Yeast dry powder as well as preparation method and application thereof

Abstract

The invention discloses yeast dry powder as well as a preparation method and application thereof, and belongs to the technical field of microorganisms. The method comprises the following steps: selecting a yeast strain, performing high-density fermentation, centrifuging for 15 minutes at the speed of 10000 r / min, and collecting yeast thalli to obtain yeast paste; weighing a yeast protective agent, adding distilled water, sterilizing at 121 DEG C for 15 minutes, fully dissolving, cooling to 50 DEG C, and fully and uniformly mixing with the yeast paste to obtain a mixture; and producing biomass particles from the obtained mixture by using a manual sterile extruder, dehydrating by using a fluidized bed dryer, and cooling to room temperature to obtain the yeast dry powder. The invention provides yeast dry powder as well as a preparation method and application thereof. The yeast dry powder can effectively inhibit gray mold, maintain grape fruit quality, enhance fruit hardness and delay fruit ripening.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to dry yeast powder and a preparation method and application thereof. Background Art

[0002] Grapes are one of the four major fruits in China and are widely grown around the world. Post-harvest preservation technology is of great significance for extending shelf life and ensuring fruit quality. During harvesting, transportation and storage, grapes are susceptible to mechanical damage, leading to problems such as browning of the stems, softening of the flesh, rotting and mildew. Because grapes contain high levels of water and sugar and have thin skins, they are susceptible to pathogens, mainly six pathogens, including Penicillium ( Penicillium spp. )、Botrytis cinerea( Botrytis cinerea )、Aspergillus ( Aspergillus spp. )、Soft rot fungi( Rhizopus spp. )、Black spot fungus( Cladosporium spp. ) and Alternaria alternata ( Alternaria spp. ). Therefore, it is of great significance to seek effective preservation methods to inhibit the occurrence of postharvest diseases.

[0003] At present, grape preservation technology can be roughly divided into three categories: physical preservation, chemical preservation and biological preservation. Physical preservation methods include refrigeration, controlled atmosphere storage, etc., while chemical preservation methods involve the use of chemical preservatives and bactericides. Biological preservation methods include plant extract preservation and microbial preservation. The latter inoculates harmless microorganisms on the surface of fruits and vegetables and uses competitive antagonism between different microorganisms to inhibit the growth of harmful microorganisms. In recent years, the use of yeast to inhibit the production of pathogens and preserve fruits and vegetables has attracted widespread attention, because yeast does not produce antibiotics or other toxic secondary metabolites in the process of resisting pathogens. Although yeast can inhibit the growth of pathogens during grape storage, free yeast is not conducive to transportation and storage, has a short survival time, and with the extension of storage time, the preservation effect gradually weakens, limiting its application in the food field. Summary of the invention

[0004] The present invention aims to provide a dry yeast powder and a preparation method and application thereof. The dry yeast powder can effectively inhibit gray mold, maintain the quality of grape fruit, enhance the hardness of the fruit, and delay the ripening of the fruit.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for preparing dry yeast powder comprises the following steps: S1. Select yeast strains, perform high-density fermentation, centrifuge at 10,000 r / min for 15 min, collect yeast cells, and obtain yeast mud; S2, weigh the yeast protectant, add distilled water, sterilize at 121°C for 15 min, fully dissolve, wait until the temperature drops to 50°C, and mix thoroughly with the yeast paste prepared in S1 to obtain a mixture; S3. The mixture obtained in step S2 is subjected to a manual sterile extruder to generate biomass particles, which are dehydrated in a fluidized bed dryer and cooled to room temperature to obtain dry yeast powder.

[0006] Preferably, in step S1, the high-density fermentation conditions are specifically: the fermentation temperature is 30° C., and the fermentation time is 120 h.

[0007] Preferably, in step S2, the yeast protective agent is sorbitan monostearate, and the addition amount is 2%.

