Low-sugar-consumption acid-resistant trametes robiniophila and application thereof

Abstract

The invention discloses a strain of low-sugar-consumption acid-resistant trametes robiniophila and application thereof.The strain of low-sugar-consumption acid-resistant trametes robiniophila is classified and named as trametes robiniophila HER-ZST-2023, the trametes robiniophila HER-ZST-2023 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 25, 2024, and the preservation number is CGMCC NO.41347. The strain of low-sugar-consumption acid-resistant trametes robiniophila has the advantages that the strain of low-sugar-consumption acid-resistant trametes robiniophila is high in yield and high in acid resistance; the low-sugar-consumption acid-resistant trametes robiniophila is applied to fermentation preparation of trametes robiniophila mycelia. The low-sugar-consumption acid-resistant trametes robiniophila can grow well in a low-sugar-consumption environment, the range of available culture medium raw materials is wider, the acid resistance is high, the trametes robiniophila can grow at a low pH value, the final hypha content can be kept, and the situation that growth is affected by acidic materials generated in the natural acidification or fermentation process of a culture medium is avoided; the pressure caused by environmental change can be resisted to a certain extent, stable growth and metabolism are maintained, the smooth proceeding of the fermentation process is guaranteed, and the risk of fermentation failure caused by environmental factors is reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a low sugar-consuming and acid-tolerant Trametes robiniophila Murr. and its application. Background Art

[0002] Trametes robiniophila Murr., also known as Tremella robiniophila, is a species in the genus Trametes of the fungal kingdom Polyporaceae. This strain mainly grows on the trunks of broad-leaved trees such as Sophora japonica and Robinia pseudoacacia, has relatively strict requirements for the growth environment, grows slowly, and is distributed in Hebei, Shaanxi, Liaoning, Hunan, Guangxi, Fujian and other places.

[0003] The polysaccharide components in Tremella robiniophila have anti-inflammatory and antibacterial effects, can inhibit inflammatory reactions and kill pathogens; its various active ingredients such as triterpenoids have an inhibitory effect on the growth of cancer cells and can be used for the adjuvant treatment of malignant tumors such as lung cancer and gastric cancer; it can stimulate the immune system to produce corresponding antibodies, improve the body's immunity, and is suitable for people who are weak and have loss of appetite after surgery or radiotherapy and chemotherapy; it has a certain effect on improving liver function, can reduce the inflammatory reaction and fibrosis process of the liver, and reduce the increase in transaminase caused by diseases such as chronic hepatitis and fatty liver.

[0004] Wild Tremella robiniophila resources are scarce, artificial cultivation is difficult, the growth cycle is long, and the biological effect is low. At present, the solid fermentation method is mainly used to produce the mycelium of Tremella robiniophila, and the liquid culture method is used to produce the mycelium. The pH value of the fermentation broth will decrease in the middle and late stages of the liquid fermentation of Tremella robiniophila. In order to increase the enrichment amount of the mycelium and obtain more mycelium, calcium carbonate needs to be added to the initial medium to neutralize the acid production. However, by neutralizing with calcium carbonate, the pH can only be controlled not lower than 5.0, while the optimal pH value for the growth of Tremella robiniophila is 6.0 - 6.5. In addition, excessive calcium ions added initially will inhibit the growth of the mycelium and affect the final fermentation effect. By adding substances such as calcium hydroxide and sodium hydroxide during the middle stage of fermentation, the pH value of the fermentation broth in the middle and late stages of fermentation can also be controlled. However, the fermentation cycle of Tremella robiniophila is relatively long, and the addition method will greatly increase the risk of bacterial contamination. In summary, it is necessary to select a strain that can grow at a lower pH value and maintain the final mycelium content. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a low sugar-consuming and acid-tolerant Trametes robiniophila Murr., which has good tolerance to low sugar consumption and acidic environment, can resist the pressure brought by environmental changes to a certain extent, maintain stable growth and metabolism, ensure the smooth progress of the fermentation process, and reduce the risk of fermentation failure caused by environmental factors.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A low-sugar and acid-tolerant Trametes robiniophila strain, named Trametes robiniophila HER-ZST-2023, was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on June 25, 2024. The address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is: CGMCC NO. 41347.

