Biocontrol bacterium strain and preparation and application of antibacterial product thereof

Abstract

The invention relates to a fungus strain with a broad-spectrum antibacterial effect, preparation of antibacterial products of the fungus strain and application of the fungus strain in control of postharvest diseases of fruits and vegetables. According to the present invention, the classification name of the fungus is Pichia kluyveri, the Latin literature name is Pichia kluyveri, and the VOCs produced by the strain can inhibit a variety of pathogenic bacteria. The antibacterial product contains at least one compound of phenethyl acetate, ethyl acetate or phenethyl alcohol.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a fungal strain with a broad-spectrum antibacterial effect and the preparation of an antibacterial product thereof and the application of the fungal strain in controlling postharvest diseases of fruits and vegetables. Background Art

[0002] Fruits and vegetables are rich in nutrients and are an indispensable part of the human diet. However, post-harvest spoilage has seriously restricted the development of the fruit and vegetable industry, causing huge economic losses every year. Pathogen invasion is one of the main factors leading to post-harvest quality decline and loss of fruits and vegetables. What is more worrying is that some post-harvest pathogens can produce mycotoxins that pose a threat to human health, increasing food safety risks.

[0003] At present, the prevention and control measures for post-harvest diseases of fruits and vegetables mainly include three approaches: physical methods, chemical methods and biotechnology. Although physical methods such as low-temperature storage and radiation treatment have certain effects, their popularity in actual agricultural production is limited due to their high cost and limited scope of application. Chemical fungicides have become the most important means of prevention and control due to their high efficiency and convenience, but excessive reliance on chemical agents will not only cause environmental pollution, but also endanger food security and human health, while promoting the development of drug resistance in pathogens, making future disease management more difficult. In contrast, biological control, as an emerging disease control strategy, is gradually gaining attention from researchers and the industry. The core of biological control is to utilize the principle of interaction between antagonistic microorganisms, introduce beneficial microorganisms to inhibit the growth and reproduction of pathogens, and thus achieve the purpose of preventing and controlling post-harvest diseases of fruits and vegetables. This method has significant safety and environmental advantages and is considered to be an ideal choice to replace chemical fungicides in the future. In recent years, hundreds of antagonistic microorganisms with potential application value have been identified worldwide, covering multiple categories such as bacteria, molds and yeasts. In particular, antagonistic yeast has become the preferred strain for controlling post-harvest diseases of fruits and vegetables due to its strong antibacterial ability, high safety, low nutritional requirements, rapid growth and reproduction rate, and strong ability to adapt to adversity.

[0004] The natural antibacterial ingredients contained in antagonistic microorganisms are not only relatively safe for humans and animals, but also easily decomposed by the environment, reducing the side effects that may be caused by chemical synthetic drugs and reducing the risk of environmental pollution. Therefore, by screening and utilizing these natural ingredients, developing efficient and safe biological control products is of great significance for improving the shelf life and safety of fruits and vegetables after harvest.

[0005] Among the numerous yeasts, Pichia spp. has attracted much attention due to its unique biological characteristics and broad industrial application prospects. For example, Pichia kluyveri has been shown to improve the aroma and flavor of wine and is widely used in the fermentation process of various beverages. However, the current research on the biological control of postharvest diseases of fruits and vegetables by P. kluyveri is still in its infancy, especially in terms of its inhibitory effect on a variety of pathogenic fungi and the acquisition of natural antibacterial ingredients with broad-spectrum resistance through this strain. There is still a lack of systematic exploration and reporting. This provides an important direction for future research, that is, how to give full play to the potential of antagonistic yeasts such as P. kluyveri and develop more efficient and safe postharvest disease control solutions for fruits and vegetables, thereby contributing to ensuring food safety and promoting sustainable agricultural development. Summary of the invention

[0006] One of the purposes of the present invention is to provide a fungal strain with a broad-spectrum antibacterial effect, and to apply the fungal strain in the biological control of pathogens in post-harvest fruits and vegetables.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] On the one hand, the present invention provides a fungal strain with a broad-spectrum antibacterial effect, characterized in that the strain is classified and named Pichia kluyveri, and its Latin name is Pichia kluyveri. The strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.32124, and the deposit date is September 30, 2024.