[0008] Preferably, in step S3, the mixture obtained in step S2 is subjected to a manual sterile extruder to generate biomass particles, which are dehydrated in a fluidized bed dryer at 50° C. for 70 min and cooled to room temperature to obtain dry yeast powder.

[0009] The invention also provides dry yeast powder prepared by the preparation method.

[0010] The present invention also provides the dry yeast powder prepared by the preparation method or the application of the dry yeast powder in inhibiting gray mold fungi.

[0011] The present invention also provides the dry yeast powder prepared by the preparation method or the application of the dry yeast powder in inhibiting gray mold fungi in grape fields and post-harvest storage.

[0012] Compared with the prior art, the present invention has the following advantages and technical effects: The present invention discloses a dry yeast powder and a preparation method and application thereof. The dry yeast powder is applied to grape fields and post-harvest storage, achieving efficient inhibition of gray mold, significantly reducing the decay rate and weight loss of grapes, and effectively extending the shelf life. In addition, the inoculated dry yeast powder can slow down fruit softening, increase fruit hardness, and delay the fruit ripening process.

[0013] The preparation method of the yeast dry powder improves the stability and antibacterial activity of yeast and enhances the survival rate and utilization rate of biological preservatives. The yeast dry powder exhibits excellent performance in grape field and post-harvest storage, not only has a significant effect on grape preservation, but also has the potential to be widely used in the preservation of other fruits, and has broad market application prospects.

[0014] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a morphological picture of the dry yeast powder provided in Example 1; Figure 2 It is a statistical chart of the inhibitory effect of the dry yeast powder provided in Example 1 and the dry yeast powder provided in the control group on the growth of Botrytis cinerea; Figure 3 The cell staining results of PI of Botrytis cinerea co-cultured with dry powder yeast at different concentrations provided in Example 1; Figure 4 The living cell count result of the yeast dry powder provided in Example 1 after being stored at room temperature for 15 days; Figure 5 The live cell count result of the yeast powder provided in Example 1 after being stored at 4° C. for 15 days; Figure 6 The inhibitory effect of the dry yeast powder provided in Example 1 on fungi in the grape field; Figure 7 A statistical diagram of microbial density incubated with dry yeast powder provided in Example 1; Figure 8 This is a statistical diagram of the inhibitory effect of the dry yeast powder provided in Example 1 on the decay of grapes during storage; Fig. 9 This is a statistical chart of sensory evaluation scores of grapes with different treatments; Fig.10 These are scanning electron microscope photos of grape wounds with different treatments; Fig.11 The effect of dry yeast powder on Botrytis cinerea provided in Example 1 is shown in FIG. Fig.11 A in the figure is the result of PI staining. Fig.11 B in the figure is the ROS result. Fig.11 C in the figure is the SEM result. Fig.11 D in the figure is the TEM B. Cinerea result; Fig.12 This is a statistical chart of the growth diameter of Botrytis cinerea treated with VOCs from dry yeast powder; Fig.13 It is a line graph of the growth area of ​​Botrytis cinerea. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0017] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0018] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0019] The yeast strain in the present invention is Saccharomyces cerevisiae NX2320, and the strain classification is named Saccharomyces cerevisiae Saccharomyces cerevisiae , which is the same yeast strain as the yeast strain in “Saccharomyces cerevisiae NX2320 inhibits Aspergillus carbonariusby cell membrane disruption and oxidative stress-mediated autophagy in invitro and during grape storage”.

[0020] Example 1 This example provides a dry yeast powder, and the preparation method includes the following steps: S1. Select Saccharomyces cerevisiae NX2320, ferment at 30℃ for 120h, centrifuge at 10000 r / min for 15min, collect yeast cells and obtain yeast mud; S2, weigh 20 g of sorbitan monostearate, add 50 mL of distilled water, sterilize at 121°C for 15 min, fully dissolve, wait until the temperature drops to 50°C, and mix thoroughly with 1000 g of yeast paste prepared in S1 to obtain a mixture; S3. The mixture obtained in step S2 was subjected to a manual sterile extruder to generate biomass particles, which were dehydrated at 50° C. for 70 min using a Mini-Glatt fluidized bed dryer, and cooled to room temperature to obtain dry yeast powder.

