Method for preparing Wickerhamomyces anomalus solid preparation by using protective agent and application of Wickerhamomyces anomalus solid preparation
By using sorbitol, skim milk powder, sodium glutamate and trehalose as protective agents during the freeze-drying process of yeast, the complex ratio was optimized, and the problem of decreased yeast survival was solved, and an efficient abnormal Wick was prepared. Ham yeast solid preparation achieves long-term storage stability and commercial application.
Patent Information
- Application Number
- CN202410535496.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-04-30
- Publication Date
- 2024-12-13
AI Technical Summary
In the prior art, yeasts are easily damaged during freeze-drying, resulting in a decrease in survival rate. The lack of lyophilized protective agent research for abnormal Wickham yeasts limits its application.
Vacuum freeze-drying method, combined with sorbitol, skim milk powder, sodium glutamate and trehalose as protective agents, the survival rate and storage stability of yeast were significantly improved by optimizing their compounding ratio, and abnormal Wick was prepared. The Ham yeast solid preparation can be stored at -20°C for 90 days, and the survival rate remains above 67%.
It significantly improves the survival rate and storage stability of yeast, meets the requirements of commercial production and use, avoids the potential harm of chemical fungicides, and complies with food safety and environmental protection standards.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of biological control of post-harvest fruit diseases, and particularly relates to a method for preparing an abnormal Wickham yeast solid preparation by using a protective agent and application thereof. Background Art
[0002] Tomato, Solanum lycopersicon L., is very susceptible to a variety of pathogens such as Alternaria, Rhizopus and Botrytis cinerea during postharvest, transportation and storage. As one of the common pathogens, Alternaria can infect a variety of fruits and vegetables, reduce tomato yields, and bring huge economic losses to my country's fruit and vegetable industry.
[0003] Wickerhamomyces anomalus is a biocontrol yeast with excellent potential. It can significantly inhibit the germination of pathogenic bacteria spores, rapidly colonize on the host, induce the activity of polyphenol oxidase, peroxidase, catalase and phenylalanine ammonia lyase in fruits, activate resistance genes in the host, and thus improve the host's resistance to pathogens. However, in the actual fruit and vegetable control process, yeast is easily affected by the environment during storage, resulting in reduced activity, which seriously restricts its application. Therefore, it is particularly important to develop and produce efficient yeast agent products.
[0004] Solid preparations have the advantages of long shelf life, low contamination, low storage and transportation costs, and are suitable for commercial development and application. At present, solid preparations of antagonistic yeast can be prepared mainly by freeze drying, spray drying, airflow drying and other methods. Among them, freeze drying is one of the most effective methods for preserving strains, which can prolong the protection time of strains and maintain the vitality of strains; but in the process of freeze drying, cells will be damaged to a certain extent, such as the formation of large ice crystals, osmotic stress, mechanical damage, solute effects, and denaturation of DNA and protein, etc., causing cell membrane rupture and changes in effective substances inside cells, and the dynamic balance of bacteria will also be destroyed. Adding a suitable freeze-drying protective agent can reduce the damage to bacteria during the freeze-drying process. For example, a composite freeze-drying protective agent for lactic acid bacteria, although some protective agents for freeze-drying of bacterial agents have been disclosed in the prior art, have a certain protective effect but the effect is unclear, and the selection and dosage of freeze-drying protective agents are different, which will cause a large fluctuation in the survival rate of lactic acid bacteria after freeze drying. With the extension of the storage period, the vitality of microorganisms will gradually decrease.
[0005] Therefore, comprehensive optimization of freeze-drying protective agents to extend the storage period of microorganisms while improving the survival rate of microorganisms has become an urgent problem to be solved. Summary of the invention
[0006] Since biocontrol products have the advantages of high efficiency, environmental protection and no side effects, biological control technology has received much attention in recent years and has evolved towards the development of commercial biological control agents. In the actual fruit and vegetable control process, yeast is easily affected by the environment during storage, resulting in reduced activity. Therefore, the research and development and production of efficient yeast agent products are particularly important. Vacuum freeze-drying is currently the most commonly used method for preparing solid bacterial agents, but the freeze-drying process will inevitably damage microbial cells, mainly including denaturation of sensitive proteins, changes in cell membrane permeability, etc.; there are currently no reports on solid preparations of abnormal Wickham yeast, and there is no research on its lyophilization protectant.
