CN109988732A - One plant of class goldbeater's skin Wei Si Salmonella and its application - Google Patents

One plant of class goldbeater's skin Wei Si Salmonella and its application Download PDF

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CN109988732A
CN109988732A CN201910368183.5A CN201910368183A CN109988732A CN 109988732 A CN109988732 A CN 109988732A CN 201910368183 A CN201910368183 A CN 201910368183A CN 109988732 A CN109988732 A CN 109988732A
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penicillium digitatum
salmonella
lactic acid
acid bacteria
goldbeater
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CN109988732B (en
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曾凯芳
马佳宏
易兰花
邓丽莉
姚世响
阮长晴
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Southwest University
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A23B7/155—Microorganisms; Enzymes; Antibiotics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

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  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Food Science & Technology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses class goldbeater's skin Wei Si Salmonella (Weissella paramesenteroides) JT13 that deposit number is CGMCC No.17539 and such goldbeater's skin Wei Si Salmonella to inhibit the drug of Penicillium digitatum (Penicillium digitatum) especially to prevent the application in the drug that the citrusfruit as caused by Penicillium digitatum adopts rear green mould in preparation.The present invention not only enriches lactic acid bacteria culturers resources bank, has widened lactic acid bacteria in the application range in preserving fruit and vegetable utilizing field, the prevention and treatment for also adopting rear green mould for citrusfruit provides new solution.

