Application of acellular fermentation broth of lactobacillus rhamnosus CLK 101 in prevention and treatment of vegetable soft rot

Abstract

The invention discloses an application of a lactobacillus rhamnosus CLK 101 cell-free fermentation broth in prevention and treatment of vegetable soft rot, and belongs to the technical field of microbial fermentation engineering. The invention provides cell-free fermentation liquor capable of being used for preventing and treating vegetable soft rot caused by pectobacterium carotovorum. A source strain of the cell-free fermentation liquor is lactobacillus rhamnosus CLK 101, and the preservation number of the cell-free fermentation liquor is CGMCC (China General Microbiological Culture Collection Center) No.30491. Thermal inactivation of the cell-free fermentation broth at 121 DEG C does not affect the use effect, and the cell-free fermentation broth can significantly inhibit growth of pectobacterium carotovorum and formation of a biofilm of the pectobacterium carotovorum, destroy completeness of cell membranes of pathogenic bacteria, reduce metabolic activity of the pathogenic bacteria and prevent and control occurrence of soft rot, and has good application potential. And a novel method is provided for biological prevention and control of soft rot of vegetables.

Description

Technical Field

[0001] The present invention relates to the application of a cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 in preventing and controlling vegetable soft rot, and belongs to the technical field of microbial fermentation engineering. Background Art

[0002] Bacterial soft rot causes serious economic losses worldwide every year and is considered a challenge in the agricultural and food sectors. Approximately 15% - 30% of agricultural product losses are caused by bacterial soft rot. The genus Pectobacterium is the main pathogen of plant bacterial soft rot. Among them, Pectobacterium carotovorum has the widest host range among all soft rot bacteria and is considered one of the most destructive plant pathogens causing soft rot globally. In recent years, Pectobacterium carotovorum has caused huge economic losses to farmers, producers, and consumers worldwide, and post-harvest vegetable spoilage and cross-contamination have been issues of great concern. However, there is currently a lack of safe and effective inhibition strategies to protect vegetables from Pectobacterium carotovorum.

[0003] Pectobacterium carotovorum is a highly diverse species, consisting of three morphologically similar but phylogenetically distinct subspecies: Pcc (Pectobacterium carotovorum subsp. carotovorum), Pco (Pectobacterium carotovorum subsp. odoriferum), and Pcb (Pectobacterium carotovorum subsp. Brasilience). The virulence of Pectobacterium carotovorum mainly lies in its ability to produce plant cell wall degrading enzymes (PCWDEs) and form biofilms. Due to the formation of biofilms, this pathogen easily adheres to and colonizes on food and abiotic surfaces, causing damage to food. At the same time, this makes the pathogen more resistant to physical and chemical treatments, and even under harsh conditions, it is more resistant and persistent than naturally surviving bacteria. Therefore, a large amount of chemical antibacterial agents need to be used to obtain a positive antibacterial effect, which has led to the abuse of chemical antibacterial agents and seriously endangers human health.

[0004] Therefore, it is very necessary to develop an antibacterial agent that can both inhibit vegetable soft rot and not affect human health. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the object of the present invention is to provide an application of a cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 in preventing and controlling vegetable soft rot, wherein the Lactobacillus rhamnosus has a preservation number of CGMCC No. 30491; the preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the preservation time is: April 30, 2024; the taxonomic name is: Lactobacillus rhamnosus.

[0006] Preferably, the preparation method of the Lactobacillus rhamnosus cell-free fermentation broth is as follows:

[0007] (1) Lactobacillus rhamnosus CLK101 was inoculated into MRS liquid culture medium and cultured at 37°C and 150 rpm for 24 h to obtain a bacterial suspension.

[0008] (2) The bacterial suspension obtained in step (1) was centrifuged at 12000 rpm and 4°C for 10 min, and the bacterial suspension was filtered using a sterile filter with a pore size of 0.22 μm to obtain a cell-free fermentation broth. The cell-free fermentation broth was vacuum freeze-dried and stored in a -80°C refrigerator.

