A culture medium for silver ear fungus and its cultivation method

Abstract

This invention proposes a culture medium and cultivation method for Tremella fuciformis, belonging to the field of Tremella fuciformis cultivation technology. It is prepared from the following raw materials in parts by weight: 0.5-1 parts antibacterial agent, 5-7 parts traditional Chinese medicine residue, 3-5 parts sugarcane bagasse, 2-3 parts fermentation extract, 30-40 parts cottonseed hulls, 7-10 parts wheat bran, 0.5-1 part gypsum, and 50-60 parts sterile water. The Tremella fuciformis culture medium obtained by this invention can significantly inhibit the growth of pathogenic bacteria during cultivation, reduce the pathogenicity rate, and increase the yield of Tremella fuciformis. Simultaneously, it increases the content and efficacy of active substances in Tremella fuciformis, producing selenium-enriched Tremella fuciformis polysaccharide. The active substances exhibit excellent antioxidant, anti-inflammatory, anti-tumor, moisturizing, hypoglycemic, hypolipidemic, and cardiovascular protective effects. Using this invention's culture medium, along with a suitable ratio of Tremella fuciformis and Clematis chinensis symbiotically, provides a new method and material for Tremella fuciformis cultivation, resulting in Tremella fuciformis with better utilization value and broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of Tremella fuciformis cultivation technology, specifically to a Tremella fuciformis culture medium and its cultivation method. Background Technology

[0002] Tremella, also known as white silver ear, snow fungus, and silver ear seed, belongs to the genus Tremella of the family Tremellaceae. It is the fruiting body of the fungus Tremella fuciformis, and is hailed as the "King of Fungi." Tremella has the effects of strengthening essence, tonifying the kidneys, moistening the intestines, benefiting the stomach, replenishing qi, harmonizing blood, strengthening the heart, invigorating the body, nourishing the brain, refreshing the mind, beautifying the skin, and prolonging life. It can improve the liver's detoxification ability and protect liver function. It can not only enhance the body's anti-tumor immunity but also enhance the tolerance of cancer patients to radiotherapy and chemotherapy. It can nourish and generate fluids, moisten the lungs and stomach, replenish lung qi, treat consumptive cough, hemoptysis, thirst due to insufficient body fluids, weakness after illness, and shortness of breath and fatigue. Tremella is also a good tonic, characterized by its moistening without being greasy. It has the functions of invigorating the spleen and stomach, replenishing qi and clearing the intestines, promoting sleep and strengthening the stomach, nourishing the brain, nourishing yin and clearing heat, and moistening dryness. It is a good tonic for patients with yin deficiency and excessive fire who cannot tolerate warm tonics such as ginseng and deer antler.

[0003] Tremella polysaccharides, the main bioactive component of Tremella fuciformis, have been proven to have effects such as inhibiting quorum sensing, enhancing immunity, lowering blood sugar, anti-tumor, anti-aging, anti-mutagenic, and anti-radiation. In addition, Tremella polysaccharides also possess excellent moisturizing activity and skin-protecting function, having a positive impact on human health and being widely used as a health supplement, moisturizer, and anti-wrinkle agent.

[0004] However, during the cultivation of white fungus, pathogenic bacteria such as Mycobacterium arachidae can severely affect the yield and quality of the fungus. Mycobacterium arachidae is not easily detected in the early stages of spawn production and is difficult to identify with the naked eye. It only becomes visible when the condition has progressed to a severe stage. The name "Mycobacterium arachidae" is simply a name given to it by mushroom farmers in our white fungus-producing region based on its morphological characteristics. According to relevant literature, it belongs to the bacterial class and is in the same category as Bacillus, Pseudomonas fluorescens, and Erwinia. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium and cultivation method for Tremella fuciformis, which can significantly inhibit the growth of pathogenic bacteria during cultivation, reduce the pathogenicity rate, and increase the yield of Tremella fuciformis. At the same time, it can increase the content and efficacy of active substances in Tremella fuciformis, and produce selenium-enriched Tremella fuciformis polysaccharide. The active substances have good antioxidant, anti-inflammatory, anti-tumor, moisturizing, hypoglycemic, hypolipidemic, and cardiovascular protective effects. Using the culture medium of this invention and a suitable ratio of Tremella fuciformis and Auricularia auricula-judae symbiotic, a new method and material for Tremella fuciformis cultivation is provided. The resulting Tremella fuciformis has better utilization value and broad application prospects.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a culture medium for *Tremella fuciformis*, prepared from the following raw materials in parts by weight: 0.5-1 parts antibacterial agent, 5-7 parts traditional Chinese medicine residue, 3-5 parts sugarcane bagasse, 2-3 parts fermentation extract, 30-40 parts cottonseed hulls, 7-10 parts wheat bran, 0.5-1 part gypsum, and 50-60 parts sterile water; the antibacterial agent is a mixture of a preservative antibacterial compound and nisin, and the structural formula of the preservative antibacterial compound is shown in Formula I.