[0009] Furthermore, the 26S rDNA sequence of the strain is shown as SEQ ID NO.3.

[0010] In another aspect, the present invention provides a composition for inhibiting pathogenic bacteria, wherein the composition comprises at least one of phenethyl acetate, ethyl acetate or phenethyl alcohol.

[0011] Preferably, the composition comprises Pichia kluyveri phenethyl acetate, ethyl acetate, isoamyl acetate, and phenylethyl alcohol.

[0012] Preferably, the main volatile substances contained in the composition are phenylethyl acetate, isoamyl acetate, ethyl acetate and phenylethanol in descending order of abundance.

[0013] Preferably, the main volatile substances contained in the composition are phenylethyl acetate, isoamyl acetate, ethyl acetate, and phenylethyl alcohol in descending order of abundance, and the abundance ratio is 59:14:7:1.

[0014] Optionally, the main volatile substances contained in the composition are phenylethanol, phenylethyl acetate, isoamyl acetate and ethyl acetate in descending order of abundance.

[0015] Optionally, the main volatile substances contained in the composition are phenylethanol, phenylethyl acetate, isoamyl acetate, and ethyl acetate in descending order of abundance, and the abundance ratio is 7:4:2:1.

[0016] Preferably, the inhibition of pathogens is inhibition of pathogen growth or inhibition of the synthesis of pathogen toxins.

[0017] In another aspect, the present invention provides a method for preparing an antibacterial substance, the method comprising the following steps:

[0018] 1) Cultivating the aforementioned strain;

[0019] 2) Collect antibacterial substances.

[0020] Preferably, the antibacterial substance is a culture containing bacteria or a culture not containing bacteria.

[0021] Preferably, the antibacterial substance is a volatile substance produced during the strain cultivation process.

[0022] Preferably, the antibacterial substance is a volatile substance produced in the 10th to 14th hour during the strain cultivation process.

[0023] Preferably, the antibacterial substance is a volatile organic compound produced in the 10th to 14th hour during the strain cultivation process.

[0024] Preferably, the antibacterial substance is a volatile organic compound produced in the 11th to 13th hour during the strain cultivation process.

[0025] Preferably, the antibacterial substance comprises phenethyl acetate, isoamyl acetate, ethyl acetate, and phenylethanol.

[0026] In another aspect, the present invention provides a method for inhibiting the synthesis of pathogenic bacteria toxins using volatile organic compounds, comprising the following steps:

[0027] 1) Collect volatile organic compounds;

[0028] 2) Use the collected volatile organic compounds to inhibit the synthesis of pathogenic bacteria toxins.

[0029] Preferably, the volatile organic compounds in step 2) are volatile substances produced in the 10th to 14th hour during the strain cultivation process.

[0030] Preferably, the volatile organic compounds in step 2) are volatile organic compounds produced in the 10th to 14th hour during the strain cultivation process.

[0031] Preferably, the volatile organic compounds in step 2) are volatile organic compounds produced in the 11th to 13th hour during the strain cultivation process.

[0032] Preferably, the volatile organic compound in step 2) is at least one of phenylethyl acetate, ethyl acetate or phenylethyl alcohol.

[0033] Preferably, step 2) is to contact the volatile substances produced by the strain with pathogens to inhibit the synthesis of pathogen toxins.

[0034] Furthermore, step 1) includes

[0035] 1.1) Cultivating the aforementioned strains;

[0036] 1.2) Collect volatile organic compounds produced during strain cultivation

[0037] In another aspect, the present invention provides a method for inhibiting the growth of pathogenic bacteria using volatile organic compounds, comprising the following steps:

[0038] 1) Collect volatile organic compounds;

[0039] 2) Use the collected volatile organic compounds to inhibit the growth of pathogens.