[0021] The effect of the dry yeast powder provided in Example 1 was verified by the following test.

[0022] 1. The appearance of yeast powder. Figure 1 .

[0023] Depend on Figure 1 It can be seen that the color of the yeast powder is granular and light yellow, with the aroma of yeast, no corruption, and no peculiar smell.

[0024] Index determination Determination of live yeast count: Determine the live yeast count by the yeast cell count method (GB / T 20886-2007). Accurately weigh 0.1g of active dry yeast with an error of 0.0002g. Dissolve the active dry yeast in 20 ml of sterile physiological saline to make a bacterial suspension and activate it in a constant temperature water bath at 37°C for 1h. Then mix the activated yeast solution and draw 0.1m of the test solution, dilute to the appropriate concentration, add 0.9 mL of methylene blue dye solution, shake well, dye at room temperature for 10 min, and inject the dyed bacterial solution into the counting chamber of the hemocytometer. Finally, observe and count under an optical microscope. The hemocytometer uses a 16x25 specification. Check whether the yeast cells on the hemocytometer are evenly dispersed. Randomly select 5 large squares (80 small squares) for counting, and take the average of three counts as the result. Live yeast cells appear colorless and transparent, while dead cells are dyed blue.

[0025] Among them, X is the viable cell rate, 100%; A is the number of yeast cells; D is the number of dead yeast cells.

[0026] Methylene blue staining solution: Add 0.025g methylene blue, 0.042g potassium chloride, 0.048g calcium chloride hexahydrate, 0.02g sodium bicarbonate, and 1.0g glucose to sterile saline to make up to 100ml. Do this in triplicate.

[0027] Determination of moisture content: Use a halogen moisture content meter to determine. Weigh about 2g of active dry yeast for brewing and place it in the tray of the moisture content meter. Spread the active dry yeast evenly on the tray. Automatically dry it at 105℃ to determine the final moisture content. Do it in triplicate.

[0028] The water content was calculated to be 4.3±0.1% and the number of viable bacteria was (1.01±0.02)×10 10 CFU / mL.

[0029] 2. Yeast powder inhibits the growth of Botrytis cinerea. The test scheme is as follows: The effect of different concentrations of dry powdered yeast on the mycelial growth of Botrytis cinerea was determined in PDB medium. The mycelial mass was determined by filtering and weighing. The control filter paper was inoculated with water. The plates were incubated at 25°C for 5 days. The results are shown in Figure 2 .

[0030] Depend on Figure 2 It can be seen that compared with the control group (0.14±0.01g), the fluidized bed dried in Example 1 (10 4 CFU / mL) and yeast powder co-cultured with the wet weight of Botrytis cinerea was significantly reduced to 0.01±0.01g. 5 and 106 CFU / mL) yeast powder can completely inhibit the growth of Botrytis cinerea after fluidized bed drying.

[0031] 3. PI staining to determine the integrity of yeast cell membrane in yeast powder: Take the yeast powder (10 6 CFU / mL) was activated at 37°C for 0.5h. The samples were incubated with 50μM PI at 37°C in the dark for 30min. After incubation, cell staining was observed under a fluorescence microscope (Nikon Eclipse 80i, Japan).

[0032] like Figure 3 As shown, the dry yeast powder dried in the fluidized bed of Example 1 was not stained with red fluorescence, indicating that most cells had a complete cell membrane structure.

[0033] 4. Stability of dry yeast powder under storage conditions The live cell counts of yeast powder stored at 4°C and room temperature were performed after 15 days of storage. Figure 4 and Figure 5 .Depend on Figure 4 and Figure 5 It can be seen that the activity of the yeast powder dried by fluidized bed drying in Example 1 during the 15-day storage period was not significantly different from that at the beginning of preparation, whether at 4°C or at room temperature.

