CN104946558A - Pediococcus pentosaceus strain P9-5, and screening identification and application thereof - Google Patents
Pediococcus pentosaceus strain P9-5, and screening identification and application thereof Download PDFInfo
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention discloses a Pediococcus pentosaceus strain which is collected by China General Microbiological Culture Collection Center, and the collection number is CGMCC No.10573. The invention also discloses application of the Pediococcus pentosaceus strain in resisting Listeria monocytogenes. Separation purification and screening of lactobacillus, and primary screening and rescreening of the Listeria monocytogenes strain are performed to obtain the safe and effective Listeria monocytogenes antagonistic strain. When being used in the stage with high tendency to contamination by the Listeria monocytogenes, the Listeria monocytogenes antagonistic strain can enhance the Listeria monocytogenes control level in food, provides a reliable and effective detection means for Listeria monocytogenes examination and quarantine inspection in import and export food, lowers the risk in imports and exports, and promotes the import and export trade and economic development.
Description
Technical field
The present invention relates to a strain microorganism, Screening and Identification and application, particularly a strain Pediococcus pentosaceus P9-5, Screening and Identification and the application in anti-Listeria Monocytogenes, belong to detection and the applied technical field of microorganism.
Background technology
Listeria Monocytogenes (Listeria monocytogenes) is extensively present in occurring in nature, meat, eggs, bird, sea-food, milk-product, vegetables etc. have been proved to be single source of infection increasing listeria spp all, this bacterium still can growth and breeding in the environment of 4 DEG C, very strong to the adaptive faculty of environment and chemical factors, high temperature resistant, acidproof, low temperature resistant, be that refrigerated food threatens one of the main pathogenic fungi of human health, after infecting, main manifestations is septicemia, meningitis and monocytosis.
The poisoning caused along with a lot of Listeria Monocytogenes occurred in world wide in recent years and the harm brought therefrom, making the list in prevention and corntrol food increase listeria spp becomes the focus that people pay close attention to.Find under study for action, some milk-acid bacteria has inhibit activities to Listeria Monocytogenes.Milk-acid bacteria is a class can produce the gram-positive microorganism of a large amount of lactic acid general designation from fermentability carbohydrate, it is the dominant bacteria in humans and animals normal intestinal flora, not only there is many useful physiological functions, and the antibacterial substances such as organic acid, hydrogen peroxide and bacteriocin can be produced.Bacteriocin be some bacterium in metabolic process by Ribosome biogenesis mechanism produce the polypeptide with bacteriostatic activity or Precursor Peptide, producing strains has immunization to the bacteriocin that himself is secreted.Find under study for action, some bacteriocin lab has strong restraining effect to single listeria spp that increases, and therefore this kind of bacteriocin causes people and pays close attention to especially, and these bacteriocins all belong to II a bacterioid element usually.Its is economical, effectively and can not cause secondary pollution to environment, be expected to pollute Listeria Monocytogenes from source controlled.
Up to the present, domesticly shaping, economic biotechnological formulation product is not also developed.The good means of one that biotechnological formulation controls as Listeria Monocytogenes are badly in need of being researched and developed.
Summary of the invention
Technical problem to be solved by this invention is for above deficiency, provides a strain Pediococcus pentosaceus P9-5 and Screening and Identification thereof and application, realizes following object:
1, by the lactic bacterium strains of the anti-Listeria Monocytogenes of screening; The screening operation of anti-Listeria Monocytogenes bacterial strain, sets up the screening method of reasonable, effective Listeria Monocytogenes antagonism lactobacillus strain.
2, the prevention and control level that in food and environment, Listeria Monocytogenes pollutes is improved.
3, more more options are provided for controlling Listeria Monocytogenes.
4, Pediococcus pentosaceus P9-5 Listeria Monocytogenes to antagonistic action is provided.
5, for the Listeria Monocytogenes inspection and quarantine of food of entering and leaving the border provides reliable and effective detection means.
This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on March 6th, 2015, be called for short CGMCC, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC No.10573, Classification And Nomenclature: Pediococcus pentosaceus (Pediococcus pentosaceus).
For solving the problems of the technologies described above, the present invention is by the following technical solutions: a strain Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC No.10573.
Based on the above Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5, the invention provides the application of described Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5 in anti-Listeria Monocytogenes.
