CN105176831A - Penicillum oxalicum, and applications thereof - Google Patents
Penicillum oxalicum, and applications thereof Download PDFInfo
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- CN105176831A CN105176831A CN201510410093.XA CN201510410093A CN105176831A CN 105176831 A CN105176831 A CN 105176831A CN 201510410093 A CN201510410093 A CN 201510410093A CN 105176831 A CN105176831 A CN 105176831A
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Abstract
The invention discloses a Penicillum oxalicum, and applications thereof. The preservation name of the Penicillum oxalicum is Penicillum oxalicum SG4, and is preserved on 30th, April, 2015, in China Center For Type Culture Collection, in Wuhan university, Wuhan, China; and preservation number is CCTCCM 2015270. It is confirmed by experiments that the Penicillum oxalicum SG4 possesses strong inhibition effects on orange Penicillium citrinum, and can be used for preparing orange biological fresh-keeping agents; a novel direction is provided for development of novel biological fresh-keeping agents and orange biological preservation; and excellent economic and social benefits are achieved.
Description
Technical field
The present invention relates to strain penicillium oxalicum and an application thereof, be mainly used in oranges and tangerines biological preservation aspect, this penicillium oxalicum can be used for antagonism oranges and tangerines Penicillium citrinum bacterium.
Background technology
Penicillium italicum causes primarily of Penicillium citrinum germ (Penicilliumitalicum), their early stage pericarp softens, and water stain shape fades, and after this goes out aerial hyphae scab director, form the white mustiness thing of thick layer, and be extended to rapidly white subcircular mildew.Main harm mature fruit in vegetative period and shelf time fruit, had a strong impact on Aspects In The Development of Citrus Industry.
Oranges and tangerines conventional are traditionally fresh-keeping refrigeration, controlled atmosphere and chemical fresh-keeping method, but refrigeration and controlled atmosphere cost is high, power consumption greatly and quality is unstable is eliminated gradually, and there is potential food safety and problem of environmental pollution in chemical fresh-keeping method, and life-time service chemicals is fresh-keeping, oranges and tangerines pathogenic bacterium can produce resistance to it.But biological way of keeping fresh overcomes unfavorable factor and the drawback of above fresh-keeping method, has the advantages such as processing target is clear and definite, cost is low, environmental protection is pollution-free.Therefore biological way of keeping fresh is more and more subject to people's attention, as: Cai Wentao isolates endophyte strain-L and strain-39 capsicum pathogenic bacterium being had to fungistatic effect from capsicum, and studies its biological control effect.Result shows: when strain-39 fermented liquid ethyl acetate extract mass concentration is 100mg/ml, and capsicum and tomato decayed fruit rate decline 51.879% and 58.47% respectively; When 75% ethanol pellet is 100mg/mL, capsicum rotting rate have dropped 47.50%, and tomato rotting rate have dropped 50.47%.Li Mei is separated G-3, G-4 of obtaining having Penicillium italicum and citrus common green mold antagonistic action from citrus orchard soil.And from its fermented liquid, isolate three kinds of activeconstituentss by silica gel column chromatography isolation technique: the inhibiting rate of activeconstituents 1 pair of Citrus, green mould is respectively 48.32%, 60.83%; The inhibiting rate of activeconstituents 2 pairs of Citrus, green mould is respectively 48.01%, 57.35%; The inhibiting rate of activeconstituents 3 pairs of Citrus, green mould is respectively 35.28%, 55.79%.
Penicillium is a kind of omnivory fungi, can grow any containing in organic matrix.The spore thermotolerance of mould is comparatively strong, and thalline the bread worm is lower, and acidic flavoring agent conventional in beverage is the carbon source that it is liked, Penicillium is applied very widely in production and having clinically.As: the microbiotic applied clinically---penicillin, extracts exactly from mould, and another important microbiotic---grisovin, is produced by Penicillium griseofulvum, is the best microbiotic of Antifungi dermatoses.
