Method for separating strains from dried diced poria cocos
By using a 35℃ low-temperature drying method and precise sterilization and soaking, the problems of high inactivation rate and high transportation cost of Poria cocos strains during the drying and preservation process were solved, achieving efficient strain separation and preservation, improving the separation success rate and reducing the contamination rate.
Patent Information
- Application Number
- CN202511076724.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-01
- Publication Date
- 2025-12-12
AI Technical Summary
Existing technologies for isolating Poria cocos strains suffer from problems such as high strain inactivation rate, high transportation costs, and the inability to obtain effective strains outside the harvest season. In particular, excessively high temperatures during drying and preservation lead to strain inactivation, and there is a lack of optimized soaking parameters.
A four-pronged control method was adopted, including a 35℃ low-temperature drying temperature, a 1.5cm³ size, 30-60s sterilization, and 30-60min soaking. This method encompassed fresh Poria cocos treatment, culture medium preparation, pre-inoculation preparation, and Poria cocos diced inoculation steps. By strictly controlling the temperature and time, the metabolic activity of the dried strain was activated.
It has enabled the dried strains to be stored at room temperature for six months, increasing the isolation success rate to 95% and reducing the contamination rate to below 5%. It has replaced the cold chain system for fresh Poria cocos and improved the preservation and transportation efficiency of the strains.
Smart Images
Figure CN121109142A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bacterial strain isolation technology, and more specifically, to a method for isolating bacterial strains from dried Poria cocos. Background Technology
[0002] In the field of edible and medicinal fungi, the isolation of *Wolfiporia cocos* strains has long relied on direct inoculation of fresh *Wolfiporia* tissue blocks. This method requires inoculation to be completed within a short period after harvesting; otherwise, the internal tissues of the sclerotia will become inactive. Furthermore, the entire process requires refrigerated transport at 4°C, and the cost accounts for more than 30% of the total production input. In addition, the concentrated production areas of *Wolfiporia* mean that effective strains cannot be obtained outside the harvest season. Existing technologies attempt to preserve the strains by drying; however, drying also has fatal technical drawbacks, such as drying temperatures exceeding 50°C leading to strain inactivation, and the lack of optimized soaking parameters reducing the strain recovery rate. Summary of the Invention
[0003] The purpose of this invention is to provide a method for isolating bacterial strains from dried Poria cocos, which can be achieved by drying at a low temperature of 35°C with a temperature of +1.5cm. 3 The four-fold control of specifications, 30-60s sterilization, and 30-60min soaking can activate the metabolic activity of dried microorganisms.
[0004] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a method for isolating bacterial strains from dried Poria cocos, comprising the following steps:
[0005] S1. Fresh Poria cocos processing and drying;
[0006] S2. Prepare the culture medium;
[0007] S3. Preparations before vaccination;
[0008] S4. Poria cocos inoculation.
[0009] The present invention is further configured as follows: the specific operation steps of processing and drying fresh Poria cocos in S1 are as follows: the surface of the collected fresh Poria cocos sclerotia is rinsed clean with pure water, the surface of the sclerotia is wiped with a cotton ball soaked in 75% alcohol, the surface of the Poria cocos sclerotia is rinsed twice again with sterile pure water, the surface moisture is absorbed with sterile paper towels, the outer skin of the sclerotia is cut open with pre-sterilized scissors and knife, and several Poria cocos cubes with a specification of 1.5cm×1.5cm are cut from the center of the inside. The Poria cocos cubes are then placed in an envelope bag and dried at a low temperature of 35°C. The temperature is strictly controlled during the process until the Poria cocos cubes are completely dry.
[0010] The present invention is further configured as follows: the specific steps for preparing the culture medium in S2 are as follows: Prepare a PDA culture medium with 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water, pH 6.5–7.0. Place the well-mixed culture medium in an Erlenmeyer flask and sterilize at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0011] The present invention is further configured as follows: the specific operation steps for the preparation before inoculation in S3 are as follows: the tools to be placed in the ultra-clean workbench include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are placed, the ultraviolet lamp is turned on for sterilization.
