CN109988732A - A strain of Weissella enterica and its application - Google Patents

Abstract

The invention discloses Weissella paramesenteroides JT13 with the deposit number of CGMCC No. 17539, and the preparation of medicines for inhibiting Penicillium digitatum, especially prevention of the Weissella paramesenteroides. Application in medicine for postharvest green mildew of citrus fruits caused by Penicillium digitorum. The invention not only enriches the resource pool of lactic acid bacteria, broadens the application scope of lactic acid bacteria in the field of fresh-keeping of fruits and vegetables, but also provides a new solution for the prevention and control of postharvest green mildew of citrus fruits.

Description

A strain of Weissella enterica and its application

technical field

The present invention relates to a strain of Weissella enterica, and also relates to the application of the Weissella enterica.

Background technique

Lactic acid bacteria are a class of Gram-positive bacilli or cocci that can ferment sugars and the main product is lactic acid. They have a good inhibitory effect on some Gram-positive or negative bacteria that are closely related to them. Fresh-keeping and other fields. However, there are few reports on the inhibitory effect of lactic acid bacteria on fungi.

Citrus is a citrus plant of the Rutaceae Citrus subfamily, and citrus fruit is the most productive fruit in the world. It is prone to rot, edema and floating skin during storage due to microbial infection and the impact of the storage environment, which seriously affects the commercial value of the fruit. . Green mold caused by the fungus Penicillium digitatum is a major invasive disease of citrus fruits during storage. At present, chemical fungicides such as carbendazim, thiophanate-methyl, and imazalil are mainly used for postharvest disease control of citrus fruits, but there are problems such as residual chemical fungicides and microbial resistance to them.

SUMMARY OF THE INVENTION

The purpose of the present invention is to screen and identify the lactic acid bacteria that has the effect of inhibiting Penicillium digitatum, especially can prevent postharvest green mildew of citrus fruit from citrus orchards, so as to enrich the resource pool of lactic acid bacteria and widen the application scope of lactic acid bacteria in the field of fruit and vegetable preservation , and at the same time provide a new solution for the prevention and control of postharvest green mildew in citrus fruits.

After research, the present invention provides the following technical solutions:

1. Weissella paramesenteroides JT13, the deposit number is CGMCCNo.17539.

2. The application of Weissella paramesenteroides JT13 in the preparation of a medicine for inhibiting Penicillium digitatum.

Further, the application of Weissella paramesenteroides JT13 in the preparation of a medicament for preventing postharvest green mildew on citrus fruits caused by Penicillium digitatum.

The Weissella paramesenteroides JT13 of the present invention was deposited on April 10, 2019 in the General Microorganism Center of the China Microorganism Culture Collection Management Committee (CGMCC for short, address: No. 1, Beichen West Road, Chaoyang District, Beijing) No. 3), the deposit number is CGMCC No.17539.

The beneficial effects of the present invention are as follows: the present invention screens out the lactic acid bacteria from the citrus orchard that has the effect of inhibiting Penicillium digitatum, especially can prevent the postharvest green mildew of citrus fruits caused by it, which not only enriches the resource pool of lactic acid bacteria, but also The scope of application of lactic acid bacteria in the field of fruit and vegetable preservation is broadened, and a new solution is provided for the prevention and control of postharvest green mildew in citrus fruits.

Description of drawings

Figure 1 shows the in vitro antibacterial effect of lactic acid bacteria on Penicillium fingertips.

Figure 2 is the in vitro antibacterial effect of lactic acid bacteria aseptic fermentation broth on Penicillium digitorum.

Figure 3 shows the control effect of lactic acid bacteria aseptic fermentation broth on postharvest green mildew disease of citrus fruits caused by Penicillium fingertips. There is no significant difference between the groups marked with the same lowercase English letters (a, b, c) in the figure (p >0.05); there is a significant difference (p<0.05) between groups marked with different lowercase English letters (a, b, c).

