CN111909884B - Lactobacillus plantarum and application thereof - Google Patents
Lactobacillus plantarum and application thereof Download PDFInfo
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- CN111909884B CN111909884B CN202011000936.6A CN202011000936A CN111909884B CN 111909884 B CN111909884 B CN 111909884B CN 202011000936 A CN202011000936 A CN 202011000936A CN 111909884 B CN111909884 B CN 111909884B
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- 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
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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
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- A23B7/155—Microorganisms; Enzymes; Antibiotics
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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Abstract
The invention provides lactobacillus plantarum, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.20197, and the preservation date is 7 and 6 days in 2020. The Lactobacillus plantarum NM22-2 and the fermentation liquid thereof have obvious inhibition effect on penicillium expansum, can effectively inhibit the infection of penicillium expansum on fruits in the application of food preservation, have the effect of preservation and fresh keeping, and prolong the storage time of the fruits.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus plantarum and application thereof.
Background
Penicillium Expansum (Penicillium Expansum) belongs to the family of the family Calycopsis, the class Deuteromycetes, the genus Penicillium, the family Calycopsis, and the genus psychrophilic mold, and mainly grows on various fruits such as apples, peaches, pears, etc., and the number of spores in the air is about 6X 103Per m3And the distribution is wide. During picking and transportation, fruits are very vulnerable to fungal infestation resulting in spoilage due to mechanical damage caused by impact and squeezing, and Penicillium expansum accounts for more than 60% of the total in fruit spoilage due to the genus Penicillium. The penicillium expansum not only causes the quality of fruits to be reduced, but also can generate a large amount of mycotoxin to cause gastrointestinal dysfunction, organ edema and bleeding of eaters, even serious injuries such as teratogenesis, carcinogenesis and the like.
At present, the prevention and control method for controlling the postharvest fungal infection of fruits is mainly physical prevention and chemical prevention and control. The physical prevention and control mainly adopts physical means such as low-temperature storage, modified atmosphere packaging, heat and microwave irradiation to inhibit the respiratory intensity of fruits so as to inhibit the growth of pathogenic bacteria, but the implementation process is relatively complicated and the cost is high. Chemical prevention and control mainly adopts chemical reagents to disinfect or sterilize microorganisms on the surfaces of fruits, the method is simple and convenient to operate, easy to implement and low in cost, but the problem of drug resistance is obvious along with long-term, large-amount or even excessive use of the chemical reagents, and more serious the residue of the chemical reagents harms human health and pollutes the natural environment. Therefore, biological control methods are receiving increasing attention as a simple, economical, safe, environmentally friendly new technology that can replace chemical agents to some extent.
The lactobacillus has various types and rich genetic diversity, is widely distributed in various fermented foods, and is a microorganism identified by European Union and having safe popularization qualification. The unique homotype and heterotype lactic acid fermentation characteristics of the lactic acid bacteria enable the pH value of the surrounding environment to be rapidly reduced in the growth and metabolism process of the lactic acid bacteria, so that the growth of various bacteria and moulds in the environment is effectively inhibited. The lactobacillus plantarum is one of lactic acid bacteria, and has the potential of becoming a biocontrol microbial inoculum due to the outstanding antibacterial property and use safety of the lactobacillus plantarum.
Disclosure of Invention
The invention aims to provide lactobacillus plantarum, which not only has obvious inhibition activity on penicillium expansum, but also can be used as a safe, green and low-cost fruit biocontrol microbial inoculum to keep fruits fresh and preserve.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides Lactobacillus plantarum named as (Lactobacillus plantarum) NM22-2, which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 20197.
The lactobacillus plantarum is characterized by being milky, smooth and moist, liquid drop-shaped, round, protruding, neat in edge, 1-2 mm in colony diameter, straight-rod-shaped, single, paired or chain-shaped, 3.0-8.0 microns multiplied by 0.9-1.2 microns in size, without flagella and spores.
In the invention, the lactobacillus plantarum is derived from a reonber yoghurt sample in inner Mongolia.
The invention also provides fermentation liquor obtained by fermenting the lactobacillus plantarum.
In the invention, the fermentation broth is a fermentation culture solution, and the preparation method of the fermentation culture solution comprises the following steps: inoculating lactobacillus plantarum into an MRS liquid culture medium, and culturing for 10-18 h at 35-40 ℃ to obtain a fermentation culture solution.
In the invention, the fermentation liquid is fermentation supernatant, and the fermentation supernatant is obtained by centrifuging the fermentation culture liquid.
