Application of bacillus tequilensis and its bacterial agent in fruit preservation

Abstract

The application provides application of a bacillus turicatus strain and a bacterial agent thereof in fruit preservation, belongs to the technical field of microorganisms, and specifically provides a bacillus turicatus strain PZB-0006 with a preservation number of CGMCC No.33085, a culture method of the bacillus turicatus strain PZB-0006, a microbial agent containing the bacillus turicatus strain PZB-0006 or metabolites of the bacillus turicatus strain PZB-0006, application of the bacillus turicatus strain PZB-0006 or the microbial agent in food preservation and biological control of plant diseases, and the bacillus turicatus strain PZB-0006 provided by the application has a wide antibacterial spectrum and strong inhibition.

Description

Technical Field

[0001] This invention relates to a strain of Bacillus tektii and its inoculant for the preservation of fruit, belonging to the field of microbial technology. Background Technology

[0002] With the rapid development of the fruit industry, post-harvest storage and transportation, as well as fruit spoilage during shelf life, have become a bottleneck restricting the industry's development. Among these issues, infectious diseases caused by microorganisms are a major cause of spoilage during post-harvest storage and transportation, resulting in losses as high as 25%-50%. Preservatives are a primary measure for reducing post-harvest losses in the fruit industry; however, poor awareness of usage guidelines and difficulties in consumer acceptance have limited the application of existing preservatives to some extent. Therefore, developing and utilizing new resources to replace chemical fungicides to meet the needs of developing green agriculture and green food is now an urgent task.

[0003] Biological control, as the most promising alternative to chemical fungicides, has been a focus of research and has made significant progress over the past four decades. Through extensive and in-depth exploration of microbial resources, numerous antagonistic bacteria with significant inhibitory effects on postharvest diseases of fruits and vegetables have been successfully isolated, such as Bacillus subtilis, Pseudomonas syringae, and Pichia pastoris. Some of these strains have been developed into commercial biocontrol agents and successfully applied in industry, such as Candifruit and BioSave. Currently, biological control, as an important component of postharvest preservation technology for fruits, plays an increasingly vital role in reducing postharvest losses, optimizing supply, and improving quality and efficiency in the fruit industry.

[0004] However, the development of biocontrol microbial agents for postharvest diseases still faces several challenges, mainly including the use of single strains, unsatisfactory application effects, and high costs, which seriously restrict the rapid development of the industry. Therefore, further exploration of microbial resources and the development of new preservative and antiseptic microbial agents are of great significance for improving and perfecting the existing biological control of postharvest diseases in fruits. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing an application of Bacillus tektii strain and its inoculant in fruit preservation.

[0006] The technical solution of the present invention is as follows:

[0007] A strain of Bacillus tekiria ( Bacillus tequilensis PZB-0006 was deposited on December 16, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33085.

[0008] The Bacillus tergenta (Bacillus tequilensis ) PZB-0006, referred to as Bacillus tequilensis PZB-0006, is isolated from fruits.

[0009] The culture method of the above-mentioned Bacillus tequilensis PZB-0006 comprises the following steps:

[0010] (1) The Bacillus tequilensis PZB-0006 is activated and cultured on a solid culture medium plate to obtain an activated strain;

[0011] (2) The activated strain obtained in step (1) is inoculated into a seed liquid culture medium for culture to obtain a seed liquid;

[0012] (3) The seed liquid obtained in step (2) is inoculated into a fermentation liquid culture medium for culture to obtain a fermentation liquid.

[0013] According to the present application, preferably, in step (1), the solid culture medium is LB solid culture medium, and the condition for the activation culture is 37±2℃ for 16-24h;

[0014] In step (2), the seed liquid culture medium is LB liquid culture medium, and the culture condition is 37±2℃, 180-200rpm for 12-18h;

[0015] In step (3), the fermentation liquid culture medium is LB liquid culture medium, and the inoculation amount is 1%-5% of the volume percentage of the fermentation medium, and the culture condition is 37±2℃, 180-200rpm for 36h-48h;

[0016] In step (1), the components of the solid culture medium are as follows by mass: 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 15.0g of agar, 1000mL of water, and pH 7.0±0.2;

[0017] In step (2) or (3), the components of the seed liquid culture medium or the fermentation liquid culture medium are as follows by mass: 10.0g of tryptone, 5.0g of yeast extract powder, 10.0g of sodium chloride, 1000mL of water, and pH 7.0±0.1.

