Vacuum cold freeze-dried powder preparation method based on eurotium cristatum

Abstract

The invention discloses a preparation method of vacuum cold freeze-dried powder based on eurotium cristatum, and belongs to the technical field of freeze-dried powder preparation. The preparation method comprises the following steps: step 1, preparation of sterile water and a culture dish: putting purified water into a glass bottle to be sealed, and putting the glass bottle into a high-pressure sterilization pot to be sterilized to obtain the sterile water; putting the culture dish into a high-pressure sterilization pot for sterilization to obtain a sterile culture dish; step 2, preparation of a golden flower bacterial suspension: weighing 5g of a tea sample of the three-hairyvein agrimony golden flower Liupu tea, putting the tea sample into sterile water, and standing to obtain a diluted bacterial suspension; 3, eurotium cristatum separation and purification: putting the diluted bacterial suspension into a constant-temperature incubator at 30 DEG C for culture; the purified eurotium cristatum bacterial colony is obtained; and step 4, preparing freeze-dried bacterial powder: performing vacuum freeze-drying on the prepared eurotium cristatum bacterial colony to obtain the freeze-dried bacterial powder after vacuum freeze-drying. The temperature and pressure changes in the freeze-drying box are monitored in real time through the controller, and the freezing speed is automatically adjusted according to the variety and the water content of eurotium cristatum, so that the freezing effect is optimized.

Description

Technical Field

[0001] The invention belongs to the technical field of freeze-dried powder preparation, and specifically is a method for preparing vacuum freeze-dried powder based on golden flower fungus. Background Art

[0002] "Golden Flower" fungus, scientifically known as Eurotium cristatum, is a dominant fungus in the "flowering" process of Fuzhuan tea. "Golden Flower" fungus is a beneficial microorganism selected from black Fuzhuan tea, which can produce small golden particles on the surface of tea leaves, hence the name "Golden Flower" fungus. "Golden Flower" belongs to the class Ascomycetes, the order Aspergillus, the family Aspergillus, and the genus Eurotium. As a probiotic, "Golden Flower" fungus has been widely used in the fields of tea, beans, cereals, and deep processing of Chinese medicinal materials.

[0003] As a microbial product widely used in the food industry, frozen bacterial powder is highly valued for its convenience in transportation, stability at low or normal temperatures, and high safety. Therefore, frozen bacterial powder has become an indispensable part of industrial production for many food manufacturers and research institutions. Vacuum freeze drying is a common method for drying bacterial powder, which consists of three stages: freezing, vacuum drying, and rehydration.

[0004] Although the existing vacuum freeze-drying technology has significant advantages in maintaining biological activity, extending shelf life and optimizing rehydration, it has the problems of cumbersome operation and complicated process. Before formal drying, the material often needs to be pre-frozen, and the drying stage requires multiple steps, which is frequent and inconvenient. Therefore, it is necessary to propose a vacuum freeze-dried powder preparation method based on golden flower bacteria. Summary of the invention

[0005] In order to solve the problem that the above-mentioned vacuum freeze-drying technology is not convenient to operate, the purpose of the present invention is to provide a method for preparing vacuum freeze-dried powder based on golden flower bacteria, which monitors the temperature and pressure changes in the freeze-drying chamber in real time through a controller, and automatically adjusts the freezing speed according to the type and water content of the golden flower bacteria. The controller realizes the automated operation of vacuum freeze-drying, reduces manual operation steps, and optimizes the freezing effect.

[0006] In order to achieve the above object, the technical scheme of the present invention is as follows: A method for preparing a vacuum freeze-dried powder based on golden flower fungus, comprising the following steps:

[0007] Step 1, preparation of sterile water and culture dishes: put purified water into a glass bottle and seal it, then put it into a high pressure sterilizer for sterilization to obtain sterile water; use a detergent to clean the culture dishes and let them dry naturally, then wrap the dried culture dishes in newspapers in groups of ten, and then put them into a high pressure sterilizer for sterilization to obtain sterile culture dishes;

[0008] Step 2, preparation of golden flower fungus suspension: weigh 5g of Sanhe Golden Flower Liubao tea sample and put it into sterile water to make 10 -1 times diluted bacterial suspension; then draw 25mL of 10 -1 Add the diluted bacterial suspension to 250 mL of sterile water to obtain 10 -2 times diluted bacterial suspension, and then pipette 10 -2 Add the diluted bacterial suspension to 9 mL of sterile water to obtain 10 -3 times diluted bacterial suspension, and so on to obtain 10 -9 times diluted bacterial suspension;

[0009] Step 3, separation and purification of golden flower bacteria: 10 -1 Up to 10 -9 Use a pipette to draw 1 mL of the diluted bacterial suspension into a sterile culture dish, then pour into the PDA culture medium and mix evenly. Repeat the operation twice for each dilution of the diluted bacterial suspension. After solidification, turn the culture medium upside down and place it in a 30°C constant temperature incubator for culture.

