of feces and local longevity code: Ba people == 1 of life r, the object includes Ba = = 23 elderly people, including 6 longevity elderly people over 100 years old. Take the longevity elderly people who are over 90 years old within 5Q kilometers around Hotan area. Only case 2: Separated from the longevity old man's stool Lactic acid bacteria strain screening After 5 years (10) (4) _, 54 stool samples from Guangxi Bama and Xinjiang Hetian Changshou = (90 years old and above) were immersed in the broth culture, and the anaerobic culture was carried out at a price. The culture was coated on the MRS _ Then, the culture is carried out for 3 days. Culture, 'collect the colonies appearing on the agar medium, and further purify, and use the colony 4 to obtain a total of ι_ multi-strain acid-producing bacteria, and obtain lactic acid bacteria by measuring the products thereof. , root growth characteristics, metabolic characteristics 'and the use of specific medium to obtain the dominant lactic acid bacteria. From the three dominant milk m towel 'according to its characteristics, obtain excellent characteristics of lactic acid bacteria - strain. The buckthorn = ritual ® has been deposited in China The National Center for the Conservation and Research of Biological Resources of the Research Institute of Food and Health Industry of the Republic of China, numbered BCRC910316, and the deposit date is May 10, 2006. It is used to detect bacterial characteristics, including physiological and biochemical reactions, as well as Gram, staining, sugar fermentation ability, etc. as described below (characteristic methods are as described below). The isolated strain was added with 10% skim milk broth culture, cultured, lyophilized, and stored at -18<t, and then used to test various characteristic tests of bacteria. 14 1316545 Example 3: Gram staining of lactic acid bacteria strains The strains were tested by Gram staining, and the strains were Gram-positive bacteria, which were non-spore, rod-shaped, and fruit: Figure 1 shows the bacillus of the present invention. Typical characteristics of the genus. The size is I 0-1. 5/zm, this is the milk. Then, the lactic acid bacteria are studied to clarify that the stalks on the stalks of the loam are 2_3_ in the diameter of the gray = special fruit, the strain is in the age of Babel, the most The culture temperature is at 37 °C. Example 4: Analysis of the strain using the KIT 50 CHL At 37 C, the strain was subjected to anaerobic conditions. Then, use the suspension; 1 stand on the table and move it...μ成& 4 "The same on the fillet plate for 48 days of τ':: two waiters. The nourishment & ocean's suspension turbidity with McFarland 2

® bl 50 CHL Seed stone K Bu 1Hr A S, covered with a layer of sterile stone in the upper layer to detect the fermentability of 49 stone water-repellent compounds. The Axis API Identification Software Program (M〇MerieUX, France) analyzed carbohydrates/q II: the results are shown in Figure 2 and Table 1. In Table 1, G represents a control tubule, GLY_5KG (a total of various carbohydrates for the fermentation of Lactobacillus rhamnosus of the present invention, corresponding to each of the small tubes in Fig. 2 (positive result: if fermented carbohydrates, due to culture = shoulder stalk purple indicator acid production from purple to yellow, esculetin determination (25 tube) from purple, black) 'soft analysis results show that the strain of the invention fermented fermenting ability and other proven buckthorn Lactobacillus saccharophila is similar and has similarity. The carbohydrates used in the table GLY 5KG can be found in the manual of the biQMerieux company in France. 1316545