Based on the above Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5, the screening method of described Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5, is characterized in that, comprise the following steps:
Step a: the separation and purification of milk-acid bacteria;
Step b: the screening of milk-acid bacteria;
Step c: the primary dcreening operation of anti-Listeria Monocytogenes bacterial strain;
Steps d: the multiple sieve of anti-Listeria Monocytogenes bacterial strain.
A kind of prioritization scheme, described step a comprises: take sample 25g, adds 225ml MRS liquid nutrient medium, cultivates 48h for 37 DEG C, mixes rear respectively with micropipet absorption 1ml nutrient solution normal saline dilution 10
-1-10
-6six gradients, get 10
-4, 10
-5, 10
-6three each 0.1ml of concentration are coated with MRS flat board, cultivate 24h, picking list bacterium colony purifying for 37 DEG C.
Further, described step b comprises: adopt the lactic acid content in the bacterium colony fermented liquid after rp-hplc determination purifying;
Bacterium colony after picking purifying is in MRS liquid nutrient medium, after 37 DEG C of cultivation 48h, get fermented liquid 5mL, the centrifugal 10min of 8000r/min, removing thalline, the aqueous sulfuric acid adding 5mL 0.01mol/L carries out acidolysis, centrifugal removing calcium sulfate, with the membrane filtration of 0.22 μm, get 1mL filtrate and dilute 10 times and namely obtain liquid to be measured and keep supplying machine testing;
By detect produce lactic acid purifying after bacterium colony carry out gram stain microscopy and hydrogen peroxide experiment, choose Gram-positive catalase negative and be required milk-acid bacteria and preserve.
Further, described step c comprises: Listeria Monocytogenes is inoculated into TSA flat board, cultivates 24h for 30 DEG C;
On picking TSA flat board single increase listeria spp bacterium colony in TSB broth culture 37 DEG C cultivate 18 hours, get after 1mL dilutes 50 times, getting 100 μ L, to be coated with TSB dull and stereotyped;
By the lactobacillus strain filtered out, be inoculated in 37 DEG C of cultivation 48h in MRS broth culture, placement 4 Oxford cups on each TSB flat board, one of them Oxford cup adds 100 μ l MRS broth cultures as blank, other three Oxford cups add 100 μ l bacterium liquid respectively, be placed in 30 DEG C of incubators and cultivate 48h, around viewing test bacterial strain, whether have inhibition zone.
Further, described steps d comprises: select the lactobacillus strain having bacteriostatic activity in primary dcreening operation process, after access MRS broth culture cultivates 3d, and the centrifugal 10min of 8000r/min;
With pH to the 6.5-7.0 of 1mol/L NaOH solution adjustment fermented liquid;
Single increase listeria spp in TSB substratum 37 DEG C cultivate 18 hours, be inoculated in 10ml TSB liquid nutrient medium by 1% inoculum size, add streptococcus acidi lactici fermented solution, be placed in 35 DEG C of cultivations, cultured continuously 8h, measure nutrient solution OD value at regular intervals, increase Listeria bacteria culture fluid for contrast with the list not adding streptococcus acidi lactici fermented solution.
Through qualification, the gene order recorded is:
1 acgctggcgg cgtgcctaat acatgcaagt cgaacgaact tccgttaatt gattatgacg
61 tacttgtact gattgagatt ttaacacgaa gtgagtggcg aacgggtgag taacacgtgg
121 gtaacctgcc cagaagtagg ggataacacc tggaaacaga tgctaatacc gtataacaga
181 gaaaaccgca tggttttctt ttaaaagatg gctctgctat cacttctgga tggacccgcg
241 gcgtattagc tagttggtga ggtaaaggct caccaaggca gtgatacgta gccgacctga
301 gagggtaatc ggccacattg ggactgagac acggcccaga ctcctacggg aggcagcagt
361 agggaatctt ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggg
421 tttcggctcg taaagctctg ttgttaaaga agaacgtggg taagagtaac tgtttaccca
481 gtgacggtat ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt
541 aggtggcaag cgttatccgg atttattggg cgtaaagcga gcgcaggcgg tcttttaagt
601 ctaatgtgaa agccttcggc tcaaccgaag aagtgcattg gaaactggga gacttgagtg
661 cagaagagga cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca
721 ccagtggcga aggcggctgt ctggtctgca actgacgctg aggctcgaaa gcatgggtag
781 cgaacaggat tagataccct ggtagtccat gccgtaaacg atgattacta agtgttggag
841 ggtttccgcc cttcagtgct gcagctaacg cattaagtaa tccgcctggg gagtacgacc