The present invention is separated and obtains a strain penicillium oxalicum from M. laxiflora leaf, and it has good restraining effect to oranges and tangerines Penicillium citrinum bacterium, provides technical support for developing new oranges and tangerines bio-preservative.
Summary of the invention
An object of the present invention is to provide a kind of penicillium oxalicum (Penicillumoxalicum) SG4 bacterial strain, this penicillium oxalicum thalline has very strong restraining effect to oranges and tangerines Penicillium citrinum bacterium, can be used for preparing oranges and tangerines bio-preservative, for developing new bio-preservative and oranges and tangerines biological preservation provides new direction.
Described penicillium oxalicum (Penicillumoxalicum) SG4 is the endogenetic fungus obtained from the root of M. laxiflora (MyricariaLaxiflora), be preserved in China typical culture collection center on April 30th, 2015, deposit number is: CCTCCM2015270.Preservation address is China, Wuhan, Wuhan University.
Penicillium oxalicum of the present invention (Penicillumoxalicum) SG4 has following Microbiological Characteristics on PDA substratum: thread shorter, a large amount of greyish-green spore of white mycelium.
The object of the invention is to provide the application on microbial inoculum that a kind of penicillium oxalicum rots at preparation biological control oranges and tangerines.Described oranges and tangerines rot disease caused by Citrus mould.
Present invention also offers a kind of penicillium oxalicum and prepare the application on preservation agent.Described penicillium oxalicum can also replace with the fermented liquid of penicillium oxalicum bacteria strain or the extract of fermented liquid.
The present invention with the DNA of penicillium oxalicum (Penicillumoxalicum) SG4 for template, upstream primer ITS1:5 '-TCCGTAGGTGAACCTGCG-3 ', downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 ', carry out colony PCR amplification, obtain the 18SrDNA partial sequence (SEQIDNO:1) of 560bp.The sequence of being included by Blast and GenBank is compared, and finds that the homology of SG4 and penicillium oxalicum is 100%.Build evolutionary tree (Fig. 4) with MEGA5, qualification bacterial strain (SG4) is nearest with the sibship of a strain P.oxalicum (HQ680452).
Accompanying drawing explanation
Fig. 1 is the colonial morphology of penicillium oxalicum of the present invention (Penicillumoxalicum) SG4.
Fig. 2 is the microscopic morphology of penicillium oxalicum of the present invention (Penicillumoxalicum) SG4.
Fig. 3 is that penicillium oxalicum of the present invention (Penicillumoxalicum) SG4 thalline is to oranges and tangerines Penicillium citrinum bacterium antagonistic action effect.
Fig. 4 is the 18SrDNA phylogenetic tree of penicillium oxalicum of the present invention (Penicillumoxalicum) SG4 bacterial strain.
Embodiment
Embodiment one: the separation of penicillium oxalicum (Penicillumoxalicum) SG4 and preservation
Within 2012, in Yichang, basin, Three Gorges, domestic kermes dam gathers M. laxiflora root, is inoculated in and adds the PDA solid medium separation fungi that working concentration is 25 μ g/ml penicillin after surface sterilization.
Surface sterilization: get M. laxiflora root tap water 5 times, after aseptic filter paper is dried, 75% alcohol-pickled 1min, aseptic water washing 3 times, then soak 60S with 3% clorox, then use aseptic water washing 5 times, aseptic filter paper washes dry root surface-moisture.
Cultivate: by sterile scissors, the root after surface sterilization is cut into 0.5*0.5cm size, be inoculated in that to add working concentration be 25 μ g/ml penicillin P DA solid mediums (potato 200g/L, glucose 20g/L, agar 20g/L, Ph6.0-6.5), cultivate at being placed in 28 DEG C.
Bacterial strain purifying: after flat board grows bacterium colony, according to form picking list colony inoculation in new PDA solid medium, cultivates at being placed in 28 DEG C.