Example 5: Extraction of total genomic DNa = This (four) ^ followed by activation of the medium, plate-aged fine MRS agar 22. Wide 4 take the early trapping Luo inoculation liquid medium 'put in C〇2 · deep thermostatic culture in the thermostat: 37 (:, wave) and 48 xiaoxiao collected lactic acid cells at the bottom of the test tube, used for the indigenous group Extraction of DNA. Take 1.5 mL, the cell culture medium in a centrifuge tube, _〇g centrifugation for 5 minutes, discard the supernatant, and then sink 3 mL of normal saline (_3 (: 1) to wash the bacterial cells 3_5 times; Shen Dian cells plus 12 (% L pumping) Lift the buffer 'and let it suspend' 25〇μ1 1〇% SDS and 75〇汕 gasification T. Mix the vibrate '6 (TC vibration water bath insulation 4〇min, the interval is 10min reverse mixing several times, can Properly extend the time to 1 hour, the effect is better; ultrasonic treatment 1 〇 min; add 750 / / L 3M NaOAc (pH 5.0), ice bath 2 〇 min; 8000g centrifuge 1 〇 ΰ collection supernatant 'plus 0 8 times the volume of isopropanol i〇〇〇〇g centrifuged i〇min sinking the hall; the obtained 7〇% ethanol strip was dissolved in 150//L of TE buffer, and RNase was added at 37. : Further RNA was removed by digestion for 1 hour. The extracted lactic acid bacteria genome DM samples were each subjected to gel electrophoresis on 1% agar [EB staining] and observed on a gel imaging system to analyze the purity and the completion of genomic DNA. 1316545 Integrity. The extracted lactic acid bacteria genomic DNA samples were then measured for OD260nm and OD280nm on an ultraviolet spectrophotometer to quantify the DNA concentration. Example 6: 16S rRNA sequencing analysis Because 16S rRNA is relatively stable for bacteria, sequence analysis of 16s is a good method for bacterial identification. The genome of the extracted experimental strain is used as a template for polymerase bond reaction ( Polymerase Chain Reaction, PCR) amplifies the i6S-rDNA sequence.