[0008]

[0009] in,

[0010] This invention further protects a method for preparing the above-mentioned culture medium for Tremella fuciformis, comprising the following steps:

[0011] S1. Water extraction of the traditional Chinese medicine composition: Poria cocos, Dendrobium officinale and lily bulb are dried, pulverized, added to water, heated to boiling and extracted to obtain a water extract mixture;

[0012] S2. Sugarcane processing: Juice fresh sugarcane and collect the sugarcane pulp and sugarcane juice;

[0013] S3. Preparation of culture medium: The water extract mixture obtained in step S1, the sugarcane juice obtained in step S2, and the selenium source are mixed evenly and sterilized to obtain the culture medium;

[0014] S4. Preparation of fermentation extract: Selenium-enriched yeast and Bacillus subtilis were inoculated into the culture medium prepared in step S3, fermented, filtered, the filter residue was Chinese medicine residue, and the filtrate was freeze-dried to obtain fermentation extract;

[0015] S5. Preparation of the preservative and antibacterial compound: Natamycin was activated and then reacted with glutamic acid to obtain an intermediate with the structure shown in Formula II; the intermediate was reacted with salicylic acid to obtain the product.

[0016]

[0017] S6. Preparation of antibacterial agent: Mix the preservative antibacterial compound and nisin evenly to prepare the antibacterial agent;

[0018] S7. Preparation of culture medium for Tremella: Sugarcane bagasse from step S2, Chinese medicine residue from step S4, fermentation extract from step S4, antibacterial agent from step S6, cottonseed hulls, wheat bran, and gypsum are added to sterile water, mixed evenly, and sterilized to obtain culture medium for Tremella.

[0019] As a further improvement of the present invention, in step S1, the mass ratio of Poria cocos, Dendrobium officinale, lily bulb and water is 2-4:1-2:3-5:120-150, and the boiling extraction time is 2-4 hours; in step S3, the mass ratio of the water extraction mixture, sugarcane juice and selenium source is 30-50:7-10:1-2, and the selenium source is selected from at least one of sodium selenite or sodium selenate; in step S4, the bacterial count of the selenium-enriched yeast and Bacillus subtilis seed solution is 10. 8 -10 9 The inoculum concentration was 1-3 v / v% and 0.5-1 v / v%, and the fermentation conditions were 45-55℃, 100-200 r / min, and fermentation culture for 48-72 h.

[0020] As a further improvement of the present invention, the preparation method of the preservative and antibacterial compound in step S5 is as follows:

[0021] T1. Natamycin was dissolved in N,N-dimethylbenzamide. At 0°C, N-hydroxysuccinimide ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, and the reaction was activated by stirring. Glutamic acid was added, and the reaction was heated and stirred. The mixture was then separated and purified to obtain the intermediate.

[0022] T2. Dissolve the intermediate and salicylic acid in toluene, add concentrated sulfuric acid, heat and stir to react, separate and purify to obtain the product.

[0023] As a further improvement of the present invention, in step T1, the molar ratio of natamycin, N-hydroxysuccinimide ester, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and glutamic acid is 1:1.3-1.5:1.3-1.5:0.9-1; the activation reaction time is 20-30 min, the heating and stirring reaction temperature is 50-60℃, and the time is 3-5 h; in step T2, the molar ratio of the intermediate and salicylic acid is 1:5.2-5.5, the amount of concentrated sulfuric acid added is 2-4 wt% of the total mass of the system, the heating and stirring reaction temperature is 110-120℃, and the time is 5-7 h.

[0024] As a further improvement of the present invention, the mass ratio of the preservative antibacterial compound and nisin in step S6 is 3-5:2; the mass ratio of sugarcane bagasse, traditional Chinese medicine residue, fermentation extract, antibacterial agent, cottonseed hull, wheat bran, gypsum, and sterile water in step S7 is 3-5:5-7:2-3:0.5-1:30-40:7-10:0.5-1:50-60.

[0025] This invention further protects a method for cultivating Tremella fuciformis, which uses the above-mentioned Tremella fuciformis culture medium for cultivation.

[0026] As a further improvement to the present invention, the following steps are included:

[0027] (1) Pack the above-mentioned tremella culture medium into bags, each bag weighing 3-3.5 kg, sterilize, cool, punch inoculation holes in the cultivation bags with a punch, inoculate the spawn, and obtain the spawn bags;

[0028] (2) After inoculation, control the temperature at 25-26℃ and the air humidity at 50-70% for 3-5 days. Then control the temperature at 20-22℃, ventilate 3-5 times a day for 20-30 minutes each time, and continue to cultivate for 7-10 days. Remove the bag, cover with paper, spray water to keep moist, and harvest when the white fungus is mature.

[0029] As a further improvement of the present invention, the sterilization method is 100-110℃, pressure of 1-1.2MPa, treatment for 30-50min, 8-12 holes are punched in each cultivation bag, the hole depth is 2-4cm, the hole diameter is 2-3cm, and the inoculum is 1-2mm above the surface of the bag during inoculation. The inoculum includes pure mycelium of Tremella fuciformis and pure mycelium of Auricularia auricula-judae, with a mass ratio of 2-3:7-10.