Description

One plant of class goldbeater's skin Wei Si Salmonella and its application
Technical field
The present invention relates to one plant of class goldbeater's skin Wei Si Salmonellas, further relate to the application of such goldbeater's skin Wei Si Salmonella.
Background technique
Lactic acid bacteria be it is a kind of can fermenting carbohydrate and primary product be lactic acid Gram-positive bacillus or coccus, to its The closer part Gram-positive of affiliation or negative bacteria have good inhibiting effect, are now widely used in food hair The fields such as ferment, fresh-keeping.But in relation to the still rare report of inhibiting effect of the lactic acid bacteria to fungi.
Citrus is Rutaceae Aurantioideae citrus plant, and citrusfruit is the fruit that yield is most in the world, is being stored In by microbial infection and storage environment influenced easily occur rot, oedema, outer skin phenomena such as, seriously affect the commodity of fruit Value.The green mould as caused by fungi Penicillium digitatum (Penicillium digitatum) is main in citrusfruit storage The infectious disease wanted.Citrusfruit postharvest disease prevention and treatment at present is mainly using chemistry such as carbendazim, thiophanate methyl, imazalils Fungicide, but the problems such as easily develop drug resistance to it there are the residual of chemical bactericide, microorganism.
Summary of the invention
Inhibit Penicillium digitatum effect especially can be pre- it is an object of the invention to screen from citrus orchard, identify to have Anti- citrusfruit adopts the lactic acid bacteria of rear green mould, to enrich lactic acid bacteria culturers resources bank, widens lactic acid bacteria in preserving fruit and vegetable utilizing field Application range, while providing new solution for the prevention and treatment that citrusfruit adopts rear green mould.
Through studying, the invention provides the following technical scheme:
Class goldbeater's skin Wei Si Salmonella 1. (Weissella paramesenteroides) JT13, deposit number CGMCC No.17539。
2. class goldbeater's skin Wei Si Salmonella (Weissella paramesenteroides) JT13 inhibits Penicillium digitatum in preparation Application in the drug of (Penicillium digitatum).
Further, class goldbeater's skin Wei Si Salmonella (Weissella paramesenteroides) JT13 is in preparation prevention by referring to Citrusfruit caused by shape mould (Penicillium digitatum) adopts the application in the drug of rear green mould.
Class goldbeater's skin Wei Si Salmonella (Weissella paramesenteroides) JT13 of the invention was April 10 in 2019 It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Beijing's southern exposure day The institute 3 of area North Star West Road 1), deposit number is CGMCC No.17539.
The beneficial effects of the present invention are: the present invention, which has filtered out to have from citrus orchard, inhibits Penicillium digitatum effect special It is not the lactic acid bacteria that the citrusfruit that can prevent to be induced by it adopts rear green mould, not only enriches lactic acid bacteria culturers resources bank, Lactic acid bacteria is widened in the application range in preserving fruit and vegetable utilizing field, the prevention and treatment for also adopting rear green mould for citrusfruit provides new solution Certainly method.
Detailed description of the invention
Fig. 1 is in vitro fungistatic effect of the lactic acid bacteria to Penicillium digitatum.
Fig. 2 is in vitro fungistatic effect of the lactic acid bacteria without fermented liquid to Penicillium digitatum.
Fig. 3 is the control effect that lactic acid bacteria without fermented liquid adopts rear green mould to citrusfruit caused by Penicillium digitatum, figure In indicate between the group of identical small English alphabet (a, b, c) that there is no significant difference (p > 0.05)；Indicate different small English There are significant difference (p < 0.05) between the group of alphabetical (a, b, c).
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with attached drawing to of the invention excellent Embodiment is selected to be described in detail.
1, the screening of lactic acid bacteria
Citrusfruit, blade, soil and peanut leaf conduct are acquired respectively from It In Beibei, Chongqing Mount Jinyun citrus orchard Sample.Each sample is totally submerged respectively in 100mL MRS broth bouillon, 37 DEG C of enrichment cultures for 24 hours, 10 times of gradient dilutions After be coated on containing 0.3%CaCO3MRS plate on, 37 DEG C of culture 48h, select the single bacterium colony with obvious molten calcium circle into Row isolates and purifies, and carries out microscopy and Gram's staining.Strain after purification is placed in -80 DEG C of refrigerators and saves.
The results are shown in Table 1, and experiment filters out 68 plants of doubtful bacterial strains altogether, wherein 16 plants come from citrusfruit, 6 plants come from mandarin orange Tangerine leaf piece, 24 plants come from citrus orchard soil, and 22 plants come from peanut leaf, and microscopy observes spherical and rod-shaped two kinds of forms, remove from office Blue Albert'stain Albert is positive.
The screening of 1 lactic acid bacteria of table
2, in vitro fungistatic effect of the lactic acid bacteria to Penicillium digitatum
Filter out 68 plants of bacterial strains are passed through into its clear in vitro fungistatic effect to Penicillium digitatum of double-layer agar technique.
Each doubtful one collarium of bacterial strain picking is inoculated in 20mL MRS broth bouillon, 37 DEG C of culture 48h obtain seed Liquid；It draws 1mL seed liquor to be inoculated in 100mL MRS broth bouillon, 37 DEG C of culture 48h；Then with transfer needle in MRS plate Intermediate parallel draws the straight line that two length are 2cm, and plate is set 37 DEG C of culture 48h；Then 10mL is added and contains 104Penicillium digitatum spore Plate is set 25 DEG C of cultures after culture medium solidifies completely by the PDA culture medium of son；Antibacterial phenomenon is observed, with non-inoculating strain MRS broth bouillon is marked as control, filters out the bacterial strain for having higher inhibitory activity to Penicillium digitatum.
As a result as shown in Figure 1, experiment filters out 10 plants of bacterial strains to Penicillium digitatum with higher inhibitory activity altogether, wherein 7 Strain comes from peanut leaf, and 2 plants come from citrus orchard soil, and 1 plant comes from Citrus leaf.
3, the identification of lactic acid bacteria
By filter out 10 plants to Penicillium digitatum have higher inhibitory activity bacterial strain by 16S rDNA sequence analyze into Row identification.
