Bacillus tequilensis and application of microbial agent of bacillus tequilensis in fruit preservation

Abstract

The invention provides bacillus tequilensis and application of a microbial agent of the bacillus tequilensis in fruit preservation, belongs to the technical field of microorganisms, and particularly provides bacillus tequilensis PZB-0006 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.33085. The invention relates to a culture method of bacillus tequilensis PZB-0006. The invention relates to a microbial agent. The microbial agent contains bacillus tequilensis PZB-0006 or a metabolite of the bacillus tequilensis PZB-0006; the invention discloses application of bacillus tequilensis PZB-0006 or a microbial agent in food preservation and biological prevention and control of plant diseases. The bacillus tequilensis PZB-0006 provided by the invention is wide in antibacterial spectrum and strong in inhibition capability.

Description

Technical Field

[0001] The present invention relates to the application of a strain of Bacillus tequilensis and its bacterial agent in fruit preservation, belonging to the field of microbial technology. Background Art

[0002] With the rapid development of the fruit industry, the rotting loss of fruits during post-harvest storage, transportation and shelf life has become one of the bottleneck problems restricting the development of the industry. Among them, the infectious diseases caused by microorganisms are one of the important reasons for the spoilage of fruits during post-harvest storage and transportation, resulting in losses as high as 25%-50%. Antiseptic and fresh-keeping agents are the main measures for reducing post-harvest losses in the fruit industry. However, due to problems such as poor awareness of usage guidelines and difficulty in consumer acceptance, the application of existing antiseptic and fresh-keeping agents is restricted to a certain extent. Therefore, it is urgent to develop and utilize new resources to replace chemical fungicides to meet the needs of developing green agriculture and green food.

[0003] As the most potential alternative to chemical fungicides, biological control has been the focus of research in the past forty years and has made great progress. Through extensive and in-depth exploration of strain resources, a large number of antagonistic bacteria with obvious inhibitory effects on post-harvest diseases of fruits and vegetables have been successfully isolated, such as Bacillus subtilis, Pseudomonas syringae, Pichia pastoris, etc. Some strains have been developed into commercial biocontrol agents and successfully applied in the industry, such as Candifruit, BioSave, etc. At present, as an important part of the post-harvest antiseptic and fresh-keeping technology system for fruits, biological control is playing an increasingly important role in reducing post-harvest losses, optimizing supply, improving quality and increasing efficiency in the fruit industry.

[0004] However, the development of post-harvest disease biocontrol microecological agents still faces various challenges, mainly reflected in problems such as single strains, unsatisfactory application effects, and high usage costs, which seriously restrict the rapid development of the industry. Therefore, further exploration of strain resources and development of new antiseptic and fresh-keeping microecological agents are of great significance for improving and perfecting the existing biological control of post-harvest diseases of fruits. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides the application of a strain of Bacillus tequilensis and its bacterial agent in fruit preservation.

[0006] The technical solution of the present invention is as follows: A strain of Bacillus tequilensis ( Bacillus tequilensis ) PZB-0006 was deposited with the China General Microbiological Culture Collection Center on December 16, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 33085.

[0007] The Bacillus tequilensis ( Bacillus tequilensis)PZB-0006, abbreviated as Bacillus tequilensis PZB-0006, was isolated from fruits.

[0008] The culturing method of the above-mentioned Bacillus tequilensis PZB-0006 includes the following steps: (1) Activate and culture Bacillus tequilensis PZB-0006 on a solid medium plate to obtain an activated strain; (2) Inoculate the activated strain obtained in step (1) into a seed liquid medium and culture it to obtain a seed solution; (3) Inoculate the seed solution obtained in step (2) into a fermentation liquid medium and culture it to obtain a fermentation broth.

[0009] Preferably according to the present invention, in step (1), the solid medium is an LB solid medium, and the conditions for activation culture are activation culture at 37 ± 2 °C for 16 - 24 h; In step (2), the seed liquid medium is an LB liquid medium, and the culture conditions are culture at 37 ± 2 °C and 180 - 200 rpm for 12 - 18 h; In step (3), the fermentation liquid medium is an LB liquid medium, inoculate the seed solution at an inoculation amount of 1% - 5% by volume of the fermentation medium, and the culture conditions are culture at 37 ± 2 °C and 180 - 200 rpm for 36 h - 48 h; In step (1), the components of the solid medium are by mass: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 15.0 g of agar, 1000 mL of water, pH 7.0 ± 0.2; In step (2) or (3), the components of the seed liquid medium or the fermentation liquid medium are by mass: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 1000 mL of water, pH 7.0 ± 0.1.