[0009] Technical effects of the present invention:

[0010] (1) The cell-free fermentation liquid of Lactobacillus rhamnosus CLK 101 provided by the present invention can inhibit the growth of Pectinobacterium carotii and the formation of biofilm, reduce the metabolic activity of the strain, destroy the integrity of its cell membrane and cause cell death. It has great application potential in preventing and controlling soft rot diseases mediated by Pectinobacterium carotii.

[0011] (2) The fermentation broth of the present invention does not contain live cells of Lactobacillus rhamnosus, thereby avoiding the adverse effects of Lactobacillus rhamnosus on vegetables. In addition, the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 does not contain active ingredients, which is more conducive to storage and use without worrying about the inactivation of ingredients. Moreover, heat inactivation at 121° C. does not affect its antibacterial function and disease prevention effect.

[0012] (3) The polypeptides and extracellular polysaccharides in the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 have good antioxidant effects. Compared with probiotics, it has the advantages of long shelf life, convenience and ease of use, strong environmental tolerance, and low activity loss. Spraying the cell-free fermentation broth of Lactobacillus rhamnosus on the surface of fruits and vegetables will not damage the quality and nutrition of fruits and vegetables, but can also inhibit harmful microorganisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the colony morphology of Lactobacillus rhamnosus CLK 101 on MRS solid medium.

[0014] Figure 2 Gram staining and microscopic observation results of Lactobacillus rhamnosus CLK 101.

[0015] Figure 3 Phylogenetic tree of Lactobacillus rhamnosus CLK 101.

[0016] Figure 4 Plate experiment diagram of the inhibition of the growth of Z3-3 by the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention.

[0017] Figure 5 Determination diagram of the minimum inhibitory concentration of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention against Z3-3.

[0018] Figure 6 Diagram of the growth effect of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations on Z3-3 within 24 hours.

[0019] Figure 7 Diagram of the determination of the cell membrane integrity of Z3-3 treated with the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations. Among them, A is the diagram of the determination of the cell membrane integrity of Z3-3 not treated with the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; B is the diagram of the determination of the cell membrane integrity of Z3-3 treated with 0.5×MIC of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; C is the diagram of the determination of the cell membrane integrity of Z3-3 treated with 1×MIC of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; D is the diagram of the determination of the cell membrane integrity of Z3-3 treated with 2×MIC of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; E is the diagram of the determination of the cell membrane integrity of Z3-3 treated with 4×MIC of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention.

[0020] Figure 8 Determination of the cell metabolic viability of Z3-3 treated with the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations. Among them, A is the XTT detection diagram of the cell metabolic viability of Z3-3 by the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; B is the diagram of the determination of the cell metabolic activity of Z3-3 by the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention.

[0021] Figure 9Determination of the effect of cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations on the biofilm formation of Z3-3. Among them, A is the crystal violet staining map of the biofilm formation of Z3-3 by the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; B is the measurement map of the biofilm formation of Z3-3 by the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention.

[0022] Figure 10 Observation map of the biofilm formation of Z3-3 treated with cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations. Among them, A is the biofilm formation map of Z3-3 without treatment with the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; B is the biofilm formation map of Z3-3 treated with 0.5×MIC of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; C is the biofilm formation map of Z3-3 treated with 1×MIC of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; D is the biofilm formation map of Z3-3 treated with 2×MIC of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention; E is the biofilm formation map of Z3-3 treated with 4×MIC of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention.

[0023] Figure 11 Disease incidence map of carrots co-inoculated with cell-free fermentation broth of Lactobacillus rhamnosus CLK 101, chlorothalonil, E. coli, and LB medium with Z3-3.

[0024] Figure 12 Disease incidence map of Chinese cabbages co-inoculated with cell-free fermentation broth of Lactobacillus rhamnosus CLK 101, chlorothalonil, E. coli, and LB medium with Z3-3. Detailed implementation manners

[0025] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, the protection scope of the present invention is not limited to the described content. Unless otherwise specified, the reagents and their reagent kits used in the embodiments are all commercially available products.

