Complex microbial inoculant and freeze-dried microbial inoculant for preventing and treating fruits and vegetables as well as preparation method and application of freeze-dried microbial inoculant

Abstract

The invention provides a complex microbial inoculant for preventing and treating fruits and vegetables, a freeze-drying preparation, a preparation method and application, and belongs to the technical field of microorganisms. The complex microbial inoculant disclosed by the invention comprises a Michelia guilliermondii (Pichia guilliermondii), a Michelia caribba (Meyerozyma caribba) and a Pichia rassica (Pichia rassica), and the complex microbial inoculant disclosed by the invention is prepared from the following raw materials: the Michelia guilliermondii, the Meyerozyma caribba, the Pichia rassica, the Pichia guilliermondii, the Pichia guilliermondii, the Pichia guilliermondii and the Pichia rassica. The complex microbial inoculant can effectively inhibit germs of various fruits and vegetables, such as gray mold, penicilliosis, aspergillosis, black spot and the like, the complex microbial inoculant and a composite protective agent form a freeze-dried preparation, transportation is convenient, rehydration operation is simple and convenient, and dip dyeing of infectious microbes in air can be avoided.

Description

Technical Field

[0001] The present disclosure belongs to the field of microbial technology, and specifically relates to a composite bacterial agent and a freeze-dried bacterial agent for fruit and vegetable prevention and control, as well as a preparation method and application thereof. Background Art

[0002] Fruit and vegetable harvesting is mostly concentrated in June to August, when the temperature and humidity are relatively high, causing latent microorganisms before harvesting and during storage and transportation to easily become diseased under suitable conditions after harvesting, eventually leading to problems such as water loss, softening, fungal infection, rot, and quality deterioration in fruits and vegetables, reducing or even losing their commercial value, and seriously hindering their industrial development.

[0003] Compared to traditional pest control methods, biological control offers advantages such as low cost, environmental friendliness, long-lasting effectiveness, and superior efficacy. Recent research has shown that using non-antibiotic-producing biocontrol yeasts instead of antibiotic-producing bacteria to treat fruit effectively controls postharvest diseases. However, the storage and transportation of liquid yeasts present significant challenges.

[0004] In order to facilitate the transportation and storage of biocontrol agents, vacuum freeze-drying technology is currently used to dry biocontrol agents into a solid form. This technology rarely changes the form of the material and can also avoid the contamination of miscellaneous bacteria in the air. At the same time, the prepared biocontrol agents are not only easy to rehydrate, but also have the advantages of convenient transportation. However, most of the current freeze-dried preparations can only achieve the inhibition of a single pathogen in a single fruit and vegetable. For example, the freeze-dried bacterial agent formed by Debaryomyces hansen can inhibit the post-harvest soft rot of strawberries. For another example, the composite bacterial agent for fermenting apples formed by Geotrichum candidum, yeast, Bacillus subtilis, etc. It can be seen that the freeze-dried preparations proposed in the current existing technical solutions cannot achieve the inhibition of the diseases of various fruits and vegetables (for example: gray mold, penicillium, black spot, aspergillosis, etc.). Therefore, it is necessary to propose a new composite bacterial agent and freeze-dried bacterial agent to achieve the prevention and control of various fruits and vegetables. Summary of the Invention

[0005] The embodiments of the present disclosure aim to solve at least one of the technical problems existing in the prior art, and disclose a composite bacterial agent for fruit and vegetable prevention and control, a freeze-dried preparation, a preparation method, and an application.

[0006] In one aspect of the present disclosure, a composite microbial agent for preventing and controlling fruits and vegetables is provided, wherein the composite microbial agent comprises: Meyerozyma guilliermondii, Meyerozyma calibbica, and Pichia rarassimilans; wherein,

[0007] The Meyer yeast (Meyerozyma guilliermondii) was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms on June 12, 2023, with the deposit number CGMCC No. 27603;

[0008] The Meyerozyma caribbica was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on June 12, 2023, with the deposit number being CGMCC No. 27604.

[0009] The Pichia rarassimilans was deposited in the General Microbiology Center of the China Culture Collection Administration on December 2, 2024, and its deposit number is CGMCC No.32875.

[0010] Optionally, the mass ratio of the Meyerozyma guilliermondii, the Meyerozyma caribbica, and the Pichia rarassimilans is 3:1:2.

[0011] Another aspect of the present disclosure provides a freeze-dried bacterial agent for the prevention and treatment of fruits and vegetables, wherein the freeze-dried bacterial agent comprises a composite bacterial agent and a composite protective agent; wherein the composite bacterial agent is the composite bacterial agent described above; wherein,

[0012] The composite protective agent comprises 15-25% of inulin, 5-15% of skim milk powder and 10-18% of xylo-oligosaccharide.

[0013] Another aspect of the present disclosure provides a method for preparing the freeze-dried bacterial agent described above, the method comprising:

[0014] Meyerozyma guilliermondii, Meyerozyma caribbica, and Pichia rarassimilans are used as a mixed seed liquid, the mixed seed liquid is purified and inoculated into a liquid culture medium, and a composite bacterial agent is formed through culturing, and the composite bacterial agent is prepared into a composite bacterial liquid using sterile water;

[0015] A composite protective agent solution is added to the composite bacterial liquid to suspend it, the suspended composite bacterial liquid is placed on a shaker for equilibrium treatment, and the treated composite bacterial liquid is poured into a culture dish, which is covered with a film and pre-frozen. After the composite bacterial liquid is completely pre-frozen, air holes are provided on the film and the culture dish is freeze-dried to obtain a freeze-dried bacterial agent.

[0016] Optionally, the liquid culture medium comprises 10-20 g / L of a nitrogen source, 10-25 g / L of a carbon source, and 1-7 g / L of an inorganic salt.

[0017] Optionally, the nitrogen source is yeast extract powder, and the concentration of the yeast extract powder is 15 g / L;

[0018] The carbon source is sucrose, and the concentration of the sucrose is 15 g / L;

[0019] The inorganic salts are magnesium sulfate and potassium dihydrogen phosphate, the concentration of the magnesium sulfate is 5 g / L, and the concentration of the potassium dihydrogen phosphate is 3 g / L.

[0020] Optionally, the inoculation amount of the mixed seed solution is 0.5-4%, the pH of the culture process is 5-9, the culture temperature is 20-36° C., and the culture time is 24-72 h.