[0040] Furthermore, step 1) includes

[0041] 1.1) Cultivating the aforementioned strains;

[0042] 1.2) Collect volatile organic compounds produced during strain cultivation

[0043] Preferably, the volatile organic compounds in step 2) are volatile substances produced in the 10th to 14th hour during the strain cultivation process.

[0044] Preferably, the volatile organic compounds in step 2) are volatile organic compounds produced in the 10th to 14th hour during the strain cultivation process.

[0045] Preferably, the volatile organic compounds in step 2) are volatile organic compounds produced in the 11th to 13th hour during the strain cultivation process.

[0046] Preferably, the volatile organic compound in step 2) is at least one of phenylethyl acetate, ethyl acetate or phenylethyl alcohol.

[0047] Preferably, step 2) is to contact the volatile substances produced by the strain with pathogens to inhibit the growth of pathogens.

[0048] On the other hand, the present invention provides an application of a compound in inhibiting the growth of pathogens or inhibiting the synthesis of pathogen toxins, characterized in that the compound is phenethyl acetate, ethyl acetate or phenethyl alcohol.

[0049] Preferably, in any of the above schemes, the pathogenic bacteria is at least one of Alternaria alternata, Trichotheciumroseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea.

[0050] Preferably, in any of the above schemes, inhibiting the synthesis of pathogenic bacteria toxins refers to inhibiting the synthesis of toxins of patulin (PAT) or alpinia spore (AOH).

[0051] Compared with the prior art, the present invention has the following advantages:

[0052] 1. The Pichia kluyveri yeast strain screened by the present invention can produce VOCs with broad-spectrum antibacterial effects and exhibit antagonistic effects on a variety of post-harvest pathogens.

[0053] 2. The antibacterial composition provided by the present invention can significantly inhibit the growth of various pathogens such as P. expansum. The antibacterial composition can be artificially prepared or produced by a screened Pichia kluyveri yeast strain.

[0054] 3. The antibacterial composition provided by the present invention can not only inhibit the growth of various pathogens, but also control the biosynthesis of fungal toxins PAT and AOH from the source, showing good application prospects.

[0055] 4. The antibacterial composition provided by the present invention is used for biological fumigation of post-harvest fruits and vegetables to prevent and control diseases. It is not only applicable to different sales stages of various products, but also to fruits and vegetables such as strawberries, blueberries and grapes that are too fragile to be treated with liquid fungicides. In addition, the fumigation of microbial volatile organic compounds will not come into direct contact with fruits and vegetables, is easy to volatilize and degrade at room temperature, and is not easy to remain on the surface of fruits and vegetables, eliminating consumers' concerns about their safety. Therefore, volatile organic compounds have broad research value and application prospects in preventing and controlling post-harvest diseases of fruits and vegetables. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] The beneficial effects of the present invention are described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0057] Figure 1A: The inhibitory effect of volatile organic compounds produced by P. kluyveri on different post-harvest pathogens. B: The growth status of pathogens on PDA medium after treatment with VOCs released at different time periods (3d). C: Statistics of pathogen colony diameter. Data are expressed as mean ± standard deviation, and different letters indicate significant differences among treatments (P<0.05).

[0058] Figure 2 Effects of volatile organic compounds produced by P. kluyveri on the production of secondary metabolites by P. expansum and A. alternata. A: Colony morphology and pigment production of P. expansum and A. alternata after 2 days of culture in CY medium. B: HPLC detection of PAT and AOH production. Data are expressed as mean ± SD, and asterisks represent significant differences (*P < 0.05, **P < 0.01, ***P < 0.001, Student's t test).

[0059] Figure 3 The inhibitory effects of three main volatile organic compounds produced by P. kluyveri on different postharvest pathogens. A: Growth status of pathogens on PDA medium after treatment with several main VOCs produced by P. kluyveri. B: Statistics of pathogen colony diameter. C: Statistics of growth inhibition rate. Data are expressed as mean ± standard deviation, and different letters indicate significant differences among treatments (P<0.05).

