CN109971687B - Lactobacillus sucicola and application thereof - Google Patents
Lactobacillus sucicola and application thereof Download PDFInfo
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- CN109971687B CN109971687B CN201910368187.3A CN201910368187A CN109971687B CN 109971687 B CN109971687 B CN 109971687B CN 201910368187 A CN201910368187 A CN 201910368187A CN 109971687 B CN109971687 B CN 109971687B
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Abstract
The invention discloses a Lactobacillus succinica JT03 with the preservation number of CGMCC No.17538 and application of the Lactobacillus in preparing a medicament for inhibiting Penicillium digitatum (Penicillium digitatum), in particular to a medicament for preventing postharvest green mold of citrus fruits caused by the Penicillium digitatum. The invention not only enriches the lactobacillus strain resource pool, widens the application range of the lactobacillus in the field of fruit and vegetable preservation, but also provides a new solution for preventing and treating the postharvest green mold of citrus fruits.
Description
Technical Field
The invention relates to a lactic acid bacterium strain and also relates to application of the lactic acid bacterium strain.
Background
Lactic acid bacteria are gram-positive bacilli or cocci which can ferment saccharides and have main products of lactic acid, have good inhibition effect on part of gram-positive or negative bacteria which are relatively close to the gram-positive bacilli or the gram-negative bacilli, and are widely used in the fields of food fermentation, fresh keeping and the like at present. However, the inhibitory action of lactic acid bacteria on fungi has not been reported yet.
Citrus is a plant of Citrus in Citrus subfamily of Rutaceae, and the citrus fruit is the fruit with the highest yield in the world, and is easily rotten, edematous, floating skin and the like under the influence of microorganism infection and storage environment in storage, so that the commodity value of the fruit is seriously influenced. The green mold caused by the fungus Penicillium digitatum is a major invasive disease during storage of citrus fruits. At present, chemical bactericides such as carbendazim, thiophanate methyl and imazalil are mainly adopted for preventing and treating the postharvest diseases of the citrus fruits, but the problems of residue of the chemical bactericides, easy generation of drug resistance of microorganisms to the chemical bactericides and the like exist.
Disclosure of Invention
The invention aims to screen and identify lactic acid bacteria which have the effect of inhibiting penicillium digitatum and can particularly prevent the postharvest green mold of citrus fruits from citrus orchards, enrich the lactic acid bacteria strain resource library, widen the application range of the lactic acid bacteria in the field of fruit and vegetable preservation, and provide a new solution for preventing the postharvest green mold of the citrus fruits.
Through research, the invention provides the following technical scheme:
1. lactobacillus succincola JT03 with the preservation number of CGMCC No. 17538.
2. Use of Lactobacillus succinica JT03 for the manufacture of a medicament for inhibiting Penicillium digitatum.
Further, the application of Lactobacillus succinica JT03 in preparing a medicine for preventing postharvest green mold of citrus fruits caused by Penicillium digitatum (Penicillium digitatum) is provided.
The Lactobacillus sucicola JT03 is preserved in the China general microbiological culture Collection center (CGMCC for short, address: No.3 Xilu-Beijing-oriented region of the Beijing market, North Chen province) within 4 months and 10 days in 2019, and the preservation number is CGMCC No. 17538.
The invention has the beneficial effects that: the invention screens out the lactic acid bacteria which have the effect of inhibiting penicillium digitatum and can especially prevent the postharvest green mold of the citrus fruits caused by the penicillium digitatum from the citrus orchard, enriches the lactic acid bacteria resource library, widens the application range of the lactic acid bacteria in the field of fruit and vegetable preservation, and provides a new solution for preventing and treating the postharvest green mold of the citrus fruits.
Drawings
FIG. 1 shows the in vitro bacteriostatic effect of lactic acid bacteria on Penicillium digitatum.
FIG. 2 shows the in vitro bacteriostatic effect of lactobacillus aseptic fermentation broth on penicillium digitatum.