[0010] According to the morphological characteristics of the initial colonies, the colonies of the golden flower fungus that do not contain other bacteria are picked up on the culture medium with an inoculation loop, and inoculated on the PDA culture medium prepared in advance, and then placed in a constant temperature fermentation box for 72 hours. Screening and purification are carried out according to the characteristics of the grown golden flower fungus colonies. The above steps are repeated many times until the golden flower fungus colonies do not contain other bacteria and the growth morphological characteristics are consistent, and purified golden flower fungus colonies are obtained;

[0011] Step 4, preparation of freeze-dried bacterial powder: adding freeze-drying protective agents to the prepared golden flower bacteria colonies respectively, and then placing them in a segmented vacuum freeze-drying mechanism for vacuum freeze-drying, and obtaining freeze-dried bacterial powder after vacuum freeze-drying;

[0012] Step 5, freeze-dried bacterial powder activity test: weigh 1g of freeze-dried bacterial powder and place it in 9ml of sterile water, shake it evenly, and get 10 -1 times freeze-dried bacterial suspension; use a pipette to draw 1 mL of 10 -1 Add 9 mL of sterile water to make 10 times the freeze-dried bacterial suspension. -2 times freeze-dried bacterial suspension, and then draw 1mL of 10 -2 Add 9 mL of sterile water to make 10 times the freeze-dried bacterial suspension. -3 times freeze-dried bacterial suspension;

[0013] 10 -1 Up to 10 -3Use a pipette to draw 1 mL of the freeze-dried bacterial suspension of each times and add it into a sterile culture dish, then pour in 40% sucrose peptone culture medium and mix evenly. Repeat the operation twice for each freeze-dried bacterial suspension. After solidification, invert the culture medium, place it in a constant temperature incubator at 30℃ and culture it for 72 hours, and count the number of live colonies.

[0014] Furthermore, in step one, the sterilization method is sterilization in a high pressure sterilizer at 121° C. for 20 minutes.

[0015] Furthermore, in step 2, the static method is to put 5g of Sanhe Jinhua Liubao tea sample into 300mL of sterile water, shake it evenly and then put it into the clean bench and let it stand for 30 minutes.

[0016] Further, in step three, the preparation method of PDA culture medium is as follows: peel 200.0g of potatoes, put them into 1000mL of pure water and boil them for 10min, filter them with gauze, add 20.0g of glucose and 20.0g of agar and stir to dissolve, add sterile water to make up to 1000mL after stirring and dissolving, and then put them into a high pressure sterilizer and sterilize them at 121°C for 20min.

[0017] Furthermore, in step 4, the lyophilization protective agent is 1 mL of 5% sucrose by mass, and the vacuum freeze-drying method is pre-freezing at -20°C for 2 to 3 hours, and then vacuum freeze-drying for 42 hours.

[0018] Furthermore, in step five, the preparation method of 40% sucrose peptone culture medium is as follows: 400.0 g sucrose, 10.0 g peptone and 15.0 g sodium chloride NaCl are stirred and dissolved with purified water, and then purified water is added to make up to 1000 mL after stirring and dissolving, and then placed in a high pressure sterilizer for sterilization at 121° C. for 20 min.

[0019] Furthermore, in step 5, the number of live bacteria in the freeze-dried bacterial colony is determined by a plate counting method.

[0020] Further, in step four, the segmented vacuum freeze-drying mechanism includes a freeze-drying box with an opening, a cover plate is hinged on one side of the opening of the freeze-drying box, a handle is fixedly connected to the outside of the cover plate, and a number of support rods are fixedly connected to the bottom of the freeze-drying box; a number of slide grooves are opened inside the freeze-drying box, and trays are slidably fitted in the slide grooves; a spiral check valve is connected to the top of the freeze-drying box, and the spiral check valve includes a number of U-shaped connecting pipes, which are interconnected, and the opening of the U-shaped connecting pipe faces the output end of the spiral check valve; the output end of the spiral check valve is connected to a vacuum component for extracting air from the freeze-drying box; the side of the freeze-drying box away from the cover plate is connected to a refrigeration component for providing cold air.

