CN103642716A - Lactobacillus plantarum and application thereof - Google Patents
Lactobacillus plantarum and application thereof Download PDFInfo
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- CN103642716A CN103642716A CN201310587738.8A CN201310587738A CN103642716A CN 103642716 A CN103642716 A CN 103642716A CN 201310587738 A CN201310587738 A CN 201310587738A CN 103642716 A CN103642716 A CN 103642716A
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Abstract
The invention relates to lactobacillus plantarum and application thereof, specifically to novel lactobacillus plantarum LP45 (YMC1005) with an accession number of CGMCC No. 8072. The invention further relates to application of the lactobacillus plantarum LP45 in reduction of cholesterol, improvement of immunity and inhibition of harmful bacteria to intestinal tracts.
Description
Technical field
The present invention relates to a kind of new bacterial strain of lactobacillus, more precisely, the invention describes a new bacterial strain LP45 of plant lactobacillus (Lactobacillus plantarum), belong to biological technical field.
Background technology
Probiotic bacterium has very great help to the mankind's health, and scientists has been carried out the research of decades to it, now proved at stomach regulate, the aspect such as anti-oxidant, decreasing cholesterol, prevention gastrointestinal cancer, immunomodulatory all plays good effect.But, the bacterial strain kind scope of probiotic bacterium is very wide, even if individual difference is all very large between the different strains of same kind, so screening operation is very difficult, after in the application of probiotic bacterium, ubiquity is taken in human body now, survival rate is not high, not strong to the adsorptive power of stomach and intestine, the performance aspect enhancing body immunity and inhibition pathogenic bacterium ability is undesirable.Therefore, whether study and screen the superior bacterial strain of prebiotic function and as probiotic bacterium application, just become the key point of nowadays probiotic bacterium research.
Summary of the invention
The object of this invention is to provide a kind of Merchant Codes is LP45, deposit number is the plant lactobacillus of YMC1005, it possesses and in human intestines and stomach, has very high survival rate, stronger stomach and intestine adhesive power, improve the function of body immunity, can significantly suppress stomach, pathogen enterobacteria, possess the potentiality that reduce cholesterol, the purposes of above-mentioned new bacterial strain is provided on the other hand.
For achieving the above object, the technical solution used in the present invention is:
A plant lactobacillus LP45, this plant lactobacillus in August, 2013 common micro-organisms DSMZ of 26 China Committee for Culture Collection of Microorganisms carried out preservation, preserving number is: CGMCC No.8072.
Plant lactobacillus LP45 has gi tract adsorptive power.
Plant lactobacillus LP45 has the function that suppresses gastrointestinal tract pathogen.
Plant lactobacillus LP45 has the function that reduces cholesterol.
Plant lactobacillus LP45 has the function that improves immunizing power.
Plant lactobacillus LP45 is for the preparation of the medicine or the food that suppress gastrointestinal tract pathogen.
Plant lactobacillus LP45 is for the preparation of the medicine or the food that reduce animals or humans body inner cholesterol concentration.
Plant lactobacillus LP45 is for the preparation of the medicine or the food that improve immunizing power.
Above-mentioned food mainly comprises bread and cheese, diet food, protective foods.
The in vitro tests providing in invention confirms, plant lactobacillus LP45(YMC1005 or L45) possess good acid resistance and bile tolerance, in gi tract, survival rate is higher, and possesses stronger gi tract adsorptive power, possesses the function that improves immunizing power, can effectively suppress the breeding of harmful bacteria, bacterial strain provided by the present invention can significantly reduce cholesterol level, in to the prevention of cardiovascular disorder or treatment, possesses larger potentiality.
Accompanying drawing explanation
Fig. 1 is plant lactobacillus LP45(YMC1005 of the present invention or L45) gene fingerprint.
In Fig. 1: M is that Maker, 1 is for plant lactobacillus LP45(YMC1005 or L45), 2 be plant lactobacillus ATCC14917.
Bacterial classification is preserved
Bacterial classification called after plant lactobacillus of the present invention (Lactobacillus plantarum) LP45, is preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms on August 26th, 2013, and preserving number is: CGMCC No.8072.Preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in more detail.
Screening and the preservation of embodiment 1 bacterial strain
Plant lactobacillus LP45(YMC1005 of the present invention or L45) separation screening obtains in the classical acid cheese looked into of Ulan, Shi Zi Wulanchabu League of Inner Mongolia city Siziwang Banner Ulan hada bush station hada loud, high-pitched sound, and concrete isolation identification step is as follows:
By Inner Mongol Siziwang Banner yoghourt sample dissolution, in sterile saline test tube, fully concussion mixes, and mixes rear use and connects collarium and dip mixing liquid streak culture on MRS flat board (37 ℃ of 72h), isolated strains.With connecing collarium from the dull and stereotyped picking list of first-generation bacterium colony, on new MRS flat board streak culture (37 ℃ of 72h), carry out purifying.Same method repurity 2 times, makes final dull and stereotyped upper for till same form bacterium colony.Picking list bacterium colony meets bacterium 37 ℃ of cultivation 18h in MRS liquid nutrient medium, in order to preservation and evaluation, uses.
