Bacillus aerophilus X41, bacterial agent and application thereof
By screening and applying the fermentation broth of Bacillus cereus X41, which produces iron carriers and decomposes inorganic phosphorus, the problem of postharvest rot in apples was solved, and the storage, preservation, and quality improvement of apples were achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANDONG AGRICULTURAL UNIVERSITY
- Filing Date
- 2026-03-18
- Publication Date
- 2026-06-05
AI Technical Summary
Existing technologies are insufficient to effectively suppress postharvest decay of apples. Physical methods are costly, chemical methods leave residues, and the application of Bacillus cereus in biological control has not yet been reported.
A strain of *Bacillus pallida* X41 was screened and isolated. It has the ability to produce siderophores, decompose inorganic phosphorus, and produce auxin. It was prepared into a fermentation broth-like inoculant and sprayed on the surface of apples to inhibit the growth of *Botrytis cinerea* and reduce rot.
It significantly reduces the size of apple rot lesions, increases the activity of antioxidant enzymes, maintains fruit quality, promotes fruit sugar conversion and increases the content of flavonoids, thus achieving apple storage and preservation.
Smart Images
Figure CN122146525A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of agricultural microbial technology, and in particular to a strain of aerobic Bacillus pallida X41, its inoculant, and its application. Background Technology
[0002] Apples are a nutritious and widely cultivated economic crop. my country, as the world's largest apple producer, accounts for more than half of the global apple production and planting area (Sun Y et al., 2021). However, apples are frequently infected by pathogens and rot during harvesting and subsequent storage, reducing their commercial value and causing huge economic losses to fruit farmers, seriously affecting the sustainable and healthy development of my country's apple industry (Gao Jiasong et al., 2025). Pathogens causing apple rot include Botrytis cinerea (…). Botrytis cinerea ), extended Penicillium ( Penicillium expansum ), Streptomyces ( Alternaria alternata Among them, Botrytis cinerea, a necrotrophic fungus, grows rapidly, quickly killing host tissue and absorbing nutrients from it, causing rapid tissue decay. Therefore, relevant technologies are needed to inhibit the growth and infection of pathogens during apple storage and preservation to achieve postharvest preservation of apples.
[0003] Currently, postharvest preservation of apples is mainly achieved through physical, chemical, and biological methods. Physical methods include rapid precooling, low-temperature storage, and controlled atmosphere storage (Wei et al., 2025). However, these methods have high requirements for equipment and storage environment, are costly, and are difficult to maintain throughout the supply chain, failing to fundamentally solve the rot problem. Chemical methods involve the use of chemical preservatives such as imazalil and prochloraz, but chemical residues are difficult to detect, and excessive use can lead to adverse reactions (Bai et al., 2025). In contrast, biological control utilizes beneficial microorganisms that compete for nutrients and space on the fruit surface or produce antimicrobial substances to inhibit pathogens. It has advantages such as being environmentally friendly, highly targeted, and less prone to developing disease resistance, making it a more sustainable and green control strategy. Therefore, screening for a beneficial biocontrol bacterium that effectively inhibits the infection and growth of apple rot fungi and maintains the storage quality of the fruit has become an important research direction in the field of postharvest apple preservation.
[0004] Pale aerobic spores ( Aeribacillus pallidus As a beneficial bacterium that has only been discovered in recent years, current research on related systems is relatively limited, mainly focusing on aerobic composting, plastic degradation, antagonism of rice bacterial blight, and the production of biological emulsifiers. There are currently no reports on the use of *Bacillus pallida* to reduce apple rot and preserve freshness. Summary of the Invention
[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide a strain of *Bacillus pallida* X41, an inoculant, and its application. This invention is the first to isolate a strain of *Bacillus pallida* from the boundary between apple rot lesions and healthy fruit. Aeribacillus pallidus X41, this strain has the ability to produce siderophores, decompose inorganic phosphorus and produce auxin, which can inhibit the growth of the pathogen Botrytis cinerea, reduce postharvest decay of apples, and thus achieve apple storage and preservation.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a strain of *Bacillus pallida* (…). Aeribacillus pallidus X41, this strain was deposited on January 6, 2026 at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 37289.
[0007] In a second aspect, the present invention provides a microbial agent containing the aforementioned *Bacillus pallida* (aerobic spores). Aeribacillus pallidus )X41.