[0060] Figure 4 Effects of three main volatile organic compounds produced by P. kluyveri on mycotoxin production by P. expansum and A. alternata. A: HPLC detection of PAT production. B: HPLC detection of AOH production. Data are expressed as mean ± SD, and different letters indicate significant differences among treatments (P < 0.05).

[0061] Figure 5 The effects of different concentrations of phenylethyl acetate on the natural incidence of blueberry and cherry fruits during storage. A: Natural incidence of blueberry and cherry during storage after treatment with different concentrations of phenylethyl acetate (5 days). B: Statistics of natural incidence of blueberry fruits during storage. C: Statistics of natural incidence of cherry fruits during storage. Data are expressed as mean ± standard deviation, and different letters indicate significant differences among treatments (P<0.05). DETAILED DESCRIPTION

[0062] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0063] Unless otherwise defined, all technical and scientific terms used in this application have the same meanings as commonly understood by ordinary technicians in the technical field to which this application belongs. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The pathogenic fungi used in this experiment, including Alternaria alternata, Trichothecium roseum, Monilinia fructicola and Penicillium expansum, were all isolated from naturally diseased fruits and fruits. After single spore separation and purification, they were stored in a -80°C refrigerator in the form of a spore suspension (containing 16% glycerol). The Botrytis cinerea strain is a haploid strain B05.10, which was obtained from Germany Provided by Professor Paul Tudzynski of Wilhelms University. The fruit materials used in this experiment were purchased from a local fruit supermarket. Apples, yellow peaches, strawberries, winter dates, blueberries and other fruits with uniform size and color, consistent maturity and no mechanical damage were selected for the experiment. Before the inoculation test, except for strawberry fruits, other fruits need to be soaked in a 2% (v / v) sodium hypochlorite solution for about 2 minutes for surface disinfection, and then the fruits are rinsed with tap water 2-3 times to remove residual sodium hypochlorite. Dry naturally and set aside. The strain was harvested from the washing liquid of ripe nectarines in Pinggu District, Beijing. The isolated yeast colonies were white, rough and dry on the surface, and serrated on the edges. The genomic DNA of the above yeast was used as a template and identified as Pichia kluyveri using universal primers for the D1 / D2 segment of the yeast 26S rDNA. The yeast strain has an inhibitory effect on Alternaria alternata, Trichothecium roseum, Monilinia fructicola, Penicillium expansum and Botrytis cinerea. The strain isolated in this example was deposited in the China General Microbiological Culture Collection (CGMCC) on September 30, 2024, with the deposit address being the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 32124, and the deposit date being September 30, 2024. The identification sequence results of the strain Pichia kluyveri NY 23 used in the present invention are as follows:

[0064] ;

[0065] Example 1 Effect of Volatile Organic Compounds on the Growth of Pathogenic Bacteria Colonies

[0066] Preparation of pathogen spore suspension: Take a 1.5mL centrifuge tube, add 1mL sterile water or potato dextrose broth (PDB), cut the pathogenic bacteria blocks that have been cultured for 7-10 days into the above sterile water or PDB, place the centrifuge tube on an oscillator and shake it thoroughly, use a gun to suck the spore suspension in the centrifuge tube, and filter it with a magic filter cloth (Merck Millipore, 475855-1R). Take 20μL of the filtrate, measure the spore concentration with a cell counter, and then dilute it to the working concentration with sterile water or PDB medium for standby use.