Upstream primer for 16S-rDNA sequencing analysis of the lactic acid bacteria of the present invention (SEQ ID NO: 2): ' (where 'Υ, Μ represents the merging primer, γ generation 5, -AGAGT TTGAT YMTGG CTCAG-3' table (CT), Μ Representative (AC)) Downstream primer (SEQ ID NO: 3): 5, -ACGGT TACCT TGTTA CGACTT-3, PCR reaction system consists of: PCR total _ 5〇μί, containing 1〇xpcR buffer 5μί ' 2. 5mm〇1/L dNTPs 4· (Η ' 5υ/μί Tag Enzyme 2 · Correction, 5〇_WL Upstream primer 4.0μί, 50pmol/VL downstream primer 4 〇, 2〇_L template Xinjiang 5. OgL, ddH20 26pL. The reaction procedure is: 95t pre-denaturation, 95t denaturation 1mm scratch annealing, 72 C extension 2min 3G secondary & '95 C denaturation lmin annealing 50s, 72. (: extension 2min 1 cycle; 72 °C extension lOmin 1 cycle. The amplified product was separated by electrophoresis, and the brominated E-bonding of Xiao G > g/mL was analyzed on the gel imaging system. The gel extraction kit (purchased from Shanghai Bocai Bioengineering 51) Recycling 15_size (10)A # segment, domain _ __τ carrier (purchased from Shenneng Gaming Company), transforming E. coli according to the method provided in the molecular cloning manual, The 16S-sequence was determined by the IPTG/X-gal LB plate field containing fresh green, and the remaining positive clones were determined by the determination of the 16S-sequence. The sequencing results are shown in Figure 3, 16S - SEQ ID NO: 1 by sequence. 1316545 Example 7: Analysis of nucleotide sequence data using BLAST The 16S-rRNA sequence determined above was compared with known various mice using the BLAST program (National Biotechnology Center website 'NCBI 'TM·ncbi.nlm.nih.gov) The homology comparison of the syrup of the syrup was carried out. 'The phylogenetic tree was established based on the obtained distance value. It was found that the 16S-rRNA sequence of the Lactobacillus rhamnosus of the present invention is identical to all other Lactobacillus rhamnosus currently known. The corresponding sequences are different, and the homology with Lactobacillus rhamnosus is the highest (more than 95%). ', Example 8: Identification of Lactobacillus rhamnosus by specific primers The present inventors according to Lactobacillus rhamnosus 16S rRNA and A genus-specific primer was designed for 23S rRNA, as shown in Table 2. The name of the primer -------- sequence LC4F ----- 5'-AGGGTGAAGTCGTAACAAGT-3, (SEQ ID NO· zn L73R — 5,-GCCAACAAGCTATGTGTTCGCTTGC -3, (SEQ ID NO. 5) The inventors used 20μ1 PCR The reaction system (PCR conditions as above) revealed that the L73R and LC4F primers were subjected to polymerase chain reaction at 73 bp and 448 bp in the test strain (lane 3) and other Lactobacillus rhamnosus (lanes 4, 5, 6 were respectively Lactobacillus rhamnosus 6〇丨2, Lactobacillus rhamnosus 6013, Lactobacillus rhamnosus grx〇5, all purchased from Yangzhou University, China, and the other bands were different. The results are shown in Figure 5. Example 9: Multiplex RAPD PCR analysis Rand〇m Amplified p〇iymorphism DNA 'RAPD' was performed on the genomic DNA of the Lactobacillus rhamnosus strain and the known Lactobacillus rhamnosus strain of the present invention. Analysis to compare the difference between this strain and other Lactobacillus rhamnosus strains in the genome. Other Lactobacillus rhamnosus strains in this example include: swim lanes are Lactobacillus rhamnosus 6012, lane 2 is Lactobacillus rhamnosus grx〇1, lane 3 is Lactobacillus rhamnosus grx02; lane 4 is rhinoceros Lactobacillus maltophilia grx〇3, lane 5 is the strain of the present invention, lane 6 is Lactobacillus rhamnosus grx05 'lane 7 is Lactobacillus rhamnosus grx〇7, and lane 8 is buckthorn 18 1316545 sugar rod g grx〇9 . (Lactobacillus rhamnosus 6〇12, Lactobacillus rhamnosus grx〇2; rhamnosus gramine grx03, Lactobacillus rhamnosus grx〇5, Lactobacillus rhamnosus grx〇7, Lactobacillus rhamnosus Grx〇9 is purchased from Yangzhou University, China). The genomic DNA of the lactic acid bacteria was extracted according to the method described above, and the integrity and concentration were analyzed. After the analysis, the lOOng gene and the DNA sample were subjected to the rapd analysis as follows. The sequence of the synthesized random primer is shown in Table 1. The optimized RAPD amplification reaction system: the total volume of the reaction is 25 μί, which contains 10×PCR buffer 2·5 μί, 2. 5_ol/L dNTP 2. Ομί, 5U/gLTag enzyme Ι.Ομί' 50pmol/pL random primer 2· 〇μΐ^, 20ng/pL template DNA 5^0μΙ^, (MH2〇12. 5μί. Reaction procedure: 9; rc2min, 36tlmin, 72O2min i ► : human follow %; 93Clmm ' 36〇Clmin, 72°C 2min, 40 Secondary cycle; 93°Clmin, 36

