CN104789497A - Acid-resistant and bile-salt-resistant Lactobacillus strain as well as screening method and application thereof - Google Patents
Acid-resistant and bile-salt-resistant Lactobacillus strain as well as screening method and application thereof Download PDFInfo
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Abstract
The invention discloses an acid-resistant and bile-salt-resistant Lactobacillus strain as well as a screening method and an application thereof. The strain is Lactobacillus salivarius L2 preserved in the CCTCC (China Center for Type Culture Collection) with the preservation number of CCTCC NO: M2014614. The acid-resistant and bile-salt-resistant Lactobacillus strain is separated from a healthy chicken intestinal tract content and can resist the acidity of pH3.0 and the bile salt concentration of 3 g/L in a simulation gastrointestinal tract environment; the logarithmic phase is longer and the acid production capacity is higher; antibacterial experiments in vitro indicate that the L2 strain has the higher inhibiting effect on Escherichia coli and can inhibit growth of salmonella enteritidis to a certain extent; the strain has acid-resistant and bile-salt-resistant characteristics, has higher Escherichia-coli-resistant and salmonella-resistant activity, grows fast, is high in acid production capacity and can be used for preparing microecological preparations suitable for poultry.
Description
Technical field
The invention belongs to microbial technology field, be specifically related to a kind of acid and bile salt tolerance lactic bacilli strains, also relate to a kind of screening method and application of acid and bile salt tolerance lactic bacilli strains simultaneously.
Background technology
Chicken colibacillosis causes one of chicken poor growth and dead major reason, and govern the development of aviculture.Microbiotic is often taked to treat in production, but microbiotic is while suppression invasive organism, also the balance of normal microbial flora in enteron aisle is destroyed, add the incidence of other digestive tract diseases on the contrary, occur once without microbiotic, the strange phenomenon that chicken group just falls ill, causes abuse of antibiotics.Abuse of antibiotics causes animal intestinal pathogenic micro-organism to produce resistance, and cause animal immunizing power to decline, the series of problems such as the drug residue increase in animal products and environmental pollution, and then the competitive power of animal products in world market is declined, and potential threat (Servin A L.Antagonistic activitiesof lactobacilli and bifidobacteria against microbial pathogens [J] .FEMS MicrobiolRev, 2004.28 (4): 405-440) is caused to human health.Therefore, develop nontoxic, to have no side effect and the Substitutes For Antibiotic of noresidue has become current study hotspot.
Using probiotic bacterium composition probiotics as Substitutes For Antibiotic first-selection approve by most practitioner.AAFCO qualification in 1998 discloses the bacterial classification that 43 kinds can be used as probiotics, and wherein more than half is milk-acid bacteria (Ewing W N.TheLiving Gut [M] .University of Notinghan.England, 1994,49).Milk-acid bacteria is as the one of probiotic bacterium, adjustable digestive tube microecological balance, antagonism pathogenic micro-organism (Sakamoto L, Igarashi M, Kimura K, et a1.Suppressiveeffect of Lactobacillus gasseri OLL 2716 (LG21) on Helicobacter pylori infection in humans [J] .JAntimicro, 2001,47 (5): 709-710, Kushal R, Anand S K, Chander H.Effect of the feedingmicroentrapped co-culture of Lactobacillus acidophilus and Bifidobacterium bifudum on theimmune response and protection of mice infected with Salmonella typhimurium [J] .Dairy SciTech, 2006, 86:387-399), promote that nutritive ingredient decomposition in animal body absorbs (Fooks L J, Full R, Gibson GR.Prebiotics, probiotics and human gut microbiology [J] .Int Dairy J, 1999, 9:53-61), reduce serum cholesterol levels (Pereira D I, Cibson G R.Cholesterol assimilation by lactic acid bacteria andbifididobacteria isolated from the human gut [J] .Appl Envir, 2002, 68 (9): 4689-4693), and antitumor action (Park H D, Rhee C H.Antimutagenic activity of lactobacillus plantarum KLAB2l isolatedfrom kimchi Korean fermented vegetables [J] .Biotech let, 2001, 23 (19): 1583-1589, Rafter J.Lacticacid bacteria and cancer:mechanistic perspective [J] .Br J Nutr, 2002,88:89-94) etc.Milk-acid bacteria is one of main fungal component in animal digestive tract, be widely used in (Wang Jing in animal productiong as feed microbe additive, Ji Haifeng, Wang Sixin, Deng. the present Research of feeding lactobacillus preparation and the application [J] in Swine Production. Animal Nutrition and Feed Science, 2010, 37 (3): 38-41), can to vie each other nutritive substance with pathogenic bacteria after it enters animal and bird intestines, prevention pathogenic bacteria obtains the nutritive element needed for its growing multiplication, organic acid as a series of in voltaile fatty acid and lactic acid etc. can also be produced, and then the pH in reduction enteron aisle, suppress propagation (the Isolauri E of the pathogenic bacterium such as intestinal bacteria, Salminen S, OuwehandA C.Microbial-gut interactions in health and disease.Probiotics [J] .Best practice & research.Clinical gastroenterology, 2004, 18 (2): 299).How to obtain resistance to gastrointestinal tract environment and growth rapidly, the fowl lactic bacterium strains of strong, the good anti-bacterial effect of acid producing ability, be urgent problem.