FIG. 3 shows the control effect of the lactobacillus-fermented sterile liquid on postharvest green mold of citrus fruit caused by Penicillium digitatum, where there is no significant difference (p > 0.05) between the groups marked with the same lower case English letters (a, b, c); there was a significant difference (p <0.05) between the groups labeled with different lower case english letters (a, b, c).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.
1. Screening of lactic acid bacteria
Orange fruits, leaves, soil and peanut leaves are respectively collected from an orange orchard in Jinyun mountain in Beiyun region of Chongqing city as samples. Completely immersing each sample in 100mL MRS broth culture medium, performing enrichment culture at 37 deg.C for 24h, diluting with 10 times gradient, and spreading on a medium containing 0.3% CaCO3Culturing the cells on the MRS plate at 37 ℃ for 48h, selecting single colonies with obvious calcium-dissolving rings, separating and purifying, and performing microscopic examination and gram staining. The purified strain is stored in a refrigerator at-80 ℃.
Results as shown in table 1, 68 suspected strains were screened out in the experiment, wherein 16 strains are from citrus fruits, 6 strains are from citrus leaves, 24 strains are from citrus orchard soil, 22 strains are from peanut leaves, both spherical and rod-shaped forms are observed through microscopic examination, and gram staining is positive.
TABLE 1 screening of lactic acid bacteria
2. In vitro antibacterial effect of lactic acid bacteria on penicillium digitatum
The screened 68 strains are subjected to double-layer plate method to determine the in vitro antibacterial effect on the penicillium digitatum.
Selecting one strain loop of each suspected strain, inoculating the strain loop into 20mL of MRS broth culture medium, and culturing at 37 ℃ for 48h to obtain seed liquid; 1mL of seed liquid is sucked and inoculated in 100mL of MRS broth culture medium, and cultured for 48h at 37 ℃; then, drawing two straight lines with the length of 2cm in parallel in the middle of an MRS plate by using an inoculating needle, and culturing the plate at 37 ℃ for 48 h; then 10mL of 104PDA culture medium of penicillium digitatum spore, after the culture medium is completely solidified, placing the flat plate at 25 deg.C for culture; and observing the bacteriostasis, and taking MRS broth culture medium streaking without inoculating strains as a control to screen out strains with higher inhibitory activity to the penicillium digitatum.
As shown in FIG. 1, 10 strains with higher inhibitory activity against Penicillium digitatum were selected in the experiment, wherein 7 strains were obtained from peanut leaves, 2 strains were obtained from citrus orchard soil, and 1 strain was obtained from citrus leaves.
3. Identification of lactic acid bacteria
The 10 screened strains with higher inhibitory activity to penicillium digitatum were identified by 16S rDNA sequence analysis.
Taking a culture solution, and extracting thallus DNA by adopting a genome DNA extraction kit; amplifying a 16s rDNA sequence by adopting a universal primer PCR, wherein a forward primer is 5'-agagtttgatcctggctcag-3' (SEQ ID No.1), and a reverse primer is 5'-ctacggctaccttgttacga-3' (SEQ ID No. 2); the reaction system comprises: 12.5. mu.L of 2 XEs Taq MasterMix (Dye), 1. mu.L of each primer and 1. mu.L of DNA template; the PCR reaction process is as follows: pre-denaturation at 94 ℃ for 2 min; then 30 cycles: denaturation, at 94 ℃ for 30 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; final elongation, 72 ℃, 2 min. The PCR amplification products were detected by 1% agarose gel electrophoresis, and sequencing was performed by Shanghai Biotech Co., Ltd. and the results of sequencing were analyzed by homology comparison using BLAST program in NCBI.
The results are shown in Table 2, and 10 strains having a higher inhibitory activity against Penicillium digitatum were identified, 8 strains being Lactobacillus succola, 1 strain being Weissella mesenteroides (Weissella parameteroides) and 1 strain being Pediococcus acidilactici.
TABLE 2 identification of lactic acid bacteria
The obtained Lactobacillus sucicola strain is named as JT03, and is preserved in China general microbiological culture Collection center (CGMCC for short, address: No.3 Xilu No.1 Beijing Kogyo Beichen) of China general microbiological culture Collection center (CGMCC), 4 months and 10 days in 2019, and the preservation number is CGMCC No. 17538. The 16S rDNA sequence is shown in SEQ ID No. 3.