[0007] In view of the deficiencies in the prior art, the present invention provides a solid preparation based on abnormal Wickham yeast and a preparation method thereof, creatively screens protective agents and the optimal compounding ratio between different protective agents, significantly improves the survival rate and storage stability of the bacteria, and the prepared abnormal Wickham yeast solid preparation can be stored at -20°C for 90 days, the survival rate of the solid preparation is above 67%, and the decay rate of the solid preparation group is stably below 24.0%; the technical problem of low survival rate of abnormal Wickham yeast in practical applications is solved, and the requirements for commercial production and use can be met.
[0008] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:
[0009] The method for preparing a solid preparation of abnormal Wickham yeast using a protective agent comprises the following steps:
[0010] (1) inoculating abnormal Wickham yeast in a culture medium NYDB, performing a first activation culture, and obtaining a first activation culture solution; then transferring the first activation culture solution to a new NYDB culture medium, and continuing a second activation culture, and obtaining an activated yeast solution; centrifuging the activated yeast solution to obtain a bacterial precipitate, and then centrifuging and washing the bacterial precipitate with sterile water, and resuspending the washed precipitate with sterile water to obtain a yeast suspension;
[0011] (2) adding sorbitol, skim milk powder, sodium glutamate and trehalose to the yeast suspension obtained in step (1), stirring evenly to obtain a mixed solution, sealing the mixed solution with a sealing film and pre-freezing it to obtain a pre-frozen mixture; finally, freeze-drying the pre-frozen mixture to obtain a solid preparation of abnormal Wickham yeast; the mass concentration of sorbitol in the mixed solution is 2-10%, the mass concentration of skim milk powder is 2.5-12.5%, the mass concentration of sodium glutamate is 1.5-6%, and the mass concentration of trehalose is 2.5-7.5%; the deposit number of the abnormal Wickham yeast is: CCTCCM2018053.
[0012] Preferably, the conditions for the first activation culture and the second activation culture in step (1) are both a temperature of 28° C., a rotation speed of 180-200 rpm, and a time of 20-24 h; and the centrifugation conditions are: 4° C., 9600×g, 5-10 min.
[0013] Preferably, the concentration of the yeast suspension in step (1) is 1×10 8 cells / mL.
[0014] Preferably, the NYDB medium described in step (1) comprises, per liter, 5 g yeast extract, 10 g glucose, 8 g beef extract, and distilled water to a volume of 1000 mL, with a natural pH and sterilized at 115° C. for 20 min.
[0015] Preferably, in the mixed solution in step (2), the mass concentration of sorbitol is 6%, the mass concentration of skim milk powder is 10%, the mass concentration of sodium glutamate is 3%, and the mass concentration of trehalose is 5%;
[0016] Preferably, the pre-freezing temperature in step (2) is -80°C for 2 to 3 hours.
[0017] Preferably, the specific operation of freeze drying in step (2) is: pre-cooling the cold trap to a temperature below -50°C, then placing the pre-frozen mixture, turning on the vacuum pump to ensure that the vacuum degree during the drying process is not less than 10Pa, and the drying time is 48 hours.
[0018] The invention also provides a solid preparation of abnormal Wickham yeast prepared based on the method.
[0019] The use of the abnormal Wickham yeast solid preparation for controlling postharvest black spot disease of tomatoes is specifically:
[0020] The abnormal Wickham yeast solid preparation was reconstituted with sterile water to obtain a bacterial suspension, in which the yeast concentration was 1×10 8 cells / mL; then the bacterial suspension is evenly sprayed on the surface of tomatoes or the tomatoes are soaked in the bacterial suspension and then taken out and dried naturally, thereby achieving the purpose of controlling post-harvest black spot disease of tomatoes.
[0021] Beneficial effects:
[0022] (1) The present invention creatively screens protective agents and optimizes the compounding ratio of different protective agents, significantly improving the survival rate and storage stability of the bacteria. The prepared abnormal Wickham yeast solid preparation can be stored at -20°C for 90 days, the survival rate of the solid preparation is above 67%, and the decay rate of the solid preparation group is stably below 24.0%. The present invention solves the technical problem of low survival rate of abnormal Wickham yeast in practical applications, can meet the requirements of commercial production and use, and is of great significance to the commercial application and development of abnormal Wickham yeast solid preparations.