[0026] The MRS liquid medium is as follows: casein peptone 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, dipotassium hydrogen phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1.08 g / L, pH 5.5 - 5.9, autoclaved at 121°C for 15 min; the formula of the MRS solid medium is to add 1.5% (w / v) agar to the liquid medium.

[0027] The LB liquid medium is: peptone 10.0 g / L, sodium chloride 5.0 g / L, yeast extract powder 5.0 g / L, glucose 1.0 g / L, pH 6.8 - 7.2, sterilized at 121 °C for 15 min; the formula of the LB solid medium is adding 1.5% (w / v) agar to the liquid medium; the formula of the LB semi-solid medium is adding 0.75% (w / v) agar to the liquid medium.

[0028] The TSB liquid medium is: tryptone 17.0 g / L, plant peptone 3.0 g / L, sodium chloride 5.0 g / L, dipotassium hydrogen phosphate 2.5 g / L, glucose 2.5 g / L, pH 7.1 - 7.5, sterilized at 121 °C for 15 min.

[0029] The pathogenic bacterium used in this example is Pectobacterium carotovorum subsp. carotovorum (Pcc) Z3 - 3.

[0030] Example 1

[0031] Isolation and purification of Lactobacillus rhamnosus CLK 101 are as follows:

[0032] Take 20 mL of the mixed fruit juice sample (apple juice and orange juice) and dilute it with sterile water in a gradient of 10 -1 to 10 -7 . Take 100 μL of each of the 5 dilutions (10 -1 , 10 -4 , 10 -5 , 10 -6 and 10 -7 ) and spread them evenly on the MRS medium supplemented with calcium carbonate, and culture at 37 °C for 48 h. To obtain pure strains, pick single colonies with calcium lysis zones on a sterile inoculation loop in a laminar flow hood, inoculate them into the MRS medium for secondary purification, and incubate at 37 °C for 48 h. The final strain is cryopreserved at -20 °C in 30% (v / v) glycerol and named CLK 101.

[0033] Example 2

[0034] Identification and phylogenetic analysis of Lactobacillus rhamnosus CLK 101 are as follows:

[0035] (1) Colony morphological characteristics: Streak the strain CLK 101 on the MRS solid medium and culture at 37 °C for 48 h. As Figure 1 shown, the colonies are round, milky white, and smooth on the surface.

[0036] (2) Bacterial cell morphological characteristics: Perform Gram staining and microscopic observation on the strain CLK 101. The results are asFigure 2 As shown, the cells of this strain are rod-shaped and it is a Gram-positive bacterium.

[0037] (3) 16S rDNA sequence and phylogenetic analysis: The DNA of CLK101 was extracted using a bacterial DNA extraction kit, and the full-length 16S rDNA sequence of this bacterium was amplified by PCR using universal primers. After the size of the PCR product was detected by 1.5% agarose gel electrophoresis, it was sent to Sangon Biotech Co., Ltd. for sequencing, and the sequencing results are shown in SEQ ID NO: 1. The 16S rDNA sequence of the strain was submitted to the GenBank database (accession number: OM370898), and BLAST homology alignment was performed and a phylogenetic tree was constructed. The results are as Figure 3 shown, the sequence similarity between CLK 101 and Lactobacillus rhamnosus strain IGM3-10 (GenBank: MT197234.1) is as high as 99.55%. Further comparison with the NCBI 16S ribosomal RNA database showed that the sequence similarity between CLK 101 and the type strain NBRC 3425 of Lactobacillus rhamnosus (NCBI reference sequence: NR_113332.1) is 99.28%. Based on the above results, CLK 101 was identified as Lactobacillus rhamnosus.

[0038] Example 3

[0039] Preparation of cell-free fermentation broth of Lactobacillus rhamnosus CLK 101, and the specific preparation steps are as follows:

[0040] (1) Inoculate Lactobacillus rhamnosus CLK 101 into MRS liquid medium, culture at 37 °C and 150 rpm for 24 h to obtain a suspension of Lactobacillus rhamnosus CLK 101.