[0008] Preferably, the bacterial agent contains *Bacillus pallida* (aerobic spores). Aeribacillus pallidus X41 exists in the form of live bacterial suspension or fermentation broth.
[0009] In a third aspect, the present invention provides the above-mentioned *Bacillus pallida* (… Aeribacillus pallidus The application of X41 or the above-mentioned microbial agents in any of the following (1)-(4): (1) Produces protease, amylase, siderophores, decomposes inorganic phosphates, and produces glutathione; (2) Inhibits the pathogenic putrefactive fungus Botrytis cinerea; (3) Reduce apple decay after harvest and achieve apple storage and preservation; (4) Prepare a preservative to reduce the decay of apples after harvest.
[0010] In a fourth aspect, the present invention provides a preservative for reducing post-harvest decay of apples, prepared by the following method: Pale aerobic Bacillus ( Aeribacillus pallidus The seed culture of X41 was inoculated into LB medium at an inoculation rate of 0.5%-1.5% (volume ratio) and cultured at 36-38℃ and 160-200rpm for 2 days. After centrifugation, the supernatant was collected to obtain the fermentation broth, which is used as a preservative to reduce the decay of apples after harvest.
[0011] As a preferred option, *Bacillus pallida* (… Aeribacillus pallidus The method for preparing seed culture of X41 is as follows: Pale aerobic Bacillus ( Aeribacillus pallidus X41 was placed in LB medium and cultured at 36-38℃ and 160-200 rpm for 1 day to obtain *Bacillus pallida* (a type of aerobic spore). Aeribacillus pallidus Seed liquid of X41.
[0012] As a preferred option, the fermentation broth contains *Bacillus pallida* (aerobic bacteria). Aeribacillus pallidus The viable count of X41 is ≥1×10⁻⁶. 8 cfu / g.
[0013] A fifth aspect of the invention provides the use of the above-described preservative for reducing post-harvest decay of apples in any of the following (a)-(e): (a) Reduce post-harvest rot in apples; (b) Used for apple storage and preservation; (c) Increase CAT activity, SOD activity and POD activity, and decrease MDA activity; (d) Stabilize the quality of postharvest apples; (e) Promotes sugar conversion in apple fruit and increases the content of flavonoids in apple fruit.
[0014] As a preferred criterion, postharvest apple fruit quality includes fruit firmness, soluble solids content, titratable acid content, soluble sugar content, and vitamin C content.
[0015] A sixth aspect of the present invention provides a method for mitigating postharvest decay of apples, comprising the following steps: Dilute the above-mentioned preservative used to reduce post-harvest rotting of apples with water, spray it on the surface of the apple fruit, and let it dry.
[0016] Preferably, the volume ratio of the preservative to water used to reduce post-harvest rot of apples is 1:(8-12).
[0017] As a preferred option, the amount of preservative used to reduce post-harvest rot of apples is 3-6 mL per apple.
[0018] The beneficial effects of this invention are: 1. This invention is the first to isolate a strain of *Bacillus pallida* (a type of aerobic spore) from the boundary between apple rotten spots and healthy fruit. Aeribacillus pallidus X41, this strain has the ability to produce siderophores, decompose inorganic phosphorus and produce auxin, which can inhibit the growth of the pathogen Botrytis cinerea, reduce postharvest decay of apples, and thus achieve apple storage and preservation.
[0019] 2. Experiments have shown that *Bacillus pallida* (a type of aerobic spores) Aeribacillus pallidusThe fermentation broth of X41 can not only significantly reduce the size of apple rot spots, increase the activity of antioxidant enzymes, and maintain the quality of apple fruits, but also promote the conversion of sugars in the fruit and increase the content of flavonoids in the fruit, thereby improving the fruit's resistance.
[0020] 3. Utilizing Pale aerobic Bacillus ( Aeribacillus pallidus The fermentation broth of X41 can be used to prepare a preservative to reduce post-harvest decay of apples. Spraying this preservative onto the surface of apples can reduce post-harvest decay and achieve apple storage and preservation. It is convenient, safe, and environmentally friendly, and has broad application prospects in apple storage and preservation. Attached Figure Description
[0021] Figure 1 Pale aerobic Bacillus ( Aeribacillus pallidus Phylogenetic tree and strain morphology diagram of X41; Figure 2 Pale aerobic Bacillus ( Aeribacillus pallidus )Graph of plate inhibition test for X41; Figure 3 Example 5: Images showing the decay state of apples treated in each step. Figure 4 In Example 7, *Bacillus pallida* (aerobic spores) Aeribacillus pallidus The effect of X41 on the metabolites of rotting apples. Detailed Implementation
[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.