901 gcaaggttga aactcaaaag aattgacggg ggcccgcaca agcggtggag catgtggttt
961 aattcgaagc tacgcgaaga accttaccag gtcttgacat cttctgacag tctaagagat
1021 tagaggttcc cttcggggac agaatgacag gtggtgcatg gttgtcgtca gctcgtgtcg
1081 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta ttactagttg ccagcattaa
1141 gttgggcact ctagtgagac tgccggtgac aaaccggagg aaggtgggga cgacgtcaaa
1201 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggatggta caacgagtcg
1261 cgagaccgcg aggttaagct aatctcttaa aaccattctc agttcggact gtaggctgca
1321 actcgcctac acgaagtcgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac
1381 gttcccgggc cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccaaagccg
1441 gtggggtaac cttttaggag ctagccgtct aaggtgg。
The present invention adopts above technical scheme, there is following technique effect: the novel method being established screening lactobacillus by the mode adopting microbial process to combine with chemical process, by Oxford cup primary dcreening operation and multiple sieve, from soil, filter out the milk-acid bacteria that a strain has antagonism Listeria Monocytogenes.Set up the screening method of reasonable, effective Listeria Monocytogenes antagonistic strain, obtain the lactobacillus strain being applicable to needs.By separation and purification and the screening of milk-acid bacteria; Primary dcreening operation and the multiple riddler of anti-Listeria Monocytogenes bacterial strain do, and screening obtains single increasing listeria spp antagonistic strain safely and effectively.The present invention is applied to easy coverlet in foodstuffs industry production and environment and increases the stage of Listeria fungi pollution, single increasing listeria spp prevention and control level in food can be improved, for the Listeria Monocytogenes inspection and quarantine of food of entering and leaving the border provides reliable and effective detection means, reduce and import and export risk, promote foreign trade and Economic development.
Below in conjunction with drawings and Examples, the present invention is described in detail.
Accompanying drawing explanation
Fig. 1 is lactate standard product 2ppm color atlas;
Fig. 2 is lactate standard product and detection sample chromatogram stacking diagram;
Fig. 3 is the lactic acid chromatographic peak figure detected in Fig. 2;
Fig. 4 is the bacteriostatic action of bacterial strain P9-5 to Listeria Monocytogenes;
Fig. 5 is the Biolog qualification result of bacterial strain P9-5.
Embodiment
Embodiment, a strain Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC No.10573.
The screening method of above Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5, comprises the following steps:
1 experiment material
1.1 experimental strain
Sample source
The soil sample of different location is gathered from the area such as Weifang, Yantai
1.2 laboratory apparatus
Vertical pressure steam sterilizer (Japanese Hirayama HVE-50); Biohazard Safety Equipment (XUN BSC-1300 II A/B3); Microscope (OLYMPUS CX21); High performance liquid chromatograph (Aglient 1200), analytical balance (Switzerland Mettler Toledo PL 602-S); Constant incubator (German MMM Incucell 111); Liquid-transfering gun (German Eppendorf); Table model high speed centrifuge (German Eppendorf 5417R);
1.3 substratum
MRS meat soup: be purchased from Beijing road and bridge technology limited liability company, article No. CM187.Take 52.3g powder and add 1L water, heated and boiled is extremely dissolved completely, packing.115 DEG C of autoclaving 15min.
MRS agar: be purchased from Beijing road and bridge technology limited liability company, article No. CM188.Take 64.25g powder and add 1L water, heated and boiled is to dissolving completely.121 DEG C of autoclaving 15min.Be cooled to about 46 DEG C and be down flat plate or inclined-plane.
TSB substratum: be purchased from Beijing road and bridge technology limited liability company, article No. CM501.Take 36g powder and add 1L water, heated and boiled is to dissolving completely.121 DEG C of autoclaving 15min.
TSA substratum: be purchased from Beijing road and bridge technology limited liability company, article No. CM502A.Take 51g powder and add 1L water, heated and boiled is to dissolving completely.121 DEG C of autoclaving 15min.Be cooled to about 46 DEG C and be down flat plate or inclined-plane.