[0030] This invention further protects a type of tremella obtained by the above-described cultivation method.

[0031] The present invention has the following beneficial effects:

[0032] This invention prepares a preservative antibacterial compound, which is obtained by the dehydration condensation reaction of natamycin and glutamic acid to obtain an intermediate, and then undergoes an esterification reaction with salicylic acid. This not only improves the water solubility of natamycin, reduces its sensitization reaction, and improves its stability (light resistance, heat resistance, etc.), but also avoids the inflammation that salicylic acid easily causes when used alone, reducing its irritation. At the same time, it improves the disinfection effect of the preservative antibacterial compound on pathogenic bacteria, especially Mycobacterium sarmentosum, thereby reducing the pathogenicity rate during the cultivation of Tremella fuciformis and increasing the yield of Tremella fuciformis. It has no effect on fungi such as Tremella fuciformis and Auricularia auricula-judae, and is safe and environmentally friendly. It works synergistically with nisin to exert its antibacterial effect by inhibiting the synthesis of bacterial cell walls, resulting in better antibacterial, preservative and antiseptic effects.

[0033] The culture medium for Tremella fuciformis of this invention also includes Chinese medicinal residues and fermentation products. These residues and fermentation products, derived from the water extraction and fermentation of Poria cocos, Dendrobium officinale, and lily, are rich in cellulose and lignin, providing abundant cellulose substances for the growth of Tremella fuciformis. At the same time, the medicinal proteins, flavonoids, saponins, triterpenes, and other substances contained therein are transformed into active substances that can be absorbed by Tremella fuciformis after being decomposed by Tremella fuciformis, greatly increasing the content of active substances such as polysaccharides and proteins in Tremella fuciformis, as well as its antioxidant, anti-inflammatory, anti-tumor, moisturizing, and cardiovascular protective effects.

[0034] This invention uses selenium-enriched yeast and Bacillus subtilis to ferment the culture medium. Under the synergistic effect of the two, on the one hand, the selenium-enriched yeast converts the inorganic selenium source into an organic selenium source. The fermentation product is absorbed by Tremella fuciformis and converted into selenium-enriched Tremella fuciformis polysaccharide, thereby greatly improving the medicinal efficacy of Tremella fuciformis. On the other hand, the by-products after fermentation, including ethanol, lactic acid, and antimicrobial lipopeptides, can effectively inhibit the generation and reproduction of pathogenic bacteria during the cultivation of Tremella fuciformis, thereby reducing the pathogenicity rate of Tremella fuciformis and increasing the yield and productivity.

[0035] The mycelium of *Tremella fuciformis* itself lacks the ability to decompose nutrients; it relies on the nutrients decomposed by *Auricularia auricula-judae* mycelium for vegetative and reproductive growth, completing its life cycle. During the growth of *Tremella fuciformis*, *Auricularia auricula-judae* mycelium first extends into the culture medium, supplying the crude nutrients in the medium for its growth. The metabolic products of *Auricularia auricula-judae* promote the stable growth of *Tremella fuciformis* mycelium. This invention, through the symbiosis of *Auricularia auricula-judae* and *Tremella fuciformis* in an appropriate ratio, can increase the activity of enzymes such as cellulase, xylanase, laccase, and amylase by producing appropriate concentrations of metabolic products from *Auricularia auricula-judae*, thereby promoting the growth of *Tremella fuciformis* and improving its yield and quality.

[0036] The culture medium for Tremella prepared by this invention can significantly inhibit the growth of pathogenic bacteria during the cultivation process, reduce the pathogenicity rate, and increase the yield of Tremella. At the same time, it increases the content and efficacy of active substances in Tremella, and produces selenium-enriched Tremella polysaccharide. The active substances have excellent antioxidant, anti-inflammatory, immune-enhancing, anti-tumor, moisturizing, blood sugar-lowering, blood lipid-lowering, blood pressure-lowering, vision-protecting, and cardiovascular-protecting effects. The culture medium of this invention, along with a suitable ratio of Tremella fuciformis and Auricularia auricula-judae symbiotic, provides a new method and material for Tremella cultivation. The resulting Tremella has better utilization value and broad application prospects. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Selenium-enriched yeast, 10 billion CFU / g, purchased from Angel Yeast Co., Ltd.; Bacillus subtilis, 10 billion CFU / g, purchased from Guangzhou Zhenwei Biotechnology Co., Ltd.

[0039] Preparation method of inoculum seed solution: Inoculate the inoculum into slant culture medium, activate and culture at 50℃ and 120 r / min for 24 h to obtain a culture with a bacterial count of 10. 8 -10 9 CFU / mL bacterial seed solution.