[0012] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0013] The present invention is further configured as follows: the specific operation steps for inoculating Poria cocos in S4 are as follows: Soak the Poria cocos pieces in a beaker containing 75% alcohol for 30-60 seconds. After the required soaking time, use sterile forceps to remove them from the alcohol and rinse them in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After rinsing three times, place the Poria cocos pieces on absorbent paper and drain for 1 minute. Soak the Poria cocos pieces in a beaker containing sterile water for 30-60 minutes. After the required soaking time, use sterile forceps to remove them from the sterile water and drain them on absorbent paper for 1 minute. Then, use sterile forceps to inoculate them into a PDA culture medium plate and seal with a sealing film to complete the inoculation. The forceps must be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0014] In summary, the present invention has the following beneficial effects:
[0015] 1. Provides a method for isolating dried Poria cocos with diced bacteria, enabling storage at room temperature for 6 months, replacing the cold chain system for fresh Poria cocos.
[0016] 2. By using 35℃ low-temperature drying (avoiding protein denaturation) and 30 minutes of sterile water soaking (precise restoration of osmotic pressure), the separation success rate is increased to 95%.
[0017] 3. Quantify 1.5cm 3 Poria cocos cubes (optimal surface area to volume ratio 0.67mm²) -1 The surface is sterilized with 75% alcohol for 30-60 seconds and the foaming window is 30-60 minutes (shorter than the growth cycle of other bacteria), reducing the contamination rate to <5%. Attached Figure Description
[0018] Figure 1 The images show 1.5cm×1.5cm Poria cocos cubes grown for the same time in the embodiments of the present invention. The left image shows surface sterilization for 15s and soaking in water for 15min, while the right image shows surface sterilization for 30s and soaking in water for 30min.
[0019] Figure 2 The images are 1cm×1cm Poria cocos pieces grown for the same time in the embodiments of the present invention. The left image shows surface sterilization for 15s and soaking in water for 15min, while the right image shows surface sterilization after soaking in water for 60s and 60min.
[0020] Figure 3 In this embodiment of the invention, surface sterilization for 15 seconds and soaking in water for 15 minutes are used for growth at the same time. The left side shows a size of 1cm×1cm, and the right side shows a size of 1.5cm×1.5cm. Detailed Implementation
[0021] The following is in conjunction with the appendix Figure 1-3 The present invention will be described in further detail below.
[0022] Example 1
[0023] 1. Processing and drying of fresh Poria cocos
[0024] Rinse the collected fresh Poria cocos sclerotia with pure water to clean the surface. Wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol. Rinse the surface of the Poria cocos sclerotia twice with sterile pure water. Use sterile paper towels to absorb the surface moisture. Take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia. Cut out several Poria cocos cubes with a size of 1.5cm × 1.5cm from the center of the inside. Then place the Poria cocos cubes in an envelope and dry them at a low temperature of 35℃. Strictly control the temperature during the process until the Poria cocos cubes are completely dry.
[0025] 2. Culture medium preparation
[0026] Prepare a PDA medium with pH 6.5–7.0 using 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water. Sterilize the mixed medium in an Erlenmeyer flask at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0027] 3. Preparation before vaccination
[0028] The tools to be placed in the clean bench beforehand include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are in place, turn on the UV lamp for sterilization.