[0067] Activate yeast and adjust the concentration to 1×10 8cell / mL, apply 100 μL of yeast suspension to YPDA plate, and incubate inverted in a 28°C constant temperature incubator for 12 h and 24 h. Collect spores according to the above method and prepare spore suspensions of different pathogens. After measuring the spore concentration with a cell counter, adjust the concentration to 1×10 with sterile water. 6 spores / mL. 5 μL of spore suspension was added to the center of the PDA medium. After drying naturally, the yeast medium plates incubated for 12 h and 24 h were sealed with the plates inoculated with pathogen spores by the method of two plates buckled. The YPDA medium without yeast suspension was buckled with the PDA plate inoculated with the same pathogen spore suspension as a control. The sealed plates were placed in a fungal incubator at 25 ° C for 3 days. The colony diameter was measured every 1 day by the cross method, and the data were statistically analyzed using SPSS software. Three replicates were set for each treatment, and the entire experiment was repeated 3 times.

[0068] During the cultivation process of the screened P. kluyveri yeast strain, we found that the yeast produced a fruity aroma that was strong and pleasant. In order to explore whether the broad-spectrum antibacterial effect of the yeast was related to the volatile organic compounds (VOCs) it produced, we used a plate-pairing test to detect the effects of volatile organic compounds produced by yeast at different time periods on the growth of various postharvest pathogens. Figure 1It can be seen that the volatile organic compounds produced by P.kluyveri yeast at different time periods have a significant inhibitory effect on the growth of T.roseum, B.cinerea, P.expansum, M.fructicola and A.alternata on PDA plates. In addition, we also found that the volatile organic compounds produced by 12h of culture have a better inhibitory effect on pathogens than those produced by 24h of culture. For T.roseum, the colony diameter of T.roseum that was not treated with volatile organic compounds reached 36.83mm after 3d of inoculation. When treated with volatile organic compounds produced by yeast culture for 12h, T.roseum could not grow on the PDA plate 3d after inoculation. However, when treated with volatile organic compounds produced by yeast culture for 24h, the colony diameter of T.roseum on the PDA plate reached 33.17mm after 3d of inoculation. This result may be related to the differences in the types and abundance of volatile organic compounds produced by the screened P.kluyveri yeast strains at different time periods. The inhibition rates of volatile organic compounds produced after 12 hours of cultivation on B. cinerea, P. expansum, M. fructicola and A. alternata reached 71.2%, 26.4%, 71.9% and 69.3% respectively 3 days after inoculation. The above results show that the volatile organic compounds produced by the screened P. kluyveri yeast strain during the cultivation process can significantly inhibit the expansion of hyphae of various postharvest pathogens.

[0069] Example 2 Effect of Volatile Organic Compounds on Toxin Production by Pathogens

[0070] P. expansum and A. alternata spores were collected according to the method of Example 1 and spore suspension was prepared. The spore concentration was measured by cell counter and adjusted to 1×10 6 spores / mL. Prepare 1×1cm cellophane, sterilize it and spread it on the PDA medium. Pipette 1μL of spore suspension and spread it evenly on the above cellophane. Culture the test yeast in advance, clasp the yeast culture medium plate that has been cultured for 12 hours and the PDA plate coated with pathogens to seal, and culture it in a 25℃ constant temperature incubator for 1.5 days. Then transfer the grown hyphae together with the cellophane to a 24-well plate that has been pre-added with 1mL Czapek Yeast Extract medium (Czapek Yeast Exatract, CY). Add 1mL of melted solid LB medium on the cover of the 24-well plate. After solidification, use a pipette to pipette 20μL of 1×10 8cell / mL of the test yeast culture solution to evenly cover the entire LB medium surface. After drying, cover the 24-well plate with a lid and seal it with a sealing film. After culturing in a 25°C constant temperature incubator for 2 days, collect the mycelium and freeze it with liquid nitrogen for later use. Collect the culture solution and filter it with a 0.22μm water filter membrane before HPLC detection.

[0071] The detection conditions of PAT are as follows: mobile phase A: water, mobile phase B: acetonitrile (ACN); detection wavelength 276 nm, injection temperature 25 °C; injection volume 10 μL; flow rate 1 mL / min; running time 12 min; isocratic elution, elution method see Table 1.