[0018] A microbial agent with a preservative and fresh-keeping function contains Bacillus tequilensis PZB-0006 or a metabolite of Bacillus tequilensis PZB-0006.

[0019] According to the present application, preferably, the microbial agent is obtained by resuspending the bacterial body with sterile water after centrifugation to remove the supernatant after fermentation of Bacillus tequilensis PZB-0006;

[0020] The bacterial body concentration of the microbial agent is (1.0-6.45)×1010 CFU / mL.

[0021] The application of the above-mentioned Bacillus tequilensis PZB-0006 or the above-mentioned microbial inoculant in any one of the following aspects, including the following:

[0022] ① Food preservation;

[0023] ② Plant disease biological control.

[0024] According to the application, preferably, the food includes fruits and vegetables;

[0025] The plant disease is a plant disease caused by pathogenic fungi.

[0026] Further preferably, the fruits include dates and cherries.

[0027] According to the application, preferably, the application uses a method of spraying Bacillus tequilensis PZB-0006 or a microbial inoculant to the surface of the fruit.

[0028] Further preferably, the concentration of the bacterial body of Bacillus tequilensis PZB-0006 or the microbial inoculant is (0.1-6.45) × 10 8 CFU / mL.

[0029] The beneficial effects of the application at least include the following:

[0030] 1. The application selects Bacillus tequilensis PZB-0006 which has broad-spectrum bacteriostatic ability to common rot-causing fungi such as Aspergillus and Alternaria, and can be used for fruit preservation and plant disease biological control.

[0031] 2. The Bacillus tequilensis PZB-0006 bacterial suspension is used by spraying, which can effectively inhibit the growth of rot-causing fungi, reduce the fruit rot rate, and prolong the postharvest storage and transportation time. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 It is a colony and bacterial body morphology diagram of Bacillus tequilensis PZB-0006;

[0033] In the figure: A is the colony morphology of PZB-0006; B is the gram staining of the bacterial body.

[0034] Figure 2 It is a phylogenetic tree of Bacillus tequilensis PZB-0006.

[0035] Figure 3 It is a plate pair test method for evaluating the bacteriostatic effect of Bacillus tequilensis PZB-0006;

[0036] In the figure: A, B, C, D, E, F, G, and H are respectivelyAspergilus 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 the corresponding colony morphology and size after treatment by Bacillus turicensis PZB-0006.

[0037] Figure 4 The picture of evaluating the bacteriostasis of Bacillus turicensis PZB-0006 by flat plate confrontation method;

[0038] In the figure: A, B, C, D, E, F are 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, F1 are the corresponding colony morphology and size after treatment by Bacillus turicensis PZB-0006.

[0039] Figure 5 The picture of preservation test of Bacillus turicensis PZB-0006 agent on fresh jujube.

[0040] Figure 6 The picture of preservation test of Bacillus turicensis PZB-0006 agent on sweet cherry. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be further described below in combination with examples and the drawings of the specification, but the scope of protection of the present application is not limited thereto.

[0042] The experimental methods in the following examples are all conventional methods, unless otherwise specified.

[0043] The experimental materials used in the following examples are all purchased from conventional biochemical reagent manufacturers, unless otherwise specified.

[0044] In the quantitative test in the following examples, three repeated experiments are set, and the average value is taken.

[0045] Example 1

[0046] Isolation and identification of Bacillus turicensis (Bacillus turicensis) Bacillus tequilensis PZB-0006

[0047] A bacterial strain, named PZB-0006, was isolated from the surface of the fruit. The colonies were irregular, wrinkled, and milky white. The bacteria were short rod-shaped and Gram-positive (see [link to relevant documentation]). Figure 1 ).

[0048] The 16S rDNA sequence of strain PZB-0006 is shown in SEQ ID NO.1, and its taxonomic position is similar to that of Bacillus tekirae (…). Bacillus tequilensis ) are in the same branch (see Figure 2 PZB-0006 strain belongs to Bacillus tekirae ( Bacillus tequilensis ).