[0008] The strain uses the strain with the deposit number of CGMCC.NO.0259 as the starting strain to cultivate fruiting bodies, and then separates and purifies the hyphae in the fruiting bodies. The obtained Trametes robiniophila hyphae are respectively inoculated on slants. After culturing for 7 days, the hyphae are picked from the slants and inoculated into a triangular flask liquid medium for culture. After culturing for 9 to 15 days, the morphology of the fungal balls is observed and the hypha content is measured. Comprehensively select a bottle of seed liquid with rapid growth, large and uniform fungal balls, and the highest hypha content when cultured for 15 days as the initial strain for the next round of purification and screening of bacteria. Repeat multiple times until a strain with good production conditions and uniform hypha morphology is selected; starting from this strain, reduce the carbon source in the medium to breed low sugar-consuming strains, and then use the ultraviolet mutagenesis method to obtain low sugar-consuming and acid-tolerant strains.

[0009] The screened low sugar-consuming and acid-tolerant strain is inoculated on a slant into a liquid medium and cultured in the dark at 28°C and 190 rpm for 9 days. After the culture is completed, the morphology of the fungal balls is observed and recorded, and the hypha content is measured. The fungal balls are uniform and dense, white in color, with a diameter of about 5 - 8 mm. The fungal liquid is light yellow and clear, with viscosity. There is no layering after standing for 10 min, and the hypha content exceeds 15 g / L. More preferably, the hypha content exceeds 20 g / L.

[0010] Application of the low sugar-consuming and acid-tolerant Trametes robiniophila strain in the fermentation preparation of Trametes robiniophila mycelium.

[0011] The low sugar-consuming and acid-tolerant Trametes robiniophila strain described in the present invention can grow well in a low sugar-consuming environment, and the range of available culture medium raw materials is wider, including but not limited to potato whole powder, wheat bran, soybean powder, glucose, maltose, soluble starch, malt extract powder, peptone, yeast powder, soybean meal powder, corn flour, etc. as culture medium components, which not only reduces costs but also realizes the effective utilization of resources.

[0012] The strain has strong acid tolerance and can grow at a lower pH value and maintain the final hypha content, avoiding the influence on growth due to the natural acidification of the culture medium or the generation of acidic substances during the fermentation process; after the pH value of the fermentation broth drops in the middle and late stages of fermentation, there is no need to add substances such as calcium hydroxide and sodium hydroxide to regulate the pH value of the fermentation broth, and it still maintains activity and growth ability. The range of lower pH values that this strain can tolerate is: it can grow and ferment normally in a culture medium with a pH not less than 3.5, and the fermentation ability has no obvious difference from that under the condition of pH 6.0.

[0013] Inoculate the Trametes robiniophila Murr. as described above into a nutrient medium and culture it in the dark at 27-30°C for 9-22 days.

[0014] The nutrient medium includes one or more of potato flour, wheat bran, soybean powder, glucose, maltose, soluble starch, malt extract powder, peptone, yeast powder, soybean meal powder, corn flour, potassium dihydrogen phosphate, magnesium sulfate, and thiamine. Its composition and reasonable proportion are determined according to the mycelium content and polysaccharide content in the product.

[0015] The nutrient medium can be, for example, potato flour 16 g / L, glucose 1 g / L, potassium dihydrogen phosphate 0.3 g / L, magnesium sulfate 0.2 g / L, with natural pH; or: wheat bran 10 g / L, soybean powder 2 g / L, maltose 1 g / L, soluble starch 1 g / L, with natural pH; or: malt extract powder 20 g / L, glucose 1 g / L, peptone 2 g / L, yeast powder 2 g / L, with natural pH; or: soybean meal powder 20 g / L, corn flour 5 g / L, glucose 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, thiamine 0.05 g / L, with natural pH; or: soybean meal powder 20 g / L, corn flour 20 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, thiamine 0.05 g / L, with natural pH.

[0016] The inoculation amount of Trametes robiniophila Murr. is 1-10%, preferably 5%.

[0017] A kind of Tremella aurantialba mycelium, which is obtained by fermenting the above-mentioned Trametes robiniophila Murr. with low glucose consumption and acid tolerance. The liquid fermentation method is preferably used, and the purpose of fermentation is to enrich the Tremella aurantialba mycelium, and the intracellular polysaccharide content of the Tremella aurantialba mycelium is relatively high at the end of fermentation.

[0018] The mycelium content in the Tremella aurantialba mycelium is not less than 15 g / L, and the mycelium polysaccharide content is not less than 2.5%. More preferably, the mycelium content in the Tremella aurantialba mycelium is not less than 20 g / L, and the mycelium polysaccharide content is not less than 3.0%.

