CN110734866B - Active freeze-dried powder for preventing and treating loquat postharvest diseases and preparation method and application thereof - Google Patents
Active freeze-dried powder for preventing and treating loquat postharvest diseases and preparation method and application thereof Download PDFInfo
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- CN110734866B CN110734866B CN201911009490.0A CN201911009490A CN110734866B CN 110734866 B CN110734866 B CN 110734866B CN 201911009490 A CN201911009490 A CN 201911009490A CN 110734866 B CN110734866 B CN 110734866B
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- 239000003223 protective agent Substances 0.000 claims abstract description 25
- 229920001202 Inulin Polymers 0.000 claims abstract description 15
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
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- A23B7/155—Microorganisms; Enzymes; Antibiotics
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/04—Preserving or maintaining viable microorganisms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention relates to an active freeze-dried powder capable of preventing and treating postharvest diseases of loquats and a preparation method and application thereof, belonging to the technical field of biological prevention and treatment of postharvest fruits; the freeze-dried powder comprises Metschnikowia pulcherrima E1, lactose, sodium glutamate, sorbitol and inulin; the method comprises the steps of firstly, inoculating activated Meiji yeast E1 into NYDB for enrichment culture, then centrifuging a culture solution to obtain bacterial sludge, then resuspending the bacterial sludge into bacterial suspension by using a protective agent solution, and performing vacuum freeze drying to obtain freeze-dried powder after subpackaging; the yeast is suspended in sterile water and adjusted to 1 × 10 concentration 8 And (5) soaking the loquats in the bacterial liquid for 5min, then taking out the loquats or directly and uniformly spraying the loquats on the surfaces of the loquats, and airing the loquats. The active freeze-dried powder can obviously reduce the incidence rate and the disease spot diameter of the loquat fruit gray leaf spot; but also can obviously reduce the rotting index of the loquat fruit; the weight loss rate of the fruits and the permeability of cell membranes of the peels are inhibited; and delays the reduction of soluble solids, titratable acid, soluble protein and Vc of the loquat fruits.
Description
Technical Field
The invention relates to active freeze-dried powder for preventing and treating postharvest diseases of loquats as well as a preparation method and application thereof, belonging to the technical field of biological prevention and treatment of postharvest fruits.
Background
Loquat (Eriobotrya japonica, iandl) belongs to Rosaceae and Eriobotrya (Eriobotrya), is a unique fruit in subtropical regions of China, has soft and juicy pulp, is sour and sweet, can play a role in health care such as clearing heat, moistening lung and the like, and is popular with consumers. The loquat peel is thin and is easy to rot caused by mechanical damage in the process of picking and transportation. Research shows that the loquat rot after picking is mainly caused by infectious diseases such as anthracnose, gray spot and the like. The loquat leaf spot can damage branches, leaves, flowers and fruits of the loquat, the loquat fruits can be damaged from young fruits to mature stages, and the diseases are caused by the pestalotiopsis fungi.
For a long time, the control of the loquat postharvest diseases mainly depends on physical methods and chemical methods. The common physical methods mainly comprise heat treatment, low-temperature storage, low-pressure storage, modified atmosphere storage, ozone treatment, irradiation treatment and the like. But the operation is complex and the cost is high, so the method is not widely applied to production practice. The common chemical method is to use chemical bactericide for treatment, and the chemical bactericide is widely applied due to the characteristics of high efficiency, convenient operation, low cost and the like, but environmental pollution is easily caused by overuse of chemical agents. In recent years, with the economic development and the improvement of consumption level, people have strengthened consciousness on food safety and environmental protection, so a new method for safely and efficiently preventing and treating the postharvest diseases of the loquats, which can replace chemical bactericides, is urgently sought.
The biological control method has the characteristics of high efficiency, safety, energy conservation, environmental protection and the like, and is concerned by more and more researchers, wherein antagonistic saccharomycetes become main research objects due to the advantages of simple nutritional requirement, rapid propagation, strong stress resistance and the like. Therefore, the antagonistic yeast is used for replacing chemical bactericide, and the application prospect of preventing and treating the postharvest diseases of the fruits is wide. At present, many researches on applying antagonistic yeast to the postharvest diseases of fruits and vegetables are carried out at home and abroad, and few researches on the gray leaf spot of the main disease of the picked loquats are carried out in the postharvest disease control of the loquats, so that the research on applying the antagonistic yeast to the control of the gray leaf spot of the picked loquats has important value in searching for an effective preservation method of the picked loquats.