[0041] (2) Centrifuge the suspension of Lactobacillus rhamnosus CLK 101 obtained in step (1) at 12000 rpm and 4 °C for 10 min, collect the supernatant, and then filter the supernatant using a sterile filter with a pore size of 0.22 μm to obtain cell-free supernatant of Lactobacillus rhamnosus. Part of the cell-free fermentation broth of Lactobacillus rhamnosus was freeze-dried under vacuum and stored in a -80 °C refrigerator, and the other part was not freeze-dried.

[0042] Example 4

[0043] Oxford cup antibacterial test of cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 against Z3-3, and the specific steps are as follows:

[0044] (1) Streak the strain Z3-3 on an LB solid medium plate and culture overnight at 37 °C. Pick a single colony and culture it overnight at 37 °C in LB liquid medium to obtain a Z3-3 bacterial solution.

[0045] (2) Pour 10 mL of LB solid medium into a petri dish. After it cools down, mix 100 μL of Z3-3 bacterial solution with 5 mL of LB semi-solid medium and pour it into the dish. After the second layer of medium cools down, place an Oxford cup on the medium and add 200 μL of cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 into the Oxford cup. Set the MRS liquid medium as the control. Place the medium in a 4°C refrigerator, take it out after 4 h, and culture it in a 37°C constant temperature incubator for 12 h.

[0046] The experimental results are as Figure 4 shown. The antibacterial diameter of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention against Z3-3 reaches 24 mm ± 1 mm. The MRS liquid medium control set in the experiment does not form an antibacterial zone against Z3-3, excluding the influence of the MRS liquid medium in the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention on Z3-3, and proving that the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention has a good antibacterial effect on Z3-3.

[0047] Example 5

[0048] Detection of the minimum inhibitory concentration of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 against Z3-3, the specific steps are as follows:

[0049] Dissolve the freeze-dried cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 in sterile water to form a solution with a concentration of 1 mg / mL, and then prepare solutions with concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.39062, 0.19531, and 0.09766 mg / mL respectively. Use the method of Example 2 to culture the Z3-3 bacterial solution. Add 100 μL of 1×10 7 CFU / mL of Z3-3 bacterial solution and 100 μL of cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 with different concentrations into each well of a 96-well plate. Use sterile water as the control. After mixing evenly, place the 96-well plate in a 37°C incubator for 48 h, and measure the change of OD 600 .

[0050] The experimental results are as Figure 5 shown. The OD of Z3-3 600The value decreased with the increase in the concentration of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention, and when the concentration was 0.39062 mg / mL, it decreased significantly. The cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at a low concentration had a small effect on Z3-3, causing them to continue growing, but at a slower rate. As the concentration increased, the effect became gradually obvious, resulting in the cessation of growth of Z3-3. Therefore, the minimum inhibitory concentration (MIC) of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 against Z3-3 was determined to be 0.39062 mg / mL.

[0051] Example 6

[0052] The growth effect of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations on Z3-3 within 24 h was determined, and the specific steps are as follows:

[0053] 1 μL of the Z3-3 bacterial suspension cells cultured overnight was inoculated into a 96-well plate containing 100 μL of fresh LB medium, and then 100 μL of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC (where MIC was 0.39062 mg / mL) was added to different wells respectively, mixed evenly, and co-cultured at 37 °C for 24 h. The OD 600 change was measured with a microplate reader every 4 h. Sterile water was set as the control group, and each treatment was repeated independently three times.

[0054] As can be seen from Figure 6 this, there was no significant difference in the growth rate and number of Z3-3 cells treated with the cell-free fermentation broth of Lactobacillus rhamnosus at 0.5×MIC compared with the control group; the growth rate of Z3-3 cells treated with the cell-free fermentation broth of Lactobacillus rhamnosus at 1×MIC and 2×MIC was slower than that of the control group; the growth rate of Z3-3 cells treated with the cell-free fermentation broth of Lactobacillus rhamnosus at 4×MIC was the slowest and the number was the least. The results showed that the cell-free fermentation broth of Lactobacillus rhamnosus inhibited the growth of Z3-3 strain cells, and as the concentration increased, the cell growth rate became slower.