The obtained Weissella mesenteroides (Weissella parameteroides) strain is named as JT13, and is preserved in China general microbiological culture Collection center (CGMCC for short, address: No.3 Xilu 1. Beichen of the rising area in Beijing city) in 2019 at 4 months and 10 days, and the preservation number is CGMCC No. 17539. The 16S rDNA sequence is shown in SEQ ID No. 4.
The obtained Pediococcus acidilactici strain is named as JY03, and is preserved in China general microbiological culture Collection center (CGMCC for short, address: Beijing city rising area Beicheng West Lu No.1 Hospital No. 3) 4-10 days in 2019, and the preservation number is CGMCC No. 17540. The 16S rDNA sequence is shown in SEQ ID No. 5.
4. In-vitro antibacterial effect of lactobacillus sterile fermentation liquid on penicillium digitatum
And (3) inspecting the in vitro bacteriostatic effect of the identified 3 strains of lactic acid bacteria on the penicillium digitatum.
1mL of lactobacillus seed liquid is absorbed and inoculated in 100mL of MRS broth culture medium, and the culture is carried out at 37 ℃; taking out a certain amount of fermentation liquor every 2h, centrifuging at 5000rpm for 15min, and passing the supernatant through a 0.22 μm microporous membrane to obtain sterile fermentation liquor with different culture times. Mixing 100 μ L fermentation liquid with 100 μ L104Root of Redfish greenishMixing the mould spore suspension (containing 5% PDB culture medium), culturing at 25 deg.C for 48 hr with MRS broth without lactobacillus as control, and determining OD600The value is obtained.
As shown in FIG. 2, the bacteria-free fermentation broth of 3 strains of lactic acid bacteria (JT03, JT13, JY03) showed various inhibitory effects on Penicillium digitatum, and the inhibitory effects were gradually increased with the increase of the culture time, and the growth of Penicillium digitatum was completely inhibited after the culture time reached 60 hours.
5. Control effect of lactobacillus sterile fermentation liquor on postharvest green mold of citrus fruits
And (3) investigating the inhibition effect of the identified sterile fermentation liquor of the 3 strains of lactic acid bacteria on the postharvest green mold of the citrus fruits through a same-hole inoculation experiment.
Inoculating 1mL of lactobacillus seed liquid into 100mL of MRS culture medium, culturing at 37 ℃ for 48h, centrifuging at 5000rpm for 15min, and passing the supernatant through a 0.22-micrometer microporous membrane to obtain lactobacillus sterile fermentation liquid; selecting healthy citrus fruits with uniform size, consistent maturity and no mechanical injury, soaking in 2% sodium hypochlorite solution for 2min for surface disinfection, washing with water and drying in the air; sterilizing equator part of fruit with 70% ethanol, punching 2 holes (diameter 3mm, depth 3mm) at equal intervals with punch, inoculating 10 μ L104Inoculating 20 μ L of lactobacillus sterile fermentation liquid after the liquid is completely absorbed, packaging the fruit with single fruit by using MRS broth culture medium without lactobacillus as control, and storing at 25 deg.C and relative humidity of 90-95%. The incidence of fruit and lesion diameter were recorded daily.
As shown in FIG. 3, the sterile fermentation broth of 3 strains of lactic acid bacteria (JT03, JT13, JY03) can inhibit the onset of citrus fruits and the increase of lesion diameter to some extent. Of these, JT03 and JT13 work better.