[0021] Optionally, the inoculation amount of the mixed seed solution is 2%, the pH of the culture process is 6, the culture temperature is 24° C., and the culture time is 60 h.

[0022] Optionally, the ratio of the composite protective agent solution to the composite bacterial solution is 2:1;

[0023] The suspended composite bacterial solution was placed on a shaker for equilibrium treatment at a temperature of 28°C, a rotation speed of 180 rpm, and a time of 30 min;

[0024] The thickness of the composite bacterial solution poured into the culture dish is 0.5 cm;

[0025] The culture dish was pre-frozen at -80°C for 3 h.

[0026] The freeze-drying temperature of the culture dish was 28° C. and the time was 30 min.

[0027] Another aspect of the present disclosure provides an application of a freeze-dried bacterial agent, wherein the freeze-dried bacterial agent described above is used to inhibit at least one of gray mold, penicillium, black spot, and aspergillosis in fruits and vegetables.

[0028] The present disclosure provides a composite bacterial agent, a freeze-dried preparation, a preparation method, and an application for the prevention and treatment of fruits and vegetables. Compared with the existing technology, the composite bacterial agent of the present disclosure can effectively inhibit various fruit and vegetable pathogens, such as gray mold, penicillium, aspergillosis, black spot, etc., and can be formed into a freeze-dried preparation by combining it with a composite protective agent, which is easy to transport and simple to rehydrate, and can prevent contamination by miscellaneous bacteria in the air.

[0029] Preservation Instructions

[0030] Depository: General Microbiology Center, China Culture Collection Administration;

[0031] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0032] Deposit date: June 12, 2023;

[0033] Deposit number: CGMCC No.27604;

[0034] Classification and nomenclature of biological materials: Meyer yeast Kalibik;

[0035] Latin name of the biomaterial: Meyerozyma caribbica.

[0036] Depository: General Microbiology Center, China Culture Collection Administration;

[0037] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0038] Deposit date: June 12, 2023;

[0039] Deposit number: CGMCC No.27603;

[0040] Classification and nomenclature of biological materials: Guillermo Mayer yeast;

[0041] Latin name of the biomaterial: Meyerozyma guilliermondii.

[0042] Depository: General Microbiology Center, China Culture Collection Administration;

[0043] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0044] Deposit date: December 2, 2024;

[0045] Deposit number: CGMCC No.32875;

[0046] Classification and nomenclature of biological materials: Pichia laris;

[0047] The Latin name of the biomaterial: Pichia rarassimilans. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The above and other features, advantages, and aspects of the various embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and that components and elements are not necessarily drawn to scale.

[0049] Figure 1 This is a flowchart of a freeze-dried bacterial preparation method according to a specific embodiment of the present disclosure;

[0050] Figure 2 Schematic diagram of the effect of carbon source and optimal carbon source concentration on mixed bacterial culture in Example 1 of the present disclosure; wherein, Figure 2 (a) is the result of mixed culture when using different carbon sources. Figure 2 (b) shows the results of mixed culture with different concentrations of sucrose;

[0051] Figure 3 Schematic diagram of the effect of nitrogen source and optimal nitrogen source concentration on mixed bacterial culture in Example 2 of the present disclosure; wherein, Figure 3 (a) is the result of mixed culture when different nitrogen sources are used. Figure 3 (b) shows the results of mixed culture when different concentrations of yeast extract powder were used;

[0052] Figure 4 Schematic diagram of the effects of inorganic salts and two optimal inorganic salt concentrations on mixed bacterial culture in Example 3 of the present disclosure; wherein, Figure 4 (a) is the result of mixed bacterial culture when different inorganic salts are used. Figure 4 (b) shows the results of mixed bacterial culture when different concentrations of potassium dihydrogen phosphate were used; Figure 4 (c) shows the results of mixed bacterial culture using different concentrations of sulfate and potassium dihydrogen phosphate;

[0053] Figure 5 Schematic diagram of the effects of pH value, inoculation amount, culture temperature and culture time on mixed culture in Example 5 of the present disclosure; wherein, Figure 5 (a) is the result of mixed culture at different pH values. Figure 5 (b) shows the results of mixed culture with different inoculation amounts; Figure 5 (c) shows the results of mixed culture at different temperatures; Figure 5 (d) shows the results of mixed culture with different culture times;

[0054] Figure 6This is a schematic diagram of the antibacterial effects of freeze-dried bacterial agents of different concentrations on strawberry gray mold and blue mold according to Example 7 of the present disclosure;

[0055] Figure 7 This is a schematic diagram of the antibacterial effects of freeze-dried bacterial agents of different concentrations on pear gray mold and black spot disease in Example 8 of the present disclosure;

[0056] Figure 8 Schematic diagram of the antibacterial effect of freeze-dried bacterial agents of different concentrations on gray mold and black spot of winter jujube according to Example 9 of the present disclosure;

[0057] Figure 9 This is a schematic diagram of the antibacterial effects of different concentrations of freeze-dried bacterial agents on gray mold and penicillium in kiwifruit according to Example 10 of the present disclosure;

[0058] Figure 10 Schematic diagram of the antibacterial effect of different concentrations of freeze-dried bacterial agents on blueberry aspergillus and gray mold in Example 11 of the present disclosure;

[0059] Figure 11 This is a schematic diagram of the antibacterial effects of freeze-dried bacterial agents of different concentrations on cucumber aspergillus and gray mold according to Example 12 of the present disclosure;

[0060] Figure 12 This is a schematic diagram of the antibacterial effect of different concentrations of freeze-dried bacterial agents on apple blue mold in Example 13 of the present disclosure;

[0061] Figure 13 This is a schematic diagram of the antibacterial effect of different concentrations of freeze-dried bacterial agents on orange Penicillium odoratum in Example 14 of the present disclosure;

[0062] Figure 14 Schematic diagram of the antibacterial effect of different concentrations of freeze-dried bacterial agents on tomato gray mold and blue mold according to Example 15 of the present disclosure. DETAILED DESCRIPTION

[0063] In order to enable those skilled in the art to better understand the technical solutions of the present disclosure, the present disclosure is further described in detail below with reference to the accompanying drawings and specific implementation methods.