The final aim of researching the biocontrol yeast is that the biocontrol preparation can be applied to actual production. The biocontrol agent should ensure that the viable count and the biocontrol effect in the finished product are high, so the biocontrol agent has certain research significance on the process optimization of the biocontrol agent. Vacuum freeze drying is carried out in low temperature and low oxygen environment, most biological reactions are stopped, and water in the treatment process is directly sublimated in a solid state, so that the original structure and shape of the material can be protected to the maximum extent, a dry product with low water content is obtained, and the storage time is long. The ice crystals are easy to cause mechanical damage to cells, cause denaturation and inactivation of proteins in the cells, reduce the number of viable bacteria and the like, so that a protective agent needs to be added to reduce the damage, various research results on the types, concentrations and proportions of the protective agent in screening of different strains at home and abroad are already provided, the problem of low survival rate of the strains in freeze-dried products still exists, and the reports on freeze-dried products of the loquat biocontrol yeast are less.
Disclosure of Invention
The invention aims to provide active freeze-dried powder for preventing and treating diseases of picked loquats and a preparation method and application thereof, so as to effectively control the occurrence of grey spot and rot phenomena of the picked loquats, reduce loss caused by the rot of the picked loquats and reduce pollution to the environment caused by chemical prevention and treatment.
The technical scheme adopted by the invention
The invention firstly provides a Meiji yeast E1 (Metschnikowia pulcherrima E1), which is stored in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 15 months, and the storage address is Wuhan university in Wuhan, china, the storage number is CCTCC NO: M2019256, the suggested classification name is as follows: the yeast Saccharomyces meijimei E1 Metschnikowia pulcherrima E1.
The invention also provides active freeze-dried powder for preventing and treating the loquat postharvest diseases, and the formula of the freeze-dried powder is as follows: 2-4 g/100mL of meiji yeast E1 (Metschnikowia pulcherrima E1), 2-4 g/100mL of lactose, 4-8 g/100mL of sodium glutamate, 0.5-2 g/100mL of sorbitol and 4-8 g/100mL of inulin.
Preferably, the formula of the active freeze-dried powder is as follows: meiji Meiqi yeast E1 (Metschnikowia pulcherrima E1) bacterial paste 2g/100mL, lactose 3.13g/100mL, sodium glutamate 5.97g/100mL, sorbitol 0.94g/100mL and inulin 5.95g/100mL.
The invention also provides a preparation method of the active freeze-dried powder for preventing and treating the loquat postharvest diseases, which comprises the following steps:
(1) Activation of the strain: selecting the Meiji yeast E1 (Metschnikowia pulcherrima E1) preserved on the inclined plane, streaking and inoculating the Meiji yeast E1 into an NYDA culture medium, and culturing at the constant temperature of 28 ℃ for 2-3 days;
(2) Enrichment culture of yeast: inoculating activated Meijimeiqi yeast E1 (Metschnikowia pulcherrima E1) into NYDB culture medium, and shake-culturing at 28 deg.C for 18-24h to obtain seed solution; then inoculating the seed solution into an NYDB culture medium with the inoculation amount of 5%, and carrying out shake-flask culture at 28 ℃ for 24-48 h to obtain a culture solution of a strain Metschnikowia pulcherrima E1;
(3) Preparation of bacterial sludge: taking the culture solution of cultured strain Meiji yeast E1 (Metschnikowia pulcherrima E1), centrifuging at 4 deg.C for 6000r/min for 10min, and pouring out the supernatant to obtain bacterial sludge;
(4) Adding a protective agent and subpackaging: resuspending the bacterial sludge with a freeze-drying protective agent solution, uniformly mixing to prepare a yeast suspension, balancing for 30min at 28 ℃,180r/min, and subpackaging in 50ml centrifuge tubes;
(5) Pre-freezing: sealing the subpackaged samples by using a preservative film, putting the samples into a refrigerator at the temperature of-80 ℃, and pre-freezing for 3-4 hours;
(6) Vacuum freeze drying: performing preservative film pricking on the pre-frozen sample, then quickly transferring the sample into a vacuum freeze dryer, and freeze-drying for 40 hours;
(7) And (4) sealing and storing: the finished samples were freeze-dried and stored in a sealed manner with a wrap film.
Wherein the NYDA culture medium in the step (1) comprises the following components: 8g of beef extract, 5g of yeast extract, 10g of glucose and 20g of agar powder, supplementing the beef extract with distilled water to 1000mL, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20min.
Wherein the NYDB culture medium in the step (2) comprises the following components: 8g of beef extract, 5g of yeast extract and 10g of glucose, supplementing to 1000mL by distilled water, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20min.
Wherein the concentration of each protective agent in the freeze-drying protective agent solution in the step (4) is as follows: 3.13g/100mL of lactose, 5.97g/100mL of sodium glutamate, 0.94g/100mL of sorbitol and 5.95g/100mL of inulin; the balance of water.