Clmin '72C10min 1 cycle (in the case of a follow-up screening towel, the annealing temperature needs to be adjusted appropriately for different bows). After the amplification was completed, it was separated by electrophoresis on a 14% agar gel and added with E:B0·5/zg/mL, and photographed on a gel imaging system. From the 30 random primers (the sequence is shown in Table 3), the primer #PG4 with good discrimination at the species and strain level was screened. As shown in Fig. 7, it is shown that there is a significant difference from other strains at 85 Qbp, and the tear can be used for screening to identify the strain of the present invention. - Subnumbered sequence in the RAPD analysis - ^__ SEQ ID NO: Numbering sequence SEQ ID NO: 5, -AATCAACTTC-3, "---------- 6 ---- — OPG-16 5,CCGCAGCCAA-3, 21 ___^,-TTCTTTAATT-3, ----------- OPG-17 5'-AACGCGC AAA-3, 22 __5,-TTTAACAACT-3, 8 *C)PG-18 5,-GTGGATGCGA-3, 23"'CTGCGCTGG A-3, 9 OPG-19 5,-GTGACGTAGG-3, 24 __5,-ACGTATCTGC-3, 10 OPG-20 5,-CAATCGCCGT-3 ' 25 GCGATCCCC A-3, 11 OPG-21 5,-GGGAGGC AAA-3, ___— -- 26 '-GTAGACCCGT-3, 12 OPG-22 5,-CCAAGAGGCT-3, 27 ^,-GTTTCCGCCC-3, 13 OPG-23 5,-TCCCCAGGAG-3, 28 5,-CTGCTGGGAC-3, 14 ----- ----- _〇PG-24 5,-AGTGCACACC-3,” 29 19 1316545 OPG-10 5, -TGCCGAGCTG-3, 15 *OPG-25 5,-AGGCATCGTG-3' 30 OPG-11 5,-GGGAACGTGT-3, 16 OPG-26 5,-ACGCGACAGA-3, 31 OPG-12 5,-AGGCGGGAAC-3, ----- 17 OPG-27 5,-AGTATGGCGG-3, 32 OPG-13 5,-CAGCACCCAC-3, 18 OPG-28 5,-GGAAGTCCTG-3, 33 OPG-14 5,-AGGTTGCAGG-3, 19 OPG-29 5,-CCAGGCTGAC-3, 34 OPG-15 5'-AGGTGACCGT-3, — — 20 ----- OPG-30 5,- GGTCGGGTCA-3, 35 Note.* indicates random primers screened for strain-level specificity.

In the experiment of the noodle, the inventors screened the random primers by using the multiplex RAPD_PCR technique to generate a strain-specific marker. The product fragment is between 0·6-2. 2kb by different combinations of 3Q random primers. In the analysis of the ra product of the combination of primer 5, acGAGGCAC-3, and 5,-ACGCG〇m, it was found that a strain-specific marker was present at 613 bp, which could be used for the scorpion scorpion _. This product was recovered in gram sequence and the primer was designed at the end of the sequence of the cloned fragment (Table 4): JL Table 4] The name of the primer _ Rhal Rha2 ---- Sequence soil £?HII^gIME^gAGAAAAC-3, (SEQ ID NO: 36) Upper ZMg^gg^g2HgCAAATGG-3, (SEQ ID NO: 37) The results are shown in Figure 6 'A fragment of about 15 Gbp and 働p was found in this strain, unlike other Lactobacilli, it was confirmed that the strain had its Unique features. Example 10: Comparison of the nucleotide sequence of the strain of the present invention and the Lactobacillus rhamnosus leg. The g strain of the present invention and the rhamnosyl milk (4), _412, rhamnock MCRF-271 (4) S_Gu nuclear _ sequence comparison, the present month The strain is in the 545th; the Western is G' and the Lactobacillus rhamnosus MCRF_412, Lactobacillus brevis MF_27i is ^ Example 11: The safety of the strain LV108 of the present invention in ICR mice In order to test the safety of LH08, choose Make 帛 ICR mice. Specifically, 20 1316545 ^ strains were inoculated into 11% skim milk for 24h (about 5xl09cfu / mL), take = 'male and female half, body weight 2Q ± 2g, rearing, day, normal activity, into her: : test Weighed after 12 hours of fasting, press 〇. 4mL/10g to fill the sample, ~: ":, 仏 $ is the animal's mental state, coat color, voluntary activities, breathing, diet, :::: secretions, etc. - The general condition and the death situation were observed, continuous, and the safety of the two were as shown in Table 5. A safety control group of the lactic acid bacteria inspected after 2 months