[0023] (2) The various protective agent components involved in the preparation of abnormal Wickham yeast solid preparations of the present invention are all of food grade origin, low cost, and meet the relevant national standards for food safety and food additives. The preparation process of the yeast solid preparation provided is scientific and reasonable, with efficient production, greatly simplified process and cost, and improved production efficiency; at the same time, it also avoids the potential harm of chemical bactericides to the human body and the environment, and is in line with the concept of energy conservation, environmental protection, and green economy promoted by the country. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The effects of different concentrations of sorbitol (A), skim milk powder (B), sodium L-glutamate (C), and trehalose (D) on the survival rate of freeze-dried abnormal Wickham yeast; different lowercase letters represent significant differences (p < 0.05).
[0025] Figure 2 Survival rate of abnormal Wickham yeast solid preparation during storage; different lowercase letters represent significant differences (p < 0.05).
[0026] Figure 3 Biocontrol effect of solid preparation of abnormal Wickham yeast during storage; different lowercase letters represent significant differences (p<0.05). DETAILED DESCRIPTION
[0027] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.
[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.
[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.
[0030] (a) Strain: Wickerhamomyces anomalus was obtained from China Center for Type Culture Collection. The specific deposit information is as follows: Wickerhamomyces anomalus; deposit number: CCTCC M2018053; deposit unit: China Center for Type Culture Collection; deposit address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, opposite the First Affiliated Primary School.
[0031] (b) NYDB medium (1 L): 5 g yeast extract, 10 g glucose, 8 g beef extract, distilled water to 1000 mL, pH natural, sterilized at 115°C for 20 min.
[0032] (c) The freeze-drying process is as follows: after sealing with plastic wrap, pre-freeze at -80°C for 2 h. At the same time, turn on the freeze dryer's refrigerator to pre-cool so that the temperature of the cold trap reaches -50°C. Put the sample in and turn on the vacuum pump to ensure that the vacuum degree during the drying process is not less than 10 Pa. The drying time is 48 h.
[0033] (d) The number of viable yeast cells before and after freeze-drying using the plate spreading method, the survival rate of yeast = (the number of viable yeast cells after freeze-drying / the number of viable yeast cells before freeze-drying) × 100%.
[0034] Example 1: Condition screening:
[0035] (1) Study on the survival rate of abnormal Wickham yeast in different concentrations of sorbitol
[0036] Sorbitol and abnormal Wickham yeast suspension are mixed evenly, and the final concentration (mass concentration) of sorbitol in the obtained mixed solution is adjusted to 0.0%, 2.0%, 4.0%, 6.0%, 8.0%, and 10.0%, respectively; then the mixed solution is added to a plate, and freeze-dried according to the freeze-drying method in step (c) to obtain a dry product.
[0037] The freeze-dried survival rate of abnormal Wickham yeast was measured when the sorbitol concentration was 0.0%, 2.0%, 4.0%, 6.0%, 8.0%, and 10.0%; Figure 1 As shown in Figure A, as the concentration of sorbitol increases, the freeze-dried survival rate of abnormal Wickham yeast also increases; but when it exceeds 6.0%, its survival rate begins to decline, indicating that excessive sorbitol not only does not promote the survival rate, but also has an adverse effect on the freeze-dried survival rate of abnormal Wickham yeast. Therefore, the optimal protective concentration of sorbitol for abnormal Wickham yeast is 6%.
[0038] (2) Study on the survival rate of abnormal Wickham yeast with different concentrations of skim milk powder
[0039] The skimmed milk powder and the abnormal Wickham yeast cells were mixed uniformly, and the final concentration (mass concentration) of the skimmed milk powder in the obtained mixed solution was adjusted to 0.0%, 2.5%, 5.0%, 7.5%, 10.0%, and 12.5%. The mixed solution was added to a plate, and freeze-dried according to the method in step (c) to obtain a dry product.
[0040] The survival rate of abnormal Wickham yeast freeze-dried was measured when the concentration of skim milk powder was 0.0%, 2.5%, 5.0%, 7.5%, 10.0%, and 12.5%. Figure 1 As shown in Figure B, with the increase of skim milk powder, the freeze-dried survival rate of abnormal Wickham yeast increased in the range of 0-7.5%, but it was not significant; when the concentration of skim milk powder was 10.0%, its survival rate was significantly improved; as the concentration of skim milk powder continued to increase, the survival rate did not continue to increase, but decreased; this shows that excessive skim milk powder will have an adverse effect on the freeze-dried survival rate of abnormal Wickham yeast. In summary, the optimal protective concentration of skim milk powder for abnormal Wickham yeast is 10%.