The invention also provides an application method of the active freeze-dried powder for preventing and treating the loquat postharvest diseases, which comprises the following steps: when in use, the active freeze-dried powder is rehydrated and activated, and the concentration of the yeast is adjusted to 1 × 10 8 And (3) soaking the loquat fruits in the bacterial liquid for 5min, then taking out the loquat fruits, or uniformly spraying the bacterial liquid on the surfaces of the loquat fruits, naturally drying the loquat fruits, then placing the loquat fruits in a plastic basket, sealing the loquat fruits by using a preservative film, and storing the loquat fruits at room temperature.
According to the invention, 3.13g/100mL of lactose, 5.97g/100mL of sodium glutamate, 0.94g/100mL of sorbitol and 5.95g/100mL of inulin are used as yeast protective agents, and the biocontrol preparation with higher activity is prepared by a freeze drying method. Test results show that the survival rate of the yeast strain after freeze-drying of the protective agent-added bacterial liquid is 94.43%, which is significantly higher than that of the yeast strain of a control group without the protective agent, by 6.04%. The active freeze-dried powder is suitable for preventing and treating the picked gray spot disease and storing and preserving the loquat after rehydration and activation, and can obviously reduce the rotting index of the loquat fruit; the weight loss rate of the fruits and the permeability of cell membranes of the peels are inhibited; and the content reduction of soluble solids, titratable acid, soluble protein and Vc of the loquat fruits is delayed.
The beneficial effects of the invention are described as follows:
according to the invention, through single-factor screening and response surface optimization, the result shows that the freeze-dried powder prepared by taking lactose, sodium glutamate, sorbitol and inulin as freeze-drying protective agents has high survival rate of yeast, namely 94.43%, and the product has a longer storage period and good rehydration performance.
According to the active freeze-dried powder prepared by the preparation method provided by the invention, the survival rate of E1 yeast cells in the freeze-dried powder can be obviously improved by adding the freeze-drying protective agent, and the preparation method can effectively improve the activity of freeze-dried yeast cells compared with a preparation method without adding the protective agent;
the active freeze-dried powder provided by the invention has a good biocontrol effect, the incidence rate and the disease spot diameter of the loquat fruit gray spot disease are obviously lower than those of a control group by yeast suspension treatment after the active freeze-dried powder is rehydrated, and significant differences exist, which indicates that the active freeze-dried powder can effectively inhibit the occurrence of the gray spot disease of the picked loquat fruit after rehydration.
The active freeze-dried powder provided by the invention has a certain fresh-keeping effect, and can obviously reduce the rotting index of loquat fruits; the weight loss rate of the fruits and the permeability of cell membranes of the peels are inhibited; and delays the reduction of soluble solids, titratable acid, soluble protein and Vc of the loquat fruits.
Drawings
FIG. 1 shows the morphological characteristics of the yeast Metronikowia pulcherrima E1; in the figure, A: colony morphology on NYDA; b: cell morphology.
FIG. 2 shows the effect of E1 active lyophilized powder of Metschnikowia pulcherrima on the rotting rate of loquat fruit gray spot. Note: the control group was treated with distilled water; the experimental group is 1X 10 8 Treating cells/mL of strain E1 freeze-dried powder bacterial suspension.
FIG. 3 shows the effect of E1 active lyophilized powder of Meiji yeast (Metschnikowia pulcherrima) on the quality of harvested loquat after storage; the rotten state of the loquat fruit is shown in the figure.
FIG. 4 shows the effect of E1 active lyophilized powder of Meiji yeast (Metschnikowia pulcherrima) on the quality of harvested loquat after storage; the graph shows the change of the weight loss ratio of the loquat fruits.
FIG. 5 shows the effect of E1 active lyophilized powder of Meiji yeast (Metschnikowia pulcherrima) on the quality of harvested loquat after storage; the graph shows the change of soluble solid content of loquat fruit.
FIG. 6 shows the effect of E1 active lyophilized powder of Meiji yeast (Metschnikowia pulcherrima) on the quality of loquat stored after harvest; the graph shows the change of titratable acid content of loquat fruit.
FIG. 7 shows the effect of E1 active lyophilized powder of Meiji yeast (Metschnikowia pulcherrima) on the quality of harvested loquat after storage; the graph shows the change of vitamin C content in loquat fruit.
FIG. 8 shows the effect of E1 active lyophilized powder of Saccharomyces meisterii (Metschnikowia pulcherrima) on the quality of loquat after harvest; the graph shows the change of soluble protein content of loquat fruit.
FIG. 9 shows the effect of E1 active lyophilized powder of Metronikowia pulcherrima on the storage quality of loquat after harvest; the figure shows the change of the cell membrane permeability of loquat pericarp.
FIG. 10 is a tree of 5.8S rDNA-ITS sequences from E1 of M.meiji (Metschnikowia pulcherrima).