[0064] In one aspect of the present disclosure, a composite microbial agent for preventing and controlling fruits and vegetables is provided, wherein the composite microbial agent comprises: Meyerozyma guilliermondii, Meyerozyma calibbica, and Pichia rarassimilans; wherein the Meyerozyma guilliermondii

[0065] guilliermondii) was deposited in the General Microbiology Center of China Culture Collection Administration Committee on June 12, 2023, and its deposit number is CGMCC No.27603; the Meyerozyma aribbica was deposited in the General Microbiology Center of China Culture Collection Administration Committee on June 12, 2023, and its deposit number is CGMCC No.27604; Pichia laras

[0066] rarassimilans) was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms on December 2, 2024, and its deposit number is CGMCC No.32875.

[0067] In this embodiment, the yeasts Kalibiker, Pichia laras, and Guillermo-Meyer yeast are antagonists and biocontrol bacteria that produce antibiotics, enzymes, and other antimicrobial substances, inhibiting cell growth and reproduction within fruits and vegetables and even killing pathogens. They rapidly colonize fruit wounds, enhancing the activity of resistance-related enzymes, occupying the wound space and inducing fruit resistance, thereby reducing the incidence of diseases. They are highly effective in preventing and controlling postharvest fruit diseases, including gray mold, penicillium, and black spot in strawberries, apples, pears, and tomatoes.

[0068] In some preferred embodiments, the mass ratio of Meyerozyma guilliermondii, Meyerozyma caribbica, and Pichiararassimilans is 3:1:2.

[0069] Another aspect of the present disclosure provides a freeze-dried bacterial agent for the prevention and treatment of fruits and vegetables, wherein the freeze-dried bacterial agent includes a composite bacterial agent and a composite protective agent; wherein the composite bacterial agent is the composite bacterial agent described above; wherein the composite protective agent includes 15-25% inulin, 5-15% skim milk powder and 10-18% xylooligosaccharides.

[0070] In some preferred embodiments, the content of inulin may be preferably 15%, 20%, 25%, etc., and more preferably 20%.

[0071] In other preferred embodiments, the content of skim milk powder may be preferably 5%, 10%, 15%, etc., and more preferably 10%.

[0072] In other preferred embodiments, the content of xylooligosaccharide may be preferably 10%, 14%, 18%, etc., and more preferably 14%.

[0073] like Figure 1As shown, another aspect of the present disclosure provides a method S100 for preparing the freeze-dried bacterial agent described above, the method comprising the following steps S110 to S120:

[0074] S110. Meyerozyma guilliermondii, Meyerozyma caribbica, and Pichia rarassimilans are used as a mixed seed liquid, the mixed seed liquid is purified and inoculated into a liquid culture medium, and a composite bacterial agent is formed through culturing. The composite bacterial agent is then prepared into a composite bacterial liquid using sterile water.

[0075] In some preferred embodiments, the inoculation amount of the mixed seed liquid is 0.5-4%, the mixed seed liquid is purified and inoculated into the liquid culture medium, the culture temperature is 20-36°C, the shaker speed is 180r / min, the pH of the culture process is 5-9, and the culture time is 24-72h.

[0076] As a further preferred solution, the inoculation amount of the mixed seed liquid is 2%, the pH of the culture process is 6, the culture temperature is 24° C., and the culture time is 60 h.

[0077] S120, adding the composite protective agent solution to the composite bacterial liquid to suspend it, placing the suspended composite bacterial liquid on a shaker for equilibrium treatment, pouring the treated composite bacterial liquid into a culture dish, covering the culture dish with a film, and pre-freezing the culture dish. After the composite bacterial liquid is completely pre-frozen, providing air holes in the film and freeze-drying the culture dish to obtain a freeze-dried bacterial agent.

[0078] In some preferred embodiments, the liquid culture medium comprises 10-20 g / L of a nitrogen source, 10-25 g / L of a carbon source, and 1-7 g / L of an inorganic salt.

[0079] As a further preferred embodiment, the nitrogen source is yeast extract powder, and the concentration of the yeast extract powder is preferably 15 g / L.

[0080] As a further preferred embodiment, the carbon source is sucrose, and the concentration of sucrose is preferably 15 g / L.

[0081] As a further preferred embodiment, the inorganic salts are magnesium sulfate and potassium dihydrogen phosphate, and the concentration of magnesium sulfate is preferably 5 g / L, and the concentration of potassium dihydrogen phosphate is preferably 3 g / L.

[0082] In other preferred embodiments, the ratio of the composite protective agent solution to the composite bacterial solution is 2:1.

[0083] In other preferred embodiments, the suspended composite bacterial liquid is placed on a shaker for equilibrium treatment at a temperature of 28° C., a rotation speed of 180 rpm, and a time of 30 minutes.

[0084] In other preferred embodiments, the thickness of the composite bacterial solution poured into the culture dish is 0.5 cm;

[0085] In other preferred embodiments, the culture dish is pre-frozen at a temperature of -80°C for 3 hours;

[0086] In other preferred embodiments, the freeze-drying temperature of the culture dish is 28° C. and the time is 30 minutes.

[0087] Another aspect of the present disclosure provides an application of a freeze-dried bacterial agent, wherein the freeze-dried bacterial agent described above is used to inhibit at least one of gray mold, penicillium, black spot, and aspergillosis in fruits and vegetables.

[0088] It should be noted that, for different fruits and vegetables, the freeze-dried bacterial agent can inhibit different pathogens. For example, when acting on strawberries, it can inhibit gray mold and blue mold of strawberries; when acting on pears, it can inhibit gray mold and black spot of pears; when acting on winter jujubes, it can inhibit gray mold and black spot of winter jujubes; when acting on kiwis, it can inhibit gray mold and blue mold of kiwis; when acting on blueberries, it can inhibit blueberry aspergillus and gray mold; when acting on cucumbers, it can inhibit cucumber aspergillus and gray mold; when acting on apples, it can inhibit apple blue mold; when acting on oranges, it can inhibit orange blue mold; when acting on tomatoes, it can inhibit gray mold and blue mold of tomatoes.

[0089] The freeze-dried bacterial agent of this embodiment can prevent and control a variety of fruits and vegetables, has a high inhibition rate, is easy to rehydrate, and is convenient to transport.

[0090] The following will describe the composite bacterial agent and freeze-dried bacterial agent with reference to specific examples:

[0091] It should be noted that Meyerozyma guilliermondii, Meyerozyma caribbica, and Pichia rarassimilans used in the following examples are biocontrol bacteria screened by the applicant in the early stage. For specific preservation information, please refer to the above records.