[0029] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0030] 4. Poria cocos inoculation
[0031] Soak the diced Poria cocos in a beaker containing 75% alcohol for 30 seconds. After the soaking time, use sterile forceps to remove it from the alcohol and rinse it in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After rinsing three times, place the Poria cocos diced Poria cocos on absorbent paper and let it drain for 1 minute. Soak the Poria cocos diced Poria cocos in a beaker containing sterile water for 30 minutes. After the soaking time, use sterile forceps to remove it from the sterile water and let it drain for 1 minute on sterile absorbent paper. Then, use sterile forceps to inoculate it into a PDA culture medium plate and seal it with sealing film to complete the inoculation. The forceps should be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0032] Example 2
[0033] 1. Processing and drying of fresh Poria cocos
[0034] Rinse the collected fresh Poria cocos sclerotia with pure water to clean the surface. Wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol. Rinse the surface of the Poria cocos sclerotia twice with sterile pure water. Use sterile paper towels to absorb the surface moisture. Take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia. Cut out several Poria cocos cubes with a size of 1.5cm × 1.5cm from the center of the inside. Then place the Poria cocos cubes in an envelope and dry them at a low temperature of 35℃. Strictly control the temperature during the process until the Poria cocos cubes are completely dry.
[0035] 2. Culture medium preparation
[0036] Prepare a PDA medium with pH 6.5–7.0 using 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water. Sterilize the mixed medium in an Erlenmeyer flask at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0037] 3. Preparation before vaccination
[0038] The tools to be placed in the clean bench beforehand include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are in place, turn on the UV lamp for sterilization.
[0039] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0040] 4. Poria cocos inoculation
[0041] Soak the diced Poria cocos in a beaker containing 75% alcohol for 30 seconds. After the soaking time, use sterile forceps to remove it from the alcohol and rinse it in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After rinsing three times, place the Poria cocos diced Poria cocos on absorbent paper and let it drain for 1 minute. Soak the Poria cocos diced Poria cocos in a beaker containing sterile water for 60 minutes. After the soaking time, use sterile forceps to remove it from the sterile water and let it drain for 1 minute on sterile absorbent paper. Then, use sterile forceps to inoculate it into a PDA culture medium plate and seal it with sealing film to complete the inoculation. The forceps should be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0042] Example 3
[0043] 1. Processing and drying of fresh Poria cocos
[0044] Rinse the collected fresh Poria cocos sclerotia with pure water to clean the surface. Wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol. Rinse the surface of the Poria cocos sclerotia twice with sterile pure water. Use sterile paper towels to absorb the surface moisture. Take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia. Cut out several Poria cocos cubes with a size of 1.5cm × 1.5cm from the center of the inside. Then place the Poria cocos cubes in an envelope and dry them at a low temperature of 35℃. Strictly control the temperature during the process until the Poria cocos cubes are completely dry.
[0045] 2. Culture medium preparation
[0046] Prepare a PDA medium with pH 6.5–7.0 using 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water. Sterilize the mixed medium in an Erlenmeyer flask at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0047] 3. Preparation before vaccination
[0048] The tools to be placed in the clean bench beforehand include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are in place, turn on the UV lamp for sterilization.
[0049] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0050] 4. Poria cocos inoculation
[0051] Soak the diced Poria cocos in a beaker containing 75% alcohol for 60 seconds. After the soaking time, use sterile forceps to remove it from the alcohol and rinse it in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After rinsing three times, place the Poria cocos diced Poria cocos on absorbent paper and let it drain for 1 minute. Soak the Poria cocos diced Poria cocos in a beaker containing sterile water for 30 minutes. After the soaking time, use sterile forceps to remove it from the sterile water and let it drain for 1 minute on sterile absorbent paper. Then, use sterile forceps to inoculate it into a PDA culture medium plate and seal it with sealing film to complete the inoculation. The forceps should be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0052] Example 4
[0053] 1. Processing and drying of fresh Poria cocos
[0054] Rinse the collected fresh Poria cocos sclerotia with pure water to clean the surface. Wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol. Rinse the surface of the Poria cocos sclerotia twice with sterile pure water. Use sterile paper towels to absorb the surface moisture. Take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia. Cut out several Poria cocos cubes with a size of 1.5cm × 1.5cm from the center of the inside. Then place the Poria cocos cubes in an envelope and dry them at a low temperature of 35℃. Strictly control the temperature during the process until the Poria cocos cubes are completely dry.