[0021] Furthermore, the vacuum component includes a vacuum pump and a controller, the vacuum pump input end is connected to the spiral check valve output end, the vacuum pump output end is connected to the vent, the vacuum pump is fixedly connected to the top of the freeze drying chamber, and the controller is used to control the operation of the vacuum pump; the controller signal is connected to a vacuum gauge, and the vacuum gauge is fixedly connected to the inner wall of the freeze drying chamber.

[0022] Furthermore, the refrigeration component includes a compressor, a microchannel condenser, a PCM evaporator and an expansion valve; the output end of the compressor is connected to the microchannel condenser, and the compressor is fixedly connected to the outer side of the bottom of the freeze drying chamber; the output end of the microchannel condenser is connected to the expansion valve, and the microchannel condenser is fixedly connected to the outside of the freeze drying chamber away from the opening; the output end of the expansion valve is connected to the PCM evaporator, and the PCM evaporator includes a number of interconnected circulation pipes, which are all located in the side wall of the freeze drying chamber in the length direction, the output end of the PCM evaporator is connected to the compressor, and the microchannel condenser is connected to an exhaust valve; the controller is used to control the operation of the compressor, the controller signal is connected to a temperature sensor, and the temperature sensor is fixedly connected to the inner wall of the freeze drying chamber.

[0023] The basic scheme has the following beneficial effects: 1. This method strictly controls the risk of microbial contamination from the initial preparation of sterile water and culture dishes to aseptic operation throughout the entire experimental process, ensuring the purity of the golden flower bacteria strain. The purified golden flower bacteria colony was inoculated into a 40% sucrose peptone medium. The high-sugar environment not only promoted the rapid growth of the golden flower bacteria, but also significantly enhanced the development and spread of mycelium, laying a solid foundation for subsequent cultivation. At the same time, the golden flower bacteria were used for liquid culture, and the freeze-dried powder of the golden flower bacteria was prepared by vacuum freeze drying, and sucrose with a mass fraction of 5% was used as a freeze-drying protective agent, which not only maintained the original biological activity and genetic characteristics of the strain, but also significantly extended its shelf life.

[0024] 2. The present invention uses pulse vacuum technology to periodically change the pressure in the vacuum chamber through the cooperation of the controller, spiral check valve, vacuum pump and vacuum gauge, which can promote the internal moisture of the material to migrate to the surface and sublimate faster, shorten the drying time and improve production efficiency. It can reduce the physical stress of the material caused by continuous high-intensity vacuum, protect the effective ingredients and biological structure integrity of the golden flower fungus, and help to form a more loose and porous dry powder structure. By accurately controlling the pressure change, pulse vacuum drying can more finely adjust the drying rate and material state, which helps to maintain or improve the color, aroma, nutritional value and biological activity of the golden flower fungus freeze-dried powder.

[0025] 3. The present invention uses a controller to monitor the temperature and pressure changes in the freeze-drying chamber in real time, and automatically adjusts the freezing speed according to the type and water content of the golden flower fungus. A microchannel condenser is used as part of its cooling system. With its high heat transfer efficiency and compact structure characteristics, it can remove heat more quickly and effectively, maintain a lower, more stable and uniform cooling temperature, thereby accelerating the sublimation process of water in the material, reducing drying time, and improving drying quality. The PCM phase change material is used to assist in the absorption and release of heat, providing more stable and accurate temperature control in the system and reducing temperature fluctuations. It responds quickly in the critical drying stage of the late freezing period, accelerates the sublimation rate of water, maintains the low temperature in the freeze-drying chamber, shortens the drying cycle, and reduces energy consumption. It can smooth temperature changes in the system, reduce thermal shock to the golden flower fungus freeze-dried powder, and help maintain the high quality and biological activity of the product.

[0026] 4. The present invention uses a specific spiral check valve as a pressure element to help adjust the pressure gradient in the vacuum pump system, optimize the pressure control at different stages, and improve the vacuuming efficiency and stability. It can accurately control the flow and direction of the gas, optimize the gas flow control, and ensure that the required vacuum degree is maintained efficiently and stably. Its design without moving parts reduces wear and maintenance requirements, improves the reliability of the system, and provides continuity of the vacuum freeze-drying process. The vacuum pump may encounter back pressure problems during operation. The one-way conduction characteristics of the spiral check valve can effectively prevent the backflow of external gas, protect the vacuum pump from unnecessary pressure shocks, and extend the service life of the equipment. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention is a flow chart of a method for preparing vacuum freeze-dried powder of golden flower fungus according to an embodiment of the present invention.

[0028] Figure 2 It is an axonometric diagram of the segmented vacuum freeze-drying mechanism according to an embodiment of the present invention.

[0029] Figure 3 It is a front cross-sectional view of the segmented vacuum freeze-drying mechanism according to an embodiment of the present invention.

