Preparation and application of biocontrol strain and its antibacterial product

Abstract

The present application relates to a kind of fungi strains with broad-spectrum bacteriostatic effect and preparation and application of bacteriostatic product thereof in the control of postharvest diseases of fruits and vegetables.The classification of the fungi of the present application is named Pichia kluyveri, and the Latin name is Pichia kluyveri.The VOCs produced by the strain have inhibitory effect on a variety of pathogenic bacteria.The bacteriostatic product contains at least one compound in phenethyl acetate, ethyl acetate or phenethyl alcohol.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a fungal strain with broad-spectrum antibacterial effects, the preparation of its antibacterial products, and their application in controlling postharvest diseases of fruits and vegetables. Background Technology

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

[0003] Currently, the control measures for postharvest diseases of fruits and vegetables mainly include three approaches: physical methods, chemical means, and biotechnology. Although physical methods such as low-temperature storage and radiation treatment have certain effects, their adoption in actual agricultural production is limited due to high costs and limited applicability. Chemical fungicides have become the most important control method due to their high efficiency and convenience; however, over-reliance on chemical agents not only leads to environmental pollution but may also endanger food security and human health, while also promoting 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 industry. The core of biological control lies in utilizing the principle of interaction between antagonistic microorganisms, introducing beneficial microorganisms to inhibit the growth and reproduction of pathogens, thereby achieving the purpose of preventing and controlling postharvest diseases of fruits and vegetables. This method has significant safety and environmental advantages and is considered an ideal alternative to chemical fungicides in the future. In recent years, hundreds of antagonistic microorganisms with potential application value have been identified globally, covering multiple categories such as bacteria, molds, and yeasts. In particular, antagonistic yeasts have become the preferred strains for controlling postharvest diseases of fruits and vegetables due to their strong antibacterial ability, high safety, low nutritional requirements, rapid growth and reproduction rate, and strong adaptability to adversity.

[0004] The natural antibacterial components contained in antagonistic microorganisms are not only relatively safe for humans and animals, but are also easily decomposed by the environment, reducing the potential side effects of chemically synthesized drugs and lowering the risk of environmental pollution. Therefore, developing highly efficient and safe biological control products by screening and utilizing these natural components is of great significance for improving the post-harvest shelf life and safety of fruits and vegetables.

[0005] Among numerous yeasts, the genus *Pichia* spp. has attracted considerable attention due to its unique biological characteristics and broad industrial application prospects. For example, *Pichia kluyveri* has been proven to improve the aroma and flavor of wine and is widely used in the fermentation processes of various beverages. However, research on the biocontrol of postharvest diseases of fruits and vegetables using *P. kluyveri* is still in its early stages, particularly regarding its inhibitory effects on various pathogenic fungi and the acquisition of broad-spectrum antibacterial components from this strain. Systematic exploration and reporting on these aspects are still lacking. This provides an important direction for future research: how to fully utilize the potential of antagonistic yeasts such as *P. kluyveri* to develop more efficient and safer postharvest disease control solutions for fruits and vegetables, thereby contributing to food safety and promoting sustainable agricultural development. Summary of the Invention

[0006] One of the objectives of this invention is to provide a fungal strain with broad-spectrum antibacterial effects and to apply it in the biological control of pathogens in postharvest fruits and vegetables.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

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

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

[0010] On the other hand, the present invention provides a composition for inhibiting pathogens, the composition comprising at least one of phenethyl acetate, ethyl acetate or phenylethanol.

[0011] Preferably, the composition comprises phenethyl ketone acetate, ethyl acetate, isoamyl acetate, and phenylethanol.

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

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

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

[0015] Optionally, the composition contains, in descending order of abundance, phenylethanol, phenethyl acetate, isoamyl acetate, and ethyl acetate, with an abundance ratio of 7:4:2:1.

[0016] Preferably, the inhibition of pathogens refers to inhibiting the growth of pathogens or inhibiting the synthesis of pathogen toxins.

[0017] On the other hand, the present invention provides a method for preparing an antibacterial substance, the method comprising the following steps:

[0018] 1) Cultivate the aforementioned strains;

[0019] 2) Collect antibacterial substances.

[0020] Preferably, the antibacterial substance is a culture containing bacteria or a culture without bacteria.

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

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

[0023] Preferably, the antibacterial substance is a volatile organic compound produced during the 10th to 14th hour of bacterial culture.