[0049] The PZB-0006 strain was preserved, and the specific information is as follows:

[0050] A strain of Bacillus tekiria ( Bacillus tequilensis PZB-0006 was deposited on December 16, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33085.

[0051] Example 2

[0052] Bacillus tekirulatus ( Bacillus tequilensis Evaluation of the antibacterial function of PZB-0006

[0053] The plate confrontation method and the plate inverting method were used to evaluate Bacillus tekirae ( Bacillus tequilensis The antibacterial function of PZB-0006 was demonstrated using a control strain, *Bacillus tekirica* C50 isolated from the surface of cherry. All control strains and pathogens used in the examples were provided by the National Germplasm Bank of Plant Protection Microorganisms (Shandong).

[0054] The evaluation of antibacterial function includes the following steps:

[0055] (1) Activation of strain: The PZB-0006 strain preserved on slant was streaked onto LB solid medium (10.0g tryptone, 5.0g yeast extract, 10.0g sodium chloride, 15.0g agar, 1000mL water, pH 7.0±0.2, autoclaved at 121℃ for 20 min) and incubated at 37℃ for 24h.

[0056] (2) Flat plate confrontation method: In the PDA medium (potato infusion powder 6.0 g, glucose 20.0 g, agar 20.0 g, water 1000 mL, pH 5.6±0.2, 115℃, 20 min high pressure sterilization), the central surface was inoculated with pathogenic bacteria cake with inoculation ring, and the activated Bacillus turkiyaensis was inoculated at the symmetrical point 1-2 cm away from the bacteria cake, and cultured at 28℃ for 5d. The flat plate without Bacillus turkiyaensis was used as the control group, and the colony diameters of the treatment group and the control group were measured with a vernier caliper, and the inhibition ability was calculated and evaluated.

[0057] (3) Flat plate against buckle method: The activated strain was inoculated in 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, 121℃, 20 min high pressure sterilization) with inoculation ring, and cultured at 37℃, 180 rpm constant temperature shaker for 48 h to obtain the fermentation liquid of the strain. The fermentation liquid was centrifuged at 12000 r / min for 2 min, and the supernatant was discarded. The bacterial cells were resuspended with an equal amount of sterile water to obtain a bacterial suspension. The pathogenic fungi were inoculated in the center of the PDA plate, and 100µL of bacterial suspension was taken with a pipette gun and spread on the LB solid plate. The PDA solid plate and the LB solid plate were buckled and fixed with paraffin sealing film. The plate without Bacillus turkiyaensis was used as the control group. Cultured at 28℃ for 5d, the growth was observed, the colony diameters of the treatment group and the control group were measured with a vernier caliper, and the inhibition ability was calculated and evaluated.

[0058] Inhibition rate (%) = (control group colony diameter - treatment group colony diameter) / control group colony diameter x 100

[0059] Table 1 Inhibition rate of PZB-0006 on different rot fungi by flat plate against buckle method

[0060]

[0061] Note: / represents no inhibition ability.

[0062] Table 2 Inhibition rate of PZB-0006 on different rot fungi by flat plate confrontation method

[0063]

[0064] Note: / represents no inhibition ability.

[0065] The results of flat plate against buckle inhibition test (Table 1, Figure 3 ) showed that Bacillus turkiyaensis PZB-0006 had obvious inhibition effect on the growth of 8 kinds of pathogenic fungi. Among them, the inhibition rate on Talaromyces stollii, Aspergillus oryzae could reach more than 90%, and the inhibition rate on Aspergillus welwitschiae, Aspergillus aflatoxiformans, Alternaria cerealis, Talaromyces annesophieae, Aspergilus flavusThe inhibition rates are all above 80%, indicating that Bacillus turicensis (PZB-0006) can achieve broad-spectrum antibacterial effect by producing high-activity volatile active substances. Bacillus tequilensis

[0066] The flat confrontation antibacterial test results (Table 2, Figure 4 ) show that Bacillus turicensis PZB-0006 has obvious inhibitory effect on the growth of 6 kinds of pathogenic bacteria. Among them, Alternaria cerealis, Talaromyces stollii, Talaromyces annesophieae The inhibition rate can reach more than 80%.

[0067] In addition, compared with the comparative strain Bacillus turicensis C50, Bacillus turicensis PZB-0006 has a wider antibacterial spectrum and stronger inhibitory capacity on pathogenic bacteria.