[0092] Example 1

[0093] This example uses the effect of carbon source on mixed bacterial culture as an example to illustrate the results:

[0094] Meyerozyma guilliermondii, Meyerozyma caribbica, and Pichia rarassimilans were used as a mixed seed liquid. After purification, the mixed seed liquid was inoculated into NYDB liquid culture medium at a ratio of 3:1:2 at a 2% inoculum amount. The composite bacterial agent was prepared by culturing at 28°C, pH = 6, and a shaking incubator at a speed of 180 r / min for 48 hours. The composite bacterial suspension was prepared into 1×108 cfu / mL with sterile water using a hemocytometer method for later use.

[0095] Among them, 15g / L YNB was used as the basic nitrogen source, and 10g / L of different carbon sources were added respectively, and screening was carried out at 28℃ and 180r / min. That is to say, the liquid culture medium included a nitrogen source and a carbon source; the best carbon source was selected to screen the carbon source concentration, among which glucose, molasses, lactose, sucrose, dextrin, and soluble starch were used as the carbon sources respectively. After selecting the best carbon source, their optimal concentrations were screened, which were 5g / L, 10g / L, 15g / L, 20g / L, and 25g / L respectively.

[0096] The effects of different types of carbon sources and optimal carbon source concentrations on mixed bacterial culture are shown in the following table. Figure 2 As shown in the results, when sucrose was used as the carbon source, the viable bacterial count in the fermentation broth was significantly higher than that of other carbon sources, reaching 8.98 lg·cfu / mL. Therefore, sucrose was selected as the carbon source for the mixed fermentation broth medium. The viable bacterial count was highest at sucrose concentrations of 15g / L and 25g / L, reaching 9.43 lg·cfu / mL. Considering the issue of saving raw materials, 15g / L sucrose was selected as the optimal concentration.

[0097] Example 2

[0098] This example uses the effect of nitrogen source on mixed bacterial culture as an example to illustrate the results:

[0099] The method of this example is the same as that of Example 1, and sucrose is used as the optimal carbon source screened out, and its concentration is 15 g / L. Different nitrogen sources and their concentrations are screened respectively. The nitrogen sources are yeast extract powder, yeast extract, beef extract, tryptone, ammonium sulfate, ammonium chloride and urea. After the optimal nitrogen source is screened out, its concentration is screened to 5 g / L, 10 g / L, 15 g / L, 20 g / L and 25 g / L, respectively.

[0100] The effects of different types of nitrogen sources and optimal nitrogen source concentrations on mixed bacterial culture are shown in the following table. Figure 3The results show that when yeast extract powder was used as the nitrogen source, the viable bacterial count in the fermentation broth was significantly higher than that of other nitrogen sources, reaching 9.29 lg·cfu / mL. Therefore, yeast extract powder was selected as the carbon source for the mixed fermentation broth. The highest viable bacterial count, reaching 9.43 lg·cfu / mL, was achieved when the yeast extract powder concentration was 15 g / L.

[0101] Example 3

[0102] This example uses the effect of inorganic salts on mixed bacterial culture as an example to illustrate the results:

[0103] The method of this example is the same as that of Example 1, and the optimal concentration of carbon source and the optimal concentration of nitrogen source are used as the basal culture medium, and 7 inorganic salts at 5 g / L are added to screen the optimal inorganic salts. The inorganic salts are potassium dihydrogen phosphate, dipotassium hydrogen phosphate, manganese sulfate, magnesium sulfate, zinc sulfate, sodium chloride and potassium sulfate. After determining the optimal inorganic salt, its concentration is screened, which are 1 g / L, 3 g / L, 5 g / L, 7 g / L, and 9 g / L, respectively. Each treatment is repeated 3 times.

[0104] Effects of different types of inorganic salts and two optimal inorganic salt concentrations on mixed bacterial culture Figure 4 The results show that when magnesium sulfate and potassium dihydrogen phosphate were used as inorganic salts, the viable bacterial count in the fermentation broth was significantly higher than that of other inorganic salts, reaching 9.46 lg cfu / mL and 9.39 lg cfu / mL, respectively. Therefore, magnesium sulfate and potassium dihydrogen phosphate were selected as inorganic salts for the mixed fermentation broth. The viable bacterial count was highest when the magnesium sulfate concentration was 5 g / L, reaching 9.48 lg cfu / mL, while the viable bacterial count was highest when the potassium dihydrogen phosphate concentration was 3 g / L, reaching 9.46 lg cfu / mL.

[0105] Example 4

[0106] This example uses the optimization of the optimal culture medium composition for mixed bacterial culture as an example to illustrate the results:

[0107] According to the above research results, the appropriate levels of carbon source (A), nitrogen source (B), first inorganic salt (C), and second inorganic salt (D) were obtained, and a 4-factor 3-level response surface optimization experiment was designed, as shown in Table 1. According to the analysis results, the optimal culture medium composition for mixed bacterial culture was finally obtained. The scheme design and results are shown in Table 2. In Table 2, A indicates that sucrose is used as the carbon source, B indicates that yeast extract is used as the nitrogen source, C indicates that magnesium sulfate is used as the first inorganic salt, and D indicates that potassium dihydrogen phosphate is used as the second inorganic salt. It can be seen from Table 2 that the highest survival rate is in Group 16, which is 9.56lg cfu / mL. The variance analysis is shown in Table 3. It can be seen from Table 3 that when the number of viable bacteria is the response value, the model P<0.01, indicating that the quadratic equation model is extremely significant. The lack of fit term P=0.1504>0.05 is not significant, indicating that the regression equation has a high degree of fit and the mathematical model is stable. The mathematical model can be used to predict the number of viable bacteria in mixed bacterial culture. As can be seen from the P value, the linear term B, the quadratic term BD, and A 2 、B 2 、C 2 and D 2 The effect on the viable bacterial survival rate of the composite bacteria was extremely significant (P < 0.01), while the linear terms A, C, and D, and the quadratic terms AB, AC, AD, BC, and CD had no significant effect (P > 0.05). Based on the F value, the order of influence of each factor on the viable bacterial count was yeast extract, magnesium sulfate, potassium dihydrogen phosphate, and sucrose, indicating that yeast extract had the most significant effect on the viable bacterial count.