[0040] Example 1

[0041] This embodiment provides a method for preparing a culture medium for Tremella fuciformis, including the following steps:

[0042] S1. Water extraction of the traditional Chinese medicine composition: 2 parts by weight of Poria cocos, 1 part by weight of Dendrobium officinale and 3 parts by weight of Lilium brownii are dried, pulverized and added to 120 parts by weight of water. The mixture is heated to boiling and extracted for 2 hours to obtain a water extract mixture.

[0043] S2. Sugarcane processing: Juice fresh sugarcane and collect the sugarcane pulp and sugarcane juice;

[0044] S3. Preparation of culture medium: 30 parts by weight of the water extract mixture obtained in step S1, 7 parts by weight of the sugarcane juice obtained in step S2, and 1 part by weight of sodium selenite are stirred and mixed for 30 minutes, and then sterilized to obtain the culture medium.

[0045] S4. Preparation of fermentation extract: Selenium-enriched yeast and Bacillus subtilis seed liquid were inoculated into the culture medium prepared in step S3 at inoculation amounts of 1 v / v% and 0.5 v / v%, respectively. The culture was fermented at 45°C and 100 r / min for 48 h. After filtration, the residue was the Chinese medicine residue. The filtrate was freeze-dried to obtain the fermentation extract.

[0046] S5. Preparation of the preservative and antibacterial compound: 0.1 mol natamycin was dissolved in 500 mL N,N-dimethylbenzamide. At 0 °C, 0.13 mol N-hydroxysuccinimide ester and 0.13 mol 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added. The mixture was stirred at 0 °C for 20 min to activate the reaction. 0.09 mol glutamic acid was added, and the mixture was heated to 50 °C and stirred for 3 h. The mixture was filtered and recrystallized from methanol to obtain the intermediate. 0.1 mol of the intermediate and 0.52 mol salicylic acid were dissolved in 300 mL toluene. Concentrated sulfuric acid was added at 2 wt% of the total mass of the system. The mixture was heated under reflux and stirred for 5 h. The mixture was filtered and recrystallized from diethyl ether to obtain the product.

[0047] Infrared spectral analysis: 3475cm -1 The peak at 3410 cm⁻¹ represents the stretching vibration of the NH bond in the amide group. -1 The absorption peak at 3325 cm⁻¹ represents the phenolic hydroxyl group. -1 The infrared absorption peak at 3211 cm⁻¹ represents that of amino groups. -1 The peak at 1662 cm⁻¹ represents the stretching vibration of the OH bond in the carboxyl group. -1 The peak of the stretching vibration at C=O is 1215 cm⁻¹. -1 The peak at 992 cm⁻¹ corresponds to the stretching vibration of the ester COC. -1 The out-of-plane bending vibration absorption peak of the CH group in the olefin; 910 cm⁻¹-1 The absorption peak at 852 cm⁻¹ is the epoxide absorption peak. -1 The peak at this location is a characteristic absorption peak of the benzene ring.

[0048] S6. Preparation of antibacterial agent: Mix 3 parts by weight of the preservative antibacterial compound and 2 parts by weight of nisin for 15 minutes to obtain the antibacterial agent;

[0049] S7. Preparation of culture medium for Tremella fuciformis: Add 3 parts by weight of sugarcane bagasse from step S2, 5 parts by weight of Chinese medicine residue from step S4, 2 parts by weight of fermentation extract from step S4, 0.5 parts by weight of antibacterial agent from step S6, 30 parts by weight of cottonseed hulls, 7 parts by weight of wheat bran, and 0.5 parts by weight of gypsum to 50 parts by weight of sterile water, stir and mix for 30 minutes, sterilize, and obtain culture medium for Tremella fuciformis.

[0050] Example 2

[0051] This embodiment provides a method for preparing a culture medium for Tremella fuciformis, including the following steps:

[0052] S1. Water extraction of the traditional Chinese medicine composition: 4 parts by weight of Poria cocos, 2 parts by weight of Dendrobium officinale and 5 parts by weight of Lilium brownii are dried, pulverized and added to 150 parts by weight of water. The mixture is heated to boiling and extracted for 4 hours to obtain a water extract mixture.

[0053] S2. Sugarcane processing: Juice fresh sugarcane and collect the sugarcane pulp and sugarcane juice;

[0054] S3. Preparation of culture medium: Mix 50 parts by weight of the water extract mixture obtained in step S1, 10 parts by weight of the sugarcane juice obtained in step S2, and 2 parts by weight of sodium selenate for 30 minutes, then sterilize to obtain the culture medium.

[0055] S4. Preparation of fermentation extract: Selenium-enriched yeast and Bacillus subtilis seed liquid were inoculated into the culture medium prepared in step S3 at inoculation amounts of 3v / v% and 1v / v%, respectively. The culture was fermented at 55℃ and 200r / min for 72h. After filtration, the residue was the Chinese medicine residue. The filtrate was freeze-dried to obtain the fermentation extract.