Bacterial strain preservation: after bacterial strain purifying, the pure inoculation of picking, in PDA slant medium, cultivates 4d for 28 DEG C, is hidden in 4 DEG C of preservations by the whiteruss envelope of sterilizing.
This bacterial strain penicillium oxalicum (Penicillumoxalicum) is carried out preservation, and preservation name is called SG4; Depositary institution: China typical culture collection center; Preservation address is China, Wuhan, Wuhan University; Preservation date: 2015-4-30; Deposit number is: CCTCCM2015270.
Embodiment two: penicillium oxalicum (Penicillumoxalicum) SG4 identifies
18SrDNA sequential analysis: adopt universal primer to carry out colony PCR amplification 18SrDNA sequence.Upstream primer ITS1:5 '-TCCGTAGGTGAACCTGCG-3 ', downstream primer ITS4:5 '-TCCTCCGCTTATTGATATGC-3 '.Reaction system is as follows:
Reaction conditions: 94 DEG C of denaturation 5min, 94 DEG C of sex change 1min, 56 DEG C of annealing 30sec, 72 DEG C extend 1.min, 33 circulations, and last 72 DEG C extend 10min.PCR primer 1% agarose gel electrophoresis detects, and the band of picking about 750bp carries out gel recovery, serves Hai Shenggong order-checking.
The sequence recorded is committed to GenBank, and obtains accession number KR024319.This sequence is carried out Blast comparison in GenBank, and chooses the sequence MEGA5 software building 18SrDNA phylogenetic tree of the higher bacterial strain of homology.
Execute example three: the preparation of penicillium oxalicum (Penicillumoxalicum) SG4 thalline
Actication of culture: go bail for deposit 4 DEG C of refrigerators the streak inoculation of penicillium oxalicum bacterial classification in PDA flat board, at 28 DEG C, cultivate 4d, obtain thalline.
Embodiment four: penicillium oxalicum (Penicillumoxalicum) SG4 thalline is to oranges and tangerines Penicillium citrinum bacterium antagonistic action.
The preparation of agar block: adopt plate streak to be inoculated on PDA substratum by M. laxiflora endogenetic fungal bacterial strain respectively, cultivate 4d at 28 DEG C, punches, in order to prepare bacterium cake on the agar plate being covered with confession examination bacterium with punch tool (diameter 10mm).Dull and stereotyped face-off method detects thalline to oranges and tangerines Penicillium citrinum bacterium bacteriostatic activity: Penicillium citrinum bacterium is inoculated in PDA slant medium, cultivate 4d for 28 DEG C, 9ml sterilized water is added in test tube, all spores are scraped with transfering loop, obtained spore suspension, get 500 μ l oranges and tangerines Penicillium citrinum bacterium spore suspensions, coating evenly, M. laxiflora endogenetic fungus bacterium cake is placed on flat board, in a district of flat board, bacterium cake forward places (bacterium faces up), another district oppositely places (bacterium faces down), cultivates 4d respectively, observations at 28 DEG C.
Antibacterial result:
SEQUENCELISTING
<110> applicant SanXia University
<120> penicillium oxalicum and application thereof
<210>SG4
<211>546
<212>DNA
SEQIDNO：1
GGCGTCGGAAGGATCATTACCGAGTGAGGGCCCTCTGGGTCCAACCTCCCACCCGTGTTTA
TCGTACCTTGTTGCTTCGGCGGGCCCGCCTCACGGCCGCCGGGGGGCATCCGCCCCCGGGC
CCGCGCCCGCCGAAGACACACAAACGAACTCTTGTCTGAAGATTGCAGTCTGAGTACTTG
ACTAAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAAGAACGCA
GCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAGTCTTTGAACGCA
CATTGCGCCCCCTGGTATTCCGGGGGGCATGCCTGTCCGAGCGTCATTGCTGCCCTCAAGC
ACGGCTTGTGTGTTGGGCTCTCGCCCCCCGCTTCCGGGGGGCGGGCCCGAAAGGCAGCGG
CGGCACCGCGTCCGGTCCTCGAGCGTATGGGGCTTCGTCACCCGCTCTGTAGGCCCGGCCG
GCGCCCGCCGGCGAACACCATCAATCTTAACCAGGTGACCTCGGATCAGGTAGGACCCGA
Claims (6)
1. penicillium oxalicum (
penicillumoxalicum) SG4, be preserved in China typical culture collection center on April 30th, 2015, deposit number is: CCTCCM2015270, and preservation address is Hubei, Wuhan, Wuhan University.