Detailed Description
The present invention will be described in more detail by the following examples, which are illustrative only, and the present invention is not limited by these examples.
Example 1: preparation of active freeze-dried powder for preventing and treating loquat postharvest diseases
Activation of the strain: selecting the Meiji yeast (Metschnikowia pulcherrima) E1 preserved on the inclined plane, streaking and inoculating the Meiji yeast to an NYDA culture medium, and culturing at the constant temperature of 28 ℃ for 2-3 days; wherein the NYDA culture medium comprises the following components: 8g of beef extract, 5g of yeast extract, 10g of glucose and 20g of agar powder, supplementing the beef extract with distilled water to 1000mL, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20min.
As for morphological characteristics of Metschnikowia pulcherrima E1, as shown in FIG. 1, after culturing for 2 days at 28 ℃ on NYDA medium, the colony is round, small, opaque, cream, neat in edge, moist and smooth in surface, easy to pick up, produces red pigment, and has reddish brown on the reverse side. The cells are observed by a microscope to be spherical or nearly spherical, and are germinated on one side or multiple sides.
The strain is stored in China Center for Type Culture Collection (CCTCC) 4-15.2019, the preservation address is Wuhan university in Wuhan, china, the preservation number is CCTCC NO: M2019256, and the suggested classification is named as Meiji Meiqi yeast E1 Metschnikowia pulcherrima E1; the evolved tree constructed from the 5.8S rDNA-ITS sequence of E1 of M.meiji (Metschnikowia pulcherrima) is shown in FIG. 10.
Enrichment culture of yeast: inoculating the activated strain E1 into an NYDB culture medium, and performing shake-flask culture at 28 ℃ for 18-24h to obtain a seed solution; then inoculating the seed solution into an NYDB culture medium in an inoculation amount of 5%, and performing shake-flask culture at 28 ℃ for 24-48 h to obtain a culture solution of the strain E1; the NYDB culture medium consists of: 8g of beef extract, 5g of yeast extract and 10g of glucose, supplementing to 1000mL by distilled water, naturally adjusting pH, and sterilizing at 121 ℃ for 20min.
Preparation of bacterial sludge: centrifuging the cultured yeast culture solution at 4 deg.C and 6000r/min for 10min, and removing supernatant to obtain bacterial sludge;
selection of centrifugation parameters: before centrifugation, the number of yeast cells in the bacterial liquid is calculated by adopting a dilution coating method, and then the bacterial liquid is centrifuged for 10 and 20min according to different centrifugation parameters (the rpm of 4000, 6000 and 8000 is respectively centrifuged). After centrifugation, the supernatant was decanted, and the pellet was resuspended in an equal volume of saline and the smear counts diluted. The optimum centrifugation condition was determined by calculating the centrifugation yield according to the formula (centrifugation yield = number of yeast cells before centrifugation/number of yeast cells after centrifugation × 100%), and finally the optimum centrifugation condition was determined for 10min of centrifugation at 6000rpm (the centrifugation yield was the highest, 89.40%).
Adding a protective agent and subpackaging: the bacterial sludge is resuspended by using a freeze-drying protective agent solution and mixed evenly to prepare a yeast suspension, so that the concentration of each component is 2g/100mL of Meiji yeast (Metschnikowia pulcherrima) E1 bacterial sludge, 3.13g/100mL of lactose, 5.97g/100mL of sodium glutamate, 0.94g/100mL of sorbitol and 5.95g/100mL of inulin. Balancing the mixed bacteria liquid at 28 ℃ for 30min at a speed of 180r/min, and subpackaging in a 50ml centrifuge tube;
pre-freezing: sealing the subpackaged samples by using a preservative film, putting the samples into a refrigerator at the temperature of-80 ℃, and pre-freezing for 3-4 hours;
vacuum freeze drying: performing preservative film perforation on the pre-frozen sample, then quickly transferring the sample into a vacuum freeze dryer, opening a vacuum pump, and performing vacuum freeze drying for 40 hours;
and (4) sealing and storing: the finished samples were freeze-dried and stored in a sealed manner with a wrap film.
Example 2: optimization of each protective agent component in active freeze-dried powder for preventing and treating loquat postharvest diseases
In the embodiment, the components and the content of the freeze-dried powder protective agent are optimized, and the specific method comprises the following steps:
1. single factor test
By consulting documents, 8 protective agents are selected to prepare protective agent solutions with different mass concentrations, then the yeast mud is resuspended to prepare bacterial suspension, freeze drying is carried out, samples before freeze drying are diluted, coated and counted, the samples after freeze drying are added with physiological saline with the same volume for rehydration and activation, then dilution, coating and counting are carried out in the same way, and the freeze drying survival rate of the strains is calculated (survival rate = number of live bacteria before freeze drying/number of live bacteria before freeze drying x 100%). The types and concentrations of the protective agents and their effects on the freeze-drying survival rate of yeast are shown in tables 1 and 2.