[0019] The strain of the present invention can, under simple fermentation conditions, by means of liquid fermentation, enrich the Tremella aurantialba mycelium, and at the same time ensure that the polysaccharide content in the mycelium remains at a relatively high level at the end of fermentation. After liquid fermentation, the mycelium is directly used as medicine in certain preparation forms after adding a certain amount of auxiliary materials.

[0020] The preparations include but are not limited to various solid dosage forms, semi-solid dosage forms, liquid dosage forms, gas dosage forms, etc.; for example: solid dosage forms such as tablets, capsules, granules, powders, pills, etc., semi-solid dosage forms such as ointments, suppositories, pastes, etc., liquid dosage forms such as aromatic waters, solutions, injections, mixtures, lotions, liniments, etc., and gas dosage forms such as aerosols, sprays, etc.

[0021] Use of the Tremella aurantialba mycelium in the preparation of a medicament for treating or adjuvantly treating tumors.

[0022] The Tremella aurantialba mycelium can improve symptoms such as liver region pain, abdominal distension, and fatigue, and can be used as an adjuvant therapeutic drug for patients with primary liver cancer who are not suitable for surgery and chemotherapy. On the basis of standard chemical drug anti-cancer treatment, it can be used for symptoms such as fatigue, lack of qi and reluctance to speak, abdominal pain or distension, poor appetite, dry or loose stools, shortness of breath, cough, excessive phlegm, pale complexion, chest pain, blood in sputum, and discomfort in the chest and hypochondrium caused by lung cancer, gastrointestinal cancer, and breast cancer, improving the quality of life of patients.

[0023] Beneficial effects:

[0024] The present invention provides a Tremella aurantialba strain with low sugar tolerance and acid tolerance, which has good tolerance to low sugar and acidic environments, can resist the stress brought about by environmental changes to a certain extent, maintain stable growth and metabolism, ensure the smooth progress of the fermentation process, and reduce the risk of fermentation failure caused by environmental factors. Using the strain in the present invention, the contents of Tremella aurantialba mycelium and Tremella aurantialba polysaccharide are effectively increased during liquid fermentation. Under a low sugar acidic culture medium and appropriate fermentation parameters, the content of Tremella aurantialba mycelium is stably increased from 15 g / L to 20 g / L. At the end of fermentation, the content of Tremella aurantialba polysaccharide that can be extracted from the Tremella aurantialba mycelium can be stably increased from 1.3% to more than 3.0%. Specific implementation manners

[0025] Example 1 Screening of the strain

[0026] I. Obtaining high-quality Tremella aurantialba fruit bodies:

[0027] 1. Starting strain: Obtained from the China General Microbiological Culture Collection Center (CGMCC)

[0028] The strain number is: CGMCC.NO.0259.

[0029] 2. Transfer the obtained strain to a slant of PDA medium and culture it in the dark at 28 °C for 9 days. The preparation method of PDA medium is: Cut 200 g of potatoes into small pieces (1 cm 3 ), add 1 liter of water and boil, then simmer for 30 min, filter to obtain the juice, add 20 g of glucose and 2 g of agar powder, make up the volume to 1000 ml with water and sterilize for standby.

[0030] 3. Select the slant culture with white and dense mycelium from the above-mentioned completed slant cultures, pick a mycelial block with a diameter of about 2 mm and inoculate it into a 1 L Erlenmeyer flask containing liquid medium. Incubate at 28 °C and 170 rpm for 9 - 15 days. The liquid medium in the Erlenmeyer flask is as follows: add 5.6 g of yeast powder, 20 g of glucose, 2.5 g of calcium carbonate, 1.5 g of potassium dihydrogen phosphate, 0.9 g of magnesium sulfate, and 0.1 g of thiamine per 1000 ml, with a natural pH.

[0031] 4. Inoculate the mycelium cultured in the Erlenmeyer flask into a solid culture flask. The medium in the solid culture flask is as follows: add 5.56 g of yeast powder, 5.56 g of glucose, 5.12 g of calcium carbonate, 231.9 g of corncob shaft, 309.3 g of wheat bran, and 442.4 g of water per 1000 g.

[0032] 5. Incubate the solid culture flask in the dark at 28 °C for 50 days.

[0033] 6. Prepare Sophora japonica tree trunks separately: Cut Sophora japonica tree trunks with a diameter of 10 - 20 cm and a length of about 20 cm, and drill several small holes with a diameter not exceeding 1 cm on the surface. Air-dry them in a dry place for 1 year. After air-drying, sterilize them with high-temperature steam at 121 °C for 1 hour and set aside.