[0041] (3) Study on the survival rate of abnormal Wickham yeast under different concentrations of L-glutamate
[0042] Evenly mix sodium L-glutamate and abnormal Wickham yeast cells, adjust the final concentration (mass concentration) of sodium L-glutamate in the obtained mixed solution to 0.0%, 1.5%, 3.0%, 4.5%, 6.0%, and 7.5%, add the mixed solution to a plate, and perform freeze-drying according to the method in step (c) to obtain a dry product.
[0043] The corresponding determination of the survival rate of abnormal Wickham yeast freeze-dried when the concentration of L-glutamate sodium is 0.0%, 2.0%, 4.0%, 6.0%, 8.0%, and 10.0%. Figure 1 As shown in Figure C, L-glutamate sodium has a significant effect on the survival rate of abnormal Wickham yeast; in the concentration range of 0-3%, with the increase of L-glutamate sodium, the freeze-dried survival rate increases rapidly, and when it increases from 3% to 7.5%, the survival rate begins to decrease with the increase of concentration, so the optimal protection concentration of L-glutamate sodium for abnormal Wickham yeast is 3%.
[0044] (4) Study on the survival rate of abnormal Wickham yeast under different concentrations of trehalose
[0045] The trehalose solution was mixed with abnormal Wickham yeast cells, and the final concentration (mass concentration) of trehalose in the obtained mixed solution was adjusted to 0.0%, 2.5%, 5.0%, 7.5%, 10.0%, and 12.5%. The mixed solution was added to a plate, and freeze-dried according to the method in step (c) to obtain a dry product.
[0046] The corresponding determination of the survival rate of abnormal Wickham yeast freeze-dried when the trehalose concentration is 0.0%, 2.5%, 5.0%, 7.5%, 10.0%, and 12.5%. Figure 1 As shown in Figure D, in the concentration range of 0-5%, the freeze-dried survival rate increased significantly with the increase of trehalose; when the trehalose concentration was between 5% and 10%, the survival rate began to decrease with the increase of concentration, so the optimal protective concentration of trehalose for abnormal Wickham yeast was 5%.
[0047] (5) Response surface analysis of the optimal formulation of abnormal Wickham yeast solid preparations under different protective agents
[0048] The optimal concentration range of single protective agents of sorbitol, skim milk powder, sodium glutamate and trehalose was further determined. The Box-Behnken Design method in Design Expert 12 software was used to design the experimental plan and determine the level of each factor. After the experiment was completed, the Design Expert 8.0.6 software was used for data analysis, and a response surface model was established to predict the optimal formula of abnormal Wickham yeast solid preparations under different protective agents.
[0049] Taking sorbitol concentration (A), skim milk powder concentration (B), sodium L-glutamate concentration (C) and trehalose concentration (D) as influencing factors and yeast survival rate (Y) as the response index, regression fitting was performed to obtain the quadratic polynomial regression equation:
[0050] Y=83.80+0.9833A+0.7750B+1.66C-0.15D+0.6AB-0.525AC-0.025AD-0.75BC-1.03BD-1.00CD-2.35A 2 2.13B 2 -5.83C 2 -3.70D 2 .
[0051] According to the model prediction results, the formula with the highest survival rate of abnormal Wickham yeast was selected as the optimal formula for subsequent experimental verification.
[0052] Table 1 shows that the response surface analysis predicted that the activity of the abnormal Wickham yeast solid preparation under the optimal protective agent compounding scheme was 84.7%.
[0053] Table 1 Response surface design scheme and experimental results
[0054]
[0055]
[0056] It can be seen from Table 2 that the F value of the model is 7.71, and the p value is 0.000239<0.05, which is significant. Among the four single factor items A, B, C, and D, sodium L-glutamate (C) has a significant effect on the survival rate of abnormal Wickham yeast (p=0.005816<0.05), and sorbitol (A), skimmed milk powder (B), and trehalose (D) have no significant effect on the survival rate of abnormal Wickham yeast.
[0057] In addition, the interaction between sorbitol (A), skim milk powder (B), sodium L-glutamate (C), and trehalose (D) had no significant effect on the freeze-dried survival rate of abnormal Wickham yeast. The p value of the lack-of-fit term was 0.059>0.05, which means that the relative size of the lack-of-fit error to the pure error is not significant. At the same time, the R 2 The value of Adeq Precision is 9.40>4, which means that the model can explain 88.53% of the response surface value variation. The coefficient of variation CV% is 2.27%<5%, which means that the model has a certain degree of credibility. Therefore, this model can be used to predict the freeze-dried survival rate of abnormal Wickham yeast under different protective agent conditions.