TABLE 1 Freeze-drying powder protectant types and concentrations
TABLE 2 Effect of different protectants on Yeast Freeze-drying survival Rate
2. PB test design and results
Two levels of each protective agent with good protection effect are selected according to a single-factor experiment, a Design-expert 8.0.6 software is used for designing a PB test, and the significance of 8 factors is compared according to the survival rate of yeast. 12 groups of protectant solutions were prepared according to the ratios in table 3. Wherein 1 and-1 respectively represent Gao Dishui flat of each factor, and-1 and 1 of the table correspond to each factor, namely 6 and 8g/100mL of sucrose, 13 and 18g/100mL of skim milk powder, 3 and 5g/100mL of lactose, 6 and 8g/100mL of glucose, 4 and 6g/100mL of sodium glutamate, 1.5 and 2.5g/100mL of polyethylene glycol, 1 and 2g/100mL of sorbitol and 4 and 6g/100mL of inulin. The PB test results and analysis are shown in tables 3 and 4.
TABLE 3 PB test design and results
TABLE 4 evaluation of the Effect of the test design 4 PB
Model Pr > F was less than 0.05, indicating that the experimental model is significant. The 'Pr > F' is less than 0.05, which indicates that the factors are significant, and the significance sequence of the factors is C-lactose > E-sodium glutamate > H-inulin > G-sorbitol > A-sucrose > D-glucose > B-skim milk powder > F-polyethylene glycol, wherein A, B, E, F and H have significant positive effect, the concentration is increased, C, D and G have significant negative effect, and the concentration is decreased. The factors in the first 4 positions of the significance sequence were selected as protectant components, namely lactose, sodium glutamate, sorbitol and inulin.
3. Steepest climbing experiment
The steepest hill climbing test was determined according to the PB test analysis results to study the corresponding interval of the maximum response value, and the test design and results are shown in table 5.
TABLE 5 steepest climb test design and results
4. Response surface optimization design results and analysis
According to the steepest climbing test, the test condition No. 4 with the largest survival rate is selected as the central point to carry out the central combination test (CCD), and the design and the result of the central combination test are shown in Table 6.
TABLE 6 center combination test design and results
The survival rate of the secondary fitting equation obtained according to the encoding factors is 94.66+0.99 a-0.64 b-0.79 c-0.037 d +0.56 a b-1.49 a c-1.65 a d-0.52 b c-0.24 b d-0.82 c-2.90 a 2 -2.62*B 2 -1.90*C 2 -1.31*D 2 (wherein A is lactose, B is sodium glutamate, C is sorbitol, and D is inulin), the coefficient of determination R of the equation 2 =94.79%, i.e. 94.79% of the test data can be interpreted, the mismatching term (p = 0.1165) is not significant, and the data fitting effect is good. The combination shows that the model has good fitting effect and can be predicted by using a response value. The survival rate is the highest condition, the optimal solution is that the mass concentrations of the four protective agents are respectively 3.13g/100mL of lactose, 5.97g/100mL of sodium glutamate, 0.94g/100mL of sorbitol and 5.95g/100mL of inulin, and the survival rate is predicted to be 94.93%. And (4) carrying out a verification test by using the optimal solution, wherein the result shows that the freeze-drying survival rate accords with the expectation.
Example 4: control effect of active freeze-dried powder for preventing and treating loquat postharvest diseases on loquat gray leaf spot
Resuspending the lyophilized powder with sterile water and adjusting the concentration to 1X 10 8 cells/mL for use; inoculating Eriobotrya japonica Botrytis pathogenic bacteria Wei Simei Pestalotiopsis victoriae P2 on PDA culture medium, culturing at 28 deg.C for 7-10 days, and preparing into 1 × 10 with sterile water 5 Conidium suspension of spores/mL for use. Wherein Wei Simei Pestalotiopsis vismaie P2 is selected by the laboratory, and has been preserved in China Center for Type Culture Collection (CCTCC) in 2019, 4 and 15 months, with the preservation address of Wuhan university in China and the preservation number of CCTCC NO: M2019257; the proposed classification was named Wei Simei Pestalotiopsis pessima P2 (Pestalotiopsis virxiae P2).