1.4 strains testeds:
Listeria Monocytogenes (Listeria monocytogenes)
2 experimental techniques
The separation and purification of 2.1 milk-acid bacterias and screening
Take sample 25g, add 225ml MRS liquid nutrient medium, cultivate 48h for 37 DEG C, mix rear respectively with micropipet absorption 1ml nutrient solution normal saline dilution 10
-1-10
-6six gradients, get 10
-4, 10
-5, 10
-6three each 0.1ml of concentration are coated with MRS flat board, cultivate 24h, picking list bacterium colony purifying for 37 DEG C.
Adopt the lactic acid content in the bacterium colony fermented liquid after rp-hplc determination purifying.Bacterium colony after picking purifying is in MRS liquid nutrient medium, after 37 DEG C of cultivation 48h, get fermented liquid 5mL, the centrifugal 10min of 8000r/min, removing thalline, the aqueous sulfuric acid adding 5mL 0.01mol/L carries out acidolysis, centrifugal removing calcium sulfate, with the membrane filtration of 0.22 μm, get 1mL filtrate and dilute 10 times and namely obtain liquid to be measured and keep supplying machine testing.
Chromatographic condition
A) chromatographic column: Aglient ZORBAX SB-C18,5 μm, 4.6 × 250mm
B) moving phase: 0.1% phosphate aqueous solution (pH2.0), acetonitrile
C) flow velocity: phosphoric acid water: acetonitrile 5:95,0.3mL/min.
D) column temperature: room temperature
E) wavelength: 210nm
F) sample size: 10 μ L
By detect produce lactic acid purifying after bacterium colony carry out gram stain microscopy and hydrogen peroxide experiment, choose Gram-positive catalase negative and be required milk-acid bacteria and preserve.
The screening of 2.2 anti-Listeria Monocytogenes strains
Listeria Monocytogenes is inoculated into TSA flat board, cultivates 24h for 30 DEG C.On picking TSA flat board single increase listeria spp bacterium colony in TSB broth culture 37 DEG C cultivate 18 hours, get after 1mL dilutes 50 times, getting 100 μ L, to be coated with TSB dull and stereotyped.By the lactobacillus strain filtered out, be inoculated in 37 DEG C of cultivation 48h in MRS broth culture, placement 4 Oxford cups on each TSB flat board, one of them Oxford cup adds 100 μ lMRS broth cultures as blank, other three Oxford cups add 100 μ l bacterium liquid respectively, be placed in 30 DEG C of incubators and cultivate 48h, around viewing test bacterial strain, whether have inhibition zone.
The multiple sieve of 2.3 anti-Listeria Monocytogenes strains
Select the lactobacillus strain having bacteriostatic activity in primary dcreening operation process, after access MRS culture medium culturing 3d, the centrifugal 10min of 8000r/min.With pH to the 6.5-7.0 of 1mol/L NaOH solution adjustment fermented liquid.Single increase listeria spp in TSB substratum 37 DEG C cultivate 18 hours, be inoculated in 10ml TSB liquid nutrient medium by 1% inoculum size, add streptococcus acidi lactici fermented solution, be placed in 35 DEG C of cultivation, cultured continuously 8h, at regular intervals mensuration nutrient solution OD600nm.Listeria bacteria culture fluid is increased for contrast with the list not adding streptococcus acidi lactici fermented solution.
2.4 identification of strains
In MRS flat board picking thalline in 50ul TaKaRa Lysis Buffer for Microorganism to Direct PCR (Code No.9164) after sex change centrifuging and taking supernatant as template.Reaction conditions: 80 DEG C, 15min.Use TaKaRa 16S rDNA Bacterial Identification PCR Kit(Code No.RR176), carry out pcr amplification object fragment.Reaction conditions: 94 DEG C of 5min; 94 DEG C of 1min, 55 DEG C of 1min, 72 DEG C of 1.5min, totally 30 circulations; 72 DEG C of 5min.
Picking bacterial classification pure growth list bacterium colony, is inoculated in BUG culture medium flat plate, 37 DEG C of constant temperature culture 24h, CO
2concentration is 6.5%, switching 1-2 generation.Aseptically, use Inoculatorz cotton swab to pick diameter from BUG flat board and be about the colony inoculation of 3mm in IF-C inoculation liquid, turbidity is adjusted to 90-98%.Bacteria suspension is poured in V-type application of sample tank, use 8 road pipettors bacteria suspension is sequentially added into microwell plate institute porose in, every hole 100 μ L.Microwell plate 37 DEG C of constant temperature culture 16-48h, use Biolog GenIII MicroStation automatic microbe identification systems to identify.