Culture bacterium solution is taken, thallus DNA is extracted using genome DNA extracting reagent kit；It is expanded using universal primer PCR 16srDNA sequence, forward primer 5 '-agagtttgatcctggctcag-3 ' (SEQ ID No.1), reverse primer 5 '- ctacggctaccttgttacga-3'(SEQ ID No.2)；Reaction system includes: 12.5 μ L 2 × Es Taq MasterMix (Dye), each primer of 1 μ L and 1 μ L DNA profiling；PCR reaction process is as follows: initial denaturation, and 94 DEG C, 2min；Then 30 recycle: becoming Property, 94 DEG C, 30s；Annealing, 55 DEG C, 30s；Extend 72 DEG C, 30s；Extend eventually, 72 DEG C, 2min.Pcr amplification product uses 1% fine jade Sepharose electrophoresis is detected, and commission Shanghai Sheng Gong Biological Co., Ltd. is sequenced, and sequencing result passes through in NCBI Blast program carry out sequence analysis analysis.
The results are shown in Table 2,10 plants has the bacterial strain of higher inhibitory activity identified Penicillium digitatum, wherein 8 plants are Lactobacillus sucicola, 1 plant is class goldbeater's skin Wei Si Salmonella (Weissella paramesenteroides), and 1 plant is Pediococcus acidilactici (Pediococcus acidilactici).
The identification of 2 lactic acid bacteria of table
It is JT03 by gained Lactobacillus sucicola Strain Designation, is preserved in China on April 10th, 2019 Microbiological Culture Collection administration committee common micro-organisms center (abbreviation CGMCC, address: BeiChen West Road, Chaoyang District, BeiJing City 1 Institute 3), deposit number is CGMCC No.17538.Its 16S rDNA sequence is as shown in SEQ ID No.3.
It is JT13 by gained class goldbeater's skin Wei Si Salmonella (Weissella paramesenteroides) Strain Designation, in Be preserved on April 10th, 2019 China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.17539.Its 16S rDNA sequence such as SEQ Shown in IDNo.4.
It is JY03 by gained Pediococcus acidilactici (Pediococcus acidilactici) Strain Designation, in April, 2019 It is preserved within 10th China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: court, Beijing The institute 3 of positive area's North Star West Road 1), deposit number is CGMCC No.17540.Its 16S rDNA sequence such as SEQ ID No.5 institute Show.
4, in vitro fungistatic effect of the lactic acid bacteria without fermented liquid to Penicillium digitatum
Investigate in vitro fungistatic effect of the without fermented liquid to Penicillium digitatum of 3 identified strains of lactic acid bacteria.
It draws 1mL lactobacillus solution to be inoculated in 100mL MRS broth bouillon, 37 DEG C of cultures；One is taken out every 2h Quantitative fermentation liquid, 5000rpm are centrifuged 15min, and supernatant crosses 0.22 μm of microporous barrier, obtains the sterile fermentation of different incubation times Liquid.By 100 μ L fermentation liquids and 100 μ L 104Penicillium digitatum spore suspension (culture medium containing 5%PDB) mixing, non-inoculating lactic acid The MRS broth bouillon of bacterium is control, and 25 DEG C of culture 48h measure OD600Value.
As a result as shown in Fig. 2, the without fermented liquid of 3 strains of lactic acid bacteria (JT03, JT13, JY03) has not Penicillium digitatum With the inhibitory effect of degree, and with the extension of incubation time, inhibitory effect is gradually increased, and can be pressed down completely after culture to 60h The growth of Penicillium digitatum processed.
5, lactic acid bacteria without fermented liquid adopts the control effect of rear green mould to citrusfruit
The without fermented liquid that 3 identified strains of lactic acid bacteria are investigated by same hole inoculation experiments adopts rear green mould to citrusfruit Inhibitory effect.
Draw 1mL lactobacillus solution be inoculated in 100mL MRS broth bouillon, 37 DEG C of cultures 48h, 5000rpm from Heart 15min, supernatant cross 0.22 μm of microporous barrier, obtain lactic acid bacteria without fermented liquid；Select that size is uniform, maturity is consistent, nothing The healthy citrusfruit of machinery wound impregnates 2min with 2% liquor natrii hypochloritis and carries out surface sterilization, and water flushing is dried；Fruit is red After 70% alcohol disinfecting of road position, 2 apertures (diameter 3mm, deep 3mm) is equidistantly made a call at fruit equator position with punch, is connect 10 μ L 10 of kind4Penicillium digitatum spore suspension inoculates 20 μ L lactic acid bacteria without fermented liquids, after liquid absorption is complete with not The MRS broth bouillon of inoculating lactic acid bacterium is control, and fruit is carried out individual packing after liquid absorption is complete, is placed in 25 DEG C, store in the environment of relative humidity 90-95%；Fruit disease incidence and lesion diameter are observed and recorded daily.
As a result as shown in figure 3, the without fermented liquid of 3 strains of lactic acid bacteria (JT03, JT13, JY03) can be to a certain extent Inhibit the morbidity of citrusfruit and the growth of lesion diameter.Wherein the effect of JT03 and JT13 is more preferable.
Finally, it is stated that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although passing through ginseng According to the preferred embodiment of the present invention, invention has been described, it should be appreciated by those of ordinary skill in the art that can To make various changes to it in the form and details, without departing from the present invention defined by the appended claims Spirit and scope.
Sequence table
<110>Southwest University
<120>one plants of class goldbeater's skin Wei Si Salmonellas and its application
<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>class goldbeater's skin Wei Si Salmonella JT13 (Weissella paramesenteroides 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. 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.
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CN110527651A (en) * 2019-09-16 2019-12-03 西南大学 One plant of lactobacillus plantarum for producing a variety of broad-spectrum antiseptic metabolins and its application
CN111690574A (en) * 2020-07-22 2020-09-22 云南省畜牧兽医科学院 Weissella mesenteroides and application thereof
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CN111690574A (en) * 2020-07-22 2020-09-22 云南省畜牧兽医科学院 Weissella mesenteroides and application thereof
CN111690574B (en) * 2020-07-22 2021-05-25 云南省畜牧兽医科学院 Weissella mesenteroides and application thereof
CN115851538A (en) * 2022-12-12 2023-03-28 华南理工大学 Weissella mesenteroides MbWp-171 and product and application thereof

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