[0030] Figure 4 It is a side cross-sectional view of the segmented vacuum freeze-drying mechanism according to an embodiment of the present invention.

[0031] Figure 5 It is a front cross-sectional view of a spiral check valve in a segmented vacuum freeze-drying mechanism according to an embodiment of the present invention. DETAILED DESCRIPTION

[0032] The following is further described in detail through specific implementation methods:

[0033] The figure marks in the drawings of the specification include: freeze drying box 1, cover plate 2, handle 3, support rod 4, tray 5, spiral check valve 6, connecting pipe 7, vacuum pump 8, compressor 9, microchannel condenser 10, PCM evaporator 11.

[0034] Embodiment 1, basically as attached Figure 1 As shown: A method for preparing vacuum freeze-dried powder based on golden flower fungus, comprising the following steps:

[0035] Step 1, preparation of sterile water and culture dishes: put pure water into a glass bottle and seal it, then put it into a high pressure sterilizer and sterilize it at 121°C for 20 minutes to obtain sterile water; use sodium hydroxide solution detergent to clean the culture dishes and let them dry naturally, and after drying, wrap the culture dishes in groups of ten with newspapers, and then put them into a high pressure sterilizer and sterilize them at 121°C for 20 minutes to obtain sterile culture dishes.

[0036] Step 2, preparation of golden flower fungus suspension: weigh 5g of Sanhe Golden Flower Liubao tea sample and put it into 300mL of sterile water, shake it evenly and put it on the clean bench to stand for 30min to make 10 -1 times diluted bacterial suspension; then draw 25mL of 10 -1 Dilute the bacterial suspension by 10 times and add 250 mL of sterile water to obtain 10 -2 times diluted bacterial suspension, and then pipette 10 -2 Add the diluted bacterial suspension to 9 mL of sterile water to obtain 10 -3 times diluted bacterial suspension, and so on to obtain 10 -9 times diluted bacterial suspension.

[0037] Step 3, separation and purification of golden flower bacteria: 10 -1 Up to 10 -9 Use a pipette to draw 1 mL of the diluted bacterial suspension into a sterile culture dish, then pour into the PDA culture medium and mix evenly. Repeat the operation twice for each dilution of the diluted bacterial suspension. After solidification, invert the culture medium and place it in a 30℃ constant temperature incubator for culture.

[0038] Among them, the preparation method of PDA culture medium is as follows: peel 200.0g of potatoes, put them into 1000mL of pure water and boil them for 10 minutes, filter them with gauze, then add 20.0g of glucose and 20.0g of agar and stir to dissolve, after stirring and dissolving, add sterile water to make up to 1000mL, and then put them into a high pressure sterilizer for sterilization at 121℃ for 20 minutes.

[0039] According to the morphological characteristics of the initial colonies, use an inoculation loop to pick up the golden flower fungus colonies that do not contain other bacteria on the culture medium, and inoculate them on the PDA culture medium prepared in advance, and then put them into a constant temperature fermentation box for culture for 72 hours. Screen and purify them according to the characteristics of the grown golden flower fungus colonies. Repeat the above steps many times until there are no other bacteria on the golden flower fungus colonies and the growth morphological characteristics are consistent, then obtain purified golden flower fungus colonies.

[0040] Step 4, preparation of freeze-dried bacterial powder: add 1 mL of 5% sucrose freeze-protectant to the prepared golden flower mushroom colonies, and then put them into a segmented vacuum freeze-drying mechanism for vacuum freeze-drying. The vacuum freeze-drying method is to pre-freeze at -20°C for 2 to 3 hours, and then vacuum freeze-dry for 42 hours to obtain freeze-dried bacterial powder.

[0041] Step 5, freeze-dried bacterial powder activity test: weigh 1g of freeze-dried bacterial powder and place it in 9ml of sterile water, shake it evenly, and get 10 -1 times freeze-dried bacterial suspension; use a pipette to draw 1 mL of 10 -1 Add 9 mL of sterile water to make 10 times the freeze-dried bacterial suspension. -2 times freeze-dried bacterial suspension, and then draw 1mL of 10 -2 Add 9 mL of sterile water to make 10 times the freeze-dried bacterial suspension. -3 times freeze-dried bacterial suspension.

[0042] 10 -1 Up to 10 -3 Use a pipette to draw 1 mL of the freeze-dried bacterial suspension into a sterile culture dish, then pour in 40% sucrose peptone culture medium and mix evenly. Repeat the operation twice for each freeze-dried bacterial suspension. After solidification, invert the culture medium and place it in a 30℃ constant temperature incubator for 72 hours, and use the plate counting method to count the number of live colonies.