[0055] Example 7

[0056] The determination of the cell membrane integrity of Z3-3 treated with the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations was carried out, and the specific steps are as follows:

[0057] Collect the bacterial liquid of Z3-3 cells in the logarithmic growth phase, wash and resuspend it in PBS, add the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at 0.5×MIC, 1×MIC, 2×MIC and 4×MIC (where MIC is 0.39062 mg / mL), and incubate the mixture at 37°C for 4 h. Then, centrifuge the cells, wash with PBS, incubate with PI (propidium iodide, a solution with a concentration of 50 μg / ml) at 4°C for 30 minutes in the dark, centrifuge the samples, wash with PBS and resuspend, and perform analysis using a flow cytometer.

[0058] Result analysis, as Figure 7 shown, the number of dead Z3-3 cells treated with the cell-free fermentation broth of Lactobacillus rhamnosus CLK101 at 0.5×MIC, 1×MIC, 2×MIC and 4×MIC was 94.6%, 97.3%, 97.7%, 98.0%, while in the control group, the number of dead cells was only 0.3%. The results indicate that the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 can cause the rupture of the Z3-3 cell membrane and cell death, and as the concentration of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention increases, the number of dead cells increases.

[0059] Example 8

[0060] Determination of the cell metabolic viability of Z3-3 treated with cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations, the specific steps are as follows:

[0061] The present invention uses an XTT detection kit to detect the effect of the cell-free fermentation broth of CLK 101 at 4 kinds of MIC (0.5×MIC, 1×MIC, 2×MIC and 4×MIC) on the cell metabolic viability of Z3-3.

[0062] Principle of the XTT detection kit: Under the action of the electron coupling agent 1-methoxy PMS, XTT is reduced by the reducing dehydrogenase produced in living cells into a water-soluble orange-yellow formazan product. The stronger the cell viability, the more formazan is produced, resulting in a deeper color. The absorbance can be detected using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm.

[0063] Add 190 μL of the overnight-cultured Z3-3 bacterial liquid into a 96-well plate, and respectively add 10 μL of the lyophilized metabolites of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at 0.5×MIC, 1×MIC, 2×MIC and 4×MIC, mix well, incubate at 37°C for 1 hour, then add 10 μL of the XTT mixture to each well, and incubate at 37°C for 2 hours. Then measure the absorbance value at 450 nm.

[0064] Cell viability (%) = (OD value of the experimental group / OD value of the control group) 450 value / control group OD450 (Value) × 100%

[0065] Result analysis: As Figure 8 shown in A, compared with the control group, as the concentration of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention increases, the color of the solution becomes lighter; as shown in Figure 8 B, the cell viabilities of Z3-3 cells treated with the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention at 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC are 86.2%, 14.2%, 8.7%, and 8.0% respectively. From the above results, it can be seen that the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention can cause a decrease in the cell metabolic activity of Z3-3, and as the concentration increases, the cell metabolic activity of Z3-3 becomes lower.

[0066] Example 9

[0067] Effect of treatment with cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations on biofilm formation of Z3-3

[0068] (I) Determination of the amount of biofilm formed by treatment with cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations on Z3-3

[0069] In this example, the biofilm of Z3-3 was quantified by the crystal violet staining method. For the crystal violet staining of the biofilm, the more the number of biofilms formed, the darker the staining. After the staining was completed, acetic acid was used to dissolve the crystal violet, and the amount of biofilm could be compared by measuring the absorbance at OD 570 nm.

[0070] 1 μL of the overnight cultured Z3-3 bacterial suspension cells were inoculated into a 96-well plate containing 100 μL of fresh LB medium, and then 100 μL of the lyophilized metabolites of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at 0.5×MIC, 1×MIC, 2×MIC, and 4×MIC were added respectively, mixed well, covered with a 96-pin plate, and cultured at 37°C for 48 hours. Then, 125 μL of crystal violet with a concentration of 0.1% was used for staining. The stained biofilm was dissolved with 125 μL of acetic acid with a concentration of 30% to dissolve the crystal violet, and the OD was measured on a microplate reader 570 nm for quantification.