[0034] 5. Field fungal growth inhibition test A field test was conducted in a grape plantation in Huailai, Hebei Province (115°N, 40°E) in 2023. The dry yeast powder provided in Example 1 was added to PBS in an Erlenmeyer flask and incubated at 37°C for 0.5 h to obtain a yeast solution. The prepared yeast solution (1%, 10 8 cfu / mL) was sprayed on the grape plants, spraying evenly on both sides of the leaves.

[0035] The first spraying time of the test was June 12, 2023, and the second spraying time was June 22, 2023. An equal amount of sterile water was used as a control. Each treatment had 48 trees, 12 trees in each group, and was repeated 4 times.

[0036] On September 14, 2023, all grape plants were surveyed, the degree of cluster damage was recorded, and the disease index and control effect were calculated. Partial level method: Grade 0: no lesions; Level 1: The lesion area accounts for less than 5% of the entire cluster; Grade 3: The lesion area accounts for 6%-15% of the entire cluster; Level 5: The lesion area accounts for 16%-25% of the entire cluster; Level 7: The lesion area accounts for 26%-50% of the entire cluster; Level 9: The lesion area accounts for more than 51% of the entire cluster. Figure 6 .

[0037] Depend on Figure 6 It can be seen that compared with the control group, the dry yeast powder provided in Example 1 has a significant inhibitory effect on grape field fungi.

[0038] 6. Effect of dry yeast powder on the quality of postharvest grapes The fungal rot of grapes was used to test the antibacterial activity of yeast powder. The experimental scheme was as follows: the fruits were disinfected, dried, and damaged, and inoculated with 10 μL of Botrytis cinerea solution. The grapes were divided into two groups (10 in each group) with the following different treatments: (1) CK: inoculated with 10 μL of sterile water; (2) 10 6 CFU / mL: inoculate 10 μL at a concentration of 10 6 CFU / mL of the yeast powder provided in Example 1; (3) 10 7 CFU / mL: inoculate 10 μL at a concentration of 10 7 CFU / mL dry yeast powder provided in Example 1; (4) 10 8 CFU / mL: inoculate 10 μL at a concentration of 10 8 CFU / mL dry yeast powder provided in Example 1; (5) 10 6 CFU / mL+1% tartaric acid: inoculate 10 μL to a concentration of 10 6 CFU / mL dry yeast powder and 1% tartaric acid provided in Example 1; (6) 10 6 CFU / mL+1% tea polyphenols: inoculate 10μL with a concentration of 10 6 CFU / mL Yeast powder and 1% tea polyphenols provided in Example 1; the fruits were dried at room temperature and stored at 20°C. The experiment was conducted twice, with 10 grapes used in each replicate. Figure 7 .

[0039] Depend on Figure 7 It can be seen that the microbial density of the dry yeast powder provided in Example 1 increased rapidly after one day of incubation, and then the population continued to increase slightly until the 4th day of the incubation period. The rapid growth of Saccharomyces cerevisiae NX2320 is that the yeast has found a favorable and appropriate environment in the fruit wound.

[0040] The grapes were stored at 20°C. The experiment was performed twice, with 10 grapes in each replicate. Visible gray mold infection, brown spots or softening of injured areas were counted as rot, and the results were expressed as the number of rotten samples divided by the total number of samples multiplied by 100 to express the percentage of rot.

[0041] About 10 g of the pulp was homogenized, mixed and filtered. The soluble solids content was determined using a refractometer. This was repeated three times. The results were as follows: Figure 8 .

[0042] Depend on Figure 8 It can be seen that all treatment groups can effectively inhibit the decay of grapes during storage. The decay rate of the CK group was 22.61±2.63% on day 3 and increased to 86.65±2.63% on day 5. 8 CFU / mL and 1×10 6 The decay rate of the CFU / mL+1% tea polyphenols treatment group remained 0 within 5 days. During the entire storage process, the decay rate of all groups was significantly lower than that of the control group. The results showed that the decay rate was concentration-dependent.