[0108] Table 1L 12 (4 3 )Response surface experiment table

[0109]

[0110] Table 2 Response surface optimization design and experimental results

[0111]

[0112]

[0113] Table 3 Variance analysis table of regression equation

[0114]

[0115] It should be noted that in Table 3, SS represents the sum of squares, DF represents the degrees of freedom, MS represents the mean square, Pr>F represents the probability of no significant effect, ** represents very significant, and * represents significant.

[0116] The response surface experimental results were subjected to multiple linear regression and binomial fitting to obtain the regression equation for Y, as shown in the following relationship (1).

[0117] Y=9.54-2.5×10-4 A+0.044B-8.667×10 -3 C-1.167×10 -3 D-

[0118] 5.25×10 -3 AB+8×10 -3 AC+0.011AD-4.5×10 -3 BC+0.059BD+0.011CD-0.21A 2 -0.11B 2 -0.11C 2 -0.15D 2 (1);

[0119] In formula (1), A represents the concentration of sucrose, B represents the concentration of yeast extract, C represents the concentration of magnesium sulfate, and D represents the concentration of potassium dihydrogen phosphate.

[0120] Example 5

[0121] This example uses the effect of physical factors on mixed bacterial culture as an example to illustrate the results:

[0122] The method of this example is the same as that of Example 1, wherein the carbon source is sucrose at a concentration of 15 g / L, the nitrogen source is yeast extract powder at a concentration of 15 g / L, the first inorganic salt is magnesium sulfate at a concentration of 5 g / L, and the second inorganic salt is potassium dihydrogen phosphate at a concentration of 3 g / L. The difference is that the pH of the culture is set to 5, 6, 7, 8, and 9 respectively. The results of the mixed culture are as follows: Figure 5 As shown in (a), the inoculation amounts are 0.5%, 1%, 2%, 3%, and 4%, respectively. The results of mixed bacterial culture are shown in FIG. Figure 5 As shown in (b), the culture temperatures were set to 20°C, 24°C, 28°C, 32°C, and 36°C, respectively. The results of the mixed bacterial culture were as follows: Figure 5 As shown in (c), the culture time is set to 24h, 36h, 48h, 60h, and 72h respectively. The results of mixed bacterial culture are as follows Figure 5 As shown in (d) in .

[0123] It should be understood that the experiments under the above different conditions are all single-factor experiments. When the pH value is optimized, the temperature is 28°C, the inoculation size is 2%, and the incubation time is 48h. When the incubation temperature is optimized, the pH is 6, the inoculation size is 2%, and the incubation time is 48h. When the incubation time is optimized, the pH is 6, the inoculation size is 2%, and the temperature is 28°C. When the inoculation size is optimized, the temperature is 28°C, the pH is 6, and the incubation time is 48h.

[0124] Effects of different pH values, inoculation amounts, culture temperatures and culture times on mixed bacterial culture Figure 5 As shown in the results, it can be seen that the viable bacteria count is the highest when the pH value is 6, which can reach 9.52lg·cfu / mL, the viable bacteria count is the highest when the inoculation amount is 2%, which can reach 9.51lg·cfu / mL, the viable bacteria count is the highest when the culture temperature is 24℃, which can reach 9.52lg·cfu / mL, and the viable bacteria count is the highest when the culture time is 60h, which can reach 9.55lg·cfu / mL.

[0125] In summary, according to Examples 1-5, during the preparation of the composite bacterial agent, the optimal carbon source is preferably sucrose, with a preferred concentration of 15 g / L. The optimal nitrogen source is preferably yeast extract powder, with a preferred concentration of 15 g / L. The inorganic salts are preferably magnesium sulfate and potassium dihydrogen phosphate, with a preferred concentration of 5 g / L for magnesium sulfate and 3 g / L for potassium dihydrogen phosphate. The pH during the culture process is preferably 6, the inoculum size is preferably 2%, the culture temperature is preferably 24°C, and the culture time is preferably 60 h. Based on the above preferred conditions, the preferred conditions for preparing the freeze-dried bacterial agent are further described. For details, please refer to the following examples.

[0126] Example 6

[0127] This example uses the effects of different protective agents on freeze-dried bacterial preparations as an example to illustrate the results:

[0128] The composite bacterial suspensions prepared in the previous examples were centrifuged at 8000 rpm for 10 minutes, and the supernatant discarded. The protective agent solutions were added to the suspensions at a 2:1 ratio according to the protective agent formulations listed in Table 4 and suspended. The suspensions were then equilibrated on a shaker set at 28°C and 180 rpm for 30 minutes. Prior to lyophilization, the viable bacterial counts in the suspensions with different protective agent formulations were measured using a dilution-coating method. These suspensions were poured into 9mm Petri dishes, with the thickness of the suspensions uniformly maintained at 0.5 cm. The dishes were then covered with plastic wrap and pre-frozen at -80°C for 3 hours. Once the suspensions were completely frozen, small air holes were punctured in the plastic wrap, and the dishes were quickly transferred to a vacuum freeze dryer set to a 48-hour freeze-drying time for lyophilization. After lyophilization, the lyophilized powders were rehydrated and activated in a 28°C water bath for 30 minutes. The viable bacterial counts of the freeze-dried suspensions with different protective agent formulations were measured using a dilution-coating method. Finally, the freeze-dried survival rates of the bacteria with different protective agent formulas were obtained according to the following relationship (2), and the optimal protective agent formula was selected. The results are shown in Table 5.

[0129] Freeze-drying survival rate (%) = (number of viable bacteria after freeze-drying / number of viable bacteria before freeze-drying) × 100 (2)

[0130] Table 4 Types and concentrations of protective agents

[0131] Protective agent concentration(%) Protective agent concentration(%) Trehalose 15 glycerin 9 sucrose 14 monosodium glutamate 8 lactose 3 Vc 1.2 glucose 11 sorbitol 20 Xylitol 5 Xylooligosaccharides 14 Mannitol 7 Inulin 20 skim milk powder 10 β-cyclodextrin 20

[0132] Table 5 Effect of protective agent on the survival rate of composite bacteria freeze-dried

[0133]

[0134]

[0135] The effects of different protective agents on the freeze-dried survival rate of the composite bacteria are shown in Table 5. According to Table 5, the survival rate was the highest when the protective agent was 20% inulin, reaching 64.34%, followed by 10% skim milk powder, with a survival rate of 63.84%, and third was 14% xylo-oligosaccharide, reaching 57.47%.