In the invention, the fermentation liquid is sterile fermentation liquid, and the sterile fermentation liquid is obtained by filtering the fermentation supernatant through a filter membrane.
The invention also provides an application of the lactobacillus plantarum or the fermentation broth in preparing products for inhibiting penicillium expansum.
The invention also provides an application of the lactobacillus plantarum or the fermentation liquor in fruit preservation and/or preservation.
The invention also provides a preservative or preservative, which comprises the lactobacillus plantarum and/or the fermentation liquor.
Compared with the prior art, the technical scheme of the invention has the beneficial effects that:
the Lactobacillus plantarum NM22-2 has a remarkable inhibition effect on penicillium expansum, can prevent fruits from decaying, can be used as a fruit biocontrol microbial inoculum to ensure the inherent quality of the fruits and prolong the storage time of the fruits, and has important significance for improving and enhancing the quality and safety of the fruits.
Biological preservation information:
the Lactobacillus plantarum NM22-2 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.20197, the preservation date of 7 and 6 days in 2020, and the preservation address of No. 3, Xilu 1 Beichen, the sunward area in Beijing.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum NM 22-2;
FIG. 2 is a microscopic examination result chart of Lactobacillus plantarum NM 22-2;
FIG. 3 is a graph showing the effect of Lactobacillus plantarum (Lactobacillus plantarum) NM22-2 in inhibiting Penicillium expansum;
FIG. 4 is a graph showing the effect of Lactobacillus plantarum NM22-2 sterile fermentation broth on inhibiting Penicillium expansum;
FIG. 5 is a diagram showing the effect of Lactobacillus plantarum NM22-2 on apple freshness preservation and preservation.
Detailed Description
The invention provides a Lactobacillus plantarum, which is named as (Lactobacillus plantarum) NM22-2 and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 20197.
In the invention, the lactobacillus plantarum is characterized by milky white, smooth and moist, liquid drop-shaped, round, raised, neat edges, 1-2 mm of colony diameter, and gram-positive bacteria which are straight rod-shaped, single, paired or chain-shaped, 3.0-8.0 microns multiplied by 0.9-1.2 microns in size, have no flagella and no spore production; the lactobacillus plantarum of the invention is derived from a sample of renberger yoghurt, inner Mongolia.
The invention provides fermentation liquor obtained by fermenting the lactobacillus plantarum NM 22-2.
In the present invention, the fermentation liquid is preferably a fermentation culture liquid, and the preparation method of the fermentation culture liquid comprises the following steps: inoculating lactobacillus plantarum into an MRS liquid culture medium, and culturing for 10-18 h at 35-40 ℃ to obtain a fermentation culture solution. Preferably, lactobacillus plantarum is inoculated into an MRS liquid medium and cultured at 37 ℃ for 12 hours to obtain a fermentation broth. The MRS liquid culture medium comprises 10g of soybean peptone, 5g of beef extract, 4g of yeast powder, 20g of glucose, 801mL of tween-801, 2g of disodium hydrogen phosphate, 5g of anhydrous sodium acetate, 2g of triamine citrate, 0.02g of manganese sulfate and 0.1g of magnesium sulfate; the preparation method of the MRS liquid culture medium optionally comprises the steps of mixing the raw materials in the ratio with distilled water, fixing the volume to 1L, adjusting the initial pH to 6.20, and sterilizing at 121 ℃ for 15 min. The sources of the soybean peptone, the beef extract, the yeast powder, the glucose, the tween-80, the disodium hydrogen phosphate, the anhydrous sodium acetate, the triammonium citrate, the manganese sulfate and the magnesium sulfate are not particularly limited, and conventional commercially available products well known by the technical personnel in the field can be adopted.
In the present invention, the fermentation liquid may be a fermentation supernatant obtained by centrifuging the fermentation culture liquid. In the invention, the rotation speed of the centrifugation is preferably 3500 Xg-4500 Xg, more preferably 4000 Xg, and the time of the centrifugation is preferably 7-13 min, more preferably 10 min.
In the invention, the fermentation liquid can be sterile fermentation liquid, and the sterile fermentation liquid is obtained by filtering the fermentation supernatant through a filter membrane. In the present invention, the filter is preferably 0.22. mu.m. The source of the filter membrane in the present invention is not particularly limited, and a conventional commercially available product well known to those skilled in the art may be used.