[0068] Some of the above rot-causing pathogenic fungi are also common pathogenic fungi causing plant diseases, such as Alternaria cerealis, Aspergilus flavus, Aspergillus welwitschiae, Fusarium solani, Aspergillus costaricensis.

[0069] Bacillus turicensis PZB-0006 has a wide antibacterial spectrum and stronger inhibitory capacity, which can not only be used for fruit preservation, but also be used for the prevention and control of plant diseases, especially plant diseases caused by pathogenic fungi.

[0070] Example 3

[0071] Preparation of Bacillus turicensis (PZB-0006) microbial inoculant Bacillus tequilensis Preparation of Bacillus turicensis (PZB-0006) microbial inoculant

[0072] The preparation method of Bacillus turicensis PZB-0006 microbial inoculant comprises the following steps:

[0073] (1) Bacillus turicensis PZB-0006 is activated and cultured on a solid culture medium (LB solid culture 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, 121℃, 20 min high-pressure sterilization) at 37℃ for 24 h to obtain an activated strain;

[0074] (2) The activated strain obtained in step (1) is inoculated into a seed liquid culture medium (LB liquid culture 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, 121℃, 20 min high-pressure sterilization) and cultured at 37℃ and 180 rpm for 24 h to obtain a seed liquid;

[0075] ​(3) The seed liquid prepared in step (2) was inoculated into a fermentation liquid medium (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, 121℃, 20 min high-pressure sterilization), and the seed liquid was inoculated at a volume percentage of 1% of the fermentation liquid medium, and cultured at 37℃, 180 rpm for 48 h to prepare a fermentation liquid; the cell concentration of the obtained fermentation liquid was 5.65×10 8 CFU / mL;

[0076] (4) The fermentation liquid was divided into 100 mL centrifuge tubes and placed in a high-speed refrigerated centrifuge, centrifuged at 12000 rpm, 4℃ for 5 min; the supernatant was discarded, and the cells were resuspended with sterile water to prepare a microbial inoculant, and the cell concentration was 6.45×10 10 CFU / mL.

[0077] Example 4

[0078] Preservation test of the microbial inoculant on fresh jujube

[0079] The fresh jujube used in this experiment was Jingtian No. 1, and a total of 5 treatments were set, which were:

[0080] (1) T1: 100-fold dilution liquid of Bacillus mojavensis PZB-0006 inoculant (microbial inoculant prepared in Example 3), cell concentration 6.45×10 8 CFU / mL;

[0081] (2) T2: 1000-fold dilution liquid of Bacillus mojavensis PZB-0006 inoculant (microbial inoculant prepared in Example 3), cell concentration 6.45×10 7 CFU / mL;

[0082] (3) Blank control CK: sterile water treatment;

[0083] (4) Positive control PC: chemical fungicide fludioxonil (0.30 g / L) treatment.

[0084] Uniformly sized, disease-free and mechanically damaged, and mature winter jujube fruits were picked and used. 30 mL of each treatment solution was accurately measured and placed in a sterilized spray bottle, and then evenly sprayed onto the surface of the winter jujube fruits. Each group had 30 fruits, and each treatment had 3 replicates. After the surface was naturally dried, it was placed in a PET preservation box, wrapped in a plastic preservation bag, sealed, and placed at room temperature 24±1℃, and the changes on the surface of the fruits were observed and recorded.

[0085] Rot rate (%) = number of rotten fruits / total number of fruits x 100

[0086] Rot inhibition rate (%) = (rot rate of control group - rot rate of treatment group) / rot rate of control group x 100

[0087] Table 3. Results of microbial inoculant in fresh jujube preservation test

[0088]

[0089] Note: The numbers in the table are mean ± standard deviation, and different letters represent significant differences p <0.05)

[0090] The results are shown in Table 3, Figure 5 After 7 days of storage, Bacillus tequilensis PZB-0006 inoculant sprayed can effectively reduce the rot rate of fresh jujube during storage. When the concentration of the inoculant is 6.45 x 10 8 CFU / mL, the fruit rot rate is 33.33%, compared with the blank control, the rot inhibition rate is 23.08%. When the concentration of the inoculant is 6.45 x 10 7 CFU / mL, the preservation effect is the best, the fruit rot rate is only 13.33%, compared with the blank control, the rot inhibition rate is 69.27%, and compared with the positive control, the rot inhibition rate is also significantly improved.