[0136] Furthermore, based on the above research results, the three optimal protective agents were selected to obtain the optimal levels of skim milk powder (A), oligoxylose (B), and inulin (C). A three-factor, three-level response surface optimization experiment was designed, as shown in Table 6. Based on the analysis results, the optimal preparation conditions for the freeze-dried inoculum were finally obtained.

[0137] The experimental design and results are shown in Table 7, and the variance analysis is shown in Table 8. Table 7 shows that the highest survival rate is in Group 1, which is 63.3%. Table 8 shows that when the survival rate is the response value, the model P < 0.01, indicating that the quadratic equation model is extremely significant. The lack of fit term P = 0.4062 > 0.05 is not significant, indicating that the regression equation has a high degree of fit and the mathematical model is stable. This mathematical model can be used to predict the number of viable bacteria in liquid inoculants. From the P value, it can be seen that the linear term C, the quadratic term BC, and A 2 、B 2 and C 2 The effect on the survival rate of viable bacteria in the inoculant was extremely significant (P<0.01). The quadratic terms AC and A had a significant effect on the survival rate of viable bacteria in the inoculant (P<0.05), while A, B, and AB had no significant effect (P>0.05). Based on the F value, the order of influence of each factor on the number of viable bacteria was inulin, skim milk powder, and oligoxylose, indicating that inulin had the most significant effect on the survival rate.

[0138] Table 6L9(3 3 )Response surface experiment table

[0139]

[0140] Table 7 Response surface optimization design and experimental results

[0141]

[0142]

[0143] Table 8 Variance analysis table of regression equation

[0144] type SS DF MS F Pr>F Significance Model 299.29 9 33.25 100.10 <0.0001 ** A 1.42 1 1.42 4.27 0.0775 B 0.85 1 0.85 2.54 0.1548 C 11.45 1 11.45 34.46 0.0006 ** AB 6.20 1 6.20 18.66 0.0035 AC 3.94 1 3.94 11.86 0.0108 * BC 23.23 1 23.23 69.93 <0.0001 ** <![CDATA[A 2 ]]> 69.74 1 69.74 209.93 <0.0001 ** <![CDATA[B 2 ]]> 107.69 1 107.69 324.16 <0.0001 ** <![CDATA[C 2 ]]> 49.10 1 49.10 147.79 <0.0001 ** residual 2.33 7 0.33 Lack of Fit 1.12 3 0.37 1.24 0.4062 Not significant Pure error 1.21 4 0.30

[0145] It should be noted that in Table 8, SS represents the sum of squares, DF represents the degrees of freedom, MS represents the mean square, Pr>F represents the probability of no significant effect, ** represents very significant, and * represents significant.

[0146] The response surface experimental results were subjected to multiple linear regression and binomial fitting to obtain the regression equation for Y, as shown in the following relationship (3).

[0147] Y=62.85+0.42A+0.33B+1.2C-1.25AB-0.99AC+2.41BC-4.07A 2 -5.06B 2 -3.41C 2 (3);

[0148] Wherein, A represents the concentration of skim milk powder, B represents the concentration of xylo-oligosaccharide, and C represents the concentration of inulin.

[0149] In summary, based on Examples 1 to 6, the optimal preparation process for this inoculant, predicted by the response surface method, is to inoculate 2% P. guilliermondii:M. caribbica:P. rarassimilans seed mixture in a 3:1:2 ratio into a medium containing 14.98 g / L sucrose, 16.06 g / L yeast extract, 4.91 g / L magnesium sulfate, and 3.07 g / L potassium dihydrogen phosphate at a pH of 6, and culture on a shaker at 24°C and 180 rpm for 60 h. The fermentation broth is then centrifuged at 8000 rpm for 10 min, the supernatant discarded, and a composite protective agent solution containing 10.07% skim milk powder, 14.31% xylo-oligosaccharides, and 21% inulin is added to the culture broth in a 2:1 ratio to suspend it. The suspended culture is then equilibrated on a shaker at 28°C and 180 rpm for 30 min. Pour the bacterial solution into a culture dish and pre-freeze it at -80℃ for 3 hours. When the bacterial solution is completely pre-frozen, poke small holes on the surface of the plastic wrap and quickly move the culture dish to a vacuum freeze dryer with the freeze-drying time set to 48 hours for freeze-drying to obtain the optimal freeze-dried bacterial agent.

[0150] Furthermore, based on the optimal conditions determined in the above examples, the in vivo inhibitory effects of different concentrations of freeze-dried bacterial agents on different fruits and vegetables are described below:

[0151] Example 7

[0152] This example uses different concentrations of freeze-dried bacteria to illustrate the in vivo inhibitory effects on strawberry gray mold and blue mold:

[0153] Healthy strawberry fruits were taken and the surface of the fruit was disinfected with 2% NaClO. Then wounds with a diameter of 4 mm and a depth of 4 mm were made at the equator of the fruit. One wound was made per fruit. 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 cfu / mL of freeze-dried bacteria, and sterile water was added as a control. 2 hours later, 1×10 4 Incubate 20 μL of B. cinerea and P. expansum at 26°C at a concentration of 100 cfu / mL. After 7 days, measure lesion diameters and calculate inhibition rates. Ten fruits were used for each treatment. The experiment was repeated three times.

[0154] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 6 As shown in Figure 2, with the increase of concentration, the inhibition rate of strawberry gray mold and blue mold increased. 5 cfu / mL, the inhibition rates were 35.61%, 30.51%, and 1×10 6 cfu / mL is 50%, 47.46%, 1×10 7 cfu / mL were 76.52%, 73.73%, and 1×10 9 cfu / mL were 85.61% and 82.2%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 86.36% and 83.9%, respectively, and the inhibition effect was the best at this time.

[0155] Example 8

[0156] This example uses different concentrations of freeze-dried bacteria to illustrate the in vivo inhibitory effects on pear gray mold and black spot:

[0157] Take pear healthy fruit, and process it in the same way as in Example 7, with 4 wounds per fruit. Add freeze-dried bacterial agent for 2 hours and inoculate 1×10 4 cfu / mL of Botrytis cinerea and 1×10 5 Incubate 20 μL of A. tenuissima at 26°C for 7 days. Measure lesion diameter and calculate inhibition rate. Ten fruits were used for each treatment. Repeat the experiment three times.