The invention also provides an application of the lactobacillus plantarum or the fermentation broth in preparing products for inhibiting penicillium expansum. The diameter of the inhibition zone of the lactobacillus plantarum NM22-2 is 16.32 +/-3.27 mm, and meanwhile, the inhibition rate of the sterile fermentation liquor prepared by the lactobacillus plantarum NM22-2 on the growth of penicillium expansum is 52.7 +/-4.7%, which shows that the lactobacillus plantarum NM22-2 or the fermentation liquor has stronger antibacterial activity on the penicillium expansum. The type and source of Penicillium expansum are not particularly limited in the present invention, and Penicillium expansum BNCC185786 used in the examples of the present invention was purchased from the institute of Biotechnology, Beijing Beinan union of Industrial science, Beijing.
The invention also provides an application of the lactobacillus plantarum or the fermentation liquor in fruit preservation and/or preservation. According to the invention, the penicillium expansum can cause the spoilage of fruits, the lactobacillus plantarum NM22-2 or the fermentation liquor has a remarkable inhibiting effect on the penicillium expansum, and meanwhile, the spoilage of the fruits is inhibited by the sterile fermentation liquor prepared by the lactobacillus plantarum NM22-2, and the infection rate is inhibited to 65.90%, which indicates that the lactobacillus plantarum or the fermentation liquor has the effects of keeping the fruits fresh and preserving the fruits. The fruit preferably comprises apple, pear, peach, grape or watermelon, and the fruit preferably has uniform size and no surface wound.
The invention also provides a preservative or preservative, which comprises the lactobacillus plantarum and/or the fermentation liquor.
The active ingredients of the preservative or preservative are the lactobacillus plantarum and/or the fermentation liquor.
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 Lactobacillus plantarum NM22-2 isolation, screening and identification
(1) Isolation of Lactobacillus plantarum NM22-2
Using normal saline to perform gradient dilution on the inner Mongolia Renbebel yoghurt sample to 10-4、10-5And 10-6Respectively coating 200 mu L of each diluent on MRS agar culture medium containing cycloheximide (0.1 wt%) and colistin sulfate (0.1 wt%), carrying out anaerobic culture at 37 ℃ for 24-72 h, selecting single colony, continuously streaking and purifying for 2 times, and selecting gram-positive bacteria and hydrogen peroxide-negative bacteria to obtain the lactobacillus plantarum.
(2) Screening of Lactobacillus plantarum NM22-2
Pouring MRS agar culture medium into a culture dish as a lower plate, performing spot grafting on 5 mu L of lactobacillus plantarum bacterial liquid after solidification, culturing at 37 ℃ for 12h to form circular bacterial plaque, and adding 10mL of bacterial liquid containing penicillium expansum fungal spores (10 mL)4spore/mL) is covered on a lower layer culture medium containing bacterial plaque of the lactobacillus plantarum, the culture is carried out for 3-5 days at 25 ℃, the lactobacillus plantarum with the strongest antibacterial activity is screened, namely the diameter of an antibacterial ring is measured by a vernier caliper, and a strain with the largest antibacterial ring width is selected, namely the finally screened lactobacillus plantarum NM 22-2.
The MRS agar culture medium comprises 10g of soybean peptone, 5g of beef extract, 4g of yeast powder, 20g of glucose, 801mL of Tween-801, 2g of disodium hydrogen phosphate, 5g of anhydrous sodium acetate, 2g of citric acid triamine, 0.02g of manganese sulfate, 0.1g of magnesium sulfate and 15g of agar powder.
The preparation method of MRS agar culture medium comprises mixing the above MRS agar culture medium with distilled water, diluting to 1L, adjusting initial pH to 6.20, and sterilizing at 121 deg.C for 15 min.
The semi-solid PDA medium was purchased from OXID.
The results in figure 3 show that Lactobacillus plantarum NM22-2 has strong antibacterial activity on Penicillium expansum, and the diameter of the inhibition zone is 16.32 +/-3.27 mm.
(3) Identification of Lactobacillus plantarum NM22-2
And identifying the lactobacillus plantarum NM22-2 according to the colony morphology characteristics, the 16S rDNA sequence and the like.
The morphological characteristics of the lactobacillus plantarum NM22-2 colony are as follows: the bacterial colony is milky white, the surface is smooth and moist, the bacterial colony is in a drop shape, circular and raised shape, the edge is neat, and the diameter of the bacterial colony is 1-2 mm (see figure 1); a single colony is picked, gram-stained and observed by a microscope, and the gram-positive bacteria are straight rod-shaped, single, paired or chain-shaped, have the size of 3.0-8.0 mu m multiplied by 0.9-1.2 mu m, have no flagellum and do not produce spores (see figure 2).