[0023] As described in the background section, maintaining post-harvest quality of apples and mitigating economic losses caused by storage spoilage are essential measures for the healthy development of the apple industry. Currently, there is no effective treatment for *Bacillus pallida* (a type of aerobic spores). Aeribacillus pallidus Reports on its application in the prevention and control of postharvest rot and the preservation of apples.
[0024] Based on this, the present invention isolated a bacterium X41 from the boundary between apple rotten spots and healthy fruit. According to morphological, physiological and biochemical characteristics and 16S-based phylogenetic analysis, this bacterium was identified as *Bacillus pallida*. Unlike previously reported *Bacillus pallida*, the *Bacillus pallida* X41 isolated in this invention is a novel biocontrol bacterium with great development potential. It simultaneously possesses the ability to produce siderophores, degrade inorganic phosphorus, and produce auxin, inhibiting the growth of the pathogen *Botrytis cinerea*. Combining multiple functions, it can be used for postharvest rot control and storage preservation of apples.
[0025] Experiments have shown that *Bacillus pallida* (aerobic spores) Aeribacillus pallidus The fermentation broth of X41 can not only significantly reduce the size of apple rot spots, increase the activity of antioxidant enzymes, and maintain the quality of apple fruits, but also promote the conversion of sugars in the fruit and increase the content of flavonoids in the fruit, thereby improving the fruit's resistance.
[0026] Pale aerobic Bacillus ( Aeribacillus pallidus The fermentation broth of X41, after being diluted with water, yields a preservative for reducing post-harvest rot in apples. Spraying this preservative onto the surface of apples can prevent post-harvest rot and has broad application prospects in apple storage and preservation.
[0027] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.
[0028] The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels.
[0029] In this invention, the components of the PDA culture medium are: 200.0 g peeled potato, 20.0 g glucose, 20.0 g agar, and 1000 mL distilled water.
[0030] The components of LB medium are: 10.0 g peptone, 5.0 g yeast extract, 10.0 g sodium chloride, and 1000 mL distilled water.
[0031] The primer synthesis and amplified fragment sequencing used in the examples were all completed by Shanghai Sangon Biotech Co., Ltd.
[0032] Example 1: Isolation and Identification of Strains 1. Isolation and purification of the strain: Apples that had naturally decayed after harvest were collected from an apple orchard in Wangshen Village, Beixin Township, Linyi County, Yuncheng City, Shanxi Province. These samples were then brought back to the laboratory, where bacterial strains were isolated using a gradient dilution plating method. The specific steps were as follows: Take 10 g of fresh sample, grind it in PBS buffer, then place it in 40 mL of sterile water and shake for 30 min before diluting to 10. -4 100 μL of the diluted solution was added to LB medium, spread, and the strains were isolated. The culture dishes were incubated at 37°C for 2 days. Single colonies were picked and cultured on new LB plates for further incubation, resulting in the isolation of more than 20 biocontrol bacteria.
[0033] 2. Screening of strains: The isolated bacterial strains were initially identified using the plate dilution plating method, revealing that they belonged to more than 10 different bacterial species. They were then screened based on their effectiveness in controlling apple rot. The specific steps are as follows: Using Fuji apples as the experimental subject, these apples were purchased from an agricultural market in Tai'an City, Shandong Province. Apples of uniform size, free from mechanical damage and pests, and at normal commercial maturity were selected for the experiment. Before use, the apples were disinfected by soaking in a 0.2% sodium hypochlorite solution for 2 minutes. The pathogen causing apple rot, *Heterocarpus spp.*, was targeted, and the inhibitory effects of all isolated strains on this pathogen were tested. A strain with the best control effect against *Heterocarpus spp.* and apple rot was obtained through screening and designated X41.
[0034] The selected strain X41 grows rapidly, forming complete colonies in 1-2 days with smooth colony surfaces. The colonies are yellow on LB medium and are Gram-negative. Both the bacterial cells and metabolites have a significant inhibitory effect on pathogenic bacteria of the genus *Hemiberlesia lataniae*. These characteristics are beneficial for the control of postharvest rot in apples with this strain, so it was decided to conduct in-depth research on this strain X41.