Liquid nutrient medium bacteria suspension is collected in sterilizing centrifuge tube, clean centrifugal 2 times, carry out the extraction of genomic dna, after extraction, utilize universal primer 27f:5'-AGAGTTTGATCCTGGCTCAG-3', 1495r:5-'CTACGGCTACCTTGTTACGA-3' carries out the PCR experiment of 16S rDNA, after completing PCR, final PCR product is checked order, and correlated series in sequencing result and Gene Bank is compared, comparative result shows LP45(YMC1005 or L45) belong to plant lactobacillus.
By LP45(YMC1005 or L45) and the pure culture liquid of ATCC14917 (plant lactobacillus) bacterial strain clean centrifugal, the thalline of acquisition is carried out to the extraction of genomic dna, after extraction, utilize random primer 5'-GACGGATCAG-3' to carry out RAPD experiment, PCR product is carried out to agarose gel electrophoresis, and carry out gel imaging, contrast the electrophoretic band of two lactobacillus plantarums, result shows, plant lactobacillus LP45(YMC1005 or L45) belong to the different strains of same kind with plant lactobacillus ATCC14917, the results are shown in accompanying drawing 1.By plant lactobacillus LP45(YMC1005 or L45) on August 26th, 2013, be preserved in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is: CGMCC No.8072.
Adopt artificial digestion liquid experiment (hydrochloric acid in gastric juice resistivity and bile acide resistivity) to study plant lactobacillus LP45(YMC1005) survivability under people's intestinal environment.
1, acid resisting test
Plant lactobacillus LP45(YMC1005 or L45) by cryopreservation tube, activated, through 3 activation, (activate for the first time 24h, activate for the second time 14h, activate for the third time 18h) after 3% be inoculated in 4 pH3.0(and use hydrochloric acid to adjust pH) liquid MRS substratum test tube, every test tube is equipped with the liquid MRS substratum of 10ml.Get after liquid MRS substratum test tube 3% inoculation of 1 pH3.0 for surveying the viable count of 0h; After liquid MRS substratum test tube 3% inoculation of another three pH3.0, for surveying the OD600 of 4h, get wherein two survey viable counts.
Survival rate=4h viable count/0h viable count * 100%
2, bile tolerance test
Plant lactobacillus LP45(YMC1005 or L45) by cryopreservation tube, activated, through 3 activation, (activate for the first time 24h, activate for the second time 14h, activate for the third time 18h) after the 3% liquid MRS substratum test tube that is inoculated in 4 0.3% cholate (sigma cholate), every test tube is equipped with the liquid MRS substratum of 10ml.Get after liquid MRS substratum test tube 3% inoculation of 1 0.3% cholate for surveying the viable count of 0h; After liquid MRS substratum test tube 3% inoculation of another three 0.3% cholate, for surveying the OD600 of 4h, get wherein two survey viable counts.
Survival rate=4h viable count/0h viable count * 100%
Result and conclusion: through test, obtain LP45(YMC1005 or L45) in liquid nutrient medium at pH3.0, the survival rate after 4 hours has reached 122.4%, higher than competing product lactobacillus rhamnosus LGG(98.9%) and Lactobacterium acidophilum NCFM(51.2%); In the liquid nutrient medium of 0.3% bile acide, after 4 hours, survival rate has reached 47.8%.Higher than competing product bacterial strain LGG(1.3%) and NCFM(2.1%).
As everyone knows, probiotic bacterium needs the test that stands Digestive system could arrive enteron aisle after being ingested, this results show LP45(YMC1005 or L45) possess higher gi tract survival rate, also shown that bacterial strain is as the potentiality of probiotic bacterium exploitation.
Embodiment 3 gi tract adsorptive power tests
Adopt LP45(YMC1005 or L45) bacterial strain absorption Caco-2 cell studies LP45(YMC1005 or L45) adsorptive power of bacterial strain to gastrointestinal tract epithelial cell, find LP45(YMC1005 or L45) adhesive power is 72.
Concrete operation steps is:
1, human intestinal cell strain Caco-2 is processed after 5min with 1ml 0.05% Regular Insulin, cell is taken gently, be sub-packed in the culture plate in 96 holes, in each hole, add 10000cells, change the DMEM substratum that 200 μ l are fresh every day.