[0056] S5. Preparation of the preservative and antibacterial compound: 0.1 mol natamycin was dissolved in 500 mL N,N-dimethylbenzamide. At 0 °C, 0.15 mol N-hydroxysuccinimide ester and 0.15 mol 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added. The mixture was stirred at 0 °C for 30 min to activate the reaction. 0.1 mol glutamic acid was added, and the mixture was heated to 60 °C and stirred for 5 h. The mixture was filtered and recrystallized from methanol to obtain the intermediate. 0.1 mol of the intermediate and 0.55 mol salicylic acid were dissolved in 300 mL toluene. Concentrated sulfuric acid was added at 4 wt% of the total mass of the system. The mixture was heated under reflux and stirred for 7 h. The mixture was filtered and recrystallized from diethyl ether to obtain the product.

[0057] S6. Preparation of antibacterial agent: Mix 5 parts by weight of the preservative antibacterial compound and 2 parts by weight of nisin for 15 minutes to obtain the antibacterial agent;

[0058] S7. Preparation of culture medium for Tremella: Add 5 parts by weight of sugarcane bagasse from step S2, 7 parts by weight of Chinese medicine residue from step S4, 3 parts by weight of fermentation extract from step S4, 1 part by weight of antibacterial agent from step S6, 40 parts by weight of cottonseed hulls, 10 parts by weight of wheat bran, and 1 part by weight of gypsum to 60 parts by weight of sterile water, stir and mix for 30 minutes, sterilize, and obtain the culture medium for Tremella.

[0059] Example 3

[0060] This embodiment provides a method for preparing a culture medium for Tremella fuciformis, including the following steps:

[0061] S1. Water extraction of the traditional Chinese medicine composition: 3 parts by weight of Poria cocos, 1.5 parts by weight of Dendrobium officinale and 4 parts by weight of Lilium brownii are dried, pulverized and added to 135 parts by weight of water. The mixture is heated to boiling and extracted for 3 hours to obtain a water extract mixture.

[0062] S2. Sugarcane processing: Juice fresh sugarcane and collect the sugarcane pulp and sugarcane juice;

[0063] S3. Preparation of culture medium: 40 parts by weight of the water extract mixture obtained in step S1, 8 parts by weight of the sugarcane juice obtained in step S2, and 1.5 parts by weight of sodium selenite were stirred and mixed for 30 minutes, and then sterilized to obtain the culture medium.

[0064] S4. Preparation of fermentation extract: Selenium-enriched yeast and Bacillus subtilis seed liquid were inoculated into the culture medium prepared in step S3 at inoculation amounts of 2 v / v% and 0.7 v / v%, respectively. The culture was fermented at 50°C and 150 r / min for 56 h. After filtration, the residue was the Chinese medicine residue. The filtrate was freeze-dried to obtain the fermentation extract.

[0065] S5. Preparation of the preservative and antibacterial compound: 0.1 mol natamycin was dissolved in 500 mL N,N-dimethylbenzamide. At 0 °C, 0.14 mol N-hydroxysuccinimide ester and 0.14 mol 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added. The mixture was stirred at 0 °C for 25 min to activate the reaction. 0.095 mol glutamic acid was added, and the mixture was heated to 55 °C and stirred for 4 h. The mixture was filtered and recrystallized from methanol to obtain the intermediate. 0.1 mol of the intermediate and 0.53 mol salicylic acid were dissolved in 300 mL toluene. Concentrated sulfuric acid was added at 3 wt% of the total mass of the system. The mixture was heated under reflux and stirred for 6 h. The mixture was filtered and recrystallized from diethyl ether to obtain the product.

[0066] S6. Preparation of antibacterial agent: Mix 4 parts by weight of the preservative antibacterial compound and 2 parts by weight of nisin for 15 minutes to obtain the antibacterial agent;

[0067] S7. Preparation of culture medium for Tremella fuciformis: Add 4 parts by weight of sugarcane bagasse from step S2, 6 parts by weight of Chinese medicine residue from step S4, 2.5 parts by weight of fermentation extract from step S4, 0.7 parts by weight of antibacterial agent from step S6, 35 parts by weight of cottonseed hulls, 8 parts by weight of wheat bran, and 0.7 parts by weight of gypsum to 55 parts by weight of sterile water, stir and mix for 30 minutes, sterilize, and obtain culture medium for Tremella fuciformis.

[0068] Comparative Example 1

[0069] The difference from Example 3 is that selenium-enriched yeast was not inoculated in step S4, and the inoculation amount of Bacillus subtilis spore liquid was 2.7 v / v.

[0070] Comparative Example 2

[0071] The difference from Example 3 is that Bacillus subtilis was not inoculated in step S4, and the inoculation amount of selenium-enriched yeast seed solution was 2.7 v / v.

[0072] Comparative Example 3

[0073] Compared with Example 3, the difference is that step S4 was not performed, and in step S7 the fermentation extract was replaced by water extraction mixture, sugarcane juice and sodium selenite in a mass ratio of 40:8:1.5.

[0074] Comparative Example 4

[0075] The difference from Example 3 is that the antibacterial agent in step S6 is a single preservative antibacterial compound.