[0010] A microbial bactericide with an anti-corrosion and fresh-keeping function contains Bacillus tequilensis PZB-0006 or the metabolites of Bacillus tequilensis PZB-0006.

[0011] Preferably according to the present invention, the microbial bactericide is obtained by centrifuging to remove the supernatant after fermentation of Bacillus tequilensis PZB-0006, and the cells are resuspended with sterile water; The cell concentration of the microbial bactericide is (1.0 - 6.45) × 10 10 CFU / mL.

[0012] The application of the above-mentioned Bacillus tequilensis PZB-0006 or the above-mentioned microbial bactericide in any of the following includes the following: ① Food fresh-keeping; ② Biological control of plant diseases.

[0013] Preferably according to the present invention, for the said application, the food includes fruits and vegetables; The plant disease is a plant disease caused by pathogenic fungi.

[0014] More preferably, the fruits include Chinese dates and cherries.

[0015] Preferably according to the present invention, for the said application, the usage method is to spray Bacillus tequilensis PZB-0006 or the microbial inoculum onto the fruit surface.

[0016] More preferably, the cell concentration of Bacillus tequilensis PZB-0006 or the microbial inoculum is (0.1~6.45)×10 8 CFU / mL.

[0017] The beneficial effects of the present invention at least include the following: 1. The present invention screens out Bacillus tequilensis PZB-0006 which has broad-spectrum antibacterial ability against common spoilage fungi such as Aspergillus and Alternaria, and can be used for the preservation of fruits and vegetables and the biological control of plant diseases.

[0018] 2. By spraying the bacterial suspension of Bacillus tequilensis PZB-0006, the growth of spoilage fungi can be effectively inhibited, the fruit rot rate can be reduced, and the post-harvest storage and transportation time can be prolonged. Description of the Drawings

[0019] Figure 1 It is the colony and cell morphology diagram of Bacillus tequilensis PZB-0006; In the figure: A is the colony morphology of PZB-0006; B is the Gram staining of the cells.

[0020] Figure 2 It is the phylogenetic tree of Bacillus tequilensis PZB-0006.

[0021] Figure 3 It is the antibacterial diagram of evaluating Bacillus tequilensis PZB-0006 by the petri dish buckling method; In the figure: A, B, C, D, E, F, G, H are respectively Aspergilus flavus , Aspergillus welwitschiae , Aspergillus oryzae , Aspergillus aflatoxiformans , Alternaria cerealis , Talaromyces stollii , Fusarium solani , Talaromyces annesophieae the colony morphology and size in the blank control; A1, B1, C1, D1, E1, F1, G1, H1 are respectively the corresponding colony morphology and size after being treated with Bacillus tequilensis PZB-0006.

[0022] Figure 4 Picture for evaluating the antibacterial activity of Bacillus tequilensis PZB-0006 by the plate confrontation method; In the figure: A, B, C, D, E, and F are respectively Aspergillus costaricensis , Alternaria cerealis , Talaromyces stollii , Fusarium solani , Talaromyces annesophieae , Aspergillus aflatoxiformans The colony morphology and size in the blank control; A1, B1, C1, D1, E1, and F1 are respectively the corresponding colony morphology and size after treatment with Bacillus tequilensis PZB-0006.

[0023] Figure 5 Pictures of the fresh-keeping test of Bacillus tequilensis PZB-0006 agent on fresh jujubes.

[0024] Figure 6 Pictures of the fresh-keeping test of Bacillus tequilensis PZB-0006 agent on sweet cherries. Specific implementation mode

[0025] The technical solution of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings of the specification, but the scope of protection of the present invention is not limited thereto.

[0026] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0027] The test materials used in the following embodiments are all obtained from conventional biochemical reagent manufacturers unless otherwise specified.

[0028] In the following embodiments, the quantitative tests are all set with three repeated experiments, and the results are averaged.

[0029] Example 1 Isolation and identification of Bacillus tequilensis ( Bacillus tequilensis ) PZB-0006 A bacterial strain was isolated from the fruit surface and named strain PZB-0006. The colony was irregular, with wrinkles on the surface, milky white in color, and the cells were short rod-shaped, Gram-positive (see Figure 1 ).

[0030] The 16S rDNA sequence of strain PZB-0006 is shown in SEQ ID NO.1, and its taxonomic status is in the same branch as Bacillus tequilensis ( Bacillus tequilensis ) (see Figure 2 ). Strain PZB-0006 belongs to Bacillus tequilensis ( Bacillus tequilensis ).

[0031] Strain PZB-0006 was preserved, and the specific information is as follows: A strain of Bacillus tequilensis ( Bacillus tequilensis ), PZB-0006, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on December 16, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 33085.