[0043] Sensory evaluation: A panel of 9 trained members from the laboratory (5 males, 4 females, aged 30-35 years) conducted the evaluation. The aroma, color and overall acceptability of the grapes were evaluated using a scale of 1 (dislike very much) to 10 (like very much). Fig. 9 .

[0044] Depend on Fig. 9 It can be seen that after 5 days of storage, the control group (1.67±0.58) had the lowest score, and the yeast powder group provided in Example 1 (10 8 CFU / mL, 8.33±0.58, followed by yeast powder (1×10 6 CFU / mL+1% tartaric acid, 8.00±1.00). 6 Compared with the CFU / mL group, the addition of tartaric acid and tea polyphenols could significantly improve the score.

[0045] 7. Scanning Electron Microscope (SEM) Specimen Processing Fruit tissues infected with fungi were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer solution (pH 7.0) at 4°C overnight. Then, the fruit tissues were prepared for SEM examination on a scanning electron microscope (Hitachi SU-8010, Tokyo, Japan). Fig.10 .

[0046] Depend on Fig.10 It can be seen that in the control group and 10 6 In the yeast treatment group, most of the conidia of Botrytis cinerea had germinated and formed thick hyphae. 7 CFU / mL yeast treatment group, both Botrytis cinerea hyphae and yeast were found. 8 CFU / mL, 10 6CFU / mL+1% tartaric acid and 10 6 Yeast colonization was clearly observed in the CFU / mL+1% tea polyphenols yeast treatment group. The results showed that the embedded yeast could rapidly multiply, colonize on the grape wound tissue, and inhibit the growth of Botrytis cinerea in a concentration-dependent manner in vivo. Tartaric acid and tea polyphenols can also enhance the activity of inhibiting fungi.

[0047] 8. Study on the mechanism of yeast powder inhibiting the growth of Botrytis cinerea The propidium iodide (PI) test was used to indicate changes in the cell membrane integrity of Botrytis cinerea. Botrytis cinerea was co-cultured with Saccharomyces cerevisiae NX2320 for 48 h. All samples were centrifuged at 8000 rpm for 5 min, washed three times, and resuspended in PBS, then incubated with PI (50 μM) at 37 °C in the dark for 30 min. Fluorescence changes of the cells were then observed using a fluorescence microscope (Nikon Eclipse 80i, Japan).

[0048] Fluorescent dye CM-H2DCFDA was used to detect intracellular ROS levels. Botrytis cinerea was co-cultured with Saccharomyces cerevisiae NX2320 for 48 hours. The cells were stained with 10 μM CM-H2DCFDA for 30 minutes in the dark at 37 °C, and then the changes in intracellular ROS levels were observed using a fluorescence microscope (Nikon Eclipse 80i, Japan).

[0049] Scanning electron microscopy was used to evaluate the surface morphological changes of co-cultures of Botrytis cinerea and Saccharomyces cerevisiae NX2320. After 48 hours of co-culture treatment with Saccharomyces cerevisiae, Botrytis cinerea was collected by centrifugation and fixed with 2.5% glutaraldehyde. The cells were gradually dehydrated in ethanol. The dehydrated suspension of Botrytis cinerea was then placed on a silica chip. The surface morphological characteristics of the samples were observed using scanning electron microscopy.

[0050] Botrytis cinerea was fixed in 2.5% glutaraldehyde for 2 hours and then transferred to embedding solution overnight. Afterwards, ultrathin sections were stained and observed using a transmission electron microscope to observe changes in the internal structure of the bacteria.

[0051] Depend on Fig.11 As shown in A, there were no cells in the control group, and the yeast microcapsules dried in the fluidized bed were stained with red fluorescence, indicating that most of the cell membranes were intact.

[0052] Depend on Fig.11 As can be seen in B, the autofluorescence of untreated cells is very weak, while the fluorescence intensity of cells co-cultured with microencapsulated yeast increases, indicating that co-culture with microencapsulated yeast leads to very high ROS levels in B. cinerea.