At present, along with the widespread use of milk-acid bacteria, people are also more and more deep to its research, milk-acid bacteria is in enhancing body immunologic function and prevent to play a significant role in digestive tract infection (Geary T M, P.H.Brooks, T.Morgan, etal.Performanceof weaner pigs fed ad lihitum with liquid feed at different drymatter concentrations [J] .Sci.FoodAgric, 1996,72:17-24; Vanwinsen R L, B A.Purlings, L A.Lipman, etal.Effect of Fermented Feedon the Microbiol Population of the Gastrointestinal Tracts of Pigs [J] .Applied and EnvironmentMicrobiology, 2001,67 (7): 3071-3076; Gunnar Fimland, Line Johnsen, Jon Nisaen-Meyer.Pediocin-like antimicrobial peptides (class 1I bacteriocins) and their iramunity proteins:biosynthesis, structure, and mode of action [J] .Peptide Sci.2005,11:688-696).Milk-acid bacteria improves IgA, IgM, IgG level in mucous membrane surface and serum by bacterium itself or cell wall constituent, strengthen humoral immunization, promote the propagation of T lymphocyte and bone-marrow-derived lymphocyte, strengthen cellular immunization (Chen Shiqiong, Li Pinglan. the application prospect [J] of property milk-acid bacteria in treatment grice diarrhoea. feed is studied, and 2003 (1): 20-22); Can competitive exclusion pathogenic bacteria adhesion, produce multiple antibacterial substance, and then play prevention intestinal tract infections effect (Zhao Hongmei, Yang Jinsheng, Yang Wentao, Deng. the impact [J] that milk-acid bacteria regulates intestinal mucosal immune. Jilin agricultural, 2011, (5): 328-329).Tiny ecosystem preparation great majority based on milk-acid bacteria on market are suitable for Mammals, and due to the Digestive tract feature of poultry uniqueness, on poultry, application has certain limitation, and clinical effectiveness is not given prominence to.Therefore fowl is developed significant with probiotics.
Summary of the invention
The object of this invention is to provide a kind of acid and bile salt tolerance lactic bacilli strains.
Second object of the present invention is to provide a kind of screening method of acid and bile salt tolerance lactic bacilli strains.
3rd object of the present invention is to provide the application of a kind of acid and bile salt tolerance lactic bacilli strains in the probiotics for the preparation of control poultry bacterial digestive tract diseases.
In order to realize above object, the technical solution adopted in the present invention is:
A kind of acid and bile salt tolerance lactic bacilli strains, described bacterial strain is saliva lactobacillus (Lactobacillus salivarius) L2, and be preserved in China typical culture collection center, deposit number is: CCTCC NO:M 2014614.
Acid and bile salt tolerance lactic bacilli strains of the present invention is saliva lactobacillus (Lactobacillus salivarius) L2, be preserved in China typical culture collection center (address is Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University China typical culture collection center in the school) on December 2nd, 2014, deposit number is: CCTCC NO:M 2014614.
Described bacterial strain filters out from poultry intestinal tract content.Security from bacterial classification and the adhesivity to host intestine are considered, lactic acid bacteria culturers is separated the dominant microflora in host intestine.
Described poultry intestinal tract content is healthy chick's cecal content.