[0034] 7. Take out the solid culture flask after the culture in step 5 in a clean environment and inoculate it onto the sterilized Sophora japonica tree trunks.

[0035] 8. Incubate the inoculated Sophora japonica tree trunks in the dark at 28 °C for 60 days.

[0036] 9. Shallowly bury the Sophora japonica tree trunks after the growth in step 8 into loose nutrient soil, and keep the humidity in the greenhouse greater than 80%.

[0037] When the natural temperature reaches about 25 °C, the fruiting bodies of Tremella aurantialba can grow.

[0038] 10. Pick the mature fruiting bodies of Tremella aurantialba and wait for separation.

[0039] II. Isolation and purification of mycelium in the fruiting body:

[0040] 1. Take the fruiting body, cut off the obviously contaminated part, soak it in 75% alcohol for 2 min after cleaning, and drain it after taking it out.

[0041] 2. Take the drained fruiting body and dry the surface with an alcohol lamp.

[0042] 3. Peel off the dried part on the surface, take a small piece of clean fruiting body and inoculate it on the surface of a petri dish.

[0043] 4. After the fruiting body is inoculated into the petri dish, once hyphae grow on the surface, immediately pick them out with an inoculation needle and inoculate them into a clean petri dish. If hyphae grow in the petri dish and there is suspected contamination at the edge, then pick out the Tremella fuciformis Berk. hyphae and inoculate them into a clean petri dish again. Repeat this process until clean Tremella fuciformis Berk. hyphae are obtained.

[0044] 5. Inoculate the obtained Tremella fuciformis Berk. hyphae onto slants respectively. After culturing for 7 days, pick the hyphae from the slants and inoculate them into a triangular flask liquid medium for culture. Observe the morphology of the fungal balls after culturing for 9 to 15 days and measure the hypha content. Comprehensively select a bottle of seed liquid with fast growth, large and uniform fungal balls, and the highest hypha content at 15 days of culture as the initial strain for the next round of purification and screening of bacteria. The triangular flask liquid medium is: add 5.6 g of yeast powder, 20 g of glucose, 2.5 g of calcium carbonate, 1.5 g of potassium dihydrogen phosphate, 0.9 g of magnesium sulfate, and 0.1 g of thiamine per 1000 ml, with natural pH.

[0045] 6. Select 10 fungal balls with larger and regular-shaped fungal balls from the strain obtained in the previous step. Place them in 100 ml of sterilized purified water and shake them on a vortex shaker for 5 min until the hyphae are dispersed.

[0046] 7. Take 1 ml of the dispersed hyphae and transfer them to 9 ml of sterilized purified water with a sterilized pipette to obtain a 10 -3 bacterial suspension. After shaking and mixing the 10 -3 bacterial suspension, take 1 ml and transfer it to 9 ml of sterilized purified water to obtain a 10 -4 bacterial suspension.

[0047] 8. Take 10 -3 uniform bacterial suspensions and 10 -4 bacterial suspensions and spread them on the surface of a PDA medium petri dish, and culture them in the dark at 28 °C for 7 days.

[0048] 9. Select several colonies with consistent colony morphology from the cultured petri dish, pick a small piece of bacterial mass with an inoculation needle respectively and inoculate it onto a PDA medium slant, and culture it in the dark at 28 °C for 7 - 9 days and then store it in a low-temperature refrigerator.

[0049] 10. Repeat steps 6 - 9 until a strain with good production conditions and uniform hypha morphology is selected.

[0050] Note: The preparation method of PDA is: Cut 200 g of potatoes into small pieces (1 cm3), add 1 liter of water and boil, then simmer for 30 min, filter to obtain the juice. Add 20 g of glucose and 2 g of agar powder, make up the volume to 1000 ml with water and sterilize for later use.

[0051] III. Breeding of low sugar-consuming strains

[0052] Selecting low sugar-consuming strains can reduce the amount of carbon source used, directly reduce production costs, and obtain higher production benefits. At the same time, reducing the carbon source in the culture medium can lower the residual sugar at the end of fermentation, and reduce the treatment pressure of tail waste in liquid fermentation. The steps for selecting low sugar-consuming strains are as follows:

[0053] 1. Use the strains obtained by the above-mentioned separation and purification as the starting strains.