[0024] Preferably, the antibacterial substance is a volatile organic compound produced during the 11th-13th hour of strain culture.

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

[0026] On the other hand, the present invention provides a method for inhibiting the synthesis of pathogenic 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 toxins.

[0029] Preferably, the volatile organic compound in step 2) is a volatile substance produced during the 10th to 14th hour of the strain culture process.

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

[0031] Preferably, the volatile organic compounds in step 2) are volatile organic compounds produced during the 11th-13th hour of strain culture.

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

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

[0034] Furthermore, step 1) includes

[0035] 1.1) Cultivate the aforementioned strains;

[0036] 1.2) Collect volatile organic compounds produced during the culture of the strain.

[0037] On the other hand, the present invention provides a method for inhibiting the growth of pathogens 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) Cultivate the aforementioned strains;

[0042] 1.2) Collect volatile organic compounds produced during the culture of the strain.

[0043] Preferably, the volatile organic compound in step 2) is a volatile substance produced during the 10th to 14th hour of the strain culture process.

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

[0045] Preferably, the volatile organic compounds in step 2) are volatile organic compounds produced during the 11th-13th hour of strain culture.

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

[0047] Preferably, step 2) involves contacting the volatile substances produced by the strain with the pathogen to inhibit the growth of the pathogen.

[0048] On the other hand, the present invention provides the 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 phenylethanol.

[0049] Preferably, in any of the aforementioned schemes, the pathogen is at least one of Alternaria alternata, Trichothecium roseum, Monilinia fructicola, Penicillium expansum, and Botrytis cinerea.

[0050] Preferably, in any of the aforementioned schemes, inhibiting pathogen toxin synthesis refers to inhibiting the synthesis of patulin (PAT) or aleurone (AOH) toxins.

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

[0052] 1. The Pichia kluyveri yeast strain screened in this invention can produce VOCs with broad-spectrum antibacterial activity, exhibiting antagonistic effects against a variety of postharvest pathogens.

[0053] 2. The antibacterial composition provided by the present invention can significantly inhibit the growth of various pathogenic bacteria such as P. expansum. This antibacterial composition can be artificially prepared or produced by screening Pichia kluyveri yeast strains.

[0054] 3. The antibacterial composition provided by the present invention can not only inhibit the growth of a variety of 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 this invention is suitable for biological fumigation of harvested fruits and vegetables to prevent diseases. It is applicable not only to different sales stages of various products but also to fruits and vegetables such as strawberries, blueberries, and grapes that are too easily damaged to be treated with liquid fungicides. Furthermore, the fumigation with microbial-derived volatile organic compounds does not involve direct contact with fruits and vegetables. It is easily volatilized and degraded at room temperature, leaving little residue on the surface of fruits and vegetables, thus eliminating consumer concerns about its safety. Therefore, volatile organic compounds have broad research value and application prospects in the prevention and control of post-harvest diseases in fruits and vegetables. Attached Figure Description

[0056] The beneficial effects of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0057] Figure 1The inhibitory effects of volatile organic compounds (VOCs) produced by *P. kluyveri* on different postharvest pathogens. Growth status of pathogens on PDA medium after VOCs release at different time points (3 days). B: Statistical analysis of pathogen colony diameter. Data are expressed as mean ± standard deviation; different letters indicate significant differences between treatments (P < 0.05).

[0058] Figure 2 The effect 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 ± standard deviation. Asterisks indicate statistical significance (*P<0.05, **P<0.01, ***P<0.001, Student's t-test).

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

[0060] Figure 4 The effects of three major volatile organic compounds produced by *P. kluyveri* on the production of mycotoxins by *P. expansum* and *A. alternata*. A: PAT production by HPLC. B: AOH production by HPLC. Data are expressed as mean ± standard deviation; different letters indicate significant differences between treatments (P < 0.05).