A screening method for above-mentioned acid and bile salt tolerance lactic bacilli strains, comprises the following steps:
1) strains separation purifying: get healthy poultry intestinal tract content, after sterilized water dilution, be coated on lactobacillus selective medium and carry out Anaerobic culturel, isolate single bacterium colony with molten calcium circle, purifying of repeatedly ruling, obtains purifying bacterium;
2) step 1 is got) gained purifying bacterium makes bacterium liquid, and be placed in acid MRS substratum respectively, MRS substratum containing bovine bile carries out Anaerobic culturel, obtain dominant strain;
3) detecting step 2) gained dominant strain to the In Vitro Bacteriostatic of intestinal bacteria and Salmonellas, the lactobacillus of Chinese People's Anti-Japanese Military and Political College enterobacteria, Salmonellas activity must be had.
Step 1) in, the lactobacillus selective medium described in 1L comprises following component: peptone 10g, beef extract powder 10g, yeast leaching powder 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, Citric Acid three ammonium 2g, magnesium sulfate 0.2g, manganous sulfate 0.05g, tween 80 1g, glucose 20g, calcium carbonate 5g, agar 25g.
Step 1) in, described Anaerobic culturel is Anaerobic culturel 24 ~ 48h under 37 DEG C of conditions.
Step 1) gained purifying bacterium forms neat in edge, smooth surface, the oyster white circular colonies that has molten calcium circle, reguarity homogeneous on described lactobacillus selective medium; This purifying bacteria strain is carried out gramstaining, and oily Microscopic observation is shaft-like gram-positive microorganism, single or in short catenation.
Step 2) in, the MRS substratum described in 1L comprises following component: beef extract 10g, peptone 10g, yeast extract 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganous sulfate 0.28g.
Step 2) in, the pH value of the MRS substratum of described acidity is 3.0.
Step 2) in, described contains in the MRS substratum of bovine bile, and the concentration of bovine bile is 3.0g/L.
Step 2) in, in described bacterium liquid, the concentration of lactobacillus is 2.0 × 10
8cfu/ml; 300 μ l bacterium liquid correspondences add containing in the MRS substratum of bovine bile of 5.0ml.
Step 3) in, described intestinal bacteria are standard E. coli strains A TCC25922; Described Salmonellas is Salmonella enteritidis.
The application of a kind of above-mentioned acid and bile salt tolerance lactic bacilli strains in the probiotics for the preparation of control poultry bacterial digestive tract diseases.
Acid and bile salt tolerance lactic bacilli strains of the present invention, is saliva lactobacillus (Lactobacillus salivarius) L2, is separated from healthy chicken intestinal contents, can tolerates acidity pH3.0 and 3g/L gallbladder salinity in Imitative gastroenteric environments; From growth curve and product love song line, L2 bacterial strain logarithmic phase is longer, and acid producing ability is stronger; Known by comparison after 16S rRNA sequence, L2 and saliva lactobacillus have very high homology, can reach 99%; Extracorporeal bacteria inhibitor test shows, L2 bacterial strain has stronger restraining effect to intestinal bacteria, also can suppress the growth of Salmonella enteritidis to a certain extent; This bacterial strain has the characteristic of acid and bile salt tolerance and has higher Chinese People's Anti-Japanese Military and Political College enterobacteria, Salmonellas activity, and growth is rapid, acid producing ability is strong, can be used for preparing novel, the efficient probiotics being suitable for fowl.
The screening method of acid and bile salt tolerance lactic bacilli strains of the present invention, lactobacillus is filtered out by selectivity culture of isolated from healthy chicken enteron aisle, through stomach juice-resistant and bile tolerance screening, and growth curve, acid producing ability mensuration are carried out to the bacterial strain filtered out, by dull and stereotyped bacteriostatic test, filter out the lactic bacilli strains had compared with high inhibition pathogenic colon bacillus characteristic; Check order finally by 16S rRNA, through Blast comparison, be accredited as saliva milk-acid bacteria; Obtained strains has the characteristic of acid and bile salt tolerance and has higher Chinese People's Anti-Japanese Military and Political College enterobacteria, Salmonellas activity, and growth is rapid, acid producing ability is strong, and this bacterial strain is that the development of fowl probiotics provides bacterial classification support.