2. the application on the microbial inoculum that rots at preparation biological control oranges and tangerines of penicillium oxalicum according to claim 1.
3. oranges and tangerines according to claim 2 rot disease caused by Citrus mould.
4. penicillium oxalicum according to claim 1 is preparing the application on preservation agent.
5. application according to claim 4, is characterized in that, described penicillium oxalicum can also replace with the fermented liquid of penicillium oxalicum bacteria strain or the extract of fermented liquid.
6. the penicillium oxalicum described in any one of claim 1-5 is the endogenetic fungus obtained from the root of M. laxiflora.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106929430A (en) * | 2017-02-10 | 2017-07-07 | 三峡大学 | One plant of penicillium oxalicum SY 15 with strong anti-oxidation and its application |
| CN107691479A (en) * | 2017-09-22 | 2018-02-16 | 北京联合大学 | A kind of penicillium oxalicum microbial inoculum and its preparation method and application |
| CN108823110A (en) * | 2018-07-26 | 2018-11-16 | 福州工微生物科技有限公司 | One plant of bacterial strain for producing griseofulvin and its application |
| CN108841889A (en) * | 2018-07-26 | 2018-11-20 | 福州工微生物科技有限公司 | The main part of Songgangmeisu --- the method for griseofulvin is produced using microbial fermentation |
| CN108841733A (en) * | 2018-07-26 | 2018-11-20 | 福州工微生物科技有限公司 | The one plant of production main part of Songgangmeisu --- bacterial strain and method of griseofulvin |
| CN109608517A (en) * | 2019-01-18 | 2019-04-12 | 三峡大学 | A kind of novel antimicrobial peptide and the application in citrus is fresh-keeping |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106929430A (en) * | 2017-02-10 | 2017-07-07 | 三峡大学 | One plant of penicillium oxalicum SY 15 with strong anti-oxidation and its application |
| CN107691479A (en) * | 2017-09-22 | 2018-02-16 | 北京联合大学 | A kind of penicillium oxalicum microbial inoculum and its preparation method and application |
| CN108823110A (en) * | 2018-07-26 | 2018-11-16 | 福州工微生物科技有限公司 | One plant of bacterial strain for producing griseofulvin and its application |
| CN108841889A (en) * | 2018-07-26 | 2018-11-20 | 福州工微生物科技有限公司 | The main part of Songgangmeisu --- the method for griseofulvin is produced using microbial fermentation |
| CN108841733A (en) * | 2018-07-26 | 2018-11-20 | 福州工微生物科技有限公司 | The one plant of production main part of Songgangmeisu --- bacterial strain and method of griseofulvin |
| CN108823110B (en) * | 2018-07-26 | 2021-07-16 | 福州工微生物科技有限公司 | Strain for producing griseofulvin and application thereof |
| CN108841733B (en) * | 2018-07-26 | 2021-09-28 | 福州工微生物科技有限公司 | Strain and method for producing griseofulvin serving as major component of tranexamycin |
| CN108841889B (en) * | 2018-07-26 | 2021-09-28 | 福州工微生物科技有限公司 | Method for producing griseofulvin serving as major component of tranexamycin by microbial fermentation |
| CN109608517A (en) * | 2019-01-18 | 2019-04-12 | 三峡大学 | A kind of novel antimicrobial peptide and the application in citrus is fresh-keeping |
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