[0035] 3. Identification of the strain: (1) Morphological identification: When strain X41 grows on LB solid medium, the colonies are light yellow and smooth. It grows rapidly and forms complete colonies in 1-2 days. It is a Gram-positive bacterium with rod-shaped cells.
[0036] (2) Sequence analysis and phylogenetic analysis: DNA was extracted from strain X41 using a bacterial genomic DNA extraction kit (Solarbio Cat#D1600). The ITS fragment was amplified using the primer sequences shown in SEQ ID No.1-SEQ ID No.2. The amplification procedure was as follows: 94℃ pre-denaturation for 1 min, 94℃ denaturation for 1 min, 55℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles, and a final extension at 72℃ for 7 min. 27F: 5'-AGA GTT TGA TCM TGG CTC AG-3' (SEQ ID No. 1), 1492R: 5'-GGT TAC CTT GTT ACG ACT T-3' (SEQ ID No. 2).
[0037] The sequencing results were compared with the BLAST database of the National Center for Biotechnology Information (NCBI). The isolated strains were sequenced using MEGA 7 software and the Neighbor-Joining (NJ) clustering method was used to construct a phylogenetic tree. The bootstrap value was tested with 1000 replicates.
[0038] The strain's rDNA-16S sequence is 1347 bp in length. Blast alignment analysis revealed that this sequence is similar to that of *Bacillus pallida*, a species already registered in the GenBank database. Aeribacillus pallidus The 16S sequence showed 100% homology. A phylogenetic tree constructed using MEGA 7.0 software and partial Bacillus sequences registered in the GenBank database revealed that strain X41 shares 100% homology with *Bacillus pallida*. Aeribacillus pallidus The highest homology was found in strain X41, which clustered into a single branch. Combined with morphological analysis, this indicates that strain X41 is a *Bacillus pallida* (a type of aerobic spore). Aeribacillus pallidus ),like Figure 1 As shown.
[0039] Based on the above morphological and functional analyses and 16S homology analysis results of strain X41, the isolated strain X41 was identified as *Bacillus pallida*. The strain was then biopreserved, and the preservation information is as follows: Bacterial strain name: Pale aerobic Bacillus Latin name: Aeribacillus pallidus ; Strain number: X41; Preservation institution: China General Microbiological Culture Collection Center, China Committee on the Preservation and Management of Microbial Cultures; The abbreviation for the depository institution is CGMCC. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: January 6, 2026; Registered with the China National Collection Center (CGMCC) No. 37289.
[0040] Example 2: Flat Plate Standoff Experiment A plate confrontation test was conducted using *Botrytis cinerea* as the test pathogen. *Botrytis cinerea* was isolated and preserved in our laboratory. The specific steps were as follows: By using Pale aerobic Bacillus ( Aeribacillus pallidus X41 and Botrytis cinerea were co-cultured on PDA medium to verify the presence of Pale aerobic Bacillus ( ). Aeribacillus pallidus The inhibitory effect of X41.
[0041] Depend on Figure 2 It can be seen that, compared with the control group (CK), *Bacillus pallida* (… Aeribacillus pallidus X41 has a significant inhibitory effect on Botrytis cinerea, a pathogen that causes apple rot.
[0042] Example 3: Pale aerobic Bacillus ( Aeribacillus pallidus Functional identification of X41 The protease, ammonia, auxin, siderophore, cellulase, and amylase production capabilities, as well as phosphorus and potassium solubilization capabilities of strain X41, were determined according to the method described by Dong and Cai (2001). The specific steps were as follows: Protease production: Perform a milk peptone test using skim milk culture medium and observe whether the milk becomes clear; Ammonia production: Use urea agar (containing phenol red indicator) to observe whether the culture medium turns red; Production of auxin: Based on the indole experiment, *Bacillus pallida* (a type of aerobic spore) was produced. Aeribacillus pallidus X41 was co-cultured with tryptophan, and then reacted with Kovac's reagent to observe whether a red color was formed; Iron-producing carrier: Blue chromium method; can the culture medium color change from blue to orange? Inorganic phosphorus decomposition: Solid NBRIP medium, inoculated with *Bacillus pallida* (aerobic spores). Aeribacillus pallidus X41 Observe whether a transparent hydrolysis zone can form on the solid culture medium; Potassium solubilization: Alexandrov medium was used as the selective medium for potassium solubilization, and the formation of transparent hydrolysis zones on the medium was observed. Cellulase production: Congo red staining method (CMC-Na plate method) to observe whether the enzymatic hydrolysis area can show a transparent hydrolysis zone; Amylase production: Lugol's iodine staining method was used to observe whether the enzymatic hydrolysis area could show a transparent hydrolysis zone after the addition of iodine solution.