Biological identification of Lactobacillus plantarum NM 22-216S rDNA:
the genome DNA of the strain Lactobacillus plantarum NM22-2 is used as a template, and the 16S rDNA of the Lactobacillus plantarum is subjected to PCR amplification by using a universal primer upstream primer 27F (5 '-NNNAGAGTTTGATCMTGGCTCAG-3') and a downstream primer 1492R (5 '-NNNACCTTGTTACGACTT-3') for amplifying the 16S rDNA of the bacteria. The PCR amplification procedure was: pre-denaturation at 94 deg.C for 5min, denaturation at 94 deg.C for 1min, annealing at 55 deg.C for 1min, extension at 72 deg.C for 1min, 30 cycles, and extension at 72 deg.C for 10 min. The PCR product is a single band, 16S rDNA (shown in a sequence table 3) is obtained through sequencing, the sequence length is 1438bp, meanwhile, homology comparison is carried out on the 16S rDNA sequence of the strain and the nucleotide sequence of a Lactobacillus plantarum model strain (Lactobacillus strain WCFS1), and the result homology is 99.99%, so that the screened Lactobacillus plantarum NM22-2 strain is determined to be Lactobacillus plantarum.
Example 2 bacteriostatic Activity of Lactobacillus plantarum NM22-2 sterile fermentation broth
Will be 5X 106Inoculating Lactobacillus plantarum NM22-2 with inoculation amount of 5% to MRS liquid culture mediumCulturing in culture medium at 37 deg.C for 12 hr, centrifuging at 4000 Xg for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain sterile fermentation liquid. Test groups: mixing Lactobacillus plantarum NM22-2 sterile fermentation broth and sterilized PDA culture medium at a ratio of 10% (v/v), pouring into a dish, cooling, solidifying, and inoculating 5 μ L fungal spore suspension (10 μ L) to the center of the dish5spores/mL); and (3) the negative control group PDA culture medium does not contain sterile fermentation liquor, the test group and the negative control group are cultured for 5-7 d at 25 ℃, the diameters of fungal colonies of the test group and the control group are measured, the hypha inhibition rate is calculated, and the test is repeated for 3 groups.
The inhibition ratio (%) - (control group fungal colony diameter-test group fungal colony diameter)/control group fungal colony diameter × 100%.
The results are shown in fig. 4, and it can be seen that lactobacillus plantarum NM22-2 sterile fermentation broth can significantly inhibit penicillium expansum, and its growth inhibition rate on penicillium expansum is 52.7 ± 4.7%.
Example 3 application of Lactobacillus plantarum NM22-2 sterile fermentation broth to control fruit spoilage
Selecting apple with uniform size and no wound on surface as research object, puncturing apple with 1mL sterile gun head to form wound with diameter of about 5mm, and mixing 5 μ L Penicillium expansum spore suspension (10 μ L)6spore/mL) into the wound of the apple, wherein a control group is only inoculated with the penicillium expansum suspension, a test group is added with 50 mu L of lactobacillus plantarum NM22-2 sterile fermentation liquor (the preparation method of the lactobacillus plantarum NM22-2 sterile fermentation liquor is the same as that of the sterile fermentation liquor in the example 2) after the penicillium expansum suspension is inoculated, the apple is placed in an incubator at 25 ℃ for 7 days, the rotting and deterioration conditions of the apple are observed, and the infection rate of the lactobacillus plantarum NM22-2 sterile fermentation liquor for inhibiting the mould expanse is calculated.
The infection inhibition rate (%) is (diameter of control putrefactive region-diameter of test putrefactive region)/diameter of control putrefactive region × 100%.