Biolog microbial identification system utilizes microorganism to carry out the difference of respiratory metabolism to different carbon source, and Biolog GenIII microwell plate carries out 94 kinds of phenotype tests (71 kinds of utilization of carbon source, 23 kinds of chemosensitivity tests) to microorganism.By the differences in turbidity (turbidity) detecting in microbial metabolism colour-change (absorbancy) that the redox materials that produces and four azole materials (as TTC, TV) react and cause and cause due to microorganism growth, generating feature finger printing, compare with reference culture spectrum data storehouse, can qualification result be drawn.
3 experimental results and analysis
The screening of 3.1 milk-acid bacterias
By the detection of high performance liquid chromatography to the lactic acid in ferment product after purifying, and gramstaining and hydrogen peroxide testing sieve select milk-acid bacteria.Detected result is shown in Fig. 1, Fig. 2, Fig. 3.
The screening of 3.2 anti-Listeria Monocytogenes bacterial strains
Filtered out 3 strains of lactic acid bacteria by Odontothrips loti and had certain bacteriostatic action to Listeria Monocytogenes, wherein 1 strain restraining effect is obvious, and the present invention has carried out primary study to it.The results are shown in Table 1.
The anti-single increasing Listeria bacteria strain primary dcreening operation result of table 1.
| Strain number | Bacteriostatic activity |
| P9-5 | ＋＋＋ |
| L4-3 | ＋＋ |
| L9-7 | ＋ |
The multiple sieve of 3.3 anti-Listeria Monocytogenes bacterial strains
Joining selecting the good lactobacillus strain fermented liquid of bacteriostatic activity in primary dcreening operation process in the substratum containing single increasing listeria spp, measuring nutrient solution OD600 value every 2h.Listeria bacteria culture fluid is increased for contrast with the list not adding streptococcus acidi lactici fermented solution.The growth of bacterial strain P9-5 to Listeria Monocytogenes has obvious restraining effect.After mixing with bacterial strain P9-5 fermented liquid, the growth of Listeria Monocytogenes is subject to obvious suppression.The results are shown in Figure 4.
3.4 identification of strains
Extract bacterial strain P9-5 genomic dna, increase its 16S rDNA gene order, checks order.
Use Biolog GenIII MicroStation automatic microbe identification systems to identify bacterial strain P9-5, draw qualification result, see Fig. 5.
The gene order recorded is:
1 acgctggcgg cgtgcctaat acatgcaagt cgaacgaact tccgttaatt gattatgacg
61 tacttgtact gattgagatt ttaacacgaa gtgagtggcg aacgggtgag taacacgtgg
121 gtaacctgcc cagaagtagg ggataacacc tggaaacaga tgctaatacc gtataacaga
181 gaaaaccgca tggttttctt ttaaaagatg gctctgctat cacttctgga tggacccgcg
241 gcgtattagc tagttggtga ggtaaaggct caccaaggca gtgatacgta gccgacctga
301 gagggtaatc ggccacattg ggactgagac acggcccaga ctcctacggg aggcagcagt
361 agggaatctt ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggg
421 tttcggctcg taaagctctg ttgttaaaga agaacgtggg taagagtaac tgtttaccca
481 gtgacggtat ttaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt
541 aggtggcaag cgttatccgg atttattggg cgtaaagcga gcgcaggcgg tcttttaagt
601 ctaatgtgaa agccttcggc tcaaccgaag aagtgcattg gaaactggga gacttgagtg
661 cagaagagga cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca
721 ccagtggcga aggcggctgt ctggtctgca actgacgctg aggctcgaaa gcatgggtag
781 cgaacaggat tagataccct ggtagtccat gccgtaaacg atgattacta agtgttggag
841 ggtttccgcc cttcagtgct gcagctaacg cattaagtaa tccgcctggg gagtacgacc
901 gcaaggttga aactcaaaag aattgacggg ggcccgcaca agcggtggag catgtggttt
961 aattcgaagc tacgcgaaga accttaccag gtcttgacat cttctgacag tctaagagat
1021 tagaggttcc cttcggggac agaatgacag gtggtgcatg gttgtcgtca gctcgtgtcg
1081 tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta ttactagttg ccagcattaa
1141 gttgggcact ctagtgagac tgccggtgac aaaccggagg aaggtgggga cgacgtcaaa
1201 tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggatggta caacgagtcg
1261 cgagaccgcg aggttaagct aatctcttaa aaccattctc agttcggact gtaggctgca
1321 actcgcctac acgaagtcgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac
1381 gttcccgggc cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccaaagccg
1441 gtggggtaac cttttaggag ctagccgtct aaggtgg。
All above-mentioned be the primary implementation method of this intellecture property, setting restriction does not implement this novel method and/or product innovation with other forms.Those skilled in the art will utilize this important information, to foregoing amendment, to realize similar implementation status.But, all based on amendment of the present invention or transformation novel method, belong to the right of reservation.