[0091] Example 5

[0092] Microbial inoculant in sweet cherry preservation test

[0093] The sweet cherry used in this experiment is Meizao, and a total of 4 treatments are set, which are

[0094] (1) T1: Bacillus tequilensis PZB-0006 inoculant (microbial inoculant prepared in Example 3) 100-fold dilution, bacterial concentration is 6.45 x 10 8 CFU / mL;

[0095] (2) T2: Bacillus tequilensis PZB-0006 inoculant (microbial inoculant prepared in Example 3) 1000-fold dilution, bacterial concentration is 6.45 x 10 7 CFU / mL;

[0096] (3) Blank control CK: treated with sterile water.

[0097] (4) Positive control PC: treated with chemical fungicide fludioxonil (0.30 g / L).

[0098] The sweet cherry fruits of uniform size, free of diseases and pests, mechanical damage and maturity were picked and prepared for use. 30 mL of each solution used in each treatment was accurately measured and filled in a sterilized spray bottle, and then sprayed on the surface of the sweet cherry fruits. 60 fruits were used in each group, and 3 repeats were used in each treatment. After the surface was naturally dried, the fruits were placed in a PET fresh-keeping box, wrapped with a plastic fresh-keeping bag, and placed in a room at 24±1℃, and the surface changes of the fruits were observed and recorded.

[0099] The rot inhibition rate (%) = (rot rate of the control group-rot rate of the treatment group) / rot rate of the control group*100

[0100] Table 4: The preservation test results of the microbial agent on sweet cherries

[0101]

[0102] Note: The numbers in the table are mean ± standard deviation, and different letters represent significant differences (P<0.05) p <0.05)

[0103] The results are shown in Table 4, Figure 6 After 8 days of storage, the test results of the Bacillus tequilensis PZB-0006 agent in the preservation of cherries after picking are shown in Table 4. The rot inhibition rate of the 6.45*10 8 CFU / mL agent (T1) spraying treatment on cherry postharvest rot was 59.09%, and the 6.45*10 7 CFU / mL agent (T2) had a preservation effect of 36.37%, which reached a significant level compared with the blank control (P<0.05). p Thus, the Bacillus tequilensis PZB-0006 agent can effectively prevent and control cherry postharvest diseases.

[0104] The Bacillus tequilensis PZB-0006 provided by the application has a wide antibacterial spectrum and stronger inhibition capacity, and can be used for fruit preservation and biological control of plant diseases.

Claims

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

2. The culture method of Bacillus turicatus PZB-0006 according to claim 1, characterized in that, The method comprises the following steps: (1) activating and culturing Bacillus tequilensis PZB-0006 on a solid culture medium plate to obtain an activated strain; (2) inoculating the activated strain obtained in step (1) into a seed liquid culture medium and culturing to obtain a seed liquid; (3) inoculating the seed liquid obtained in step (2) into a fermentation liquid culture medium and culturing to obtain a fermentation liquid.

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

1.

4. A microbial inoculant, characterized in that, The Bacillus tequilensis PZB-0006 according to claim 1.

5. The microbial inoculant of claim 4, wherein The microbial agent is obtained by resuspending the bacterial body with sterile water after removing the supernatant by centrifugation after fermentation of the Bacillus tequilensis PZB-0006; The microbial agent has a cell concentration of (1.0-6.45)×10 10 CFU / mL.

6. The Bacillus tequilensis PZB-0006 according to claim 1 or the microbial agent according to claim 4 is applied in food preservation.

7. Use according to claim 6, wherein The food comprises fruits and vegetables.

8. Use according to claim 7, wherein the compound is ###0002### The fruits comprise dates and cherries.

9. The use according to claim 6, wherein the compound is ###0002### The use method is to spray the Bacillus tequilensis PZB-0006 or the microbial agent onto the surface of the fruits.

10. Use according to claim 9, wherein the compound is ###0002### The concentration of the bacterial body of Bacillus tequilensis PZB-0006 or microbial inoculant is (0.1-6.45) x 10 8 CFU / mL.

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