[0076] Comparative Example 5

[0077] The difference from Example 3 is that the antibacterial agent in step S6 is a single lactic acid nisin.

[0078] Comparative Example 6

[0079] The difference from Example 3 is that no antibacterial agent was added in step S7.

[0080] Comparative Example 7

[0081] The difference from Example 3 is that no Chinese herbal medicine residue was added in step S7.

[0082] Comparative Example 8

[0083] The difference from Example 3 is that no fermentation extract was added in step S7.

[0084] Test Example 1

[0085] The antibacterial agents in Examples 1-3 and Comparative Examples 4 and 5 of this invention were subjected to antibacterial experiments.

[0086] A 5 μg / mL antibacterial agent solution was prepared, and 10 μL of each solution was spotted sequentially onto culture media containing bacteria. An equal volume of sterile water for injection was added to the negative control group, and 5 μg / mL fluconazole for injection was added to the positive control group. The media were incubated at 28°C for 24 h. After incubation, the culture media were removed, and the diameter of the inhibition zone on each media was measured using a steel ruler. The experiment was repeated three times, and the average value was taken. The results are shown in Table 1.

[0087] Table 1

[0088]

[0089] As can be seen from the table above, the antibacterial agents in Examples 1-3 of the present invention have good antibacterial and antimicrobial effects.

[0090] Example 4

[0091] A method for cultivating silver ear fungus includes the following steps:

[0092] (1) Pack the culture medium of Tremella fuciformis prepared in Example 1 into bags, with each bag weighing 3.2 ± 0.2 kg. Sterilize (100℃, 1 MPa pressure, 30 min), cool, and punch inoculation holes on the cultivation bags with a puncher. Punch 8 holes in each cultivation bag, with a hole depth of 2 cm and a hole diameter of 2 cm. Inoculate with spawn, with the spawn 1 mm above the surface of the bag. The spawn includes pure mycelium of Tremella fuciformis and pure mycelium of Clerodendrum trichotomum, with a mass ratio of 2:7. Disinfect and sterilize in time during the inoculation process (the spawn is cleaned with benzalkonium chloride, and the inoculation personnel are disinfected with 75% ethanol) to obtain the spawn bags.

[0093] (2) After inoculation, the temperature is controlled at 25.5±0.5℃ and the air humidity is 60±10% for 3 days. Then, the temperature is controlled at 21±1℃, and ventilation is carried out 3 times a day for 20 minutes each time. Continue to cultivate for 7 days, remove the bag, cover with paper, spray water to keep moist, and harvest when the white fungus is mature.

[0094] Example 5

[0095] A method for cultivating silver ear fungus includes the following steps:

[0096] (1) Pack the culture medium of Tremella fuciformis prepared in Example 2 into bags, with each bag weighing 3.2 ± 0.2 kg. Sterilize (110℃, pressure 1.2 MPa, treatment for 50 min), cool, and punch inoculation holes in the cultivation bags with a puncher. Punch 12 holes in each cultivation bag, with a hole depth of 4 cm and a hole diameter of 3 cm. Inoculate with spores, with the spores 2 mm above the surface of the bag. The spores include pure mycelium of Tremella fuciformis and pure mycelium of Clerodendrum trichotomum, with a mass ratio of 3:10. Disinfect and sterilize in time during the inoculation process (the spores are washed with benzalkonium chloride, and the inoculation personnel are disinfected with 75% ethanol) to obtain the spore bags.

[0097] (2) After inoculation, the temperature is controlled at 25.5±0.5℃ and the air humidity is 60±10% for 3 days. Then, the temperature is controlled at 21±1℃ for 30 minutes each time, and the culture is continued for 10 days. Remove the bag, cover with paper, spray water to keep moist, and harvest when the white fungus is mature.

[0098] Example 6

[0099] A method for cultivating silver ear fungus includes the following steps:

[0100] (1) Pack the culture medium of Tremella fuciformis prepared in Example 3 into bags, with each bag weighing 3.2 ± 0.2 kg. Sterilize (105℃, pressure 1.1 MPa, treatment for 40 min), cool, and punch inoculation holes in the cultivation bags with a puncher. Punch 10 holes in each cultivation bag, with a hole depth of 3 cm and a hole diameter of 2.5 cm. Inoculate with spawn, with the spawn 1.5 mm above the surface of the bag. The spawn includes pure mycelium of Tremella fuciformis and pure mycelium of Clerodendrum trichotomum, with a mass ratio of 2.5:8. Disinfect and sterilize in time during the inoculation process (the spawn is washed with benzalkonium chloride, and the inoculation personnel are disinfected with 75% ethanol) to obtain the spawn bags.

[0101] (2) After inoculation, the temperature is controlled at 25.5±0.5℃ and the air humidity is 60±10% for 3 days. Then, the temperature is controlled at 21±1℃, and ventilation is carried out 4 times a day for 25 minutes each time. Continue to cultivate for 8 days, remove the bag, cover with paper, spray water to keep moist, and harvest when the white fungus is mature.