Finally, it is noted that the above-mentioned embodiments illustrate rather than limit the invention, and that, while the invention has been described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Sequence listing
<110> university of southwest
<120> Lactobacillus succincola and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctacggctac cttgttacga 20
<210> 3
<211> 1032
<212> DNA
<213> Lactobacillus sucicola JT03
<400> 3
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
tagtccatgc cgtaaacgat gaatgctaag tgttggaggg tttccgccct tcagtgctgc 840
agctaacgca ttaagcattc cgcctgggga gtacgatcgc aagattgaaa ctcaaaggaa 900
ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960
cttaccaggt cttgacatct tctgccaatc tgagagatca gatgttccct tcggggacaa 1020
gaatgacacg tg 1032
<210> 4
<211> 1034
<212> DNA
<213> Weissella mesenteroides JT13(Weissella parameteroides JT13)
<400> 4
tggcatgcgg tgctatacat gcagtcgaac gctttgtctt taattgatct gacgagcttg 60
ctctgatttg attttatctg acaaagagtg gcgaacgggt gagtaacacg tgggtaacct 120
acctcttagc aggggataac atttggaaac aagtgctaat accgtataat accaacaacc 180
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
cgaaggcggc tttctggact gtaactgacg ttgaggctcg aaagtgtggg tagcaaacag 780
gattagatac cctggtagtc cacaccgtaa acgatgagtg ctagatgttc gagggtttcc 840
gcccttgagt gtcgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt 900
tgaaactcaa aggaattgac ggggacccgc acaagcggtg gagcatgtgg tttaattcga 960
agcaacgcga agaaccttac caggtcttga catcccttgc taatcctaga aataggacgt 1020
tcccttcggg gaca 1034
<210> 5
<211> 895
<212> DNA
<213> Pediococcus acidilactici JY03(Pediococcus acidilactici JY03)
<400> 5
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. Lactic acid bacteriaLactobacillus sucicolaJT03 with preservation number CGMCC No. 17538.
2. The lactic acid bacterium according to claim 1Lactobacillus sucicolaJT03 application in preparation of medicines for inhibiting penicillium digitatum (Penicillium digitatum) The use of (1) in medicine.
3. Use according to claim 2, characterized in that lactic acid bacteriaLactobacillus sucicola JT03 use in preparation of medicine for preventing and treating penicillium digitatum (A) and BPenicillium digitatum) Application of the drug for causing the postharvest green mold of citrus fruits.
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Citations (3)
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| CN105357979A (en) * | 2013-03-13 | 2016-02-24 | 有机平衡有限公司 | Novel lactobacillus strains and the uses thereof |
| KR20170050527A (en) * | 2015-10-30 | 2017-05-11 | 대상 주식회사 | Vegetable Lactobacillus plantarum DSR KF15 having Activities on Antimicrobial And Antifungal for keeping freshness and Use Thereof |
| CN108611299A (en) * | 2018-05-10 | 2018-10-02 | 南京师范大学 | One plant of lactobacillus plantarum for producing bacteriostatic peptide and its application |
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| CN106070576B (en) * | 2016-06-29 | 2020-03-17 | 浙江大学 | Method for inhibiting postharvest disease of fruit by inducing resistance and preparation used in method |
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|---|---|---|---|---|
| CN105357979A (en) * | 2013-03-13 | 2016-02-24 | 有机平衡有限公司 | Novel lactobacillus strains and the uses thereof |
| KR20170050527A (en) * | 2015-10-30 | 2017-05-11 | 대상 주식회사 | Vegetable Lactobacillus plantarum DSR KF15 having Activities on Antimicrobial And Antifungal for keeping freshness and Use Thereof |
| CN108611299A (en) * | 2018-05-10 | 2018-10-02 | 南京师范大学 | One plant of lactobacillus plantarum for producing bacteriostatic peptide and its application |
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| Title |
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| "Production and Characterization of Antifungal Compounds Produced by Lactobacillus plantarum IMAU10014";HaiKuan Wang等;《Plos One》;20120119;第7卷(第1期);第1-7页 * |
| "拮抗意大利青霉和指状青霉的柑橘生防用乳酸菌筛选";李昆太等;《乳酸菌与营养健康：第九届乳酸菌与健康国际研讨会摘要汇编》;20140521;第51页 * |
| "柑橘绿霉病菌（Penicillium digitatum）MFS1基因的克隆和功能初步分析";王继业等;《中国植物病理学会2008年学术年会论文集》;20080701;第150页 * |
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