[0158] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 7 As shown in Figure 2, with the increase of concentration, the inhibition rate of pear gray mold and black spot increased. 5cfu / mL, the inhibition rates were 37.25%, 41.19%, and 1×10 6 cfu / mL is 50.42%, 53.57%, 1×10 7 cfu / mL were 68.63%, 69.29%, and 1×10 9 cfu / mL were 85.15% and 82.62%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 88.24% and 84.76%, respectively, and the inhibition effect was the best at this time.

[0159] Example 9

[0160] This example uses different concentrations of freeze-dried bacterial agents to illustrate the in vivo inhibitory effects on gray mold and black spot of winter jujube:

[0161] Take healthy winter jujube fruits and treat them in the same way as in Example 7. Each fruit has one wound. Add freeze-dried bacterial agent for 2 hours and inoculate 1×10 4 cfu / mL of Botrytis cinerea and 1×10 5 Incubate 20 μL of A. tenuissima at 26°C for 7 days. Measure lesion diameter and calculate inhibition rate. Ten fruits were used for each treatment. Repeat the experiment three times.

[0162] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 8 As shown in Figure 2, with the increase of concentration, the inhibition rate of gray mold and black spot of winter jujube increased. 5 cfu / mL, the inhibition rates were 38.89%, 33.33%, and 1×10 6 cfu / mL is 55.56%, 50%, 1×10 7 cfu / mL were 66.67%, 61.11%, and 1×10 9 cfu / mL were 77.78% and 72.22%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 86.11% and 77.78%, respectively, and the inhibition effect was the best at this time.

[0163] Example 10

[0164] This example uses the in vivo inhibitory effects of freeze-dried bacteria at different concentrations on gray mold and penicillium mold in kiwi fruit as an example:

[0165] Take healthy kiwifruit, disinfect the surface of the fruit with 2% NaClO, and make wounds with a diameter of 4 mm and a depth of 4 mm at the equator of the fruit. Each fruit should have two wounds. Add 1×10 5 , 1×106 , 1×10 7 , 1×10 8 , 1×10 9 cfu / mL of freeze-dried bacteria, and sterile water was added as a control. 2 hours later, 1×10 4 Incubate 20 μL of B. cinerea and P. expansum at 26°C at a concentration of 100 cfu / mL. After 7 days, measure lesion diameters and calculate inhibition rates. Ten fruits were used for each treatment. The experiment was repeated three times.

[0166] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 9 As shown in the figure, with the increase of concentration, the inhibition rate of kiwifruit gray mold and blue mold increased. At 1×105 cfu / mL, the inhibition rates were 45%, 39.31%, and 1×10 6 cfu / mL is 62.5%, 53.56%, 1×10 7 cfu / mL were 72.5%, 67.57%, and 1×10 9 cfu / mL were 85%, 84.03%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 87.5% and 86.98%, respectively, and the inhibition effect was the best at this time.

[0167] Example 11

[0168] This example uses different concentrations of freeze-dried bacteria to illustrate the in vivo inhibitory effects on blueberry aspergillus and gray mold:

[0169] Take healthy blueberry fruits, disinfect the fruit surface with 2% NaClO, and make a wound with a diameter of 4mm and a depth of 4mm at the equator of the fruit. Each fruit should have one wound. Add 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 cfu / mL of freeze-dried bacteria, and sterile water was added as a control. 2 hours later, 1×10 5 cfu / mL of Aspergillus niger and 1×10 4 Incubate 20 μL of B. cinerea at 26°C with a concentration of 100 cfu / mL. After 7 days, measure the lesion diameter and calculate the inhibition rate. Ten fruits were used for each treatment. Repeat the experiment three times.

[0170] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 10As shown in the figure, with the increase of concentration, the inhibition rate of blueberry aspergillus and gray mold increased. At 1×105 cfu / mL, the inhibition rates were 27.97% and 29.17%, respectively. At 1×10 6 cfu / mL is 53.39%, 49.17%, 1×10 7 cfu / mL were 72.03%, 70.83%, and 1×10 9 cfu / mL were 79.66% and 76.67%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 83.9% and 85%, respectively, and the inhibition effect was the best at this time.

[0171] Example 12

[0172] This example uses the in vivo inhibitory effects of freeze-dried bacterial agents of different concentrations on cucumber aspergillus and gray mold as examples to illustrate:

[0173] Take healthy cucumber fruits, disinfect the fruit surface with 2% NaClO, and make wounds with a diameter of 4 mm and a depth of 4 mm at the equator of the fruit. Each fruit should have two wounds. Add 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 cfu / mL of freeze-dried bacteria, and sterile water was added as a control. 2 hours later, 1×10 5 Incubate 20 μL of 1×104 cfu / mL of Aspergillus niger (A. niger) and 1×104 cfu / mL of Botrytis cinerea (B. cinerea) at 26°C. After 7 days, measure the lesion diameter and calculate the inhibition rate. Ten fruits were used for each treatment. The experiment was repeated three times.

[0174] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 11 As shown in Figure 2, with the increase of concentration, the inhibition rate of cucumber aspergillus and gray mold increased. 5 cfu / mL, the inhibition rates were 32.71%, 36.54%, and 1×10 6 cfu / mL is 44.39%, 48.56%, 1×10 7 cfu / mL were 55.61%, 59.62%, and 1×10 9 cfu / mL were 59.81% and 68.75%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 69.63% and 78.37%, respectively, and the inhibition effect was the best at this time.

[0175] Example 13

[0176] This example uses different concentrations of freeze-dried bacteria to illustrate the inhibitory effect on apple blue mold:

[0177] Take healthy apples and treat them in the same way as in Example 7. Make 4 wounds per fruit. Add freeze-dried bacteria for 2 hours and inoculate 1×10 4 20 μL of Penicillium expansum (P. expansum) containing 100 cfu / mL was incubated at 26°C. After 7 days, the diameter of the lesions was measured and the inhibition rate was calculated. Ten fruits were used for each treatment. The experiment was repeated three times.

[0178] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 12 As shown in Figure 2, the inhibition rate of apple blue mold increased with the increase of concentration. 5 cfu / mL, the inhibition rate was 30.67%, 1×10 6 cfu / mL is 35.89%, 1×10 7 cfu / mL was 49.08%, 1×10 9 cfu / mL was 59.82%, and at a concentration of 1×10 8 cfu / mL, the inhibition rate was 62.27%, and the inhibition effect was the best at this time.