Accompanying drawing explanation
Fig. 1 is the colonial morphology of lactobacillus on lactobacillus selective medium filtered out from poultry intestinal tract;
Fig. 2 is the microscopical determination form of the purifying bacteria strain be separated;
Fig. 3 is the growing state (OD of lactic bacilli strains on the MRS substratum of acidity
600) detected result;
Fig. 4 is the growing state (OD of lactic bacilli strains on the MRS substratum containing bovine bile
600) detected result;
Fig. 5 is lactic bacilli strains resistance to Gradient acid Characteristics Detection result;
Fig. 6 is the lactic bacilli strains cholate of resistance to gradient Characteristics Detection result;
Fig. 7 is lactic bacilli strains 48h growth curve;
Fig. 8 is that lactic bacilli strains 48h produces love song line;
Fig. 9 is the bacteriostatic activity detected result of dominant strain L2, and wherein, a is the fungistatic effect to intestinal bacteria ATCC25922, and b is the inhibition to Salmonella enteritidis;
Figure 10 is the bacteriostatic activity detected result of dominant strain L4, and wherein, c is the fungistatic effect to intestinal bacteria ATCC25922, and d is the inhibition to Salmonella enteritidis;
Figure 11 is the bacteriostatic activity detected result of dominant strain L10, and wherein, e is the fungistatic effect to intestinal bacteria ATCC25922, and f is the inhibition to Salmonella enteritidis;
Figure 12 is lactobacillus L2 bacterial strain 16Sr RNA PCR primer electrophorogram;
Figure 13 is the evolutionary tree of lactobacillus L2 bacterial strain and homology higher bacterial 16 S r RNA.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
In embodiment, MRS meat soup (MRS substratum), lactobacillus selective medium, pGEM-T carrier and pfu enzyme are all biological purchased from treasured match; The agents useful for same such as calcium carbonate, bovine bile, hydrochloric acid, sodium hydroxide are chemically pure reagent.
Data processing in embodiment: process data and the growth curve of lactobacillus acid and bile salt tolerance characteristic with Excel 2013 and produce sour curve data; Lactobacillus 16S rRNA sequencing result Blast is carried out sequence analysis.The sequence higher to the homology obtained adopts MEGA5.0 to build evolutionary tree.
Embodiment 1
Screening method and the qualification process of the acid and bile salt tolerance lactic bacilli strains of the present embodiment are as follows:
1. strains separation purifying:
Get healthy chick cecal content 1.00g, after the dilution of 5ml sterilized water, by filtered through gauze to remove the solid residue in cecal content, obtain filtrate; Get 0.5ml filtrate and use sterilized water doubling dilution to 10 successively
-7, get 10
-5, 10
-6, 10
-7three each 100 μ l of extent of dilution, are uniformly coated on lactobacillus selective medium flat board, 37 DEG C of Anaerobic culturel 24h, isolate single bacterium colony with molten calcium circle, and purifying of repeatedly ruling, until obtain the bacterium colony of purifying bacterium L1 ~ L10; Described line purifying be lactobacillus selective medium.
Wherein, the lactobacillus selective medium described in 1L comprises following component: peptone 10g, beef extract powder 10g, yeast leaching powder 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, Citric Acid three ammonium 2g, magnesium sulfate 0.2g, manganous sulfate 0.05g, tween 80 1g, glucose 20g, calcium carbonate 5g, agar 25g.
2. identification of morphology:
Carry out colony morphological observation to being separated the purifying bacterium obtained, and gramstaining is carried out, the form of oily Microscopic observation bacterium and staining conditions to the single bacterium colony be separated, and identify according to " uncle Jie Shi bacteriological surveillance handbook ";
Morphologic observation result: after 37 DEG C of Anaerobic culturel 24h, lactobacillus selective medium is formed neat in edge, smooth surface, the oyster white circular colonies (as shown in Figure 1) that has molten calcium circle, reguarity homogeneous; The purifying bacteria strain of separation is carried out gramstaining, visible in shaft-like gram-positive microorganism under oily mirror, single or in short catenation (as shown in Figure 2).
3. the resistance of primary dcreening operation bacterial strain in Gradient acid and cholate environment (Imitative gastroenteric environments):
Get MRS substratum: the MRS substratum described in 1L comprises following component: beef extract (beef extract powder) 10g, peptone 10g, yeast extract 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganous sulfate 0.28g; After water-soluble for above-mentioned each component, with pressure kettle at 121 DEG C of sterilizing 15min, regulate pH to be 6.2 ~ 6.4, to obtain final product.