[0054] 2. Inoculate the strains into liquid medium A and culture them at 28°C and 190 rpm for 9 days. A total of 6 parallel controls are prepared, with the glucose content remaining unchanged in two of them, and the glucose content gradually decreasing in the remaining bottles. After 9 days of culture, select the bottle of bacterial liquid with the best mycelial morphology and the highest mycelial content. After dilution and separation to obtain single mycelia, coat them on the low sugar-consuming petri dish medium and culture at 28°C for 5 days. Low sugar-consuming petri dish medium: Add 5 g of glycerol, 5 g of yeast powder, 5 g of peptone, 1 g of sodium nitrate, 1 g of dipotassium hydrogen phosphate, g of sodium chloride, 0.5 g of magnesium sulfate heptahydrate, 0.1 g of manganese sulfate monohydrate, 0.1 g of thiamine, and 20 g of agar powder to 1000 ml, and adjust the pH to 7.0. After sterilization, dispense into petri dishes.

[0055] 3. Select colonies with white mycelial color, dense mycelia, and an aerial mycelial height of about 1 mm from the petri dishes after 5 days of culture in step 2. Pick a small piece of the colony and inoculate it into liquid medium B, and inoculate two controls into liquid medium A in the same way. Culture at 28°C and 190 rpm for 9 days. After 9 days of culture, select the bottle of bacterial liquid with the best mycelial morphology and the highest mycelial content. After dilution and separation to obtain single mycelia, coat them on the low sugar-consuming petri dish medium and culture at 28°C for 5 days.

[0056] 4. Select colonies with white mycelial color, dense mycelia, and an aerial mycelial height of about 1 mm from the petri dishes after 5 days of culture in step 3. Pick a small piece of the colony and inoculate it into liquid medium C, and inoculate two controls into liquid medium A in the same way. Culture at 28°C and 190 rpm for 9 days. After 9 days of culture, select the bottle of bacterial liquid with the best mycelial morphology and the highest mycelial content. After dilution and separation to obtain single mycelia, coat them on the low sugar-consuming petri dish medium and culture at 28°C for 5 days.

[0057] 5. Select colonies with white mycelial color, dense mycelia, and an aerial mycelial height of about 1 mm from the petri dishes after 5 days of culture in step 4. Pick a small piece of the colony and inoculate it into liquid medium D, and inoculate two controls into liquid medium A in the same way. Culture at 28°C and 190 rpm for 11 days. After 11 days of culture, select the bottle of bacterial liquid with the best mycelial morphology and the highest mycelial content. After dilution and separation to obtain single mycelia, coat them on the low sugar-consuming petri dish medium and culture at 28°C for 5 days.

[0058] 6. Select colonies with white mycelium color, dense mycelium, and aerial mycelium height of about 1 mm from the petri dishes after culturing for 5 days in 4. Pick a small piece of the colony and inoculate it into liquid medium E. Inoculate two control bottles into liquid medium A according to the same operation. Culture for 15 days at 28°C and 190 rpm. After culturing for 15 days, select the bottle of bacterial liquid with the best mycelium morphology and the highest mycelium content.

[0059] 7. Take the bacterial liquid with basically the same mycelium morphology and content at the end of the culture of the hypoglycemic medium and the non-hypoglycemic parallel sample in 6 as the starting strain for the next step. Dilute and isolate the strain to obtain single mycelium and then coat it on the low sugar consumption petri dish medium. Culture at 28°C for 5 days.

[0060] 8. After culturing for 5 days in the low sugar consumption petri dish medium in 7, select colonies with white mycelium color, dense mycelium, and aerial mycelium height of about 1 mm and transfer them to the slant medium. Store at below 4°C for later use as the starting strain for the next step. Slant medium: PDA medium is dispensed into test tubes with a diameter of 20 mm, sterilized, placed into a slant, and used after cooling and solidifying.

[0061] Note: The composition of the triangular flask liquid medium with each sugar gradient is shown in Table 1.