[0061] Figure 5 This study investigated the effects of different concentrations of phenethyl acetate treatment on the natural disease incidence of blueberries and cherries during storage. A: Natural disease incidence of blueberries and cherries during storage (5 days) after treatment with different concentrations of phenethyl acetate. B: Statistics on the natural disease incidence of blueberries during storage. C: Statistics on the natural disease incidence of cherries during storage. Data are expressed as mean ± standard deviation. Different letters indicate significant differences between treatments (P < 0.05). Detailed Implementation

[0062] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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 meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments were purchased from conventional biochemical reagent stores. The pathogenic fungi used in this experiment, including *Alternaria alternata*, *Trichothecium roseum*, *Monilinia fructicola*, and *Penicillium expansum*, were all isolated from naturally diseased fruit fruits, purified by single-spore isolation, and stored in a -80°C freezer as spore suspensions (containing 16% glycerol). The *Botrytis cinerea* strain was a haploid strain B05.10, obtained from Germany. The fruit materials used in this experiment were provided by Professor Paul Tudzynski of Wilhelms University. Fruits of uniform size and color, consistent ripeness, and without mechanical damage, including apples, yellow peaches, strawberries, jujubes, and blueberries, were selected for the experiment. Before inoculation, except for strawberries, other fruits were soaked in a 2% (v / v) sodium hypochlorite solution for approximately 2 minutes for surface disinfection, followed by rinsing with tap water 2-3 times to remove residual sodium hypochlorite. They were then air-dried for later use. The yeast strain was obtained from the washing liquid of ripe nectarines from Pinggu District, Beijing. The isolated yeast colonies were white, with a rough and dry surface and serrated edges. Using the genomic DNA of the above yeast strain as a template, it was identified as *Pichia kluyveri* using universal primers for the 26S rDNA D1 / D2 region. This yeast strain exhibited inhibitory effects against *Alternaria alternata*, *Trichothecium roseum*, *Monilinia fructicola*, *Penicillium expansum*, and *Botrytis cinerea*. The strain isolated in this embodiment was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 30, 2024. The deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32124 and deposit date of September 30, 2024. The identification sequence results of the strain Pichia kluyveri NY 23 used in this invention are as follows:

[0064] ;

[0065] Example 1: Effects of Volatile Organic Compounds on the Growth of Pathogenic Bacterial Colonies

[0066] Preparation of pathogen spore suspension: Take a 1.5 mL centrifuge tube and add 1 mL of sterile water or Potato Dextrose Broth (PDB). Cut pathogenic bacteria blocks that have been cultured for 7-10 days and add them to the sterile water or PDB. Place the centrifuge tube on a shaker and shake thoroughly. Aspirate the spore suspension from the centrifuge tube using a pipette tip and filter using a Merck Millipore (475855-1R) filter cloth. Take 20 μL of the filtrate and measure the spore concentration using a cell counter. Then dilute with sterile water or PDB to the working concentration for later use.

[0067] Activate the yeast and adjust the concentration to 1×10 8Cells / mL: 100 μL of yeast suspension was spread onto YPDA plates and incubated upside down at 28°C for 12 h and 24 h. Spores were collected using the above method, and spore suspensions of different pathogens were prepared. After determining the spore concentration using a cell counter, the concentration was adjusted to 1 × 10⁻⁶ cells / mL with sterile water. 6 spores / mL. 5 μL of spore suspension was added dropwise to the center of a PDA medium. After air-drying, yeast culture plates incubated for 12 h and 24 h were sealed with plates inoculated with pathogen spores using a two-plate sealing method. YPDA medium without yeast suspension was sealed with PDA plates inoculated with the same pathogen spore suspension as a control. The sealed plates were incubated in a fungal incubator at 25°C for 3 days. Colony diameter was measured every day using the cross-hatching method. Data were statistically analyzed using SPSS software. Each treatment was repeated in triplicate, and the entire experiment was repeated three times.

[0068] During the cultivation of the selected *P. kluyveri* yeast strain, we observed that it produced a fruity aroma, which was strong and pleasant. To investigate whether the broad-spectrum antibacterial effect of this yeast was related to the volatile organic compounds (VOCs) it produced, we used a plate-to-plate assay to detect the effects of VOCs produced by the yeast at different time points on the growth of various postharvest pathogens. Figure 1It was found that volatile organic compounds (VOCs) produced by *P. kluyveri* yeast at different culture time points significantly inhibited the growth of *T. roseum*, *B. cinerea*, *P. expansum*, *M. fructicola*, and *A. alternata* on PDA plates. Furthermore, we discovered that VOCs produced after 12 hours of culture had a better inhibitory effect on pathogens than those produced after 24 hours. For *T. roseum*, the colony diameter reached 36.83 mm after 3 days of inoculation without VOC treatment. Treatment with VOCs produced after 12 hours of yeast culture resulted in no growth of *T. roseum* on PDA plates after 3 days, while treatment with VOCs produced after 24 hours of yeast culture resulted in a colony diameter of 33.17 mm on PDA plates after 3 days. This result may be related to the differences in the types and abundance of VOCs produced by the selected *P. kluyveri* yeast strains at different time points. The volatile organic compounds produced during 12 hours of cultivation showed inhibition rates of 71.2%, 26.4%, 71.9%, and 69.3% against *B. cinerea*, *P. expansum*, *M. fructicola*, and *A. alternata*, respectively, three days after inoculation. These results indicate that the volatile organic compounds produced by the selected *P. kluyveri* yeast strain during cultivation can significantly inhibit the spread of mycelia from various postharvest pathogens.