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

1. Screening of lactic acid bacteria

Citrus fruits, leaves, soil and peanut leaves were collected from Jinyun Mountain Citrus Orchard in Beibei District, Chongqing City. Each sample was completely immersed in 100 mL of MRS broth medium, enriched and cultured at 37 °C for 24 h, 10-fold serially diluted and spread on MRS plates containing 0.3% CaCO ₃ , incubated at 37 °C for 48 h, and selected with obvious calcium solubility. Individual colonies in the circle were isolated and purified, and subjected to microscopy and Gram staining. The purified strains were stored in -80°C refrigerator.

The results are shown in Table 1. A total of 68 suspected strains were screened in the experiment, of which 16 were from citrus fruits, 6 from citrus leaves, 24 from citrus orchard soil, and 22 from peanut leaves. Spherical and rod-shaped were observed under microscope. Both morphologies were Gram-positive.

Table 1 Screening of lactic acid bacteria

2. In vitro antibacterial effect of lactic acid bacteria on Penicillium digitorum

The 68 strains screened out were confirmed by the double-layer plate method to determine their antibacterial effect on Penicillium digitorum in vitro.

Pick a loop of each suspected strain and inoculate it in 20mL MRS broth medium, cultivate at 37°C for 48h to obtain seed solution; inoculate 1mL seed solution into 100mL MRS broth medium, inoculate at 37°C for 48h; then use an inoculating needle Draw two lines with a length of 2 cm in parallel in the middle of the MRS plate, and incubate the plate at 37°C for 48 hours; then add 10 mL of PDA medium containing 10 ⁴ Penicillium fingertips spores, and after the medium is completely solidified, place the plate at 25°C Culture; observe the antibacterial phenomenon, take the MRS broth medium without inoculated strains as the control, and screen out the strains with higher inhibitory activity against Penicillium digitorum.

The results are shown in Figure 1. A total of 10 strains with high inhibitory activity against Penicillium digitorum were screened in the experiment, of which 7 were from peanut leaves, 2 were from citrus orchard soil, and 1 was from citrus leaves.

3. Identification of lactic acid bacteria

The 10 strains screened out with higher inhibitory activity against Penicillium fingertips were identified by 16S rDNA sequence analysis.

Take the culture liquid and extract the bacterial DNA with a genomic DNA extraction kit; use the universal primer PCR to amplify the 16srDNA sequence, the forward primer is 5'-agagtttgatcctggctcag-3' (SEQ ID No. 1), and the reverse primer is 5' -ctacggctaccttgttacga-3'(SEQ ID No.2); the reaction system includes: 12.5μL 2×Es Taq MasterMix (Dye), 1μL each primer and 1μL DNA template; PCR reaction process is as follows: pre-denaturation, 94°C, 2min; then 30 cycles: denaturation, 94°C, 30s; annealing, 55°C, 30s; extension, 72°C, 30s; final extension, 72°C, 2min. PCR amplification products were detected by 1% agarose gel electrophoresis, and entrusted to Shanghai Sangon Biological Co., Ltd. for sequencing. The sequencing results were analyzed by BLAST program in NCBI for homology comparison.

The results are shown in Table 2. 10 strains with high inhibitory activity against Penicillium digitorum were identified, 8 of which were Lactobacillus sucicola, 1 was Weissella paramesenteroides, and 1 was lactic acid tablet. Coccus (Pediococcus acidilactici).

Table 2 Identification of lactic acid bacteria

The obtained Lactobacillus sucicola strain was named JT03, and was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (abbreviated as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on April 10, 2019, and the preservation number It is CGMCC No.17538. Its 16S rDNA sequence is shown in SEQ ID No.3.

The obtained Weissella paramesenteroides strain was named JT13, and was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (CGMCC for short) on April 10, 2019, address: Beichen West Road, Chaoyang District, Beijing No. 1 Courtyard No. 3), the deposit number is CGMCC No.17539. Its 16S rDNA sequence is shown in SEQ ID No.4.

The obtained Pediococcus acidilactici strain was named JY03, and was deposited in the General Microbiology Center of the China Microorganism Culture Collection Management Committee (CGMCC for short) on April 10, 2019. Address: 3, No. 1, Beichen West Road, Chaoyang District, Beijing No.), the deposit number is CGMCC No.17540. Its 16S rDNA sequence is shown in SEQ ID No.5.