[0102] Comparative Example 9

[0103] The difference from Example 3 is that the culture medium for Tremella fuciformis was prepared from Comparative Example 1.

[0104] Comparative Example 10

[0105] The difference from Example 3 is that the culture medium for Tremella fuciformis was prepared from Comparative Example 2.

[0106] Comparative Example 11

[0107] The difference from Example 3 is that the culture medium for Tremella fuciformis was prepared from Comparative Example 3.

[0108] Comparative Example 12

[0109] The difference from Example 3 is that the culture medium for Tremella fuciformis was prepared from Comparative Example 4.

[0110] Comparative Example 13

[0111] The difference from Example 3 is that the culture medium for Tremella fuciformis was prepared from Comparative Example 5.

[0112] Comparative Example 14

[0113] The difference from Example 3 is that the culture medium for Tremella fuciformis was prepared from Comparative Example 6.

[0114] Comparative Example 15

[0115] The difference from Example 3 is that the culture medium for Tremella fuciformis was prepared from Comparative Example 7.

[0116] Comparative Example 16

[0117] The difference from Example 3 is that the culture medium for Tremella fuciformis was prepared from Comparative Example 8.

[0118] Comparative Example 17

[0119] Compared with Example 3, the difference is that the strains include pure mycelium of Tremella fuciformis and pure mycelium of Auricularia auricula-judae, with a mass ratio of 1:1.

[0120] Comparative Example 18

[0121] Compared with Example 3, the difference is that the strains include pure mycelium of Tremella fuciformis and pure mycelium of Auricularia auricula-judae, with a mass ratio of 2.5:20.

[0122] Test Example 2

[0123] The *Tremella fuciformis* obtained from Examples 4-6 and Comparative Examples 9-18 of this invention were evaluated. The results are shown in Table 2.

[0124] Table 2

[0125]

[0126]

[0127] Note: The more "+" signs in the same column, the more likely the mycelium is to grow vigorously, appearing thick and dense; the faster the color change is; and the more uniform the flower shape and the more large flowers there are.

[0128] As can be seen from the table above, the tremella cultured by the cultivation methods in Examples 4-6 of the present invention has good growth, is thick and dense, changes color quickly, has a neat shape, has a large number of large tremella, is almost disease-free, and has a high yield.

[0129] Test Example 3

[0130] The Tremella fuciformis cultured in Examples 4-6 and Comparative Examples 9-18 of this invention were harvested and dried. 10g of dried Tremella fuciformis was pulverized, added to 100mL of deionized water, heated to boiling and extracted 2mL. Ethanol was added until the ethanol content of the system was 80wt%. After precipitation for 2h, the solid was collected by centrifugation, washed, and dried to obtain Tremella fuciformis polysaccharide (commercially available Tremella fuciformis polysaccharide was used as a control).

[0131] 1. Test the yield of polysaccharides in each group of Tremella fuciformis and calculate the polysaccharide content (g / 100g dry weight).

[0132] 2. The selenium content in the prepared Tremella polysaccharide was tested (in accordance with GB 5009.93—2017 "National Food Safety Standard - Determination of Selenium in Food").

[0133] 3. The obtained Tremella polysaccharide was prepared into a sample solution with a concentration of 50 mg / L, and its antioxidant activity was tested using the following method:

[0134] Mix 1 mL of sample solution with 2 mL of 0.2 mmol / L DPPH solution dissolved in anhydrous ethanol, shake well, and react in the dark at room temperature for 20 min. Filter and measure the absorbance A of the filtrate at 517 nm. i The absorbance value A was measured by replacing the DPPH solution with 1 mL of anhydrous ethanol. j The absorbance value A0 was measured by replacing the enzyme hydrolysate with 1 mL of water, with vitamin C as a positive control. Each group was set up in triplicate.

[0135] DPPH free radical scavenging rate (%) = (1-A) i -A j / A0)×100%

[0136] The results are shown in Table 3.

[0137] Table 3

[0138]