Selecting loquat fruits with uniform size and no mechanical damage, washing the loquat fruits with clear water, soaking and sterilizing the loquat fruits with 0.1% sodium hypochlorite solution for 10min, washing the loquat fruits with clear water for two or three times, and naturally airing the loquat fruits. Perforating a hole (3 mm × 3 mm) at equator of fructus Eriobotryae with sterilized perforator, and adding 20 μ L of 1 × 10 8 cfu/mL freeze-dried powder bacteriumStanding for 2 hr, adding 20 μ L of 1 × 10 5 The Wei Simei pestalotiopsis conidium suspension of spores/mL, and sterile water is used for replacing freeze-dried powder bacteria liquid as a control. Putting the loquat fruits in a plastic basket in order, sealing the loquat fruits by using a preservative film, storing the loquat fruits at room temperature, observing and recording the rotting rate and the disease spot diameter of the loquat fruits after 5 days, and evaluating the inhibition effect of the meiji yeast E1 active freeze-dried powder on the gray leaf spot of the loquat.
The formula for the incidence of disease is as follows:
incidence (%) of disease = fruit/total number of fruits affected × 100%
The test result is shown in figure 2, in the 5d control group, the loquat fruit incidence rate is only 23.60% in the E1 yeast active freeze-dried powder treatment group, which is significantly lower than the control group, and the lesion diameter is also significantly smaller than the control group (P < 0.05). Therefore, the E1 saccharomycete active freeze-dried powder can effectively retain the cell activity and the biocontrol effect of the saccharomycete E1, and the freeze-dried powder can effectively control the post-harvest grey speck disease of the loquat caused by Wei Simei pestalotiopsis sp.
Example 5: influence of active freeze-dried powder for preventing and treating loquat postharvest diseases on loquat postharvest storage quality
Resuspending the lyophilized powder with sterile water and adjusting the concentration to 1X 10 8 cells/mL, ready for use. And (3) soaking: soaking the loquat fruit in the bacterial liquid for 5min, taking out and naturally drying; and (3) spraying group: spraying the loquat fruits with the bacterial liquid, and naturally drying; control group: soaking the loquat fruit in sterile water for 5min, taking out and naturally drying. Putting the groups of loquat fruits into a plastic basket, sealing the plastic basket by using a preservative film, storing the plastic basket at room temperature, measuring the rot index and related indexes of fruit quality of the fruits every 3 days, measuring the rot index and related indexes of fruit quality for 18 days totally, and repeating the treatment for 3 times. The method for measuring the storage quality of the loquat fruits by using the E1 saccharomycete active freeze-dried powder comprises the following steps:
(1) Rot index:
dividing the loquat fruit according to the rotten condition into 5 grades, 0 grades: no rot exists; level 1: the rotten area is less than 25% of the total area of the fruit; and 2, stage: the rotten area accounts for 25 to 50 percent of the total area of the fruits; and 3, level: the rotten area accounts for 50 to 75 percent of the total area of the fruits; and 4, stage 4: the rotten area is more than 75% of the total area of the fruit, and the rotten index is calculated according to the following formula:
rot index = Σ (level × number of fruits of this level)/(total number of fruits × highest level) × 100%
The rotting condition of the loquat fruits is shown in figure 3, the weight loss rate of the fruits of each treatment group is increased along with the prolonging of the storage days, the rotting indexes of the loquats of the soaked and sprayed treatment groups are obviously lower than those of the aseptic water treatment group, from 3d, the rotting indexes of the loquats soaked and sprayed by the active freeze-dried powder are obviously lower than those of the control group, when the loquats are stored to 18d, the rotting indexes of the two treatment groups are respectively 11.94% and 12.77%, and are obviously lower than (P < 0.05) 27.22% of the control group, and the active freeze-dried powder can effectively inhibit the rotting of the loquat fruits in the storage process of the loquats.
(2) Weight loss rate:
weighing the initial weight and the sampling weight of the loquat fruits, and calculating the weight loss ratio according to the following formula:
weight loss rate = (weight before treatment-weight after storage)/weight before treatment × 100%
The change of the weight loss rate of the loquat fruits during storage is shown in fig. 4, the weight loss rate of the fruits of each treatment group is increased along with the prolonging of the storage days, and the weight loss rate of the loquat fruits of the treatment group which is soaked in the active freeze-dried powder and sprayed 12 days before storage is not obviously different from that of the control group, but is basically lower than that of the control group; after the loquat is stored for 15 days, the weight loss rate of the soaking treatment group and the spraying treatment group is obviously lower than that of the control group, which shows that the active freeze-dried powder can slow down the loss of water and the like in the storage period of the loquat and has positive protection effect on the quality of the loquat.
(3) Soluble solid content:
the results were averaged for 6 loquat fruits measured using a hand-held refractometer.
The change of the soluble solid content of the loquat fruits during storage is shown in fig. 5, the soluble solid content of the fruits of each treatment group is reduced along with the prolonging of the storage days, the soluble solid content of the loquat fruits of the treatment group soaked by the active freeze-dried powder and sprayed by the active freeze-dried powder is basically higher than that of the control group, and the significant difference exists between the 15 th day and the 18 th day, which shows that the active freeze-dried powder plays a certain role in protecting the soluble solid content of the loquat fruits, and the effect of the spraying group is more obvious.