[0179] Example 14

[0180] This example uses different concentrations of freeze-dried bacteria to illustrate the inhibitory effect on orange Penicillium mold:

[0181] Take healthy orange fruits and treat them in the same way as in Example 7. Each fruit has one wound. Add freeze-dried bacteria agent for 2 hours and inoculate 1×10 4 20 μL of Penicillium citrinum (CFU / mL) was incubated at 26°C. After 7 days, the lesion diameter was measured and the inhibition rate was calculated. Ten fruits were used for each treatment. The experiment was repeated three times.

[0182] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 13 As shown in Figure 2, the inhibition rate of orange Penicillium mold disease increases with the increase of concentration. 5 cfu / mL, the inhibition rate was 31.02%, 1×10 6 cfu / mL is 37.55%, 1×10 7 cfu / mL was 46.94%, 1×10 9 cfu / mL was 54.69%, and at a concentration of 1×10 8 cfu / mL, the inhibition rate was 61.63%, and the inhibition effect was the best at this time.

[0183] Example 15

[0184] This example uses different concentrations of freeze-dried bacteria to illustrate the in vivo inhibitory effects on tomato gray mold and blue mold:

[0185] Take healthy tomato fruits and treat them in the same way as in Example 7. Each fruit has one wound. Add freeze-dried bacteria agent for 2 hours and inoculate 1×10 4 Incubate 20 μL of B. cinerea and P. expansum at 26°C at a concentration of 100 cfu / mL. After 7 days, measure lesion diameters and calculate inhibition rates. Ten fruits were used for each treatment. The experiment was repeated three times.

[0186] Furthermore, after inoculation with different concentrations of freeze-dried bacteria, the statistical results of the inhibition rate are as follows: Figure 14 As shown in Figure 2, with the increase of concentration, the inhibition rate of tomato gray mold and blue mold increased. 5 cfu / mL, the inhibition rates were 39.2%, 46.62%, and 1×10 6 cfu / mL is 48%, 54.73%, 1×10 7 cfu / mL were 74.4%, 75.68%, and 1×10 9 cfu / mL were 92.8% and 90.54%, and at a concentration of 1×10 8 cfu / mL, the inhibition rates were 95.2% and 92.57%, respectively, and the inhibition effect was the best at this time.

[0187] In summary, when the concentration of freeze-dried bacteria is 1×10 8 cfu / mL, the inhibition rate of pathogens in fruits and vegetables is the highest.

[0188] It is understood that the above embodiments are merely exemplary embodiments for illustrating the principles of the present disclosure, and the present disclosure is not limited thereto. Those skilled in the art may make various modifications and improvements without departing from the spirit and substance of the present disclosure, and such modifications and improvements are also considered to be within the scope of protection of the present disclosure.

Claims

1. A composite bacterial agent for the prevention and treatment of fruits and vegetables, characterized in that: The composite bacterial agent includes: Meyerozyma guilliermondii, Meyerozyma caribbica, and Pichia rarassimilans; wherein, The Meyerozyma guilliermondii was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on June 12, 2023, with a deposit number of CGMCC No. 27603. The Meyerozyma caribbica was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms on June 12, 2023, with a deposit number of CGMCC No. 27604. The Pichia rarassimilans was deposited in the General Microbiology Center of the China Culture Collection Administration on December 2, 2024, and its deposit number is CGMCC No.32875.

2. The composite bacterial agent according to claim 1, characterized in that The mass ratio of the Meyerozyma guilliermondii, the Meyerozyma caribbica, and the Pichia rarassimilans is 3:1:

2.

3. A freeze-dried bacterial agent for the prevention and treatment of fruits and vegetables, characterized in that: The freeze-dried bacterial agent comprises a composite bacterial agent and a composite protective agent; wherein the composite bacterial agent is the composite bacterial agent according to claim 1 or 2; wherein, The composite protective agent comprises 15-25% of inulin, 5-15% of skim milk powder and 10-18% of xylo-oligosaccharide.

4. A method for preparing the freeze-dried bacterial agent according to claim 3, characterized in that: The method comprises: Meyerozyma guilliermondii, Meyerozyma caribbica, and Pichia rarassimilans are used as a mixed seed liquid, the mixed seed liquid is purified and inoculated into a liquid culture medium, and a composite bacterial agent is formed through culturing, and the composite bacterial agent is prepared into a composite bacterial liquid using sterile water; A composite protective agent solution is added to the composite bacterial liquid to suspend it, the suspended composite bacterial liquid is placed on a shaker for equilibrium treatment, and the treated composite bacterial liquid is poured into a culture dish, which is covered with a film and pre-frozen. After the composite bacterial liquid is completely pre-frozen, air holes are provided on the film and the culture dish is freeze-dried to obtain a freeze-dried bacterial agent.

5. The method according to claim 4, characterized in that The liquid culture medium comprises 10-20 g / L of nitrogen source, 10-25 g / L of carbon source, and 1-7 g / L of inorganic salt.

6. The method according to claim 5, characterized in that The nitrogen source is yeast extract powder, and the concentration of the yeast extract powder is 15 g / L; The carbon source is sucrose, and the concentration of the sucrose is 15 g / L; The inorganic salts are magnesium sulfate and potassium dihydrogen phosphate, the concentration of the magnesium sulfate is 5 g / L, and the concentration of the potassium dihydrogen phosphate is 3 g / L.

7. The method according to claim 4, characterized in that The inoculation amount of the mixed seed liquid is 0.5-4%, the pH value during the culture process is 5-9, the culture temperature is 20-36° C., and the culture time is 24-72 hours.

8. The method according to claim 7, characterized in that The inoculation amount of the mixed seed liquid is 2%, the pH value during the culture process is 6, the culture temperature is 24° C., and the culture time is 60 h.

9. The method according to claim 4, characterized in that The content ratio of the composite protective agent solution to the composite bacterial solution is 2:1; The suspended composite bacterial solution was placed on a shaker for equilibrium treatment at a temperature of 28°C, a rotation speed of 180 rpm, and a time of 30 min; The thickness of the composite bacterial solution poured into the culture dish is 0.5 cm; The culture dish was pre-frozen at -80°C for 3 h. The freeze-drying temperature of the culture dish was 28° C. and the time was 30 min.

10. An application of a freeze-dried bacterial agent, characterized in that: The freeze-dried bacterial agent according to claim 3 is used to inhibit at least one of gray mold, penicillium, black spot, and aspergillosis of fruits and vegetables.

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