[0062] Table 1

[0063]

[0064] IV. Breeding of acid-tolerant strains

[0065] During the middle and late stages of the liquid fermentation of Tremella aurantialba, the pH value of the fermentation broth will decrease. To increase the enrichment amount of mycelium and thus obtain more mycelium, calcium carbonate needs to be added to the initial medium to neutralize the acid production. However, by neutralizing with calcium carbonate, the pH can only be controlled not lower than 5.0, while the optimal pH value for the growth of Tremella aurantialba is 6.0 - 6.5. In addition, excessive calcium ions added initially will inhibit the growth of mycelium and affect the final fermentation effect. By adding substances such as calcium hydroxide and sodium hydroxide during the middle stage of fermentation, the pH value of the fermentation broth in the middle and late stages of fermentation can also be controlled. However, the fermentation cycle of Tremella aurantialba is relatively long, and the addition method will largely increase the risk of contamination. In summary, strains that can grow at a lower pH value and maintain the final mycelium content need to be bred. Attempts to obtain strains that can grow under lower pH conditions by gradually reducing the initial pH of the medium were unsuccessful. Later, the ultraviolet mutagenesis method was used to obtain better strains. The breeding steps are as follows:

[0066] 1. Ultraviolet mutagenesis

[0067] 1) Take 30 ml of liquid culture medium in a triangular flask with a magnetic stirrer, and gently scrape the plate spores

[0068] Into an empty Erlenmeyer flask, shake at 220 rpm on a shaker for 10 min, then filter through a sterile single-layer lens paper funnel to obtain the bacterial suspension to be treated, denoted as 10 0 ;

[0069] 2) Use a 10 ml pipette to pipette 9 ml of sterile water into n dilution tubes respectively to prepare n dilution tubes with different gradients;

[0070] 3) Place the bacterial suspension at each dilution gradient in a sterilized petri dish, place a magnetic stir bar, and perform ultraviolet irradiation at 15 w and 30 cm. The irradiation time is shown in Table 2;

[0071] 4) Place the treated solution after ultraviolet irradiation in ice water and ice-bath for 1 hour;

[0072] 5) Incubate the bacterial suspension after ice-bathing at 28 °C and 170 rpm for 2 hours;

[0073] 6) Spread the bacterial suspension after ice-bathing on the low pH value plate medium and incubate at 28 °C.

[0074] The low pH value plate medium is as follows: Place 500 ml of distilled water in a 1000 ml Erlenmeyer flask, add

[0075] 1.25 g of yeast powder, 2.50 g of peptone, 6.50 g of agar powder, 5 g of glucose and 0.5 ml of Tween

[0076] 80, dissolve it, gently cover the lid of the Erlenmeyer flask, sterilize at 121 °C for 25 min. In a small beaker, add 10 ml of distilled water and 1.25 g of malic acid, stir until dissolved, filter the aqueous solution of malic acid through a 0.45 um filter membrane, and add the treated aqueous solution of malic acid

[0077] to the sterilized medium, stir evenly to make the pH value reach 3.7 ± 0.1. When cooled to about 50 °C, pour it into a petri dish and wait for it to solidify for use.

[0078] 2. Screening and activation of the dominant strain after mutagenesis

[0079] After culturing the mutagenized bacterial solution on the low pH value plate medium for 7 days, some colonies can be picked out from each petri dish with growing colonies. The selection criteria are that the hyphae are white in color, the hyphae are dense, the aerial hyphae are about 1 mm in height, and there is a relatively obvious demarcation line between the colony edge and other colonies.

[0080] 3. Inoculate the above colonies on the slant medium respectively and incubate in the dark at 28 °C for 7 days for later use.

[0081] 3. Inoculate the above colonies on the slant medium respectively and incubate in the dark at 28 °C for 7 days for later use.

[0082] 4. Pick colonies with a diameter of approximately 2 mm from the inclined planes in 3 and inoculate them into a low-sugar, low-pH liquid medium. Inoculate one Erlenmeyer flask for each inclined plane. Incubate at 28 °C and 170 rpm for 9 days. Low-sugar, low-pH liquid medium: 4 g of glycerol, 2 g of glucose, 5 g of yeast powder, 5 g of peptone, 1.5 g of dipotassium hydrogen phosphate, 0.5 g of sodium chloride, 0.5 g of magnesium sulfate heptahydrate, 0.1 g of thiamine. Add water to make up to 1000 ml and sterilize at 121 °C for 25 min. Dissolve 1.25 g of malic acid in 10 ml of distilled water, filter through a 0.45-μm filter membrane, and add it to the sterilized medium. Mix well and set aside.

[0083] 5. For the bacterial liquid after the cultivation in 4, select a bacterial suspension with large and uniform bacterial balls and high viscosity of the bacterial liquid, dilute it 1000 times, shake until the hyphae are evenly dispersed, and then spread it on the surface of the PDA medium. Incubate at 28 °C for 6 days, and then select colonies with white color, dense hyphae, and aerial hyphae with a height of about 1 mm and inoculate them into a low-sugar, low-pH liquid medium. Incubate at 28 °C and 170 rpm for 9 days.