The above is only preferred embodiment of the present invention, and be not restriction the present invention being made to other form, any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the Equivalent embodiments of equivalent variations.But everyly do not depart from technical solution of the present invention content, any simple modification, equivalent variations and the remodeling done above example according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.
Claims (7)
1. a strain Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC No.10573.
2. the application of Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5 in anti-Listeria Monocytogenes as claimed in claim 1.
3. a screening method of Pediococcus pentosaceus (Pediococcus pentosaceus) P9-5 as claimed in claim 1, is characterized in that, comprise the following steps:
Step a: the separation and purification of milk-acid bacteria;
Step b: the screening of milk-acid bacteria;
Step c: the primary dcreening operation of anti-Listeria Monocytogenes bacterial strain;
Steps d: the multiple sieve of anti-Listeria Monocytogenes bacterial strain.
4. a screening method as claimed in claim 3, it is characterized in that, described step a comprises: take sample 25g, adds 225ml MRS liquid nutrient medium, cultivate 48h for 37 DEG C, mix rear respectively with micropipet absorption 1ml nutrient solution normal saline dilution 10
-1-10
-6six gradients, get 10
-4, 10
-5, 10
-6three each 0.1ml of concentration are coated with MRS flat board, cultivate 24h, picking list bacterium colony purifying for 37 DEG C.
5. a screening method as claimed in claim 4, is characterized in that, described step b comprises: adopt the lactic acid content in the bacterium colony fermented liquid after rp-hplc determination purifying;
Bacterium colony after picking purifying is in MRS liquid nutrient medium, after 37 DEG C of cultivation 48h, get fermented liquid 5mL, the centrifugal 10min of 8000r/min, removing thalline, the aqueous sulfuric acid adding 5mL 0.01mol/L carries out acidolysis, centrifugal removing calcium sulfate, with the membrane filtration of 0.22 μm, get 1mL filtrate and dilute 10 times and namely obtain liquid to be measured and keep supplying machine testing;
By detect produce lactic acid purifying after bacterium colony carry out gram stain microscopy and hydrogen peroxide experiment, choose Gram-positive catalase negative and be required milk-acid bacteria and preserve.
6. a screening method as claimed in claim 5, is characterized in that, described step c comprises: Listeria Monocytogenes is inoculated into TSA flat board, cultivates 24h for 30 DEG C;
On picking TSA flat board single increase listeria spp bacterium colony in TSB broth culture 37 DEG C cultivate 18 hours, get after 1mL dilutes 50 times, getting 100 μ L, to be coated with TSB dull and stereotyped;
By the lactobacillus strain filtered out, be inoculated in 37 DEG C of cultivation 48h in MRS broth culture, placement 4 Oxford cups on each TSB flat board, one of them Oxford cup adds 100 μ l MRS broth cultures as blank, other three Oxford cups add 100 μ l bacterium liquid respectively, be placed in 30 DEG C of incubators and cultivate 48h, around viewing test bacterial strain, whether have inhibition zone.
7. a screening method as claimed in claim 6, is characterized in that, described steps d comprises: select the lactobacillus strain having bacteriostatic activity in primary dcreening operation process, after access MRS broth culture cultivates 3d, and the centrifugal 10min of 8000r/min;
With pH to the 6.5-7.0 of 1mol/L NaOH solution adjustment fermented liquid;
Single increase listeria spp in TSB substratum 37 DEG C cultivate 18 hours, be inoculated in 10ml TSB liquid nutrient medium by 1% inoculum size, add streptococcus acidi lactici fermented solution, be placed in 35 DEG C of cultivations, cultured continuously 8h, measure nutrient solution OD value, increase Listeria bacteria culture fluid for contrast with the list not adding streptococcus acidi lactici fermented solution.
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