[0043] The experimental results are shown in Table 1.
[0044] Table 1. Pallor aerobic Bacillus ( Aeribacillus pallidus Functional identification results of X41 Table 1 shows that *Bacillus pallida* (aerobic spores) Aeribacillus pallidus X41 has the ability to produce glutenin, amylase, siderophores, and inorganic phosphorus.
[0045] Example 4: Preservative for reducing post-harvest decay of apples (1) Bacillus pallida ( Aeribacillus pallidus X41 was placed in LB medium and cultured at 37°C and 180 rpm for 1 day to obtain *Bacillus pallida* (a type of aerobic spore). Aeribacillus pallidus Seed liquid of X41; (2) Bacillus pallida ( Aeribacillus pallidus The seed culture of X41 was inoculated into LB medium at a volume ratio of 1%, and cultured at 37°C and 180 rpm for 2 days. The supernatant was collected by centrifugation to obtain *Bacillus pallida*. Aeribacillus pallidusThe fermentation liquid of X41 is a preservative used to reduce the spoilage of apples after harvest. Among them, pale aerobic spores ( Aeribacillus pallidus The viable cell count in the fermentation broth of X41 was 1×10⁻⁶. 8 cfu / g.
[0046] Example 5: A method for reducing post-harvest decay of apples. The preservative for reducing post-harvest decay of apples prepared in Example 4 was mixed with deionized water at a volume ratio of 1:10, and then sprayed onto the surface of the apples and dried.
[0047] Example 6: Fruit Infection Experiment Apples of uniform size, free from mechanical damage and pests, and at normal commercial maturity were selected for the experiment. Before the experiment, the apples were disinfected by immersing them in a 0.2% sodium hypochlorite solution for 2 minutes. The disinfected apples were then randomly divided into three groups of five apples each. The specific treatment groups are as follows: Blank control group (CK1 group): Use a sterile punch to make holes (5 mm) on the surface of the disinfected fruit. After removing the peel, inoculate the sterile PDA culture medium mycelium into the holes and spray 5 mL of sterile water evenly onto the fruit surface. The pathogenic bacterium Botrytis cinerea group (CK group): Use a sterile punch to make holes (5mm) on the surface of the disinfected fruit. After removing the peel, inoculate the Botrytis cinerea cake into the holes and spray 5 mL of sterile water evenly onto the fruit surface. The pathogenic bacterium *Botrytis cinerea* + *Bacillus pallida* X41 group (T1 group): Holes (5 mm) were punched in the sterilized fruit surface using a sterile puncher. After removing the peel, *Botrytis cinerea* mycelium cake was inoculated into the holes, and then the *Bacillus pallida* prepared in Example 4 was added. Aeribacillus pallidus Spray 5 mL of the fermentation liquid of X41 (i.e., a preservative used to reduce post-harvest decay of apples) evenly onto the surface of the fruit and let it dry.
[0048] After each group was treated, the fruit was left to stand for 7 days, and the degree of rotting was observed. The results were as follows: Figure 3 As shown in the table. Simultaneously, the diameter of apple rot lesions, the activity of antioxidant enzymes in the fruit, and other fruit quality-related indicators were measured. The results are shown in Tables 2-4. The detection methods for each indicator are as follows: (1) Apple rot inhibition rate: The diameter of rot lesions appearing at the inoculation site of pathogens on the fruit surface was measured using a ruler; (2) Antioxidant enzyme activity of fruit: Peroxidase (POD) activity was detected by guaiacol method, catalase (CAT) activity was detected by ultraviolet absorption method, superoxide dismutase (SOD) activity was detected by nitroblue tetrazolium (NBT) photochemical reduction inhibition method, and malondialdehyde (MDA) activity was detected by thiobarbituric acid (TBA) method. (3) Fruit quality related indicators: including fruit firmness, soluble solids, titratable acid, soluble sugar and vitamin C content. Among them, fruit firmness is the resistance encountered when a hardness tester is used to penetrate or squeeze the fruit pulp to a specified depth under certain pressure, expressed as force value; soluble solids are detected using a handheld refractometer (saccharimeter); soluble sugar content is detected using acid-base neutralization titration; soluble sugar content is detected using anthrone colorimetric method; and vitamin C content is detected using 2,6-dichlorophenolindophenol titration.