[0032] Example 2 Evaluation of the antibacterial function of Bacillus tequilensis ( Bacillus tequilensis ), PZB-0006 The antibacterial function of Bacillus tequilensis ( Bacillus tequilensis ), PZB-0006 was evaluated by the plate confrontation method and the plate sandwich method, using a strain of Bacillus tequilensis C50 isolated from the surface of cherries as the control strain. The control strains and pathogenic bacteria used in the examples were provided by the National Plant Protection Microbial Germplasm Resource Bank (Shandong).

[0033] The evaluation of the antibacterial function includes the following steps: (1) Strain activation: Streak the PZB-0006 strain preserved on the slant on an LB solid medium (tryptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, agar 15.0 g, water 1000 mL, pH 7.0 ± 0.2, autoclaved at 121 °C for 20 min), and incubate at 37 °C for 24 h.

[0034] (2) Plate confrontation method: Inoculate a pathogenic bacteria cake in the center of the surface of a PDA medium (potato powder 6.0 g, glucose 20.0 g, agar 20.0 g, water 1000 mL, pH 5.6 ± 0.2, autoclaved at 115 °C for 20 min) with an inoculation loop. At the same time, symmetrically inoculate the activated Bacillus tequilensis at a point 1-2 cm away from the bacteria cake, and incubate at 28 °C for 5 d. Use the plate without inoculating Bacillus tequilensis as the control group. Measure the colony diameters of the treatment group and the control group with a vernier caliper, and calculate and evaluate the antibacterial ability.

[0035] (3)Petri dish buckling method: Use an inoculation loop to pick up the activated strain and inoculate it into LB liquid medium (tryptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, water 1000 mL, pH 7.0 ± 0.1, autoclaved at 121 °C for 20 min). Incubate in a constant temperature shaker at 37 °C and 180 rpm for 48 h to obtain the fermentation broth of the strain. Centrifuge the fermentation broth at 12000 r / min for 2 min, discard the supernatant, and resuspend the cells with an equal volume of sterile water to obtain a cell suspension. Inoculate the pathogenic fungus in the center of a PDA plate, and use a pipette to aspirate 100 µL of the cell suspension and spread it on an LB solid plate. Buckle the PDA solid plate and the LB solid plate together and fix them with paraffin sealing film. Use the plate without inoculating Bacillus tequilensis as the control group. Incubate at 28 °C for 5 d, observe the growth situation, measure the colony diameter of the treatment group and the control group with a vernier caliper, and calculate and evaluate the antibacterial ability.

[0036] Inhibition rate (%) = (Colony diameter of the control group - Colony diameter of the treatment group) / Colony diameter of the control group × 100 Table 1 Inhibition rate of PZB-0006 against different spoilage fungi determined by the Petri dish buckling method

[0037] Note: / represents no antibacterial ability.

[0038] Table 2 Inhibition rate of PZB-0006 against different spoilage fungi determined by the plate confrontation method

[0039] Note: / represents no antibacterial ability.

[0040] The results of the Petri dish buckling antibacterial test (Table 1, Figure 3 ) show that Bacillus tequilensis PZB-0006 has an obvious inhibitory effect on the growth of 8 pathogenic bacteria. Among them, the inhibition rate against Talaromyces stollii, Aspergillus oryzae can reach over 90%, and the inhibition rates against Aspergillus welwitschiae, Aspergillus aflatoxiformans, Alternaria cerealis, Talaromyces annesophieae, Aspergilus flavus are all above 80%, indicating that Bacillus tequilensis ( Bacillus tequilensis ) PZB-0006 can achieve a broad-spectrum antibacterial effect by producing highly active volatile substances.

[0041] The results of the plate confrontation antibacterial test (Table 2, Figure 4 ) show that Bacillus tequilensis PZB-0006 has an obvious inhibitory effect on the growth of 6 pathogenic bacteria. Among them, the inhibition rate against Alternaria cerealis, Talaromyces stollii, Talaromyces annesophieae can reach over 80%.

[0042] In addition, compared with the reference strain Bacillus tequilensis C50, Bacillus tequilensis PZB-0006 has a broader antibacterial spectrum and stronger inhibitory ability against pathogenic bacteria.

[0043] Some of the above spoilage-causing pathogenic fungi are also common pathogenic bacteria that cause plant diseases, such as Alternaria cerealis, Aspergilus flavus, Aspergillus welwitschiae, Fusarium solani, Aspergillus costaricensis.

[0044] Bacillus tequilensis PZB-0006 has a broad antibacterial spectrum and strong inhibitory ability. It can not only be used for the preservation of fruits and vegetables, but also for the prevention and control of plant diseases, especially plant diseases caused by pathogenic fungi.