[0043] The preparation method of 40% sucrose peptone medium is as follows: 400.0 g sucrose, 10.0 g peptone and 15.0 g sodium chloride NaCl are stirred and dissolved with purified water, and then purified water is added to make up the volume to 1000 mL after stirring and dissolving, and then placed in a high pressure sterilizer for sterilization at 121°C for 20 minutes.

[0044] The above method uses strict microbiological techniques to separate and purify golden flower fungi from the original sample to ensure that there is no contamination from foreign bacteria. Through multi-stage dilution and PDA culture medium screening, combined with careful observation of morphological characteristics, the effective separation and purification of golden flower fungi is achieved, the possible interference of foreign bacteria is removed, and the singleness and stability of the prepared bacterial powder are ensured. It shows significant superiority in ensuring the purity of the strain, improving the stability and activity of the bacterial powder, and promoting the efficient utilization of bacterial resources.

[0045] Example 2

[0046] The difference from the above embodiment is that, as shown in the attached Figure 2 -Attached Figure 5 As shown: the segmented vacuum freeze-drying mechanism comprises a freeze-drying box 1 with an opening, a cover plate 2 is hingedly connected to one side of the opening of the freeze-drying box 1, a handle 3 is fixedly connected to the outer side of the cover plate 2 with bolts, and a plurality of support rods 4 are fixedly connected to the bottom of the freeze-drying box 1 with bolts; a plurality of slide grooves are opened inside the freeze-drying box 1, and trays 5 are slidably fitted in the slide grooves; a spiral check valve 6 is connected to the top of the freeze-drying box 1, and the spiral check valve 6 includes a plurality of U-shaped connecting pipes 7, which are connected to each other, and the opening of the U-shaped connecting pipe 7 faces the output end of the spiral check valve 6; the output end of the spiral check valve 6 is connected to A vacuum assembly is provided for extracting air from the freeze drying chamber 1; the vacuum assembly includes a vacuum pump 8 and a controller, the controller model is preferably DSC-3, the vacuum pump 8 model is preferably 2RH008, the input end of the vacuum pump 8 is connected to the output end of the spiral check valve 6, the output end of the vacuum pump 8 is connected to a vent, the vacuum pump 8 is bolted and fixedly connected to the top of the freeze drying chamber 1, and the controller is used to control the operation of the vacuum pump 8; the controller signal is connected to a vacuum gauge, the vacuum gauge model is preferably APG100-XLC, and the vacuum gauge is bolted and fixedly connected to the inner wall of the freeze drying chamber 1.

[0047] The side of the freeze drying box 1 away from the cover plate 2 is connected to a refrigeration component for providing coldness, and the refrigeration component includes a compressor 9, a microchannel condenser 10, a PCM evaporator 11 and an expansion valve; the compressor 9 is preferably of model K270CY1, the output end of the compressor 9 is connected to the microchannel condenser 10, and the compressor 9 is bolted and fixedly connected to the outside of the bottom of the freeze drying box 1; the output end of the microchannel condenser 10 is connected to the expansion valve, and the microchannel condenser 10 is bolted and fixedly connected to the side of the freeze drying box 1 away from the opening; the output end of the expansion valve is connected to the PCM evaporator 11, and the PCM evaporator 11 includes a number of interconnected circulation pipes, which are all located in the side wall of the freeze drying box 1 in the length direction, the output end of the PCM evaporator 11 is connected to the compressor 9, and the microchannel condenser 10 is connected to an exhaust valve; the controller is used to control the operation of the compressor 9, and the controller signal is connected to a temperature sensor, the temperature sensor model is preferably DS18B20, and the temperature sensor is bolted and fixedly connected to the inner wall of the freeze drying box 1.

[0048] The specific implementation process is as follows: First, when performing vacuum freeze drying, open the cover plate 2, and place the materials to be vacuum frozen on several trays 5 installed in the freeze drying box 1, and turn on the refrigeration component through the controller to pre-freeze the materials. At this time, the low-temperature and low-pressure refrigerant gas is sucked in by the compressor 9. After the refrigerant absorbs the heat of the material or the environment in the PCM evaporator 11, it becomes a low-temperature and low-pressure gas, and then the gas is sucked in and compressed by the compressor 9. The compression process increases the temperature and pressure of the refrigerant to form a high-temperature and high-pressure gas. The high-temperature and high-pressure refrigerant gas then enters the microchannel condenser 10, where the refrigerant releases heat to exchange heat with the external cooling medium, and the refrigerant is condensed into a high-pressure liquid after heat release. After the high-pressure liquid refrigerant leaves the condenser, it enters the next link through the expansion valve. The expansion valve quickly reduces the pressure of the refrigerant. At the same time, as the temperature drops, the refrigerant is converted into a mixture of low-temperature and low-pressure liquid and gas. The low-temperature and low-pressure mixture then enters the PCM evaporator 11. At this stage, the refrigerant absorbs the heat in the material, causing the material to freeze or maintain a low-temperature state, thereby completing the pre-freezing of the material. At this time, the water in the material is completely frozen into ice crystals. This step helps to reduce cell damage and keep the material structure intact.