[0069] Example 2: The effect of volatile organic compounds on toxin production by pathogens

[0070] Spores of *P. expansum* and *A. alternata* were collected according to the method in Example 1, and spore suspensions were prepared. After determining the spore concentration using a cell counter, the concentration was adjusted to 1 × 10⁻⁶ using sterile water. 6 spores / mL. Prepare 1×1cm cellophane, sterilize it, and place it on PDA medium. Spread 1μL of spore suspension evenly onto the cellophane. Culture the test yeast in advance. Seal the yeast culture plate (cultured for 12 hours) and the PDA plate containing the pathogen onto the plate. Incubate at 25℃ for 1.5 days. Transfer the grown hyphae along with the cellophane to a 24-well plate pre-filled with 1mL of Czapek Yeast Extract (CY). Add 1mL of melted solid LB medium to the top of the 24-well plate. After solidification, pipette 20μL of a 1×10⁻⁶ spore suspension. 8The test yeast culture medium was prepared at a concentration of 1 cell / mL and evenly covered the entire surface of the LB medium. After drying, the 24-well plate was covered and sealed with sealing film. It was then incubated statically in a constant temperature incubator at 25℃ for 2 days. The mycelia were collected and frozen in liquid nitrogen for later use. The culture medium was collected and filtered through a 0.22μm aqueous filter membrane before HPLC analysis.

[0071] The detection conditions for PAT were 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, and the elution method is shown in Table 1.

[0072] Table 1. PAT isocratic elution conditions

[0073]

[0074] The detection conditions for AOH were 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 ℃; injection volume: 10 μL; flow rate: 1 mL / min; running time: 30 min; gradient elution, and the elution method is shown in Table 2.

[0075] Table 2 AOH gradient elution conditions

[0076]

[0077] Patulin (PAT) produced by *P. expansum* and aleurone (AOH) produced by *A. alternata* are two fungal toxins that seriously contaminate fruits and vegetables. Long-term exposure carries risks of teratogenicity, carcinogenicity, and mutagenicity. Figure 2 As shown in Figures A and C, VOCs treatment inhibited mycelial growth and significantly reduced mycelial quantity. After removing the mycelia, the CY medium inoculated with *P. expansum* in the VOCs-treated and untreated groups showed a clear color difference; the untreated group's medium was orange-yellow, while the treated group's medium was light yellow. However, the color of the CY medium inoculated with *A. alternata* did not change significantly. These results suggest that VOCs treatment may affect pigment synthesis in *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, reaching only 0.9% of that in the untreated group, and the AOH content produced by *A. alternata* was only 39% of that in the control group. These results indicate that VOCs treatment can affect the biosynthesis of fungi by influencing the growth of pathogenic mycelia, making it an effective method for controlling fungal toxins at their source.

[0078] Example 3: Main components of volatile organic compounds

[0079] Prepare a 20 mL headspace vial, sterilize it at high temperature, and add 5 mL of YPDA solid culture medium to prepare a culture slant. After activation of the test yeast, determine the concentration using a cell counter and dilute to 1×10⁻⁶ with sterile water. 8 cell / mL. Take 50 μL of yeast suspension and spread it evenly on the above slant culture medium. Tighten the screw cap of the headspace vial and place it at an angle in a 28℃ constant temperature incubator for 12 h and 24 h before performing headspace extraction.

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

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

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

[0083] Table 3. Major VOCs identified by GC-MS after 12 hours of culture of *P. kluyveri*.

[0084]

[0085] Table 4. Major VOCs identified by GC-MS after 24 hours of *P. kluyveri* culture.