[0049] Table 2. Effects of *Bacillus pallida* X41 on apple rot caused by *Botrytis cinerea*. The inhibitory effect of *Bacillus cereus* X41 on *Botrytis cinerea* infection was observed by inoculating apples with holes to induce rot. The results showed that *Bacillus cereus* X41 effectively inhibited *Botrytis cinerea* infection and significantly reduced the size of apple rot lesions. On the third and fifth days after inoculation, compared with the untreated treatment (CK), the diameter of rot lesions treated with *Bacillus cereus* X41 (T1) was significantly reduced by 43.15% and 50.40%, respectively.
[0050] Table 3 Effects of *Bacillus pallida* X41 on antioxidant enzyme activity in fruits As shown in Table 3, spraying with *Bacillus cereus* X41 significantly enhanced the activity of antioxidant enzymes in apple fruits. This enhanced antioxidant enzyme activity indicates that *Bacillus cereus* X41 can effectively induce resistance in the fruit itself and enhance its ability to resist pathogen infection.
[0051] Specifically, spraying the surface of apples with the fermentation broth of *Bacillus cereus* X41 can increase the CAT, SOD, and POD activities of apples, while decreasing MDA activity. Compared to the fermentation broth without *Bacillus cereus* X41 (CK), CAT activity increased by 16.75%, SOD activity increased by 13.62%, POD activity increased by 26.42%, and MDA activity decreased by 35.30%.
[0052] Table 4. Effects of *Bacillus pallida* X41 on postharvest fruit quality. Five days after inoculation with the pathogen *Botrytis cinerea*, fruit samples were taken to test relevant quality parameters. The results showed that compared to the control (CK1), inoculation with *Botrytis cinerea* (CK) reduced fruit firmness, soluble solids, soluble sugar, and vitamin C by 31.66%, 17.48%, 33.38%, and 22.49%, respectively. Compared to CK, treatment with the fermentation broth of *Bacillus cereus* X41 (T1) increased fruit firmness, soluble solids, soluble sugar, and vitamin C by 20.73%, 28.10%, 18.14%, and 19.79%, respectively. This indicates that *Bacillus cereus* X41 can effectively reduce the impact of *Botrytis cinerea* on fruit rot and maintain fruit quality to a certain extent.
[0053] In addition, by Figure 3 It can be seen that spraying the fermentation broth of *Bacillus cereus* X41 can significantly reduce the size of fruit rot lesions on the surface of apple fruits. This indicates that spraying the fermentation broth of *Bacillus cereus* X41 can inhibit *Botrytis cinerea*.
[0054] Example 7: Effects of *Bacillus pallida* X41 on the metabolites of rotting apples Take 0.1g of pulp from 1cm outside the lesion of the fruit, grind it, extract with ethyl acetate, and reconstitute with methanol after rotary evaporation. Then, pipette 200 μL of the methanol-reconstituted liquid sample into a 1.5 mL centrifuge tube, add 5 μL of extraction buffer (water:methanol = 1:1 (v:v)), vortex for 30 s, and then perform low-temperature ultrasonic extraction for 30 min (5 ℃, 40 kHz). Place the ultrasonically treated sample at -20 ℃ for 30 min, then centrifuge at 4 ℃ and 13000 g for 15 min. Collect the supernatant after centrifugation, dry it with nitrogen, reconstitute it with 100 µL of reconstitution solution (acetonitrile:water = 1:1 (v:v)), perform low-temperature ultrasonic extraction for 5 min (5 ℃, 40 kHz), centrifuge at 4 ℃ and 13000 g for 5 min, and transfer the supernatant to a vial with an inner tube for analysis.
[0055] 2 μL of the supernatant was separated using an HSS T3 column and then analyzed by mass spectrometry. The chromatographic conditions were as follows: Mobile phase A consisted of 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B consisted of 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). Separation gradient: 0–0.1 min, mobile phase B increased from linearity of 0% to 5%; 0.1–2 min, mobile phase B increased from linearity of 5% to 25%; 2–9 min, mobile phase B increased from linearity of 25% to 100%; 9–13 min, mobile phase B maintained linearity of 100%; 13.0–13.1 min, mobile phase B decreased from linearity of 100% to 0%; 13.1–16 min, mobile phase B maintained linearity of 0%. The flow rate was 0.40 mL / min, and the column temperature was 40 °C.