[0049] After the pre-freezing is completed, the vacuum pump 8 is used to reduce the vacuum degree in the freeze drying box 1 to a certain degree, and the vacuum pump 8 quickly discharges the gas through the spiral check valve 6. Because the spiral check valve 6 is interconnected by a number of U-shaped connecting pipes, and the opening of the U-shaped connecting pipe is facing the output end of the spiral check valve 6, when the gas flows through from the input end, it can pass quickly; on the contrary, when the gas flows back from the output end of the spiral check valve 6, it will be blocked by the bend of the U-shaped connecting pipe. When the gas is blocked by the bend many times, the kinetic energy of the gas gradually decreases, thereby reducing the situation of gas backflow in the spiral check valve 6. Therefore, a pulse airflow is generated by the action of the spiral check valve 6 and the vacuum pump to dry the material. At this time, the temperature is still kept below the freezing point, which prompts the ice crystals in the material to sublimate directly into water vapor without passing through the liquid state, and the moisture in the material evaporates and is discharged by the vacuum pump 8, so as to ensure efficient drying while protecting the quality of the material. After the drying process is completed, the vacuum pump 8 is turned off, the pulse airflow input is stopped, and the crystal temperature is reduced to a natural state. Finally, the cover plate 2 is opened, the material is taken out, and the freeze-dried bacterial powder is obtained.

[0050] Experiments and Results

[0051] 1. How to use

[0052] 1.1 Preparation of culture dishes and sterile water

[0053] Preparation of culture dishes: Use detergent to clean the culture dishes and let them dry naturally. After drying, wrap the culture dishes in newspapers in groups of ten and sterilize them in an autoclave at 121°C for 20 minutes.

[0054] Preparation of sterile water: Pour purified water into a glass bottle or conical flask, seal it, and place it in a high pressure sterilizer and sterilize it at 121°C for 20 minutes to obtain sterile water.

[0055] 1.2 Culture medium preparation

[0056] PDA culture medium: 200.0g peeled potatoes, 1000mL pure water, boil for 10min, filter with gauze, add 20.0g glucose and 20.0g agar, stir to dissolve, add sterile water to make up to 1000mL, put in a high pressure sterilizer and sterilize at 121℃ for 20min.

[0057] Improved PDA medium: 150g peeled potatoes, 1000mL pure water, boil for 10min, filter with gauze, add 20g sucrose, 15g agar powder, 5g yeast extract, stir to dissolve, add sterile water to make up to 1000mL, put in an autoclave and sterilize at 121℃ for 20min.

[0058] Soluble starch culture medium: sucrose 150g, soluble starch 15g, agar 15g, yeast extract 5g, stir thoroughly with purified water to dissolve, add sterile water to make up to 1000mL, and sterilize in an autoclave at 121℃ for 20min.

[0059] Soluble starch tea culture medium: 10g of modern process Liubao tea, 1000mL of pure water, boil for 10min, filter with gauze, add 150g of sucrose, 15g of soluble starch, 15g of agar, 5g of yeast paste, stir to dissolve, add sterile water to make up to 1000mL, put in a high pressure sterilizer and sterilize at 121℃ for 20min.

[0060] 40% sucrose peptone medium: sucrose 400.0g, peptone 10.0g, sodium chloride NaCl5.0g, agar 20.0g, stir thoroughly with purified water to dissolve, add sterile water to make up to 1000mL after stirring and dissolving, put in a high pressure sterilizer and sterilize at 121℃ for 20min.

[0061] 1.3 Isolation and purification of “Golden Flower” bacteria

[0062] Preparation of bacterial suspension: Weigh 5g of Sanhe Jinhua Liubao tea sample into 300mL of sterile water and let stand for 30min to make 10 -1 When using on a clean bench, shake thoroughly and pipette 25 mL of 10 -1 Add the bacterial suspension to 250 mL of sterile water to obtain 10 -2 times diluted bacterial suspension, and then pipette 10 -2 Add 9 mL of sterile water to obtain 10 -3times diluted bacterial suspension, and so on until it is diluted to 10 -9 times diluted bacterial suspension.