[0139] As shown in the table above, when the culture medium for tremella prepared using the materials from Examples 1-3 of this invention is used to cultivate tremella, the polysaccharide content is high, the selenium content is high, and the antioxidant effect is good.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A culture medium for Tremella fuciformis, characterized in that, It is prepared from the following raw materials in parts by weight: 0.5-1 parts antibacterial agent, 5-7 parts Chinese herbal medicine residue, 3-5 parts sugarcane bagasse, 2-3 parts fermentation extract, 30-40 parts cottonseed hulls, 7-10 parts wheat bran, 0.5-1 parts gypsum, and 50-60 parts sterile water; the antibacterial agent is a mixture of a preservative antibacterial compound and nisin, and the structural formula of the preservative antibacterial compound is shown in Formula I: Formula I; Where R= ; The method for preparing the culture medium for the white fungus includes the following steps: S1. Water extraction of the traditional Chinese medicine composition: Poria cocos, Dendrobium officinale and lily bulb are dried, pulverized, added to water, heated to boiling and extracted to obtain a water extract mixture; S2. Sugarcane processing: Juice fresh sugarcane and collect the sugarcane pulp and juice; S3. Preparation of culture medium: Mix the water extract mixture obtained in step S1, the sugarcane juice obtained in step S2, and the selenium source evenly, and sterilize to obtain the culture medium; S4. Preparation of fermentation extract: Selenium-enriched yeast and Bacillus subtilis were inoculated into the culture medium prepared in step S3, fermented, filtered, the filter residue was Chinese medicine residue, and the filtrate was freeze-dried to obtain the fermentation extract; S5. Preparation of the preservative and antibacterial compound: Natamycin was activated and reacted with glutamic acid to obtain an intermediate with the structure shown in Formula II; the intermediate was reacted with salicylic acid to obtain the product; Formula II; The specific method for preparing the preservation and antibacterial compound is as follows: T1. Natamycin was dissolved in N,N-dimethylbenzamide. At 0°C, N-hydroxysuccinimide ester and 1-ethyl-(3-dimethylaminopropyl)carbodiimide were added, and the reaction was activated by stirring. Glutamic acid was added, and the reaction was heated and stirred. The mixture was then separated and purified to obtain the intermediate. T2. Dissolve the intermediate and salicylic acid in toluene, add concentrated sulfuric acid, heat and stir to react, separate and purify to obtain the product; S6. Preparation of antibacterial agent: Mix the preservative antibacterial compound and nisin evenly to prepare the antibacterial agent; S7. Preparation of culture medium for Tremella: Sugarcane bagasse from step S2, Chinese medicine residue from step S4, fermentation extract from step S4, antibacterial agent from step S6, cottonseed hulls, wheat bran, and gypsum are added to sterile water, mixed evenly, and sterilized to obtain culture medium for Tremella.

2. The culture medium for Tremella fuciformis according to claim 1, characterized in that, In step S1, the mass ratio of Poria cocos, Dendrobium officinale, lily bulb, and water is 2-4:1-2:3-5:120-150, and the boiling extraction time is 2-4 hours. In step S3, the mass ratio of the water extract mixture, sugarcane juice, and selenium source is 30-50:7-10:1-2, and the selenium source is selected from at least one of sodium selenite or sodium selenate. In step S4, the bacterial count of the selenium-enriched yeast and Bacillus subtilis seed solution is 10. 8 -10 9 The inoculum concentration was 1-3 v / v% and 0.5-1 v / v%, and the fermentation conditions were 45-55℃, 100-200 r / min, and fermentation culture for 48-72 h.

3. The culture medium for Tremella fuciformis according to claim 1, characterized in that, In step T1, the molar ratio of natamycin, N-hydroxysuccinimide ester, 1-ethyl-(3-dimethylaminopropyl)carbodiimide, and glutamic acid is 1:1.3-1.5:1.3-1.5:0.9-1; the activation reaction time is 20-30 min, and the heating and stirring reaction temperature is 50-60℃ for 3-5 h; in step T2, the molar ratio of the intermediate and salicylic acid is 1:5.2-5.5, the amount of concentrated sulfuric acid added is 2-4 wt% of the total mass of the system, and the heating and stirring reaction temperature is 110-120℃ for 5-7 h.

4. The culture medium for Tremella fuciformis according to claim 1, characterized in that, The mass ratio of the preservative antibacterial compound and nisin in step S6 is 3-5:2; the mass ratio of sugarcane bagasse, traditional Chinese medicine residue, fermentation extract, antibacterial agent, cottonseed hull, wheat bran, gypsum, and sterile water in step S7 is 3-5:5-7:2-3:0.5-1:30-40:7-10:0.5-1:50-60.

5. A method for cultivating Tremella fuciformis, characterized in that, The culture medium for *Tremella fuciformis* as described in claim 1 was used for cultivation.

6. The cultivation method according to claim 5, characterized in that, Includes the following steps: (1) Pack the culture medium of the white fungus described in claim 1 into bags, each bag weighing 3-3.5 kg, sterilize, cool, punch inoculation holes in the cultivation bags with a punch, inoculate the fungus, and obtain the fungus bags; (2) After inoculation, control the temperature at 25-26℃ and the air humidity at 50-70% for 3-5 days. Then control the temperature at 20-22℃, ventilate 3-5 times a day for 20-30 minutes each time, and continue to cultivate for 7-10 days. Remove the bag, cover with paper, spray water to keep moist, and harvest when the white fungus is mature.

7. The cultivation method according to claim 6, characterized in that, The sterilization method is 100-110℃, pressure 1-1.2MPa, treatment for 30-50min, 8-12 holes are punched in each cultivation bag, the hole depth is 2-4cm, the hole diameter is 2-3cm, and the inoculum is 1-2mm above the surface of the bag during inoculation. The inoculum includes pure mycelium of Tremella fuciformis and pure mycelium of Auricularia auricula-judae, with a mass ratio of 2-3:7-10.

8. A type of tremella obtained by the cultivation method as described in claim 5 or 6.

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