[0056] Mass spectrometry conditions: Sample mass spectrometry signal acquisition used positive and negative ion scanning mode, with a mass scan range of m / z: 70-1050. Ion spray voltage: positive ion voltage 3500 V, negative ion voltage 2800 V; sheath gas 40 psi, auxiliary heating gas 10 psi; ion source heating temperature 400 ℃; cyclic collision energy 20-40-60 V; MS1 resolution 70000; MS2 resolution 17500. Results are as follows: Figure 4 As shown.
[0057] Depend on Figure 4 It can be seen that spraying *Bacillus cereus* X41 (T3) on apple fruits infected with *Botrytis cinerea* can effectively alter the metabolic components of the apple fruits. The highest levels of differentially expressed substances were found in the biosynthetic pathways of flavonoids and flavanols, the interconversion pathway between pentose and gluconic acid, and the metabolism of ascorbate and adalatate. This indicates that spraying *Bacillus cereus* X41 can effectively promote sugar conversion in the fruit, increase the content of flavonoids in the fruit, and improve fruit resistance.
[0058] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.
Claims
1. A strain of pale aerobic Bacillus ( Aeribacillus pallidus X41, characterized in that, Its accession number is CGMCC No.37289.
2. A microbial agent, characterized in that, The bacterial agent contains the aforementioned Aeribacillus spallidus X41.
3. The microbial agent as described in claim 2, characterized in that, The bacterial agent contains *Bacillus pallida* (a type of aerobic spores). Aeribacillus pallidus X41 exists in the form of live bacterial suspension or fermentation broth.
4. The *Bacillus pallida* as described in claim 1 (… Aeribacillus pallidus The use of the microbial agent according to claim 41 or claim 2 in any one of the following (1)-(4): (1) Produces protease, amylase, siderophores, decomposes inorganic phosphates, and produces glutenin; (2) Inhibits the pathogenic putrefactive fungus Botrytis cinerea; (3) Reduce apple decay after harvest and achieve apple storage and preservation; (4) Prepare a preservative to reduce the decay of apples after harvest.
5. A preservative for reducing post-harvest decay of apples, characterized in that, It is prepared by the following method: Pale aerobic Bacillus ( Aeribacillus pallidus The seed culture of X41 was inoculated into LB medium at an inoculation rate of 0.5%-1.5% (volume ratio) and cultured at 36-38℃ and 160-200 rpm for 2 days. After centrifugation, the supernatant was collected to obtain the fermentation broth, which is used as a preservative to reduce the decay of apples after harvest.
6. The preservative for reducing post-harvest decay of apples as described in claim 5, characterized in that, Pale aerobic spores ( Aeribacillus pallidus The method for preparing seed culture of X41 is as follows: Pale aerobic Bacillus ( Aeribacillus pallidus X41 was placed in LB medium and cultured at 36-38℃ and 160-200 rpm for 1 day to obtain *Bacillus pallida* (a type of aerobic spore). Aeribacillus pallidus Seed liquid of X41.
7. The preservative for reducing post-harvest decay of apples as described in claim 5, characterized in that, In the fermentation broth, *Bacillus pallida* (aerobic bacteria) Aeribacillus pallidus The viable count of X41 is ≥1×10⁻⁶. 8 cfu / g.
8. The use of the preservative according to any one of claims 5-7 in any one of the following (a)-(e): (a) Reduce post-harvest rot in apples; (b) Used for apple storage and preservation; (c) Increase CAT activity, SOD activity and POD activity, and decrease MDA activity; (d) Stabilize the quality of postharvest apples; (e) Promotes sugar conversion in apple fruit and increases the content of flavonoids in apple fruit.
9. A method for reducing postharvest decay of apples, characterized in that, Includes the following steps: The preservative for reducing post-harvest decay of apples as described in any one of claims 5-7 is diluted with water, sprayed onto the surface of the apples, and then dried.
10. The method for reducing postharvest decay of apples as described in claim 9, characterized in that, The volume ratio of preservative to water used to reduce post-harvest rot of apples is 1:(8-12), and the dosage of preservative used to reduce post-harvest rot of apples is 3-6 mL / apple.