Prepare that bovine bile mass concentration is 0.0g/L (contrast) respectively, the MRS substratum of 1.0g/L, 2.0g/L, 3.0g/L and pH value be 3.0,4.0,5.0,6.3 (contrasts), the MRS substratum of 7.0.
Get step 1 gained purifying bacterium L1 ~ L10 and make lactobacillus bacterium liquid respectively, than turbid instrument, lactobacillus bacterial concentration is adjusted to 2.0 × 10 with Maxwell
8cfu/ml, obtains bacteria suspension; Get respectively pH that the corresponding bacteria suspension of 300 μ l adds 5.0ml be 3.0,4.0,5.0,6.3 (contrasts), 7.0 MRS substratum and bovine bile concentration be the MRS substratum of 0.0 (contrast), 1.0g/L, 2.0g/L, 3.0g/L, often organize three repetitions, after 37 DEG C of Anaerobic culturel 4h, with UV spectrophotometer measuring bacterium liquid OD
600value, result as shown in Figure 3, Figure 4.
As can be seen from Fig. 3, Fig. 4, lactobacillus dominant strain L2, L4, L10 are the MRS substratum of 3.0, the middle well-grown of MRS substratum (Imitative gastroenteric environments) containing the bovine bile of 3.0g/L in pH value.
Investigate the characteristic of the lactobacillus resistance to Gradient acid of dominant strain L2, L4, L10 and cholate, result as shown in Figure 5, Figure 6.As can be seen from Fig. 5, Fig. 6, lactobacillus dominant strain L2, L4, L10 Imitative gastroenteric environments has higher tolerance.
4. acid and bile salt tolerance strain growth curve and product love song line:
(lactobacillus concentration is 2.0 × 10 to the bacteria suspension of difference extracting lactic acid bacillus dominant strain L2, L4, L10
8cfu/ml) 300 μ l, are placed in the MRS substratum of 100ml, 37 DEG C of anaerobism quiescent culture, and every 2h sampling once, get 48h continuously, with ultraviolet spectrophotometer survey different time sections get the OD of bacterium liquid
600value (result is as shown in Figure 7), with precision acidity meter detect different time sections get the pH value (result as shown in Figure 8) of bacterium liquid.
As can be seen from Figure 7, the latent period of lactobacillus dominant strain L2, L4, L10 is all very short, enters logarithmic phase soon; Before L2, L10 bacterial strain, 14h growth rapidly, OD
600value straight line rises, and 14 ~ 30h, cell density tends to balance substantially, OD
600value stabilization is about 2.5; Before L4 bacterial strain, 12h growth is very fast, 12 ~ 48h period OD
600value is basically stable at about 2.2; Bacterial strain L10 comparatively L2 enters stationary phase a little earlier, and 30 ~ 48h, L2 bacterial strain has the trend of slowly growth, and L10 bacterial strain OD
600value maintains about 2.5 substantially.
Fig. 8 display be the changing conditions of lactobacillus dominant strain L2, L4, L10 nutrient solution pH in 48h.As can be seen from Figure 8, the initial pH of MRS liquid nutrient medium is 6.37, and adding bacterial concentration is 2.0 × 10
8after L2, L4, L10 bacteria suspension 300ul of cfu/ml, pH becomes 6.01,6.21,6.17 respectively; 37 DEG C of Anaerobic culturel, before three strain bacterium, 16h thalline enters logarithmic phase very soon, and accretion rate is accelerated, and producing acid amount significantly increases, and pH value declines rapidly, and wherein L2 bacterial strain is the most obvious; After 16h, L4, L10 bacterial strain almost no longer produces acid, and pH is stabilized in about 4.0, and L2 bacterial strain produces the obviously minimizing of acid amount, and pH value declines slowly, and after 36h, thalline almost no longer produces acid, and pH tends towards stability, and maintains about 3.7.
5. the mensuration of dominant strain In Vitro Bacteriostatic:
Standard E. coli strains A TCC25922 and Salmonella enteritidis are inoculated in nutrient broth respectively, and bacterial concentration is diluted to 1.0 × 10 with Maxwell than turbid instrument after cultivating by 200r/min incubator overnight
8cfu/ml, getting 100ul is spread evenly across on LB agar plate, after 37 DEG C of dry 30min, flat board evenly places 3 sterilizing Oxford cups, the advantage lactobacillus bacteria suspension cultivating 16h is added Oxford cup to just reaching, be placed in after 37 DEG C of constant incubators cultivate 12, measure the diameter of inhibition zone, with aseptic MRS in contrast.Result is as shown in Fig. 9, Figure 10, Figure 11.