(4) Titratable acid content:
selecting 6 loquat fruits randomly by an acid-base titration method, weighing 10g pulp samples, adding 40mL distilled water for homogenate, cleaning the pulp samples with an appropriate amount of distilled water into a 100mL volumetric flask, leaching for 30min in a constant-temperature water bath at 80 ℃, cooling, fixing the volume to a scale mark, and filtering. And (3) putting 20mL of filtrate into a 100mL conical flask, adding 2-4 drops of phenolphthalein indicator, titrating with 0.1mol/L NaOH solution until the solution is reddish, and taking a titration end point within 30s without fading, recording the volume of used NaOH, and expressing the result as the percentage of malic acid, wherein each treatment is repeated for 3 times.
The change of the titratable acid content of the loquat fruits during storage is shown in fig. 6, the titratable acid content of the fruits of each treatment group is reduced along with the prolonging of the storage days, the titratable acid content of the loquat fruits of the treatment group soaked and sprayed by the active freeze-dried powder is obviously higher than that of the control group at the 6 th day, and the titratable acid content of the loquat fruits of the treatment group is close to that of the control group at the later storage period but basically higher than that of the control group, so that the active freeze-dried powder plays a certain role in protecting the titratable acid content of the loquat fruits.
(5) The content of vitamin C:
6 loquat fruits are randomly selected by adopting an ultraviolet rapid determination method, 5g of pulp sample is weighed, 1% hydrochloric acid of 10mL is added for homogenate, and a 25ml volumetric flask is used for volume fixing. Centrifuge at 5000r/min for 15min and supernatant was used for assay. 0.4mL of the supernatant was taken, and added to a 10mL graduated tube containing 0.8mL of 10% HCl, diluted to the graduation with water, shaken, and the absorbance of the solution was measured at 243nm with a blank correction of distilled water. Separately taking 0.4mL of supernatant, adding 4mL of distilled water and 1.6mL of 1mol/L NaOH to a 10mL graduated test tube, shaking uniformly, standing for 15min, adding 1.6mL of 10% HCl, mixing uniformly, diluting to a graduated scale by adding distilled water, correcting blank by using distilled water, and measuring the absorbance value at 243 nm. From the difference in the above absorbance values, the Vc standard curve was looked up to calculate the Vc content in the sample, and each treatment was repeated 3 times.
The change of the vitamin C content of the loquat fruits during storage is shown in figure 7, the vitamin C content of the fruits of each treatment group is reduced along with the prolonging of the storage days, the vitamin C content of the loquat fruits of the treatment group soaked and sprayed by the active freeze-dried powder is higher than that of the control group, and the significant difference exists from the 9 th day. The active freeze-dried powder plays a certain positive role in maintaining the vitamin C content of the loquat fruits.
(6) Soluble protein:
selecting 3 loquat fruits randomly by adopting a Coomassie brilliant blue method, weighing 1g of pulp sample, adding 8ml of distilled water, grinding into homogenate, centrifuging at 8000r/min for 10min, and using the supernatant for determination. Taking 0.1ml of supernatant, adding 0.9ml of distilled water and 2ml of Coomassie brilliant blue G-250 reagent, mixing uniformly in a test tube with a plug, standing for 2min, measuring absorbance value at 595nm, checking protein content by a standard curve, and repeating each treatment for 3 times.
The change of the soluble protein content of the loquat fruits during storage is shown in fig. 8, the soluble protein content of the fruits of each treatment group is reduced along with the prolonging of the storage days, and the soluble protein content of the loquat fruits of the groups soaked and sprayed by the active freeze-dried powder is obviously higher than that of the control group. The active freeze-dried powder plays a certain positive role in maintaining the content of soluble protein in the loquat fruits.
(7) Relative conductivity:
accurately weighing 0.5g pericarp, placing into centrifuge tube, washing with deionized water for 3 times, adding 20mL deionized water, shaking and soaking on shaking table for 20min, and measuring initial conductivity L 0 Boiling for 10min, cooling, and measuring the conductivity L 1 In L of 1 /L 0 X 100% represents the relative conductivity, and each treatment was repeated 3 times.
The relative conductivity is an important index for evaluating the integrity of cell membranes of the pericarps, the greater the relative conductivity, the greater the cell membrane permeability, the greater the change of the relative conductivity of the pericarps during the storage period of the loquat fruits is as shown in fig. 9, the relative conductivity of the fruits of each treatment group is increased along with the extension of the storage days, the relative conductivity of the pericarps of the loquats soaked by the active freeze-dried powder and sprayed by the active freeze-dried powder is obviously lower than that of the control group, and the soaking group has more obvious effect than the spraying group. The active freeze-dried powder has a certain protection effect on a cell membrane system of loquat pericarp.