[0071] The experimental results are as Figure 9 shown. Figure 9 A shows that as the concentration of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention increases, the color of the solution gradually becomes lighter; Figure 9Group B showed the OD of Z3-3 cells treated with cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of 0.5×MIC, 1×MIC, 2×MIC and 4×MIC with the crystal violet staining solution for biofilm. 570 The values were 0.16, 0.11, 0.06 and 0.05 respectively, which were significantly lower than the OD 570 of 0.2 in the control group, and showed a decreasing trend.

[0072] (II) Observation on the formation of biofilm of Z3-3 treated with cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 at different concentrations

[0073] In this example, the formation of biofilm of Z3-3 was observed by laser confocal microscopy.

[0074] The overnight cultured Z3-3 bacterial suspension cells were inoculated into TSB culture medium at a ratio of 1%, mixed evenly, 500 μL of the mixed solution was added into a laser confocal dish, and then equal amounts of cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of 0.5×MIC, 1×MIC, 2×MIC and 4×MIC were added as experimental groups, and MRS culture medium was added as a control, and cultured at 37 °C for 24 h. Subsequently, the culture medium was removed, fixed with methanol for 20 min, and finally the methanol was discarded. The biofilm was stained using the Cell-CheckT cell viability / toxicity kit. The samples were observed under an Eclipse Ti laser confocal scanning microscope. Green emission fluorescence was detected at 500 - 550 nm in the AlexaFluor 488 dye channel.

[0075] The experimental results were as Figure 10 shown. Compared with the control group, with the increase in the concentration of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention, fewer biofilms of Z3-3 were observed.

[0076] In summary, the above results indicate that the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 of the present invention can inhibit the formation of biofilm of Z3-3, and the higher the concentration of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101, the fewer the number of biofilms formed by Z3-3.

[0077] Example 10

[0078] Study on the biocontrol effect of the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 against soft rot, the specific process is as follows:

[0079] The strains Z3-3 and E. coli were respectively streaked and activated on LB solid medium, cultured at 37 °C for 24 h, then single colonies on the plate were respectively picked and inoculated into LB liquid medium, and cultured overnight at 37 °C until the OD of the bacterial liquid 600> 2.0; Meanwhile, configure the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 cultured overnight using the method of Example 1.

[0080] In the biocontrol experiment, 4 groups of experiments were set up. Among them, the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 (CLK101) + Z3-3 was the experimental group, chlorothalonil + Z3-3 was the positive control group, E. coli + Z3-3 was the negative control group, and LB + Z3-3 was the blank control group.

[0081] Select fresh Chinese cabbages and carrots as the materials for the biocontrol experiment. Cut the stalks of Chinese cabbages into pieces of 4 cm × 5 cm, and cut the carrots into thick slices with a thickness of 0.3 cm. Wash the materials in tap water, and then continuously disinfect the surfaces with 44% sodium hypochlorite and 75% ethanol; mix the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 (CLK 101), chlorothalonil, E. coli, and LB medium with the Z3-3 bacterial solution at a ratio of 1:1 respectively. The working concentration of the mixed bacterial solution was OD 600 = 0.1. Subsequently, use a pipette to inoculate 2 μL of the mixed bacterial solution in the center of the experimental materials (Chinese cabbages and carrots).

[0082] The results are as Figure 11 and Figure 12 shown. In the biocontrol experiment of Chinese cabbages and carrots, no obvious disease symptoms were observed in the group of cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 (CLK 101) + Z3-3 and the chlorothalonil + Z3-3 group, but the diseased areas in the E. coli + Z3-3 group and the LB + Z3-3 group were larger, with obvious waterlogging phenomena, and the disease conditions were more serious. The above experimental results indicate that the cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 has a significant biocontrol effect on the soft rot of vegetables caused by Pectobacterium carotovorum subsp. carotovorum Z3-3.

Claims

1. Application of cell-free fermentation broth of Lactobacillus rhamnosus CLK 101 in preventing and controlling soft rot of vegetables.

2. The application according to claim 1, characterized in that: The preservation number of the Lactobacillus rhamnosus CLK 101 is CGMCC No. 30491.

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