[0045] Example 3 Preparation of the microbial agent of Bacillus tequilensis ( Bacillus tequilensis ) PZB-0006 The preparation method of the microbial agent of Bacillus tequilensis PZB-0006 includes the following steps: (1) Bacillus tequilensis PZB-0006 was cultured on a solid medium (LB solid medium: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 15.0 g of agar, 1000 mL of water, pH 7.0 ± 0.2, autoclaved at 121 °C for 20 min) plate at 37 °C for 24 h to obtain an activated strain; (2) The activated strain obtained in step (1) was inoculated into a seed liquid medium (LB liquid medium: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 1000 mL of water, pH 7.0 ± 0.1, autoclaved at 121 °C for 20 min), and cultured at 37 °C and 180 rpm for 24 h to obtain a seed liquid; (3) The seed liquid obtained in step (2) was inoculated into a fermentation liquid medium (LB liquid medium: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 1000 mL of water, pH 7.0 ± 0.1, autoclaved at 121 °C for 20 min). The seed liquid was inoculated at an inoculation amount of 1% by volume of the fermentation liquid medium, and cultured at 37 °C and 180 rpm for 48 h to obtain a fermentation broth; the cell concentration of the obtained fermentation broth was 5.65×10 8 CFU / mL; (4) The fermentation broth was aliquoted into 100 mL centrifuge tubes and placed in a high-speed refrigerated centrifuge, centrifuged at 12000 rpm and 4 °C for 5 min; the supernatant was discarded, and the cells were resuspended with sterile water to prepare a microbial agent, and the cell concentration was 6.45×10 10 CFU / mL.

[0046] Example 4 Antiseptic and Fresh-keeping Test of Microbial Inoculum on Fresh Jujubes The fresh jujube variety used in this experiment was Jingcang No. 1, and 5 treatments were set up, namely: (1) T1: 100-fold dilution of the Paenibacillus tequilensis PZB-0006 inoculum (the microbial inoculum prepared in Example 3), with a cell concentration of 6.45×10 8 CFU / mL; (2) T2: 1000-fold dilution of the Paenibacillus tequilensis PZB-0006 inoculum (the microbial inoculum prepared in Example 3), with a cell concentration of 6.45×10 7 CFU / mL; (3) Blank control CK: Treated with sterile water; (4) Positive control PC: Treated with the chemical fungicide fludioxonil (0.30 g / L).

[0047] Select Dongzao fruits with uniform size, no pests, diseases and mechanical damage, and consistent maturity after picking for standby. Accurately measure 30 mL of the solution used in each treatment, put it into a sterilized sprayer, and then evenly spray it on the surface of the Dongzao fruits. There are 30 fruits in each group, and each treatment has 3 replicates. After the surface is naturally dried, put it into a PET fresh-keeping box, seal it with an outer plastic fresh-keeping bag, and place it at room temperature of 24±1°C to observe and record the changes on the fruit surface.

[0048] Rot rate (%) = Number of rotten fruits / Total number of fruits × 100 Rot inhibition rate (%) = (Rot rate of control group - Rot rate of treatment group) / Rot rate of control group × 100 Table 3 Results of the antiseptic and fresh-keeping test of microbial inoculum on fresh jujubes

[0049] Note: The numbers in the table are mean ± standard deviation, and different letters represent significant differences ( p <0.05) The results are shown in Table 3, Figure 5 , after 7 days of storage, the Paenibacillus tequilensis PZB-0006 inoculum can effectively reduce the rot rate of fresh jujubes during storage. When the inoculum concentration is 6.45×10 8 CFU / mL, the fruit rot rate is 33.33%, and compared with the blank control, the rot inhibition rate is 23.08%. When the inoculum concentration is 6.45×10 7 CFU / mL, the antiseptic effect is the best, the fruit rot rate is only 13.33%, compared with the blank control, the rot inhibition rate can reach 69.27%, and compared with the positive control, the corruption inhibition rate is also significantly improved.

[0050] Example 5 Antiseptic and Fresh-keeping Experiment of Microbial Inoculum on Sweet Cherries The sweet cherry variety used in this experiment was Meizao. A total of 4 treatments were set up, namely (1)T1: 100-fold dilution of Bacillus tequilensis PZB-0006 inoculum (the microbial inoculum prepared in Example 3), with a cell concentration of 6.45×10 8 CFU / mL; (2)T2: 1000-fold dilution of Bacillus tequilensis PZB-0006 inoculum (the microbial inoculum prepared in Example 3), with a cell concentration of 6.45×10 7 CFU / mL; (3)Blank control CK: Sterile water treatment.