[0058] Table 2 Analysis of variance of response surface regression model
[0059]
[0060] Embodiment 2:
[0061] The abnormal Wickham yeast was inoculated into the culture medium NYDB, and the first activation culture was carried out at 28°C and 180rpm for 20 hours to obtain the first activation culture solution; then the first activation culture solution was transferred to a new NYDB culture medium (inoculation amount 1%), and the second activation culture was continued at 28°C and 180rpm for 20 hours to obtain the activated yeast solution; the activated yeast solution was centrifuged (4°C, 9600×g, 5min) to obtain the bacterial precipitate, and then the bacterial precipitate was centrifuged and washed with sterile water, and the washed precipitate was resuspended with sterile water to obtain a yeast suspension with a concentration of 1×10 8 cells / mL;
[0062] Obtain abnormal Wickham yeast suspension, add sorbitol, skim milk powder, sodium glutamate and trehalose therein, wherein the mass concentration of sorbitol in the obtained mixed solution is 6%, the mass concentration of skim milk powder is 10%, the mass concentration of sodium glutamate is 3%, and the mass concentration of trehalose is 5%; then pre-freeze at -80°C for 2h; when the temperature of the cold trap of the freeze dryer reaches below -50°C, put the sample in, the vacuum degree of the drying process is not higher than 10pa, and freeze-dry for a total of 48h. After the freeze-drying is completed, take out the freeze-dried powder, which is the abnormal Wickham yeast solid preparation.
[0063] Embodiment 3:
[0064] The abnormal Wickham yeast solid preparation in Example 2 was weighed, reconstituted with sterile water to obtain a bacterial suspension, and activated in a 37° C. water bath for 0.5 h, then the bacterial suspension was diluted and spread, placed in a constant temperature incubator (28° C.), the number of live bacteria on the plate was counted, and the survival rate per gram of abnormal Wickham yeast solid preparation was calculated.
[0065] Depend on Figure 2 It can be seen that the survival rate of the abnormal Wickham yeast solid preparation on the 0th day was 85.9%, which was close to the response surface prediction value, indicating that the optimal compound method obtained by this method is highly reliable and can provide a theoretical reference for the commercialization of abnormal Wickham yeast solid preparations in the future.
[0066] From the 0th day to the 90th day of storage, the survival rate of the abnormal Wickham yeast solid preparation decreased with the increase of time, which was 85.9%, 81.0%, 75.7% and 67.0%, respectively; therefore, the abnormal Wickham yeast solid preparation still maintains strong activity during long-term storage and has the potential to prepare commercial solid preparations.
[0067] Embodiment 4:
[0068] Select mature tomatoes without pests and diseases, uniform color and size, and no mechanical damage as test tomatoes, wash them with clean water, soak them in 1% sodium hypochlorite solution for 2 minutes, then take them out and rinse the sodium hypochlorite remaining on the surface of the tomatoes with running water, and randomly place them in plastic baskets to dry for later use.
[0069] Grouping: divided into control group, positive control group and yeast solid preparation group;
[0070] Control group (CK): sterile water;
[0071] Positive control group (P): Add sorbitol, skim milk powder, sodium glutamate and trehalose to NYDB medium, the mass concentration of sorbitol in the obtained mixed solution is 6%, the mass concentration of skim milk powder is 10%, the mass concentration of sodium glutamate is 3%, and the mass concentration of trehalose is 5%; then pre-freeze at -80℃ for 2h; when the temperature of the freeze dryer cold trap reaches below -50℃, put the sample in, the vacuum degree of the drying process is not higher than 10pa, and freeze-dry for a total of 48h. After the freeze-drying is completed, take out the freeze-dried powder; reconstitute it with sterile water to obtain the freeze-dried protective agent solution;
[0072] Yeast solid preparation group (Y): The abnormal Wickham yeast solid preparation in Example 2 was weighed and reconstituted with sterile water to obtain a bacterial suspension with a concentration of 1×10 8 cells / mL.
[0073] Biocontrol experiment: A wound with a diameter of 3 mm and a depth of 3 mm was made at the equator of the tomato with a sterile puncher, and 30 μL of sterile water, freeze-dried protective agent solution, and freeze-dried yeast suspension (1×10 8 cells / mL), and then 30 μL of pathogenic bacteria Alternaria suspension (1×10 5 spores / mL). Then, the culture medium was placed at 25°C to observe the incidence of tomato black spot disease.