Bacteriostatic activity criterion: antibacterial circle diameter≤5mm is that biocidal property is weak; Antibacterial circle diameter 5 ~ 10mm is that biocidal property is more weak, and antibacterial circle diameter 10 ~ 15mm is that biocidal property is comparatively strong, and antibacterial circle diameter >=15mm is that biocidal property is strong.
Experimental result shows, and lactobacillus dominant strain L2, L4, L10 are respectively 18mm, 9mm, 8mm to the diameter that intestinal bacteria ATCC25922 forms inhibition zone, and diameter Salmonella enteritidis being formed to inhibition zone is respectively 6mm, 0mm, 8mm.From bacteriostatic test, L2 bacterial strain has stronger restraining effect to intestinal bacteria ATCC25922, and L4, L10 bacterial strain also has restraining effect to a certain degree to intestinal bacteria, but bacteriostatic action is more weak; Three strain bacterium are to the inhibition of Salmonella enteritidis all not as the fungistatic effect to intestinal bacteria ATCC25922, and wherein L2, L10 bacteriostatic action to Salmonella enteritidis is more weak, and L4 to Salmonella enteritidis almost without bacteriostatic action.Therefore filter out lactobacillus L2 bacterial strain.
6.16S rRNA identifies and systematic evolution tree:
With 16S rRNA universal primer 16S 27f (AGAGTTTGATCCTGGCTCAG, as shown in SEQ ID No.1) and 16S 1525r (AAGGAGGTGATCCAGCCGCA, as shown in SEQ ID No.2) to increase the 16S rRNA fragment of lactobacillus L2 bacterial strain; PCR reacts use 10 μ l system: 2 × pfu Mix 5uL, 16S 27f (10 μm of ol/L) 0.2 μ l, 16S 1525r (10 μm of ol/L) 0.2 μ l, bacterium liquid 1 μ l, sterilized water 3.6 μ l.PCR response procedures is: 95 DEG C of denaturation 10min; 95 DEG C of sex change 30s, 56.2 DEG C of annealing 30s, 72 DEG C extend 30s, 30 circulations; 72 DEG C extend 10min.PCR primer with 1% agarose gel electrophoresis, DNA Green dyes, and detects PCR primer size.
DNA glue reclaims test kit and carries out purifying to bacterium liquid PCR primer, after detecting purified product concentration with micro-ultraviolet spectrometry degree meter, purified product is connected with pGEM-T carrier, proceed to bacillus coli DH 5 alpha competence subsequently, after blue hickie screening, (M13f:TGTAAAACGACGGCCAGT, as shown in SEQ ID No.3 to choose positive colony bacterium universal primer M13; M13r:CAGGAAACAGCTATGACC, as shown in SEQ ID No.4) carry out pcr amplification, and choose positive colony and check order.
Sequence through pcr amplification is about 1500bp, the 16S rRNA PCR primer of L2 bacterial strain is after order-checking, Blast gene comparision is carried out in GeneBank GenBank in NCBI, result shows, and the homology of L2 bacterial strain and saliva lactobacillus (Lactobacillus salivarius) 16S rRNA sequence reaches 99%.Combining form feature, can be judged to be of the same race.The 16SrRNA sequence of L2 bacterial strain is 1475bp (Figure 12), makes a concrete analysis of as shown in SEQ ID No.5.Evolutionary tree (Figure 13) shows to have very high homology from people, chicken, pig GI saliva lactobacillus.