As can be seen from the graph 5, after the surface of the apple is punctured and inoculated with penicillium expansum and placed at 25 ℃ for 7 days, the diameter of a large-area rotten area of the apple in a control group is 39.82 +/-5.30 mm, the rotten area of the apple in a test group added with lactobacillus plantarum NM22-2 sterile fermentation liquor is obviously smaller, the diameter of the rotten area of the apple is 13.58 +/-1.67 mm, the infection inhibition rate of the apple is 65.90%, and the test result shows that the apple can be obviously inhibited from being infected by the penicillium expansum by adding the lactobacillus plantarum NM22-2 sterile fermentation liquor, and the storage time of the apple is prolonged.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of inner Mongolia agriculture
<120> lactobacillus plantarum and application thereof
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<170> SIPOSequenceListing 1.0
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<213> upstream primer (Artificial Sequence)
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nnnagagttt gatcmtggct cag 23
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<212> DNA
<213> downstream primer (Artificial Sequence)
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nnnaccttgt tacgactt 18
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<213> 16S rDNA(Artificial Sequence)
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gcctaataca tgcaagtcga acgaactctg gtattgattg gtgcttgcat catgatttac 60
atttgagtga gtggcgaact ggtgagtaac acgtgggaaa cctgcccaga agcgggggat 120
aacacctgga aacagatgct aataccgcat aacaacttgg accgcatggt ccgagtttga 180
aagatggctt cggctatcac ttttggatgg tcccgcggcg tattagctag atggtggggt 240
aacggctcac catggcaatg atacgtagcc gacctgagag ggtaatcggc cacattggga 300
ctgagacacg gcccaaactc ctacgggagg cagcagtagg gaatcttcca caatggacga 360
aagtctgatg gagcaacgcc gcgtgagtga agaagggttt cggctcgtaa aactctgttg 420
ttaaagaaga acatatctga gagtaactgt tcaggtattg acggtattta accagaaagc 480
cacggctaac tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggatt 540
tattgggcgt aaagcgagcg caggcggttt tttaagtctg atgtgaaagc cttcggctca 600
accgaagaag tgcatcggaa actgggaaac ttgagtgcag aagaggacag tggaactcca 660
tgtgtagcgg tgaaatgcgt agatatatgg aagaacacca gtggcgaagg cggctgtctg 720
gtctgtaact gacgctgagc tcgaaagtat gggtagcaaa caggattaga taccctggta 780
gtccataccg taaacgatga atgctaagtg ttggagggtt tccgcccttt cagtgctgca 840
gctaacgcat taagcattcc gcctggggag tacggccgca aggctgaaac tcaaaggaat 900
tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgaagctac gcgaagaacc 960
ttaccaggtc ttgacatact atgcaaatct aagagattag acgttccctt cggggacatg 1020
gatacaggtg gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc 1080
aacgagcgca acccttatta tcagttgcca gcattaagtt gggcactctg gtgagactgc 1140
cggtgacaaa ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg 1200
gctacacacg tgctacaatg gatggtacaa cgagttgcga actcgcgaga gtaagctaat 1260
ctcttaaagc cattctcagt tcggattgta ggctgcaact cgcctacatg aagtcggaat 1320
cgctagtaat cgcggatcag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg 1380
cccgtcacac catgagagtt tgtaacaccc aaagtcggtg gggtaacctt ttaggaac 1438
Claims (10)
1. The lactobacillus plantarum is characterized in that the lactobacillus plantarum is named as lactobacillus plantarum (A)Lactobacillus plantarum) NM22-2, deposited in China general microbiological culture Collection center with the preservation number of CGMCC No. 20197.
2. The lactobacillus plantarum of claim 1, wherein the lactobacillus plantarum is characterized by being milky white, smooth and moist, droplet-shaped, round, ridged, neat-edged, 1-2 mm in colony diameter, gram-positive bacteria in the shape of a straight rod, single, paired or chain-shaped, 3.0-8.0 μm x 0.9-1.2 μm in size, flagellate-free, and non-sporulating.
3. The Lactobacillus plantarum of claim 1, wherein the Lactobacillus plantarum is derived from a sample of Hurenbel yogurt, inner Mongolia.
4. A fermentation broth obtained by fermentation of the Lactobacillus plantarum strain defined in claim 1.
5. The fermentation broth according to claim 4, wherein the fermentation broth is a fermentation culture solution, and the preparation method of the fermentation culture solution comprises the following steps: inoculating lactobacillus plantarum into an MRS liquid culture medium, and culturing for 10-18 h at 35-40 ℃ to obtain a fermentation culture solution.
6. The fermentation broth of claim 5, wherein the fermentation broth is a fermentation supernatant obtained by centrifuging the fermentation broth.
7. The fermentation broth of claim 6, wherein the fermentation broth is a sterile fermentation broth obtained by filtering the fermentation supernatant with a filter membrane.
8. Use of a lactobacillus plantarum according to any one of claims 1-3 or a fermentation broth according to any one of claims 4-7 for the preparation of a product inhibiting penicillium expansum.
9. Use of a lactobacillus plantarum according to any one of claims 1-3 or a fermentation broth according to any one of claims 4-7 for the preservation and/or preservation of fruit.
10. An antistaling agent or preservative comprising the Lactobacillus plantarum according to any one of claims 1 to 3 and/or the fermentation broth according to any one of claims 4 to 7.
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