The influence of E1 active freeze-dried powder of Metschnikowia pulcherrima on the storage quality of loquat after harvest is shown in figures 3-9, and the results show that the active freeze-dried powder biocontrol agent prepared by the preparation method can obviously reduce the rot index of loquat after harvest and inhibit the weight loss rate of fruits and the increase of cell membrane permeability of pericarp; and delays the reduction of soluble solids, titratable acid, soluble protein and Vc of the loquat fruits. Therefore, the E1 active freeze-dried powder biocontrol agent of the meiji yeast (Metschnikowia pulcherrima) can keep higher activity and biocontrol efficiency of the yeast, has positive effect on the aspects of storage and preservation of the picked loquats, and is worthy of further research and popularization.
Claims (7)
1. An active freeze-dried powder for preventing and treating loquat postharvest diseases, which comprises: 2-4 g/100mL of E1 bacterial mud of Meiji yeast (Metschnikowia pulcherrima), 2-4 g/100mL of lactose, 4-8 g/100mL of sodium glutamate, 0.5-2 g/100mL of sorbitol and 4-8 g/100mL of inulin; the Meiji yeast E1 (Metschnikowia pulcherrima E1) has the preservation number of CCTCC NO: M2019256.
2. The active freeze-dried powder for controlling loquat postharvest diseases according to claim 1, further comprising: meiji Meiqi yeast (Metschnikowia pulcherrima) E1 bacterial mud 2g/100mL, lactose 3.13g/100mL, sodium glutamate 5.97g/100mL, sorbitol 0.94g/100mL and inulin 5.95g/100mL.
3. The preparation method of the active freeze-dried powder for controlling the loquat postharvest diseases as claimed in claim 1, is characterized by comprising the following steps:
(1) Activation of the strain: selecting and streaking the E1 of the Meiji yeast (Metschnikowia pulcherrima) preserved on the inclined plane into an NYDA culture medium, and culturing at the constant temperature of 28 ℃ for 2-3 d;
(2) Enrichment culture of yeast: inoculating the activated strain E1 into an NYDB culture medium, and performing shake-flask culture at 28 ℃ for 18-24h to obtain a seed solution; then inoculating the seed solution into an NYDB culture medium in an inoculation amount of 5%, and performing shake-flask culture at 28 ℃ for 24-48 h to obtain a culture solution of the strain E1;
(3) Preparation of bacterial sludge: centrifuging the cultured yeast culture solution at 4 deg.C and 6000r/min for 10min, and removing supernatant to obtain bacterial sludge;
(4) Adding a protective agent and subpackaging: resuspending the bacterial sludge with a freeze-drying protective agent solution, uniformly mixing to prepare a yeast suspension, balancing at 28 ℃ at 180r/min for 30min, and subpackaging in a 50ml centrifuge tube;
(5) Pre-freezing: sealing the subpackaged samples by using a preservative film, putting the samples into a refrigerator at the temperature of-80 ℃, and pre-freezing for 3-4 hours;
(6) Vacuum freeze drying: performing preservative film perforation on the pre-frozen sample, then quickly transferring the sample into a vacuum freeze dryer, and freeze-drying for 40h;
(7) And (4) sealing and storing: the freeze-dried sample is sealed and stored by a preservative film;
wherein the NYDA culture medium in the step (1) comprises the following components: 5g of yeast extract, 8g of beef extract, 10g of glucose and 20g of agar powder, supplementing the yeast extract, the beef extract, the glucose and the agar powder to 1000mL by using distilled water, naturally adjusting the pH value, and sterilizing at 121 ℃ for 20min;
wherein the NYDB culture medium in the step (2) comprises the following components: 5g of yeast extract, 8g of beef extract and 10g of glucose, supplementing to 1000mL by distilled water, naturally adjusting pH, and sterilizing at 121 ℃ for 20min.
4. The preparation method of active freeze-dried powder for preventing and treating loquat postharvest diseases according to claim 3, wherein the concentration of the saccharomycete mud in the step (4) is 2-4 g/100mL.
5. The preparation method of active freeze-dried powder for controlling loquat postharvest diseases according to claim 3, characterized in that the concentration of the yeast bacterial sludge in step (4) is 2g/100mL.
6. The preparation method of active freeze-dried powder for controlling loquat postharvest diseases according to claim 3, wherein the concentration of each protective agent in the protective agent solution in the step (4) is as follows: lactose 3.13g/100mL, sodium glutamate 5.97g/100mL, sorbitol 0.94g/100mL and inulin 5.95g/100mL.
7. The application of the active freeze-dried powder for preventing and treating loquat postharvest diseases according to claim 1, which is used for preventing and treating loquat postharvest diseases.
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