[0051] (4)Positive control PC: Treatment with the chemical fungicide fludioxonil (0.30 g / L).

[0052] Pick out sweet cherry fruits with uniform size, no pests, diseases and mechanical damage, and consistent maturity after picking for standby. Accurately measure 30 mL of the solutions used in each treatment, respectively put them into sterilized sprayers, and then evenly spray them on the surface of sweet cherry fruits. There are 60 fruits in each group, and each treatment has 3 replicates. After the surface is naturally dried, put them into PET fresh-keeping boxes, seal them with plastic fresh-keeping bags outside, and place them at room temperature of 24±1℃, and observe and record the changes on the fruit surface.

[0053] Rot inhibition rate (%) = (control group rot rate - treatment group rot rate) / control group rot rate × 100 Table 4 Results of Antiseptic and Fresh-keeping Experiment of Microbial Inoculum on Sweet Cherries

[0054] Note: The numbers in the table are mean ± standard deviation, and different letters represent significant differences ( p <0.05) The results are shown in Table 4 Figure 6 , after 8 days of storage, the results of the antiseptic and fresh-keeping experiment of Bacillus tequilensis PZB-0006 inoculum on postharvest cherries are shown in Table 4. The inhibition rate of spraying treatment with 6.45×10 8 CFU / mL inoculum (T1) on postharvest cherry spoilage was 59.09%, and the antiseptic effect of 6.45×10 7 CFU / mL inoculum (T2) was 36.37%, both of which reached a significant level compared with the blank control ( p <0.05). This shows that Bacillus tequilensis PZB-0006 inoculum can effectively control postharvest diseases of cherries.

[0055] The Bacillus tequilensis PZB-0006 provided by the present invention has a broad antibacterial spectrum and stronger inhibitory ability, and can be used for the fresh-keeping of fruits and vegetables and the biological control of plant diseases.

Claims

1. A strain of Bacillus tequilensis ( Bacillus tequilensis ), designated as PZB-0006, was deposited on December 16, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 33085.

2. The culture method of Bacillus tequilensis PZB-0006 according to claim 1, characterized in that, It includes the following steps: (1) Activate and culture Bacillus tequilensis PZB-0006 on a solid medium plate to obtain an activated strain; (2) Inoculate the activated strain obtained in step (1) into a seed liquid medium and culture it to obtain a seed solution; (3) Inoculate the seed solution obtained in step (2) into a fermentation liquid medium and culture it to obtain a fermentation broth.

3. The culturing method according to claim 2, wherein In step (1), the solid medium is an LB solid medium, and the conditions for activation culture are activation culture at 37±2°C for 16 - 24 h; In step (2), the seed liquid medium is an LB liquid medium, and the culture conditions are culture at 37±2°C and 180 - 200 rpm for 12 - 18 h; In step (3), the fermentation liquid medium is an LB liquid medium, inoculate the seed solution at an inoculation amount of 1% - 5% by volume of the fermentation medium, and the culture conditions are culture at 37±2°C and 180 - 200 rpm for 36 h - 48 h; In step (1), the components of the solid medium are by mass: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 15.0 g of agar, 1000 mL of water, pH 7.0±0.2; In step (2) or (3), the components of the seed liquid medium or the fermentation liquid medium are by mass: 10.0 g of tryptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 1000 mL of water, pH 7.0±0.

1.

4. A microbial inoculant, characterized in that, Containing Bacillus tequilensis PZB-0006 as claimed in claim 1 or the metabolite of Bacillus tequilensis PZB-0006.

5. The microbial inoculant according to claim 4, characterized in that, The microbial inoculant is obtained by centrifuging the supernatant after fermentation of Bacillus tequilensis PZB-0006 and resuspending the thallus with sterile water; The cell concentration of the microbial inoculum is (1.0~6.45)×10 10 CFU / mL.

6. The application of Bacillus tequilensis PZB-0006 as claimed in claim 1 or the microbial inoculant as claimed in claim 4 in any one of the following, including: ① Food preservation; ② Biological control of plant diseases.

7. The application according to claim 6, characterized in that, The food includes fruits and vegetables; The plant diseases are plant diseases caused by pathogenic fungi.

8. The application according to claim 7, wherein The fruits include jujubes and cherries.

9. The application according to claim 6, characterized in that The usage method is to spray Bacillus tequilensis PZB-0006 or the microbial inoculant onto the fruit surface.

10. The application according to claim 9, characterized in that, The cell usage concentration of Bacillus tequilensis PZB-0006 or microbial inoculum is (0.1~6.45)×10 8 CFU / mL.

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