[0072] Table 1 PAT isocratic elution conditions

[0073]

[0074] The detection conditions of AOH are as follows: mobile phase A: water (containing 1 mM oxalic acid), mobile phase B: acetonitrile (containing 1 mM oxalic acid); detection wavelength 256 nm, injection temperature 35 °C; injection volume 10 μL; flow rate 1 mL / min; running time 30 min; gradient elution, elution method see Table 2.

[0075] Table 2 AOH gradient elution conditions

[0076]

[0077] Patulin (PAT) produced by P. expansum and alkalinomycin (AOH) produced by A. alternata are two serious mycotoxins that contaminate fruits and vegetables. Long-term exposure has the risk of teratogenicity, carcinogenicity, and mutagenicity. Figure 2 As shown in A and C, after VOCs treatment, hyphae growth was inhibited and the amount of hyphae was significantly reduced. After removing the hyphae, the CY culture medium inoculated with P. expansum in the VOCs-treated and untreated groups showed obvious color differences. The culture medium of the untreated group was orange-yellow, while the culture medium of the treated group was light yellow. The color of the CY culture medium inoculated with A. alternata did not change significantly. The above results indicate that VOCs treatment may affect the pigment synthesis of P. expansum. The culture medium was collected and the synthesis of PAT and AOH was detected by HPLC. Figure 2 As shown in B and D, the PAT content produced by P. expansum was significantly reduced after VOCs treatment, only 0.9% of the untreated group, and the AOH content produced by A. alternata was only 39% of the control group. The above results show that VOCs treatment can affect the biosynthesis of fungal toxins by affecting the growth of pathogenic fungi hyphae, and VOCs treatment is an effective method to achieve source control of fungal toxins.

[0078] Example 3 Main components of volatile organic compounds

[0079] Prepare a 20 mL headspace injection bottle, sterilize it at high temperature, and add 5 mL of YPDA solid medium to prepare a culture medium slant. After the test yeast is activated, the concentration is measured using a cell counter and diluted to 1 × 10 8 Take 50 μL of yeast suspension and spread it evenly on the above slant culture medium, tighten the screw cap of the headspace bottle, place it tilted in a 28°C constant temperature incubator and culture it for 12 h and 24 h before headspace extraction.

[0080] Volatile organic compounds were collected by headspace solid phase microextraction (SPME) and analyzed by gas chromatography-mass spectrometry (GC-MS). The headspace injection vial of the cultured yeast was preheated at 50°C for 30 min, and then the extraction head was inserted to extract volatile organic compounds at 50°C for 30 min.

[0081] VOCs were analyzed using an Agilent 7890B / 7000C GC-MS instrument, with a chromatographic column of HP–5MS UI (30m×0.25mm×0.25μm; Agilent). The temperature program was as follows: the starting temperature was 35°C, maintained for 2 min, then increased to 100°C at a rate of 5°C / min, maintained at 100°C for 1 min, and then increased to 230°C at a rate of 7°C / min; helium flow rate was 4.0 mL / min as carrier gas. Mass spectrometry conditions: injection temperature: 230°C, electron bombardment energy: 70 eV, ion source temperature: 230°C, quadrupole temperature: 280°C, solvent delay 4 min, mass scan range m / z 40-450. The sample was split 3:1. The NIST mass spectrum search database was used to identify unknown substances through a computer search system. Six replicates were set for each sample.

[0082] The volatile organic compounds with high abundance identified by GC-MS after 12h and 24h of P. kluyveri yeast cultivation are shown in Tables 3 and 4, respectively. The main volatile substances produced by 12h cultivation are phenylethyl acetate, isoamyl acetate, ethyl acetate, and phenylethanol in descending order of abundance, with an abundance ratio of 59:14:7:1. The main volatile substances produced by 24h cultivation are phenylethanol, phenylethyl acetate, isoamyl acetate, and ethyl acetate in descending order of abundance, with an abundance ratio of 7:4:2:1.