[0074] Depend on Figure 3 It can be seen that from the 0th day to the 90th day, the rot rate of the group treated with abnormal Wickham yeast agent slowly increased from 14.1% to 24.0%, and the rot rate of the positive control treatment continued to be 100%; during the storage period, the rot rate of the group treated with abnormal Wickham yeast agent was significantly lower than that of the control group (p<0.05), indicating that the abnormal Wickham yeast agent has a stable biocontrol effect. Therefore, the abnormal Wickham yeast solid preparation still has a strong biocontrol effect during storage, significantly reducing the incidence of tomato black spot disease, and the abnormal Wickham yeast is suitable for preparation into a fungal agent, with prospects for commercial application.
[0075] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; therefore, although this specification has described the present invention in detail with reference to the above embodiments, a person skilled in the art should understand that the present invention can still be modified or replaced by equivalents; and all technical solutions and improvements thereof that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.
Claims
1. A method for preparing a solid preparation of abnormal Wickham yeast using a protective agent, characterized in that: The specific steps are as follows: (1) inoculating abnormal Wickham yeast in a culture medium NYDB, performing a first activation culture, and obtaining a first activation culture solution; then transferring the first activation culture solution to a new NYDB culture medium, and continuing a second activation culture, and obtaining an activated yeast solution; centrifuging the activated yeast solution to obtain a bacterial precipitate, and then centrifuging and washing the bacterial precipitate with sterile water, and resuspending the washed precipitate with sterile water to obtain a yeast suspension; (2) adding sorbitol, skim milk powder, sodium glutamate and trehalose to the yeast suspension obtained in step (1), stirring evenly to obtain a mixed solution, sealing the mixed solution with a sealing film and pre-freezing it to obtain a pre-frozen mixture; finally, freeze-drying the pre-frozen mixture to obtain a solid preparation of abnormal Wickham yeast; the mass concentration of sorbitol in the mixed solution is 2-10%, the mass concentration of skim milk powder is 2.5-12.5%, the mass concentration of sodium glutamate is 1.5-6%, and the mass concentration of trehalose is 2.5-7.5%; the deposit number of the abnormal Wickham yeast is: CCTCCM2018053.
2. The method for preparing a solid preparation of abnormal Wickham yeast using a protective agent according to claim 1, characterized in that: The NYDB medium described in step (1) is composed of 5 g yeast extract, 10 g glucose, 8 g beef extract, and distilled water to 1000 mL, with a natural pH value and sterilized at 115° C. for 20 min.
3. The method for preparing a solid preparation of abnormal Wickham yeast using a protective agent according to claim 1, characterized in that: The conditions for the first activation culture and the second activation culture in step (1) are: temperature 28° C., rotation speed 180-200 rpm, and time 20-24 h.
4. The method for preparing a solid preparation of abnormal Wickham yeast using a protective agent according to claim 1, characterized in that: The centrifugation conditions are: 4°C, 9600×g, 5-10 min.
5. The method for preparing a solid preparation of abnormal Wickham yeast using a protective agent according to claim 1, characterized in that: The concentration of the yeast suspension in step (1) is 1×10 8 cells / mL.
6. The method for preparing a solid preparation of abnormal Wickham yeast using a protective agent according to claim 1, characterized in that: In the mixed solution in step (2), the mass concentration of sorbitol is 6%, the mass concentration of skim milk powder is 10%, the mass concentration of sodium glutamate is 3%, and the mass concentration of trehalose is 5%.
7. The method for preparing a solid preparation of abnormal Wickham yeast using a protective agent according to claim 1, characterized in that: The pre-freezing temperature in step (2) is -80°C for 2 to 3 hours.
8. The method for preparing a solid preparation of abnormal Wickham yeast using a protective agent according to claim 1, characterized in that: The specific operation of freeze drying in step (2) is: pre-cooling the cold trap to a temperature below -50°C, then placing the pre-frozen mixture, turning on the vacuum pump to ensure that the vacuum degree during the drying process is not less than 10Pa, and the drying time is 48 hours.
9. A solid preparation of abnormal Wickham yeast prepared according to the method according to any one of claims 1 to 8.
10. Use of the abnormal Wickham yeast solid preparation according to claim 9 for controlling postharvest black spot disease of tomatoes.