[0084] 6. Repeat the operation in 5 three times, inoculate the strain with passage stability onto an inclined plane, and incubate at 28 °C for 7 days for later use.

[0085] Table 2: Ultraviolet irradiation schedule

[0086]

[0087] V. Comparison of the growth conditions between low-sugar-tolerant and acid-tolerant strains and the original strains

[0088] Randomly select 3 inclined planes from the inclined planes of the strains obtained by fruiting body isolation and purification, numbered A, B, and C. Randomly select 3 inclined planes from the inclined planes of the low-sugar-tolerant and acid-tolerant strains obtained through breeding, numbered D, E, and F. Inoculate the above 6 inclined planes into a low-sugar, low-pH liquid medium respectively, and incubate in the dark at 28 °C and 190 rpm for 9 days. The initial 6 bottles of low-sugar, low-pH liquid medium need to be accurately weighed and made up to volume to ensure that the total amount and the content of each component of each bottle of medium are the same. After the cultivation is completed, observe and record the morphology of the bacterial balls, measure and record the hypha content, pulverize the hyphae, and measure and record the polysaccharide content by the ganoderma polysaccharide content determination method. The comparison results are shown in Table 3.

[0089] Table 3

[0090]

[0091]

[0092] Note: 1. The method for measuring the mycelium content is as follows: Place the Erlenmeyer flask containing the cultured bacterial suspension in a 90°C water bath and heat it statically for 2 hours. At this time, the bacterial balls have naturally precipitated. After pouring out the supernatant liquid, add 500 ml of distilled water to the Erlenmeyer flask again. Shake well and let it precipitate naturally for 30 minutes. Then pour out the supernatant liquid again and add 500 ml of distilled water and shake well. Pour the mixture into a suction flask for suction filtration. After filtering out the water, put the mycelium together with the filter paper into the oven. Dry it at 50°C for 6 hours. Take it out and weigh it, subtract the weight of the filter paper, and record the weight of the mycelium.

[0093] 2. The method for measuring the polysaccharide content in the mycelium is the same as the method for measuring the Ganoderma lucidum polysaccharide content in the Pharmacopoeia. Before measurement, the mycelium needs to be dried to a constant weight, crushed, and sieved through a 100-mesh sieve before being tested.

[0094] 3. Low-sugar-consuming and low-pH liquid medium: 4 g of glycerol, 2 g of glucose, 5 g of yeast powder, 5 g of peptone, 1.5 g of dipotassium hydrogen phosphate, 0.5 g of sodium chloride, 0.5 g of magnesium sulfate heptahydrate, 0.1 g of thiamine. Add water to make up to 1000 ml and sterilize at 121°C for 25 minutes. Dissolve 1.25 g of malic acid in 10 ml of distilled water, filter it through a 0.45-μm filter membrane, and add it to the sterilized medium, mix well and set aside.

[0095] After the above mutagenesis, comparison, and genetic stability investigation, the finally obtained strain was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the preservation number is: CGMCC NO. 41347.

[0096] Example 2 Study on the fermentation parameters of low-sugar-consuming and acid-tolerant Trametes robiniophila Murr

[0097] 1. Selection of medium formula

[0098] Several different medium formulas were selected, formulated according to the formula and made up to 650 ml, placed in a 1-L Erlenmeyer shaking flask, sterilized at 121°C and waiting for inoculation. The inoculated strain was the above-mentioned low-sugar-consuming and acid-tolerant Trametes robiniophila Murr. After inoculation at the same time, it was cultured at 28°C and 180 rpm for 14 days. After the culture was completed, the mycelium content and the mycelium polysaccharide content were measured, and the results are shown in Table 4.

[0099] Table 4

[0100]

[0101]

[0102] In the above table, Formula A is: 16 g of potato flour, 1 g of glucose, 0.3 g of potassium dihydrogen phosphate, 0.2 g of magnesium sulfate. Add drinking water to make up to 1000 ml, and the pH is natural.

[0103] Formula B: Add 10 g of wheat bran, 2 g of soybean powder, 1 g of maltose, and 1 g of soluble starch per 1000 ml, and make up to 1000 ml with drinking water. The pH is natural.

[0104] Formula C: 20 g of malt extract powder, 1 g of glucose, 2 g of peptone, and 2 g of yeast powder, and make up to 1000 ml with drinking water. The pH is natural.

[0105] Formula D: 20 g of soybean meal powder, 5 g of corn flour, 1 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 0.05 g of thiamine, and make up to 1000 ml with drinking water. The pH is natural.