[0083] Table 3 Main VOCs identified by GC-MS after 12 h of culture of P. kluyveri

[0084]

[0085] Table 4 Main VOCs identified by GC-MS after 24 h of culture of P. kluyveri

[0086]

[0087] Example 4 Effect of the main components of volatile organic compounds on the growth of pathogenic bacteria colonies

[0088] Prepare a 5.5L Lock & Lock box and soak it in sodium hypochlorite for disinfection. Use double-sided tape to stick a 3cm×3cm aromatherapy volatile paper on the Lock & Lock box lid. Prepare a spore suspension according to the method in Example 1, and take 5μL to inoculate on a PDA plate with a diameter of 90mm. Place the inoculated PDA plate open in the Lock & Lock box (6 per box), and according to the volume of the Lock & Lock box, absorb the volatile substance standard onto the aromatherapy volatile paper to make the final fumigation concentration of 10μL / L, and quickly close the Lock & Lock box lid. Using the plate not treated with the volatile substance standard as the control, all samples were cultured at 22-25℃. When differences in colony growth appeared, the colony diameters were counted and photographed. The growth inhibition rate was calculated according to the following formula:

[0089]

[0090] like Figure 3 As shown in the figure, it can be seen that the three main volatile substances (10 μL / L) showed obvious inhibitory effects on the growth of different pathogens, among which phenylethyl acetate had a significantly stronger inhibitory effect on P. expansum, B. cinerea, M. fructicola and A. alternaria than the other two volatile substances. After 7 days of culture, the colony diameter of P. expansum in the control group was 43.30 mm, while after fumigation with 10 μL / L phenylethyl acetate, the colony diameter of P. expansum was only 31.40 mm. B. cinerea, M. fructicola and A. alternaria almost stopped growing on PDA medium after fumigation with phenylethyl acetate. In addition to phenylethyl acetate, ethyl acetate also showed relatively strong inhibitory effects on T. roseum and phenylethanol on A. alternaria. After 3 days of cultivation on PDA plates, the colony diameter of T. roseum in the control group reached 50.50 mm, while the colony diameter of the ethyl acetate treatment group was only 20.33 mm, and the inhibition rate reached 60%. After 7 days of cultivation, the colony diameter of A. alternaria in the control group reached 48.30 mm, while the colony diameter of the phenylethanol treatment group was only 14.20 mm, and the inhibition rate reached 71%. The above results show that the several major VOCs produced by P. kluyveri yeast can also effectively control the growth of various postharvest pathogens when used alone.

[0091] Example 5 Effect of the main components of volatile organic compounds on the synthesis of PAT and AOH

[0092] Prepare a 5.5L Lock box and soak it in sodium hypochlorite for disinfection. Use double-sided tape to stick a 3cm×3cm aromatherapy volatile paper on the cover of the Lock box. Collect P. expansum and A. alternata spores and prepare a spore suspension according to the method of Example 1. Add 1μL of a concentration of 1×10 6 spores / mL spore suspension was evenly spread on a PDA plate covered with 1×1cm glass paper. The inoculated PDA plate was placed in a lock-and-lock box with the lid open. According to the volume of the lock-and-lock box, the volatile substance standard was absorbed on the aromatherapy volatile paper to make the final fumigation concentration 10μL / L, and the lock-and-lock box lid was quickly closed. The pathogens cultured in the lock-and-lock box without the volatile substance standard were used as the control. All samples were cultured at 22-25℃ for 1.5 days, and then the lock-and-lock box was opened. The hyphae grown on the PDA plate and the glass paper were transferred to a 24-well plate pre-added with 1mL of Chad-yeast extract medium (CY). The 24-well plate was placed back in the lock-and-lock box with the lid open. The volatile substance standard was added again according to the above method and the lid was quickly closed. The pathogens cultured in the lock-and-lock box without the volatile substance standard were used as the control. All samples were cultured at 22-25℃ for 2 days. The culture solution was collected and filtered through a 0.22 μm water filter membrane before HPLC detection.