2, lactobacillus plantarum LP45(YMC1005 or L45) respectively with after twice of MRS activation, be seeded to 5mlMRS broth and cultivate, after 24 hours, get 1ml with 6000rpm after centrifugal 10 minutes, with PBS(pH 7.2) clean and obtain lactic acid bacterial liquid twice.Add 20 μ l lactic acid bacterial liquids in the cell of step 1, then cultivate and milk-acid bacteria was adhered in 1 hour,
3, milk-acid bacteria adhered to after 1 hour, fall except substratum, with PBS damping fluid, wash 5 times to remove the milk-acid bacteria not adhering to, add 100 μ l, 10% formalin, 30 minutes, fixed cell and bacterium, then wash 3 times with PBS damping fluid, finally with 100 μ l Viola crystallinas, dye, after five minutes, with a small amount of 75% alcohol, get express developed, remove the staining agent on cell.
4, utilize phase contrast microscope to observe the adsorptivity of milk-acid bacteria to intestinal epithelial cell, random under different fields of microscope, the milk-acid bacteria of observing and calculate 50 cells is adsorbed situation, finally calculates the lactic acid bacterium number that each cell on average adsorbs.
, of the present invention plant lactobacillus LP45(YMC1005 or L45 known through test-results) adhesive power 72, adhesive power is stronger.An important indicator for screening probiotic bacterium is exactly its adhesive power to gastrointestinal tract epithelial cell and mucous membrane.The strong probiotic bacterium of adhesive power also correspondingly extended to some extent in the time of growing of determining of intestinal mucosa, and it is significant that this immunoregulation effect for performance probiotic bacterium and stimulating is repaired intestinal tract injury position.
Embodiment 4 immunity tests
Adopt Caco-2 cell, the RPIM1640 substratum of 10%FBS is at 37 ℃, under 5%CO2 condition with LP45(YMC1005 or L45) bacterial strain cultivates altogether, finds LP45(YMC1005 or L45) no matter bacterial strain be the expression that can both significantly regulate IL-6 and IL-8 in Caco-2 cell after viable bacteria or heated and inactivated.
Adopt mouse macrophage (J774.1), the RPIM1640 substratum of 10%FBS is at 37 ℃, under 5%CO2 condition with LP45(YMC1005 or L45) bacterial strain cultivates altogether, find LP45(YMC1005 or L45) bacterial strain obviously promoted the secretion of IL-6, makes IL-6 expression amount bring up to 110ng/ml by 0.
From test-results, plant lactobacillus LP45(YMC1005 or L45) possess the function of strengthening immunity.
Embodiment 5 Determination of Cholesterol Content tests
1, drawing standard curve
Accurate measuring cholesterol reference liquid 0.2 ml, 0.4 ml, 0.6 ml, 0.8 ml, 1.0 ml, 1.2 ml, 1.4ml, 1.6ml are placed in dry test-tube, and be settled to 2.0ml with dehydrated alcohol, along wall, slowly add 2.0ml ferric ammonium sulfate nitrite ion again, concussion mixes, to be cooled to room temperature, measure absorbancy with ultraviolet spectrophotometer 560nm place.Take cholesterol concentration as X-coordinate, and OD560 is ordinate zou, drawing standard curve.
2, cholesterol determination
A. thing lactobacillus LP45(YMC1005) in MRS liquid nutrient medium, activated for 3 generations, by Maxwell turbidimetry, adjusting bacteria concentration is 3.0 * 10
9cFU/ml is inoculated into containing 37 ℃ of cultivations in the MRS liquid nutrient medium of cholesterol by 2% inoculum size, cultivates respectively 24 h, 48h.
B. get nutrient solution 0.2 mL and be placed in dry centrifuge tube, first add 0.8 mL dehydrated alcohol, after shaking up, (dehydrated alcohol divides 2 times and adds to add 4 mL dehydrated alcohols again, object makes protein to disperse very thin Precipitation), add a cover, vortex mixer mixes fast, centrifugal (10000 r/min, 15 min).
C. with micropipet, accurately pipetting supernatant liquor 2 mL joins in colorimetric cylinder, add again 2 mL ferric ammonium sulfate nitrite ions, add fashionable will slowly adding along tube wall, make to keep sharp interface between two liquid, add rear shake well and mix (can not limit edged shake), to produce high heat (being greater than 80 ℃), promote colour developing.
D. each pipe is placed to 15 min, treat that inner reaction tube liquid is cooled to room temperature, in 30～60 min, on spectrophotometer, in 560 nm places, measure absorbancy.It is blank tube that the 2 mL dehydrated alcohols of take add 2 mL ferric sulfate reagent, take that what do not connect bacterium is standard value containing the MRS liquid culture A0560 of cholesterol.