The result of autopsy of the parenchyma (heart and kidney): normal digestive organs (normal stomach spleen, lung parenchymal organs (heart, liver, spleen and kidney): normal digestion 1 § official (stomach, small intestine, large intestine): normal small intestine, large intestine): Recovering a large amount of sputum in the small intestine and large intestine of the stomach bacteria is visible. The maximum amount of the mouse to the yeast is 8QmL/kg. Feeding secretions, movement: i? The amount of activity is free,: suction hook, no abnormalities in the nose and mouth, weekly weight, no abnormal urine. After observing - after the week, weighed the body, and after 2 months, the cervical vertebra sacrificed the animal, and all the animals observed the heart, liver, spleen, lungs, kidneys, etc. without any abnormality. Upper, second, small intestine!: The neonate in the large intestine is applied to the MRS fat-filled plate but in the control group, the towel is not = 'i her towel class _ lactic acid coffee 8 strain, the embodiment 12 · the hair (4) strain LV1Q8 acid and In the ability to resist bile salts, Γί people have the ability to resist acid and anti-biliary salts of Lactobacillus bisporus LV108, and adjust the MRS medium to ρΗ2·G MRS medium, pH 3. 〇&

21 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ In the medium of the same soil, the culture solution of the test bacteria was inoculated with 2% of the inoculation amount, and 37 = 1 Μ,,,,,, -, :: The results showed that the strain (4) can be at pH 2, 3. _ _ _ _ The middle number is checked out. The survival rate was higher at pH 3 and 3. H2 && Finance: The strain of this 1 has strong acid and bile resistance. =; Lisu yoghurt on the cell colony of human colonic adenocarcinoma cell line... The inventors performed a detailed study of human colonic adenocarcinoma 8) η National Institute of Chinese Academy of Sciences Shanghai Biochemical Institute) See the 发 发 发 在 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人 人Lactobacillus is about 6. 〇/cell. It is a competitive inhibition of the pathogenic Escherichia coli by the strain of the present invention, ~, Lactobacillus brevisii 2, Lactobacillus rhamnosus 6〇 ^Competitiveness_Inhibition_Strong. Quantum Luna Example 14: Antioxidant ability of Lactobacillus rhamnosus LV108 The present inventors studied Lactobacillus rhamnosus 8 pairs of experimental mice (10) mice, although each I 'weight · 20 ± 2g 'eye from the Nanjing Jiangning Tangshan Qinglong Experimental Animal Center, China) anti-oxidation month b9 force binding. The normal mice were divided into two groups: the high-low and low-dose groups (the high and low dose groups), and the control group and the positive control group (Vascin E9mg (kg), Each country and 10 mice were irrigated at a concentration of 4 〇 mL.kg-ld-work, etc., and the vitamin E of the positive control and the positive control for 8 consecutive days. Physiological salt = ° at the end, 1 hour after gavage, the eyeball was taken for blood, and the serum was tested for SOD activity and MDA s. According to (4) 吟 oxidase method (3) 卩 reagent Wei Zijiao Nanjing Juli Biological Products Co., Ltd. 1316545 determination of serum SOD content; MDA in serum was determined by thiobarbituric acid method (MDA kit purchased from Nanjing Juli Biological Products Co., Ltd.). The results are shown in Table 6. Lactobacillus rhamnosus fermented milk and positive for vitamins £ increased the activity of SOD and decreased the content of MDA, which was significantly different from the control group. It indicated that Lactobacillus rhamnosus LV108 can enhance the antioxidant capacity of mice and reduce the production of lipid peroxidation products in vivo. Anti-oxidation function test of Lactobacillus rhamnosus fermented milk Group control group positive group Lactobacillus rhamnosus fermented milk dose (0.4mL/10g)