[0106] Formula E: 20 g of soybean meal powder, 20 g of corn flour, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, and 0.05 g of thiamine, and make up to 1000 ml with drinking water. The pH is natural.

[0107] 2. Single factor of liquid fermentation inoculation amount

[0108] Add 2800 g of soybean meal powder, 1000 g of corn flour, 140 g of glucose, 140 g of potassium dihydrogen phosphate, 70 g of magnesium sulfate, 7 g of thiamine, and 5 ml of antifoaming agent into a 200 L fermenter, and make up to 140 L with drinking water. Sterilize at 118 °C for 30 minutes.

[0109] A total of 4 batches of experiments were carried out. The bacterial liquid cultured in a triangular shaking flask for 9 days was inoculated. The inoculation amounts for each batch were 1400 ml (1%), 2800 ml (2%), 7000 ml (5%), and 14000 ml (10%) respectively.

[0110] Fermenter culture conditions: The stirring speed is 170 revolutions per minute, and compressed air is introduced at the bottom to maintain the pressure in the tank at 0.5 bar. No pH regulator or medium is added during the fermentation process.

[0111] During the fermentation process of each batch, samples were taken on the 5th, 7th, 9th, and 13th days of fermentation to measure the mycelium content, and samples were taken on the 13th, 16th, 19th, and 22nd days of fermentation to measure the mycelium content and the polysaccharide content of the dried mycelium after drying the mycelium. The results are shown in Table 5.

[0112] Table 5

[0113]

[0114]

[0115] Considering the economic cost of the culture medium, its availability, and the fermentation effect, the most suitable culture medium formula can be considered as follows: add 20 g of soybean meal powder, 5 g of corn starch, 1 g of glucose, 1 g of potassium dihydrogen phosphate, 0.5 g of magnesium sulfate, 0.05 g of thiamine, and a small amount of antifoaming agent per 1000 ml.

[0116] Considering the difficulty of preparing the seed liquid, the scale-up arrangement of the liquid fermentation equipment, etc., the optimal inoculation amount can be considered to be about 5%.

[0117] In a 200 L fermentation tank, the stirring speed should be 170 revolutions per minute.

[0118] The sterile compressed air inlet volume should be controlled for bottom aeration, and the inlet volume should be controlled to maintain a tank pressure of about 0.5 bar.

[0119] According to the above optimal fermentation parameters, the fermentation period is 16 - 20 days. At the end of fermentation, the mycelium content in the fermentation broth can be stably obtained at about 20 g per liter, and the polysaccharide content in the dried mycelium after drying the mycelium is about 3%.

Claims

1. A strain of low sugar consumption and acid-tolerant Trametes sophorae, which is classified and named Trametes sophorae ( Trametes robiniophila )HER-ZST-2023 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number: CGMCCNO.41347.

2. Use of the low-sugar-consuming and acid-resistant Trametes sophorae according to claim 1 in fermenting and preparing Sophora auricularia mycelium.

3. The use according to claim 2, characterized in that: The Trametes sophorae described in claim 1 is inoculated into a nutrient medium and cultured at 27-30° C. in the dark for 9-22 days.

4. The use according to claim 3, characterized in that: The nutrient medium comprises one or more of potato flour, wheat bran, soybean flour, glucose, maltose, soluble starch, malt extract powder, peptone, yeast powder, soybean meal powder, corn flour, potassium dihydrogen phosphate, magnesium sulfate and thiamine.

5. The use according to claim 3, characterized in that: The inoculation amount of Trametes sophorae is 1~10%.

6. The use according to claim 3, characterized in that: The nutrient medium is 20 g / L soybean meal, 5 g / L corn meal, 1 g / L glucose, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate, and 0.05 g / L thiamine.

7. A Sophora auricula mycelium, characterized in that: The Sophora auricularia mycelium is obtained by fermenting the low-sugar-consuming and acid-resistant Trametes sophorae fungi described in claim 1.

8. The Sophora japonica mycelium according to claim 7, characterized in that The mycelium content in the Sophora auricularia mycelium exceeds 15 g / L, and the mycelium polysaccharide content is not less than 2.5%.

9. Use of the Sophora japonica mycelium according to claim 7 in the preparation of a drug for treating tumors.

10. The use according to claim 9, characterized in that: The mycelium of Sophora japonica fungus can be directly used as medicine in the form of preparation after adding a certain amount of auxiliary materials.

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