E. degrading rate of cholesterol=(A0560-A560)/A0560 * 100%.
Result and conclusion: through test, LP45(YMC1005 or L45) degrading rate of cholesterol is 80.2%, a little less than competing product bacterial strain NCFM(89.6%), higher than bacterial strain LGG(34.7%), illustrate that it has very strong removing cholesterol ability.
Embodiment 6 bacteriostatic tests
The bacteriostatic experiment that comprises intestinal bacteria, streptococcus aureus.Intestinal bacteria, streptococcus aureus are used after LB liquid nutrient medium activation 18h, are diluted to 10
5-10
6cfu/ml gets 200ul and is evenly applied in LB solid medium culture dish, after half an hour, put Oxford cup, each culture dish is put 3, is denoted as A, B, C, and A represents that nutrient solution stoste, B representative adjusts nutrient solution stoste, C representative after pH5.5 to adjust 10 times of nutrient solution stoste dilutions after pH5.5.Each Oxford cup adds 300ul, after 37 ℃ of cultivation 24h, measures inhibition zone.
After testing, this bacterial strain intestinal bacteria inhibition zone is 11.98mm, to streptococcus aureus inhibition zone, is 24.8mm.Result shows LP45(YMC1005 or L45) intestinal bacteria and streptococcus aureus are had to good bacteriostatic action.
The above embodiment is only the preferred embodiments of the present invention, and is not the exhaustive of the feasible enforcement of the present invention.For persons skilled in the art, any apparent change of under the prerequisite that does not deviate from the principle of the invention and spirit, it having been done, within all should being contemplated as falling with claim protection domain of the present invention.
Yi Ran bio tech ltd, <110> Hebei
<120> plant lactobacillus and application thereof
<130> 2013
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1000
<212> DNA
<213> plant lactobacillus LP45
<400> 1
catctgtcac ttagcggctg gttcctaaaa ggttacccca ccgactttgg gtgttacaaa 60
ctctcatggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gcggcatgct 120
gatccgcgat tactagcgat tccgacttca tgtaggcgag ttgcagccta caatccgaac 180
tgagaatggc tttaagagat tagcttactc tcgcgagttc gcaactcgtt gtaccatcca 240
ttgtagcacg tgtgtagccc aggtcataag gggcatgatg atttgacgtc atccccacct 300
tcctccggtt tgtcaccggc agtctcacca gagtgcccaa cttaatgctg gcaactgata 360
ataagggttg cgctcgttgc gggacttaac ccaacatctc acgacacgag ctgacgacaa 420
ccatgcacca cctgtatcca tgtccccgaa gggaacgtct aatctcttag atttgcatag 480
tatgtcaaga cctggtaagg ttcttcgcgt agcttcgaat taaaccacat gctccaccgc 540
ttgtgcgggc ccccgtcaat tcctttgagt ttcagccttg cggccgtact ccccaggcgg 600
aatgcttaat gcgttagctg cagcactgaa gggcggaaac cctccaacac ttagcattca 660
tcgtttacgg tatggactac cagggtatct aatcctgttt gctacccata ctttcgagcc 720
tcagcgtcag ttacagacca gacagccgcc ttcgccactg gtgttcttcc atatatctac 780
gcatttcacc gctacacatg gagttccact gtcctcttct gcactcaagt ttcccagttt 840
ccgatgcact tcttcggttg agccgaaggc tttcacatca gacttaaaaa accgcctgcg 900
ctcgctttac gcccaataaa tccggacaac gcttgccacc tacgtattac cgcggctgct 960
ggcacgtagt tagccgtggc tttctggtta aataccgtca 1000
Claims (8)
1. a plant lactobacillus LP45, this plant lactobacillus in August, 2013 common micro-organisms DSMZ of 26 China Committee for Culture Collection of Microorganisms carried out preservation, preserving number is: CGMCC No.8072.
2. a kind of bacterium plant lactobacillus LP45 according to claim 1, it has gi tract adsorptive power.
3. a kind of bacterium plant lactobacillus LP45 according to claim 1, it has the function that suppresses gastrointestinal tract pathogen.
4. a kind of bacterium plant lactobacillus LP45 according to claim 1, it has the function that reduces cholesterol.
5. a kind of bacterium plant lactobacillus LP45 according to claim 1, it has the function that improves immunizing power.
6. an application of the plant lactobacillus LP45 as described in claim 1-5 any one, is characterized in that: for the preparation of the medicine or the food that suppress gastrointestinal tract pathogen.
7. an application of the plant lactobacillus LP45 as described in claim 1-5 any one, is characterized in that: for the preparation of the medicine or the food that reduce animals or humans body inner cholesterol concentration.
8. an application of the plant lactobacillus LP45 as described in claim 1-5 any one, is characterized in that: for the preparation of the medicine or the food that improve immunizing power.
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