NS

VitE 0.4mL (stock solution) 0.4mL (after dilution) SOD(U/mL) 80.3+7.84 89.0+2.61* 89.4+1.65= 87.7+2.05 Compared with the control group * p&lt;〇. 〇5,** ρ<〇. 〇ι MDA(nM/mL) 10.00 il.66 6.80+0.93** 8.05+1.46* 8.17+1.35* Example 15: Inhibition of Escherichia coli by Lactobacillus rhamnosus LV108. Escherichia coli 0157:H7 (purchased from Yangzhou University, China) was inoculated into the nutrient broth, and the number of viable cells was counted by the plate count method after 18 h. Control the number of live bacteria about 1 〇 6 coffee / this, this / agricultural degree is easy to cause disease. Pipette the bacterial solution 55mL on the corresponding solid medium plate = bacteria operation _ through sterile air, place the bacteria liquid on the surface 嶋 = ί, = t. Hole depth 3_, will adjust the concentration of lactic acid bacteria 3 ^ hole, can not overflow outside the hole, culture in their respective growth environment = diameter, · with the same coffee rhamnolipid _12 (purchased from = 2 = inhibition) 12. Figure 2, and 1 = _12 = _ is 12 as shown on the side of the curry side = Helicobacter pylori after the inhibition of Helicobacter pylori Ln〇8 on Helicobacter pylori (purchased from Chinese disease prevention and control) Central infectious disease pre-23 1316545 1 take a small amount on the surface of the garnished medium, slightly push it, put it in iLic __ microenoxidation environment (85% N2, ig% melon, 5% (Γ), elute, The neck standard is more turbid than the word, determine _, make 1 〇 5cfu / iiiL. Pipette 5mL of bacteria liquid on the corresponding solid medium plate, the bacteria are not rod holes, can not overflow the diacid bacteria solution to the full diameter; Buckwheat __Caf 5=Quantity inhibition zone, the diameter of the inhibition zone of Helicobacter pylori against Helicobacter pylori is 12.7 (Figure 9), 卩 is only 9·5 coffee, the age of rhamnose milk Helicobacter pylori Example 17. The raw material composition of the food composition containing w-ribose milk stalks is shown in Table 7. [Table 7] Raw material ___% by weight (%) Fresh milk __ 92.5 White granulated sugar _ ____ 7.5 ~~ According to the above formula ratio, the fresh milk is heated to above ah, adding ί:ϊ ioTM 6::C ? Mpa^ ^ -- i 6035(^^,: llg University Ή )) Turn 6G32 (constructed from Yangzhou

Af) l-10〇xl〇 Cfu &gt; LV108 l-50xl06cfu 〇 40-42t fermentation to a pH of 4. 2_4. 5, swimming active rodent Wei rod _ fermented milk. Ρ^δ Example 18: Containing rat rooster bar g (four) composition 1316545 The raw material is formulated as shown in Table 8. [Table 8] Heavy ^ in %) 10%