Research report, milk-acid bacteria is (ROBINSONA P H while raising body growth performance, ERASMUS LJ.Effects of analyzable diet components on responses of lactating dairy cows to Saccharomycescerevisiae based yeast products:A systematic review of the literature [J] .Anim Feed SciTechnol, 2009, 149:185-198), obvious reducing effect is had in the field planting level at each position of enteron aisle to enterobacteriaceae lactobacteriaceae, as Van Schie reports, the lactic acid that lactobacillus metabolism produces and voltaile fatty acid can reduce field planting level (the Magnusson J of Salmonellas at enteron aisle, Strum K, Roos S, et a1.Broad and complex antifungal activity amongenvironmental isolates of lactic acid bacteria [J] .FEMS Microbiol Lett, 2003, 219:129-135), and then have influence on the balance of intestinal microflora.The meta-bolitess such as lactobacillin, lactic acid, propionic acid are that milk-acid bacteria secretes generation in metabolic process, at present to the acidic substance of the more lactobacillin of the research of milk-acid bacteria antagonism pathogenic bacterium mechanism and generation thereof.Compared with L4, L10 bacterial strain, lactobacillus L2 bacterial strain has relevant with its stronger acid producing ability compared with the mechanism of action of high inhibition effect to intestinal bacteria ATCC25922.Pediocin is the one of the bacteriocin that milk-acid bacteria produces, and finds its three-dimensional structure research, and its N holds middle part to form unique YGNGVxCxxXXCXV aminoacid sequence, and C holds α spiral then closely related with its anti-microbial activity.
Claims (10)
1. an acid and bile salt tolerance lactic bacilli strains, it is characterized in that: described bacterial strain is saliva lactobacillus (Lactobacillussalivarius) L2, be preserved in China typical culture collection center, deposit number is: CCTCC NO:M 2014614.
2. acid and bile salt tolerance lactic bacilli strains according to claim 1, is characterized in that: described bacterial strain filters out from poultry intestinal tract content.
3. a screening method for acid and bile salt tolerance lactic bacilli strains as claimed in claim 1, is characterized in that: comprise the following steps:
1) strains separation purifying: get healthy poultry intestinal tract content, after sterilized water dilution, be coated on lactobacillus selective medium and carry out Anaerobic culturel, isolate single bacterium colony with molten calcium circle, purifying of repeatedly ruling, obtains purifying bacterium;
2) step 1 is got) gained purifying bacterium makes bacterium liquid, and be placed in acid MRS substratum respectively, MRS substratum containing bovine bile carries out Anaerobic culturel, obtain dominant strain;
3) detecting step 2) gained dominant strain to the In Vitro Bacteriostatic of intestinal bacteria and Salmonellas, the lactobacillus of Chinese People's Anti-Japanese Military and Political College enterobacteria, Salmonellas activity must be had.
4. the screening method of acid and bile salt tolerance lactic bacilli strains according to claim 3, it is characterized in that: step 1) in, the lactobacillus selective medium described in 1L comprises following component: peptone 10g, beef extract powder 10g, yeast leaching powder 2g, dipotassium hydrogen phosphate 2g, sodium acetate 5g, Citric Acid three ammonium 2g, magnesium sulfate 0.2g, manganous sulfate 0.05g, tween 80 1g, glucose 20g, calcium carbonate 5g, agar 25g.
5. the screening method of acid and bile salt tolerance lactic bacilli strains according to claim 3, is characterized in that: step 1) gained purifying bacterium forms neat in edge, smooth surface, the oyster white circular colonies that has molten calcium circle, reguarity homogeneous on described lactobacillus selective medium; This purifying bacteria strain is carried out gramstaining, and oily Microscopic observation is shaft-like gram-positive microorganism, single or in short catenation.
6. the screening method of acid and bile salt tolerance lactic bacilli strains according to claim 3, it is characterized in that: step 2) in, the MRS substratum described in 1L comprises following component: beef extract 10g, peptone 10g, yeast extract 5g, glucose 20g, sodium-acetate 5g, dibasic ammonium citrate 2g, tween 80 0.1g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganous sulfate 0.28g.
7. the screening method of acid and bile salt tolerance lactic bacilli strains according to claim 6, is characterized in that: step 2) in, described contains in the MRS substratum of bovine bile, and the concentration of bovine bile is 3.0g/L.
8. the screening method of the acid and bile salt tolerance lactic bacilli strains according to claim 6 or 7, is characterized in that: step 2) in, in described bacterium liquid, the concentration of lactobacillus is 2.0 × 10
8cfu/ml; 300 μ l bacterium liquid correspondences add containing in the MRS substratum of bovine bile of 5.0ml.
9. the screening method of acid and bile salt tolerance lactic bacilli strains according to claim 3, is characterized in that: step 3) in, described intestinal bacteria are standard E. coli strains A TCC25922; Described Salmonellas is Salmonella enteritidis.
10. the application of acid and bile salt tolerance lactic bacilli strains as claimed in claim 1 in the probiotics for the preparation of control poultry bacterial digestive tract diseases.
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