4. In vitro antibacterial effect of lactic acid bacteria aseptic fermentation broth on Penicillium digitorum

The in vitro bacteriostatic effect of the identified three strains of lactic acid bacteria on Penicillium fingertips was investigated.

Inoculate 1 mL of lactic acid bacteria seed liquid into 100 mL of MRS broth medium and cultivate at 37 °C; take out a certain amount of fermentation broth every 2 h, centrifuge at 5000 rpm for 15 min, and pass the supernatant through a 0.22 μm microporous membrane to obtain aseptic fermentation with different incubation times. liquid. Mix 100 μL of fermentation broth with 100 μL of 10 ⁴ Penicillium fingertips spore suspension (containing 5% PDB medium), MRS broth medium without lactic acid bacteria as control, culture at 25°C for 48 h, and measure OD ₆₀₀ value.

The results are shown in Figure 2, the aseptic fermentation broth of the three strains of lactic acid bacteria (JT03, JT13, JY03) had different degrees of inhibitory effect on Penicillium fingertips, and with the prolongation of the culture time, the inhibitory effect gradually increased, and the culture to After 60h, the growth of Penicillium digitorum was completely inhibited.

5. Control effect of lactic acid bacteria aseptic fermentation broth on postharvest green mildew of citrus fruits

The inhibitory effect of the sterile fermentation broth of the identified three strains of lactic acid bacteria on postharvest green mold of citrus fruits was investigated by inoculation experiments in the same hole.

Inoculate 1 mL of lactic acid bacteria seed liquid into 100 mL of MRS broth medium, cultivate at 37°C for 48 h, centrifuge at 5000 rpm for 15 min, and pass the supernatant through a 0.22 μm microporous membrane to obtain a sterile fermentation broth of lactic acid bacteria. Mechanically injured healthy citrus fruits were soaked in 2% sodium hypochlorite solution for 2 minutes for surface disinfection, rinsed with water and air-dried; after the equatorial part of the fruit was disinfected with 70% alcohol, 2 small holes (diameter 3mm, depth 3mm), inoculate 10 μL 10 ⁴ Penicillium dactylus spore suspension, and then inoculate 20 μL of lactic acid bacteria sterile fermentation broth after the liquid is completely absorbed. Take the MRS broth medium without lactic acid bacteria as the control, and after the liquid is completely absorbed The fruits were packaged individually and stored in an environment of 25°C and a relative humidity of 90-95%; the incidence of fruit and the diameter of disease spots were observed and recorded every day.

The results are shown in Figure 3, the aseptic fermentation broth of the three strains of lactic acid bacteria (JT03, JT13, JY03) can inhibit the onset of citrus fruit and the growth of the diameter of the lesions to a certain extent. Among them, the effect of JT03 and JT13 is better.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described with reference to the preferred embodiments of the present invention, those of ordinary skill in the art should Various changes in the above and in the details may be made therein without departing from the spirit and scope of the present invention as defined by the appended claims.

sequence listing

<110> Southwest University

<120> A strain of Weissella enterica and its application

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agagtttgat cctggctcag 20

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ctacggctac cttgttacga 20

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<212> DNA

<213> Lactobacillus sucicola JT03

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ggagggcggc agctatacat gcagtcgtac gcaaaacttt ctctgagtgt tcgcactcac 60

tgaaagattt gagtggcgaa cgggtgagta acacgtgggt aacctgccca gaagaagggg 120

ataacacttg gaaacaggtg ctaataccgt ataacaacaa agaccgcatg gtctttgttt 180

aaaagatggt tctgctatca cttctggatg gacccgcggc gtattagcta gttggtgagg 240

taatggctca ccaaggcaat gatacgtagc cgaactgaga ggttgatcgg ccacattggg 300

actgagacac ggcccaaact cctacgggag gcagcagtag ggaatcttcc acaatggacg 360

aaagtctgat ggagcaacgc cgcgtgagtg aagaaggttt tcggatcgta aaactctgtt 420

gttagagaag aacgtgtgtg agagtaactg ttcatgcagt gacggtatct aaccagaaag 480

ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttgtccggat 540

ttattgggcg taaagggaac gcaggcggtt ttttaagtct gatgtgaaag ccttcggctt 600

aaccgaagtc gtgcattgga aactggaaga cttgagtgca gaagaggaga gtggaactcc 660

atgtgtagcg gtgaaatgcg tagatatatg gaagaacacc agtggcgaaa gcggctctct 720

ggtctgtaac tgacgctgag gttcgaaagc atgggtagca aacaggatta gataccctgg 780

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agctaacgca ttaagcattc cgcctgggga gtacgatcgc aagattgaaa ctcaaaggaa 900

ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960

cttaccaggt cttgacatct tctgccaatc tgagagatca gatgttccct tcggggacaa 1020

gaatgacacg tg 1032

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gcatggttgt tggttgaaag atggttctgc tatcactaag agatggaccc gcggtgcatt 240

agctagttgg taaggtaacg gcttaccaag gcaatgatgc atagccgagt tgagagactg 300

atcggccaca atgggactga gacacggccc atactcctac gggaggcagc agtagggaat 360

cttccacaat gggcgcaagc ctgatggagc aacgccgcgt gtgtgatgaa gggtttcggc 420

tcgtaaaaca ctgttataag agaagaacgg cactgagagt aactgttcag tgtgtgacgg 480

tatcttacca gaaaggaacg gctaaatacg tgccagcagc cgcggtaata cgtatgttcc 540

aagcgttatc cggatttatt gggcgtaaag cgagcgcaga cggttattta agtctgaagt 600

gaaagccctc agctcaactg aggaatggct ttggaaactg gatgacttga gtgcagtaga 660

ggaaagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg 720

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gcccttgagt gtcgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt 900

tgaaactcaa aggaattgac ggggacccgc acaagcggtg gagcatgtgg tttaattcga 960

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aaggatgcgc atgctataca tgcagtcgaa cgaacttccg ttaattgatt atgacgtgct 60

tgcactgaat gagattttaa cacgaagtga gtggcggacg ggtgagtaac acgtgggtaa 120

cctgcccaga agcaggggat aacacctgga aacagatgct aataccgtat aacagagaaa 180

accgcctggt tttcttttaa aagatggctc tgctatcact tctggatgga cccgcggcgc 240

attagctagt tggtgaggta acggctcacc aaggcgatga tgcgtagccg acctgagagg 300

gtaatcggcc acattgggac tgagacacgg cccagactcc tacgggaggc agcagtaggg 360

aatcttccac aatggacgca agtctgatgg agcaacgccg cgtgagtgaa gaagggtttc 420

ggctcgtaaa gctctgttgt taaagaagaa cgtgggtgag agtaactgtt cacccagtga 480

cggtatttaa ccagaaagcc acggctaact acgtgccagc agccgcggta atacgtaggt 540

ggcaagcgtt atccggattt attgggcgta aagcgagcgc aggcggtctt ttaagtctaa 600

tgtgaaagcc ttcggctcaa ccgaagaagt gcattggaaa ctgggagact tgagtgcaga 660

agaggatagt ggaactccat gtgtagcggt gaaatgcgta gatatatgga agaacaccag 720

tggcgaaggc ggctgtctgg tctgtaactg acgctgaggc tcgaaagcat gggtagcgaa 780

caggattaga taccctggta gtccatgccg taaacgatga ttactaagtg ttggagggtt 840

tccgcccttc agtgctgcag ctaacgcatt aagtaatccg cctggggagt acgac 895

Claims (3)

1. Class goldbeater's skin Wei Si Salmonella 1. (Weissella paramesenteroides) JT13, deposit number CGMCC No.17539。
2. 2. class goldbeater's skin Wei Si Salmonella (Weissella paramesenteroides) JT13 described in claim 1 presses down in preparation Application in the drug of Penicillium digitatum (Penicillium digitatum) processed.
3. 3. application as claimed in claim 2, which is characterized in that class goldbeater's skin Wei Si Salmonella (Weissella Paramesenteroides) JT13 prevents the citrus fruit as caused by Penicillium digitatum (Penicillium digitatum) in preparation The application in the drug of rear green mould is adopted in fact.