[0086]

[0087] Example 4: Effects of major components of volatile organic compounds on the growth of pathogenic bacterial colonies

[0088] Prepare a 5.5L Lock & Lock container and disinfect it by soaking in sodium hypochlorite. Attach a 3cm x 3cm aromatherapy evaporation paper to the Lock & Lock container lid using double-sided tape. Prepare a spore suspension according to the method in Example 1, and inoculate 5μL onto a 90mm diameter PDA plate. Place the inoculated PDA plate open in each Lock & Lock container (6 plates per container). Based on the volume of the Lock & Lock container, add volatile substance standards to the aromatherapy evaporation paper to achieve a final fumigation concentration of 10μL / L. Quickly close the Lock & Lock container lid. Use plates without volatile substance standards as a control. Incubate all samples at 22-25℃. After observing differences in colony growth, count the colony diameter and photograph it. The growth inhibition rate is calculated using the following formula:

[0089]

[0090] like Figure 3 As shown, the three main volatile substances (10 μL / L) all exhibited significant inhibitory effects on the growth of different pathogens. Phenylephrine acetate showed 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 phenylephrine acetate, the colony diameter of *P. expansum* was only 31.40 mm. *B. cinerea*, *M. fructicola*, and *A. alternaria* almost ceased to grow on PDA medium after fumigation with phenylephrine acetate. Besides phenylephrine 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-treated group was only 20.33 mm, with an inhibition rate of 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-treated group was only 14.20 mm, with an inhibition rate of 71%. These results indicate that several major VOCs produced by *P. kluyveri* yeast can effectively control the growth of various postharvest pathogens when used alone.

[0091] Example 5: Effects of major volatile organic compound components on the synthesis of PAT and AOH

[0092] Prepare a 5.5L Lock & Lock container and sterilize it by soaking in sodium hypochlorite. Attach a 3cm x 3cm aromatherapy evaporation paper to the Lock & Lock container lid using double-sided tape. Collect *P. expansum* and *A. alternata* spores according to the method in Example 1 and prepare a spore suspension. Add 1 μL of a 1×10⁻⁶ spore suspension. 6 Spread a spore suspension of spores / mL evenly onto a PDA plate lined with 1×1cm cellophane. Place the inoculated PDA plate open in a Lock & Lock container. Add volatile standard to the fumigation paper according to the container's volume, achieving a final fumigation concentration of 10 μL / L. Quickly close the Lock & Lock container lid. Using pathogens cultured in a Lock & Lock container without volatile standard treatment as a control, incubate all samples at 22-25℃ for 1.5 days. After incubation, open the Lock & Lock container and transfer the mycelia grown on the PDA plate, along with the cellophane, to a 24-well plate pre-filled with 1 mL of Czapek's yeast extract medium (CY). Return the 24-well plate open to the Lock & Lock container, re-add volatile standard as described above, and quickly close the lid. Using pathogens cultured in a Lock & Lock container without volatile standard treatment as a control, incubate all samples at 22-25℃ for 2 days. The culture medium was collected, filtered through a 0.22 μm aqueous filter membrane, and then analyzed by HPLC.

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

[0094] Example 6: The Influence of Major volatile Organic Compound Components on Natural Disease Development in Fruits

[0095] Fresh fruit was purchased from the market and transported to the laboratory for selection and repackaging. The repackaged fruit was immediately placed in Lock & Lock containers that had been pre-sterilized with sodium hypochlorite. A 3cm x 3cm aromatherapy volatile paper was attached to the lid of the Lock & Lock container using double-sided tape. Volatile substance standards were then applied to the aromatherapy volatile paper, and the lid was quickly closed. Fruits without the volatile substance standards served as a control. All samples were incubated at 22-25℃. Disease incidence was observed at different storage times, and the proportion of rotten fruit to the total number of fruits was calculated to determine the natural disease rate.

[0096] We statistically analyzed the effects of phenethyl acetate fumigation treatment on the natural disease incidence in blueberries and cherries, and the results are as follows: Figure 5 As shown in the figure, compared with the control group, phenethyl acetate treatment can significantly reduce the incidence of disease in fruits during storage, and the natural incidence of disease in fruits shows a decreasing trend with increasing treatment concentration. Treating fruits with a concentration of 12 μL / L can reduce the natural incidence of disease by more than 50%.

[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A type of Kluyveromyces yeast ( Pichia kluyveri NY23, characterized in that, The strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 32124 and deposit date of September 30, 2024.

2. The application of Kluyveromyces NY23 as described in claim 1 in the preparation of antibacterial substances, characterized in that, The antibacterial substance is a volatile substance produced by Pichia pastoris NY23 during cultivation, and the volatile substance includes phenethyl acetate, ethyl acetate or phenylethanol.

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