[0093] The synthesis of PAT and AOH after fumigation of several major VOCs, including phenylethyl acetate, phenylethanol, and ethyl acetate, was tested. The results are as follows: Figure 4 As shown in Figure A, PAT was almost undetectable in the phenylethyl acetate treatment group, and the synthesis of patulin in the phenylethyl alcohol and ethyl acetate treatment groups was significantly reduced compared with the control group, about 67% and 85% of the control group. After the above three substances were treated, the synthesis of AOH was reduced by about 50% compared with the control group, but there was no significant difference in the synthesis of AOH among the three treatment groups.

[0094] Example 6 Effects of the main components of volatile organic compounds on the natural disease of fruits

[0095] Fresh fruits were purchased from the market, and the fruits were selected and packaged after being transported to the laboratory. The packaged fruits were immediately placed in a Lock & Lock box that had been soaked and disinfected with sodium hypochlorite in advance. A 3cm×3cm aromatherapy volatile paper was attached to the Lock & Lock box lid with double-sided tape, and the volatile substance standard was absorbed onto the aromatherapy volatile paper, and the Lock & Lock box lid was quickly closed. The fruits without volatile substance standard treatment were used as controls, and all samples were cultured at 22-25℃. The disease incidence of the fruits was observed at different storage times, and the proportion of rotten fruits to the total number of fruits was counted to calculate the natural incidence rate.

[0096] We conducted a statistical analysis of the natural disease incidence of blueberry and cherry fruits after fumigation with phenylethyl acetate. The results are as follows: Figure 5 It can be seen that compared with the control group, phenylethyl acetate treatment can significantly reduce the incidence of fruit during storage, and with the increase of treatment concentration, the natural incidence of fruit shows a downward trend. Using a concentration of 12 μL / L to treat the fruit can reduce the natural incidence by more than 50%.

[0097] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A fungal strain with broad-spectrum antibacterial effect, characterized in that: The 26S rDNA sequence of the strain is shown as SEQ ID NO.

3.

2. Use of the strain according to claim 1 in preparing antibacterial substances, characterized in that: The strain is classified and named Pichia kluyveri, and its Latin name is Pichia kluyveri. The strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No 32124, and the deposit date is September 30, 2024.

3. A composition for inhibiting pathogenic bacteria, characterized in that: The composition comprises at least one of phenethyl acetate, ethyl acetate or phenethyl alcohol.

4. A method for preparing an antibacterial substance, characterized in that: The method comprises the following steps: 1) Cultivating the strain of claim 1, the strain is classified and named Pichia kluyveri, the Latin name is Pichia kluyveri, the strain is deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, the deposit number is CGMCC No32124, and the deposit date is September 30, 2024; 2) Collect antibacterial substances.

5. The use according to claim 4, characterized in that The antibacterial substances collected in step 2) are volatile substances produced during the strain cultivation process.

6. Use of a compound in inhibiting the growth of pathogenic bacteria or inhibiting the synthesis of pathogenic bacteria toxins, characterized in that: The compound is at least one of phenylethyl acetate, ethyl acetate or phenylethyl alcohol.

7. A method for inhibiting the synthesis of pathogenic bacteria toxins using volatile organic compounds, comprising the following steps: 1) Collect volatile organic compounds; 2) Use the collected volatile organic compounds to inhibit the synthesis of pathogenic bacteria toxins.

8. The method according to claim 7, characterized in that The volatile organic compound is a single compound or a combination comprising at least one of phenethyl acetate, ethyl acetate or phenethyl alcohol.

9. A method for inhibiting the growth of pathogenic bacteria using volatile organic compounds, comprising the following steps: 1) Collect volatile organic compounds; 2) Use the collected volatile organic compounds to inhibit the growth of pathogens.

10. The method according to claim 9, characterized in that The volatile organic compound is a single compound or a combination comprising at least one of phenethyl acetate, ethyl acetate or phenethyl alcohol.

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