[0063] Diluted 10 -1 Up to 10 -9 Use a pipette to draw 1 mL of bacterial suspension into a sterile culture dish, then pour in the culture medium and mix evenly. Repeat the operation twice for each bottle of bacterial suspension. After solidification, turn the culture medium upside down and place it in a 30°C constant temperature incubator for culture.

[0064] Purification: According to the morphological characteristics of the initial colonies, use an inoculation loop to pick the "golden flower" colonies that do not contain other bacteria on the culture medium, inoculate them on the prepared culture medium, and culture them in a constant temperature fermentation box for 72 hours. Screen and purify according to the characteristics of the grown colonies, and repeat the above steps many times until there are no other bacteria on the colonies and the growth morphological characteristics are consistent.

[0065] 1.4 Determination of culture medium

[0066] Using the one-point culture method, the colonies of "Golden Flower" bacteria with good growth were picked with an inoculation loop and inoculated in the center of PDA medium, modified PDA medium, soluble starch medium, soluble starch tea medium and 40% sucrose peptone medium plates, and marked respectively. Inverted and placed in a 30℃ constant temperature incubator for culture, the color, shape and texture of the colonies were observed and recorded every day, and the colony diameter was measured.

[0067] 2. Experimental results

[0068] The growth of the "Golden Flower" fungus under five different culture media was studied. The growth rate of the strain on different culture media can be seen (Table 1), and the colony morphology of the strain on different culture media is shown in (Table 2). The results show that the strain can grow on culture media with different components, but the colony morphology will vary with the different components of the culture media. It can be seen that the composition of the culture medium has a certain influence on the growth of the strain, and the growth rate of the same strain on different culture media is different, but the colony morphology and color are roughly the same. After culturing to the sixth day, the strain grew fastest in 40% sucrose peptone culture medium. Therefore, it can be seen that the strain is conducive to mycelial growth in a culture medium with a high sugar content.

[0069] Table 1 Changes in colony diameter when strains were cultured on five media

[0070]

[0071] Table 2 Effects of different culture media on bacterial growth morphology

[0072]

[0073]

[0074] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0075] The above is only an embodiment of the present invention. The common sense such as the known specific structure and characteristics in the scheme is not described in detail here. The ordinary technicians in the relevant field know all the common technical knowledge in the technical field of the invention before the application date or priority date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical known structures or known methods should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, several deformations and improvements can be made, which should also be regarded as the protection scope of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing vacuum freeze-dried powder based on golden flower fungus, characterized in that: The following steps are involved: Step 1, preparation of sterile water and culture dishes: put purified water into a glass bottle and seal it, then put it into a high pressure sterilizer for sterilization to obtain sterile water; use a detergent to clean the culture dishes and let them dry naturally, then wrap the dried culture dishes in newspapers in groups of ten, and then put them into a high pressure sterilizer for sterilization to obtain sterile culture dishes; Step 2, preparation of golden flower fungus suspension: weigh 5g of Sanhe Golden Flower Liubao tea sample and put it into sterile water to make 10 -1 times diluted bacterial suspension; then draw 25mL of 10 -1 Add the diluted bacterial suspension to 250 mL of sterile water to obtain 10 -2 times diluted bacterial suspension, and then pipette 10 -2 Add the diluted bacterial suspension to 9 mL of sterile water to obtain 10 -3 times diluted bacterial suspension, and so on to obtain 10 -9 times diluted bacterial suspension; Step 3, separation and purification of golden flower bacteria: 10 -1 Up to 10 -9 Use a pipette to draw 1 mL of the diluted bacterial suspension into a sterile culture dish, then pour into the PDA culture medium and mix evenly. Repeat the operation twice for each dilution of the diluted bacterial suspension. After solidification, turn the culture medium upside down and place it in a 30°C constant temperature incubator for culture. According to the morphological characteristics of the initial colonies, the colonies of the golden flower fungus that do not contain other bacteria are picked up on the culture medium with an inoculation loop, and inoculated on the PDA culture medium prepared in advance, and then placed in a constant temperature fermentation box for 72 hours. Screening and purification are carried out according to the characteristics of the grown golden flower fungus colonies. The above steps are repeated many times until the golden flower fungus colonies do not contain other bacteria and the growth morphological characteristics are consistent, and purified golden flower fungus colonies are obtained; Step 4, preparation of freeze-dried bacterial powder: adding freeze-drying protective agents to the prepared golden flower bacteria colonies respectively, and then placing them in a segmented vacuum freeze-drying mechanism for vacuum freeze-drying, and obtaining freeze-dried bacterial powder after vacuum freeze-drying; Step 5, freeze-dried bacterial powder activity test: weigh 1g of freeze-dried bacterial powder and place it in 9ml of sterile water, shake it evenly, and get 10 -1 times freeze-dried bacterial suspension; use a pipette to draw 1 mL of 10 -1 Add 9 mL of sterile water to make 10 times the freeze-dried bacterial suspension. -2 times freeze-dried bacterial suspension, and then draw 1mL of 10 -2 Add 9 mL of sterile water to make 10 times the freeze-dried bacterial suspension. -3 times freeze-dried bacterial suspension; 10 -1 Up to 10 -3 Use a pipette to draw 1 mL of the freeze-dried bacterial suspension of each times and add it into a sterile culture dish, then pour in 40% sucrose peptone culture medium and mix evenly. Repeat the operation twice for each freeze-dried bacterial suspension. After solidification, invert the culture medium, place it in a constant temperature incubator at 30℃ and culture it for 72 hours, and count the number of live colonies.

2. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 1, characterized in that: In step 1, the sterilization method is to sterilize in an autoclave at 121°C for 20 minutes.

3. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 2, characterized in that: In step 2, the static method is to put 5g of Sanhe Jinhua Liubao tea sample into 300mL of sterile water, shake it evenly, and then put it on the clean bench to stand for 30 minutes.

4. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 3, characterized in that: In step three, the preparation method of PDA culture medium is as follows: peel 200.0g of potatoes, put them into 1000mL of pure water and boil them for 10min, filter them with gauze, then add 20.0g of glucose and 20.0g of agar and stir to dissolve, after stirring and dissolving, add sterile water to make up to 1000mL, and then put them into a high pressure sterilizer and sterilize them at 121°C for 20min.

5. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 4, characterized in that: In step 4, the freeze-drying protective agent is 1 mL of 5% sucrose by mass, and the vacuum freeze-drying method is pre-freezing at -20°C for 2 to 3 hours, and then vacuum freeze-drying for 42 hours.

6. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 5, characterized in that: In step 5, the preparation method of 40% sucrose peptone medium is as follows: 400.0 g sucrose, 10.0 g peptone and 15.0 g sodium chloride NaCl are stirred and dissolved with purified water, and then purified water is added to make up to 1000 mL after stirring and dissolving, and then placed in a high pressure sterilizer for sterilization at 121° C. for 20 min.

7. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 6, characterized in that: In step 5, the number of live bacteria in the freeze-dried bacterial colonies is determined by a plate count method.

8. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 7, characterized in that: In step four, the segmented vacuum freeze-drying mechanism includes a freeze-drying box with an opening, a cover plate is hinged on one side of the opening of the freeze-drying box, a handle is fixedly connected to the outside of the cover plate, and a number of support rods are fixedly connected to the bottom of the freeze-drying box; a number of slide grooves are opened inside the freeze-drying box, and trays are slidably fitted in the slide grooves; a spiral check valve is connected to the top of the freeze-drying box, and the spiral check valve includes a number of U-shaped connecting pipes, which are interconnected, and the opening of the U-shaped connecting pipe faces the output end of the spiral check valve; the output end of the spiral check valve is connected to a vacuum component for extracting air from the freeze-drying box; the side of the freeze-drying box away from the cover plate is connected to a refrigeration component for providing cold air.

9. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 8, characterized in that: The vacuum assembly includes a vacuum pump and a controller. The input end of the vacuum pump is connected to the output end of the spiral check valve. The output end of the vacuum pump is connected to a vent. The vacuum pump is fixedly connected to the top of the freeze drying chamber. The controller is used to control the operation of the vacuum pump. The controller signal is connected to a vacuum gauge, and the vacuum gauge is fixedly connected to the inner wall of the freeze drying chamber.

10. The method for preparing vacuum freeze-dried powder based on golden flower fungus according to claim 9, characterized in that: The refrigeration components include a compressor, a microchannel condenser, a PCM evaporator, and an expansion valve; The output end of the compressor is connected to the microchannel condenser, and the compressor is fixedly connected to the outer side of the bottom of the freeze drying chamber; the output end of the microchannel condenser is connected to the expansion valve, and the microchannel condenser is fixedly connected to the side of the freeze drying chamber away from the opening; the output end of the expansion valve is connected to the PCM evaporator, and the PCM evaporator includes a number of interconnected circulation pipes, which are all located in the side wall of the freeze drying chamber in the length direction. The output end of the PCM evaporator is connected to the compressor, and the microchannel condenser is connected to an exhaust valve; the controller is used to control the operation of the compressor, and the controller signal is connected to a temperature sensor, and the temperature sensor is fixedly connected to the inner wall of the freeze drying chamber.

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