[0055] 2. Culture medium preparation
[0056] Prepare a PDA medium with pH 6.5–7.0 using 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water. Sterilize the mixed medium in an Erlenmeyer flask at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0057] 3. Preparation before vaccination
[0058] The tools to be placed in the clean bench beforehand include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are in place, turn on the UV lamp for sterilization.
[0059] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0060] 4. Poria cocos inoculation
[0061] Soak the diced Poria cocos in a beaker containing 75% alcohol for 60 seconds. After the soaking time, use sterile forceps to remove it from the alcohol and rinse it in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After three rinses, place the Poria cocos diced Poria cocos on absorbent paper and let it drain for 1 minute. Soak the Poria cocos diced Poria cocos in a beaker containing sterile water for 60 minutes. After the soaking time, use sterile forceps to remove it from the sterile water and let it drain for 1 minute on sterile absorbent paper. Then, use sterile forceps to inoculate it into a PDA culture medium plate and seal it with sealing film to complete the inoculation. The forceps should be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0062] Example 5
[0063] 1. Processing and drying of fresh Poria cocos
[0064] Rinse the collected fresh Poria cocos sclerotia with pure water to clean the surface. Wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol. Rinse the surface of the Poria cocos sclerotia twice with sterile pure water. Use sterile paper towels to absorb the surface moisture. Take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia. Cut out several Poria cocos cubes with a size of 1cm×1cm from the center of the inside. Then place the Poria cocos cubes in an envelope and dry them at a low temperature of 35℃. Strictly control the temperature during the process until the Poria cocos cubes are completely dry.
[0065] 2. Culture medium preparation
[0066] Prepare a PDA medium with pH 6.5–7.0 using 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water. Sterilize the mixed medium in an Erlenmeyer flask at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0067] 3. Preparation before vaccination
[0068] The tools to be placed in the clean bench beforehand include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are in place, turn on the UV lamp for sterilization.
[0069] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0070] 4. Poria cocos inoculation
[0071] Soak the diced Poria cocos in a beaker containing 75% alcohol for 30 seconds. After the soaking time, use sterile forceps to remove it from the alcohol and rinse it in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After rinsing three times, place the Poria cocos diced Poria cocos on absorbent paper and let it drain for 1 minute. Soak the Poria cocos diced Poria cocos in a beaker containing sterile water for 30 minutes. After the soaking time, use sterile forceps to remove it from the sterile water and let it drain for 1 minute on sterile absorbent paper. Then, use sterile forceps to inoculate it into a PDA culture medium plate and seal it with sealing film to complete the inoculation. The forceps should be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0072] Example 6
[0073] 1. Processing and drying of fresh Poria cocos
[0074] Rinse the collected fresh Poria cocos sclerotia with pure water to clean the surface. Wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol. Rinse the surface of the Poria cocos sclerotia twice with sterile pure water. Use sterile paper towels to absorb the surface moisture. Take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia. Cut out several Poria cocos cubes with a size of 1cm×1cm from the center of the inside. Then place the Poria cocos cubes in an envelope and dry them at a low temperature of 35℃. Strictly control the temperature during the process until the Poria cocos cubes are completely dry.
[0075] 2. Culture medium preparation
[0076] Prepare a PDA medium with pH 6.5–7.0 using 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water. Sterilize the mixed medium in an Erlenmeyer flask at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0077] 3. Preparation before vaccination
[0078] The tools to be placed in the clean bench beforehand include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are in place, turn on the UV lamp for sterilization.