[0053] Depend on Fig.11From the C in Figure 1, we can see that the mycelium of the control group, Botrytis cinerea, is plump and smooth, with a complete structure. The mycelium of the mycelium co-cultured with Microcystis saccharomyces cerevisiae is smaller, with deeper wrinkles and folds. Fig.11 As shown in D, transmission electron microscopy showed that the cytoplasm of untreated hyphae was evenly distributed and contained visible organelles and ribosomes, while the vesicles in the mycelium co-cultured with Microcystis were fewer and smaller. The cell wall and plasma membrane of the control group were uniform and intact. However, the cells were highly deformed, the chromatin was uneven and locally aggregated in the mycelium co-cultured with Microcystis, and the organelles were unclear. The intracellular material of Botrytis cinerea was reduced, the organelles and endomembrane system could no longer be distinguished, and the cell membrane and cell wall began to shrink and became irregular in the mycelium co-cultured with Microcystis.

[0054] 9. Determination of the inhibitory effect of volatile organic compounds (VOCs) on fungal growth The effect of yeast producing volatile antifungal compounds was evaluated by mouth-to-mouth method. 5 spores / mL, 10 μL) was dropped onto a filter paper with a diameter of 5 mm in the center of the upper plate. The yeast powder provided in Example 1 was added to PBS in an Erlenmeyer flask and incubated at 37°C for 0.5 h to obtain a yeast solution. The yeast solution (10 6 CFU / mL, 100µL) was spread on the lower plate. After drying, the two plates were placed face up and sealed with sealing film. The culture was incubated at 25°C. The mouth-to-mouth plate without yeast on the lower plate was used as a control. Each treatment was repeated 3 times. The results are shown in Figure 12-13 .

[0055] Depend on Fig.12 It can be seen that after 6 days of cultivation, the growth diameter of Botrytis cinerea treated with VOCs in Example 1 was smaller than that of the control.

[0056] Depend on Fig.13 It can be seen that the growth area rate of fungi is also affected by the presence of yeast. Compared with the control, the growth area rate of fungi co-cultured with the VOCs of Example 1 decreased during the 6-day culture. These results show that the VOCs produced in Example 1 have an inhibitory effect on the hyphae growth and conidia germination of Botrytis cinerea in vitro.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing dry yeast powder, characterized in that: The steps include: S1. Select yeast strains, perform high-density fermentation, centrifuge at 10,000 r / min for 15 min, collect yeast cells, and obtain yeast mud; S2, weigh the yeast protectant, add distilled water, sterilize at 121°C for 15 min, fully dissolve, wait until the temperature drops to 50°C, and mix thoroughly with the yeast paste prepared in S1 to obtain a mixture; S3. The mixture obtained in step S2 is subjected to a manual sterile extruder to generate biomass particles, which are dehydrated in a fluidized bed dryer and cooled to room temperature to obtain dry yeast powder.

2. The preparation method according to claim 1, characterized in that: In step S1, the high-density fermentation conditions are specifically as follows: the fermentation temperature is 30° C. and the fermentation time is 120 h.

3. The preparation method according to claim 1, characterized in that: In step S2, the yeast protective agent is sorbitan monostearate, and the addition amount is 2%.

4. The preparation method according to claim 1, characterized in that: In step S3, the mixture obtained in step S2 is subjected to a manual sterile extruder to generate biomass particles, which are dehydrated in a fluidized bed dryer at 50° C. for 70 min and cooled to room temperature to obtain dry yeast powder.

5. The dry yeast powder prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the dry yeast powder prepared by the preparation method according to any one of claims 1 to 4 or the dry yeast powder according to claim 5 in inhibiting Botrytis cinerea.

7. Use of the dry yeast powder prepared by the preparation method according to any one of claims 1 to 4 or the dry yeast powder according to claim 6 in inhibiting Botrytis cinerea in grape fields and post-harvest storage.

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