= Proportion of fine milk powder, New Zealand, yeast powder, egg peak loss = 65 C ' 2GMPa pressure homogenization bin c kill g 2q_3q points -= c - ^^^a#i„i(1_5〇xl〇6cfu/mL) , 36^, ^ΓρηΪΪ =· 5, from ... cold silk to the tree, in 3%, that is (four) money milk = object. Pure G · 5 money Li Wei (four) cold secret mixed with her mouth after filling into the capsule Medium's preparation of a pharmaceutical composition containing Lactobacillus rhamnosus. The rhamnose lactocarp preserved in this strain g-On the syllabus rW_) LV Lake has been preserved in the preservation and research of biological resources of the Huamin Food Industry Development Institute _ heart (team 肊), 穸 number is BCRC 910316, the date of registration is May 1st, 2006. It should be understood that after reading the ____ 容, the person skilled in the art: to the present invention For a variety of more accustomed limbs, these equivalent forms are defined by the same book. Month 25 1316545 [Simplified illustration of the drawings] Figure 1 depicts the situation of the rhamnose lactoplasts of the present invention after dyeing Gram stains. The carbohydrates of the invented Lactobacillus rhamnosus using API KIT 50 CHL should be analyzed, among 50 small tubes (No. 1 - 49), Q means a control tube, and a yellow tube means The internal carbon hydration 3 changed from purple to yellow due to fermentation of Lactobacillus rhamnosus, and the eschar was determined (25 tubes) from purple to black. Figure 3 depicts the sequencing of nucleotides in the lactobacillus of the present invention. Results Figure 4 depicts the nuclear thief sequence of 16S ritual in Lactobacillus of the present invention. Figure 5_ depicts the electrophoresis results of the serotypes of Phytophthora serrata ([top and LG4F)]. 1 is the molecular weight of 醒ker and -(6)(10)^, the object 'lane 2 is the negative control; the lane 3 is the strain of the invention, the lanes 4, 5, 6 respectively, the Lactobacillus fermentum 6012, the Lactobacillus rhamnosus 6013, the buckthorn Lactobacillus saccharophilus grx〇5. Figure 6 depicts the results of PCR identification of Lactobacillus rhamnosus primers _ and Rha2 of the present invention; swimming ^, DL2 deletion 'lanes 丨 is blank, lane 2 is negative control, lane 3 is the invention of Li The results of the lactic acid lactobacillus m. The results of RAPD_PCR amplification of Lactobacillus rhamnosus using primer 0PG4, lane M is, 'nong mti.1 f rhamnose lactorod 6012, lane 2 is Lactobacillus rhamnosus grx01, For ^路日(四) Mito ritual bar 囷 8ΓΧ〇2; Lane 4 is Lactobacillus rhamnosus grx03, lane 5 srx07, and sound swimming force 6 is buckthorn Lactobacillus maltophilia grX〇5, lane 7 is Lactobacillus rhamnosus stagnation, and ice channel 8 is Lactobacillus rhamnosus _9. Sexuality can be depleted to the human colon adenocarcinoma cell line Caco-2 cells in vitro The inhibitory effect of the additive test is the inhibitory effect of cognac on Escherichia coli.

Claims (1)

1316545 好修(更).本本/ Patent Application---------------- 1. -mrniWactohacUlm ^ Lactobacillus lactis in the process of cutting the people η secret exhibition ===, the rat Li Xin, the deposit Gu is BCRC91G316.用途 及 及 研究 研究 研究 · 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种 一种Or drugs. Dry bacteria Dal dry money other intestines 3. = Please = _ Di 丨 的 — 种 种 种 种 种 种 种 种 种 种 种 种 种 途 途 途 途 途 途 途 途 途 途 途 途 途 途 途 途 10 10 10 10 10 10 10 10 10 10 10 10 It is characterized in that the bacterial digestive tract is characterized by gastritis, monthly ulceration, duodenal inflammation, intestine, * &lt; 5. a Lactobacillus rhamnosus as described in the scope of the patent application, and/or Metabolism|Material use, which is characterized in that it is used for preparing anti-oxidation foods and/or medicines: ..., 6·-food-feeding conjugates, the ship is in, and the shot contains an effective amount of the towel. The gage g and / or its test substance, the food amount of w can be used. 7. == Ϊ Ϊ 6 6 6 6 6 6 6 6 6 食物 食物 食物 食物 食物 食物 食物 食物 食物 食物 食物 食物 食物 食物 食物 食物 食物, solution products, powder products, or suspension products. 8. A pharmaceutical composition characterized in that it contains a rhamnose lactoglobin g and/or a metabolite thereof as described in item i of the patent application, to enable (4) a carrier. 9_ The pharmaceutical composition according to Item 8 of the patent application, wherein the special dosage form is selected from the group consisting of granules, capsules, tablets, powders, oral liquids, suspensions or according to the patent of the Shenqing patent. The food composition according to the invention, characterized in that the composition is as follows: 1 〇 ^ cfu / mL or loM 〇 12 cfu / g of Lactobacillus rhamnosus and / or its metabolites; and a small amount of food A carrier that can be used. The pharmaceutical composition according to claim 8, characterized in that the composition is as follows: 1 〇M (Pcfu/mL or 1 (M〇12 cfu / g of Lactobacillus rhamnosus and / or its metabolites; and a small amount of a pharmaceutically acceptable carrier.