[0079] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0080] 4. Poria cocos inoculation
[0081] Soak the diced Poria cocos in a beaker containing 75% alcohol for 30 seconds. After the soaking time, use sterile forceps to remove it from the alcohol and rinse it in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After rinsing three times, place the Poria cocos diced Poria cocos on absorbent paper and let it drain for 1 minute. Soak the Poria cocos diced Poria cocos in a beaker containing sterile water for 60 minutes. After the soaking time, use sterile forceps to remove it from the sterile water and let it drain for 1 minute on sterile absorbent paper. Then, use sterile forceps to inoculate it into a PDA culture medium plate and seal it with sealing film to complete the inoculation. The forceps should be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0082] Example 7
[0083] 1. Processing and drying of fresh Poria cocos
[0084] Rinse the collected fresh Poria cocos sclerotia with pure water to clean the surface. Wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol. Rinse the surface of the Poria cocos sclerotia twice with sterile pure water. Use sterile paper towels to absorb the surface moisture. Take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia. Cut out several Poria cocos cubes with a size of 1cm×1cm from the center of the inside. Then place the Poria cocos cubes in an envelope and dry them at a low temperature of 35℃. Strictly control the temperature during the process until the Poria cocos cubes are completely dry.
[0085] 2. Culture medium preparation
[0086] Prepare a PDA medium with pH 6.5–7.0 using 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water. Sterilize the mixed medium in an Erlenmeyer flask at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0087] 3. Preparation before vaccination
[0088] The tools to be placed in the clean bench beforehand include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are in place, turn on the UV lamp for sterilization.
[0089] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0090] 4. Poria cocos inoculation
[0091] Soak the diced Poria cocos in a beaker containing 75% alcohol for 60 seconds. After the soaking time, use sterile forceps to remove it from the alcohol and rinse it in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After rinsing three times, place the Poria cocos diced Poria cocos on absorbent paper and let it drain for 1 minute. Soak the Poria cocos diced Poria cocos in a beaker containing sterile water for 30 minutes. After the soaking time, use sterile forceps to remove it from the sterile water and let it drain for 1 minute on sterile absorbent paper. Then, use sterile forceps to inoculate it into a PDA culture medium plate and seal it with sealing film to complete the inoculation. The forceps should be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0092] Example 8
[0093] 1. Processing and drying of fresh Poria cocos
[0094] Rinse the collected fresh Poria cocos sclerotia with pure water to clean the surface. Wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol. Rinse the surface of the Poria cocos sclerotia twice with sterile pure water. Use sterile paper towels to absorb the surface moisture. Take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia. Cut out several Poria cocos cubes with a size of 1cm×1cm from the center of the inside. Then place the Poria cocos cubes in an envelope and dry them at a low temperature of 35℃. Strictly control the temperature during the process until the Poria cocos cubes are completely dry.
[0095] 2. Culture medium preparation
[0096] Prepare a PDA medium with pH 6.5–7.0 using 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4, and 1000mL water. Sterilize the mixed medium in an Erlenmeyer flask at 121℃ for 20 minutes. After sterilization, pour the mixture into 9cm diameter plates for inoculation.
[0097] 3. Preparation before vaccination
[0098] The tools to be placed in the clean bench beforehand include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are in place, turn on the UV lamp for sterilization.
[0099] After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
[0100] 4. Poria cocos inoculation
[0101] Soak the diced Poria cocos in a beaker containing 75% alcohol for 60 seconds. After the soaking time, use sterile forceps to remove it from the alcohol and rinse it in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After three rinses, place the Poria cocos diced Poria cocos on absorbent paper and let it drain for 1 minute. Soak the Poria cocos diced Poria cocos in a beaker containing sterile water for 60 minutes. After the soaking time, use sterile forceps to remove it from the sterile water and let it drain for 1 minute on sterile absorbent paper. Then, use sterile forceps to inoculate it into a PDA culture medium plate and seal it with sealing film to complete the inoculation. The forceps should be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.
[0102] The experimental results are as follows:
[0103] ① After drying Poria cocos at a low temperature of 35℃, sterilizing it with alcohol, and soaking it in water, it still retains its activity and can grow normally in the culture medium.
[0104] ② Comparing the same specifications of Poria cocos pieces treated with three surface sterilization times of 10s, 30s, and 60s, it was found that the Poria cocos pieces sterilized for 30s and 60s grew better than those sterilized for 10s.
[0105] ③ Comparing the same size Poria cocos pieces treated for 15 min, 30 min, and 60 min, it was found that the Poria cocos pieces soaked for 30 min and 60 min grew better than those soaked for 15 min.
[0106] ④ Comparing two different sizes of Poria cocos cubes, 1cm×1cm and 1.5cm×1.5cm, but with the same surface sterilization time and soaking time, it was found that the 1.5cm×1.5cm Poria cocos cubes grew better after surface sterilization and soaking than the 1cm×1cm Poria cocos cubes.
[0107] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.
Claims
1. A method for isolating bacterial strains from dried Poria cocos, characterized in that: Includes the following steps: S1. Fresh Poria cocos processing and drying; S2. Prepare the culture medium; S3. Preparations before vaccination; S4. Poria cocos inoculation.
2. The method for isolating bacterial strains from dried Poria cocos diced according to claim 1, characterized in that: The specific steps for processing and drying fresh Poria cocos in S1 are as follows: Rinse the surface of the collected fresh Poria cocos sclerotia with pure water, wipe the surface of the sclerotia with a cotton ball soaked in 75% alcohol, rinse the surface of the Poria cocos sclerotia twice with sterile pure water, use sterile paper towels to absorb the surface moisture, take out pre-sterilized scissors and a knife to cut open the outer skin of the sclerotia, cut out several Poria cocos cubes with a size of 1.5cm×1.5cm from the center of the inside, then place the Poria cocos cubes in an envelope bag and dry them at a low temperature of 35℃. The temperature is strictly controlled during the process until the Poria cocos cubes are completely dry.
3. The method for isolating bacterial strains from dried Poria cocos diced according to claim 1, characterized in that: The specific steps for preparing the culture medium in S2 are as follows: Prepare PDA culture medium with 200g potato, 20g glucose, 18g agar, 2g KH2PO4, 0.5g MgSO4 and 1000mL water, pH 6.5-7.0; place the well-mixed culture medium in an Erlenmeyer flask and sterilize at 121℃ for 20min. After sterilization, pour it into a 9cm diameter plate for inoculation.
4. The method for isolating bacterial strains from dried Poria cocos diced according to claim 1, characterized in that: The specific steps for preparing before inoculation in S3 are as follows: Tools to be placed in the ultra-clean workbench include an alcohol lamp, a lighter, a small blue bottle filled with alcohol, a marker, PDA culture medium, and sealing film (to minimize contamination after inoculation). After the tools are placed, turn on the ultraviolet lamp for sterilization. After sterilizing the tweezers, absorbent paper, pure water, and beaker at 121℃ for 20 minutes, place them in a clean bench with UV light pre-activated until the pure water cools to room temperature. Finally, turn off the UV light and place the poria cocos pieces to be inoculated into the workbench.
5. The method for isolating bacterial strains from dried Poria cocos diced according to claim 1, characterized in that: The specific steps for inoculating Poria cocos in S4 are as follows: Soak Poria cocos pieces in a beaker containing 75% alcohol for 30-60 seconds. After the required soaking time, use sterile tweezers to remove them from the alcohol and rinse them in a beaker containing sterile water. After rinsing, pour out the water and rinse twice more with sterile water. After rinsing three times, place the Poria cocos pieces on absorbent paper and let them drain for 1 minute. Soak the Poria cocos pieces in a beaker containing sterile water for 30-60 minutes. After the required soaking time, use sterile tweezers to remove them from the sterile water and let them drain on absorbent paper for 1 minute. Then, use sterile tweezers to inoculate them into a PDA culture medium plate and seal with a sealing film to complete the inoculation. The tweezers must be soaked in alcohol after each inoculation and sterilized and cooled over an alcohol lamp before reuse.