Method for preparing tremella aurantialba polyphenol by utilizing tremella aurantialba TA58.21 fungus chassis cell fermentation

Abstract

The invention belongs to the technical field of biological pharmacy, and relates to a method for preparing tremella aurantialba polyphenol by utilizing tremella aurantialba TA58.21 fungus chassis cell fermentation, which comprises the following steps: adding a cooked soybean meal culture medium and a tremella aurantialba seed solution into a fermentation tank, introducing sterile oxygen, controlling the stirring rotating speed of the fermentation tank and the culture temperature to be 25-30 DEG C, continuously culturing for 5-8 days, and when the fermentation liquor becomes red, stopping the fermentation; when a large amount of foam is generated, closing an exhaust valve, keeping the tank pressure at 0.1 MPa, increasing the fermentation temperature to 50 DEG C, continuing to culture for 24 hours, increasing the stirring rotating speed of the fermentation tank, heating the fermentation liquor to 80-90 DEG C, keeping the temperature for 4-8 hours, stopping stirring, continuing to heat to 120 DEG C, and keeping the temperature for 30 minutes; centrifuging the fermentation liquor to remove precipitates and mycelia; collecting supernate, and pre-treating macromolecular substances and thallus fragments; collecting permeate liquid, and intercepting through a nanofiltration membrane to obtain a concentrated solution; and carrying out spray drying or freeze drying treatment on the concentrated solution to obtain a high-purity product. According to the invention, the tremella aurantialba polyphenol product with good water solubility, heat resistance, acid resistance and molecular weight of less than 10000 Da can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine technology, and in particular relates to a method for preparing auricularia polyphenols by fermentation of the TA58.21 fungal chassis cells. Background Technology

[0002] Golden ear fungus (Tremella aurantialba) belongs to the kingdom Fungi, phylum Basidiomycota, class Tremella, order Tremellales, family Auriculariaceae, genus Aurantialba, and species Aurantialba. The fruiting body of golden ear fungus is rich in nutrients such as protein, dietary fiber, fat, β-carotene, various amino acids, vitamins (VB, VC, VD), and minerals (zinc, iron, potassium, magnesium), as well as bioactive substances such as polysaccharides, polyphenols, flavonoids, and coumarins. It has effects such as lowering blood sugar and blood lipids, anti-oxidation, anti-inflammation, antithrombosis, anticoagulation, anti-radiation, antiviral, expectorant and antitussive, and enhancing immunity. It is a rare medicinal and edible fungus and a nourishing tonic, widely used in the food, pharmaceutical, and cosmetic fields.

[0003] Golden ear polyphenols are natural compounds found in golden ear fungus and have various biological activities, including antioxidant, hypoglycemic, hypolipidemic, immune-enhancing, anti-inflammatory, anticoagulant and antitumor effects.

[0004] Currently, there is limited research on the fermentation of polyphenols using *Auricularia auricula-judae* mycelium, and there are also few reports on the extraction of polyphenols from the fruiting bodies. *Auricularia auricula-judae* polyphenols are mostly extracted from the fruiting bodies, which are products of the symbiotic relationship between *Auricularia auricula-judae* and *Auricularia spp.* These fruiting bodies have a very high content of gelatinous substances and are highly viscous. Extraction of polyphenols from these fruiting bodies is cumbersome, and the resulting concentrations and yields are low. Furthermore, since the fruiting bodies are produced by symbiotic fungi, the hydrolysis products are complex mixtures. Therefore, extracting polyphenols from the fruiting bodies is not a viable method. Summary of the Invention

[0005] The purpose of this invention is to provide a biomanufacturing method for preparing *Auricularia auricula-judae* polyphenols by fermentation of the basal cells of the *Auricularia auricula-judae* fungus TA58.21. The method involves pure culture of *Auricularia auricula-judae* mycelium using a liquid submerged fermentation mode, and the synthesis of polyphenols from the mycelial cells. This polyphenol exhibits high temperature and acid resistance, significant antioxidant and free radical scavenging effects; each μg of *Auricularia auricula-judae* polyphenol can scavenge 5 μg of DPPH, achieving a DPPH free radical scavenging capacity of over 90%, and an antioxidant capacity of over 92%. In terms of thermal stability, it remains intact at 120℃ and its biological activity is not easily inactivated at pH=2.

[0006] This invention provides a strain of *Auricularia auricula-judae* TA58.21, with accession number CGMCC No. 42090 and microbiological classification name *Auricularia auricula-judae*. Naematelia aurantialbaIt is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on August 28, 2025.

[0007] Another aspect of the present invention provides a method for preparing *Auricularia auricula-judae* polyphenols by fermentation of the TA58.21 fungal basal cells, comprising the following steps: 1) Add cooked soybean powder culture medium to a 200L fermenter. After high-temperature sterilization and cooling, inoculate with Auricularia auricula-judae seed liquid. Introduce sterile oxygen at an aeration ratio of 0.5:1. Initially control the stirring speed of the fermenter at 150 rpm and the culture temperature at 25-30℃. Culture continuously for 5-8 days. When the fermentation liquid turns red and a large amount of foam is produced, close the exhaust valve, maintain the pressure inside the tank above 0.1MPa, raise the fermentation temperature to 50℃, and continue to culture for 24 hours. The strain in the Auricularia auricula-judae seed liquid is Auricularia auricula-judae TA58.21. 2) Increase the stirring speed of the fermenter to 350 rpm, heat the fermentation liquid to 80-90℃, keep it at this temperature for 4-8 hours, stop stirring, continue to raise the temperature to 120℃, keep it at this temperature for 30 minutes, causing the cell walls of the auricularia auricula mycelium to break down and release polyphenols. 3) Centrifuge the mycelium to remove precipitates; 4) Collect the supernatant (polyphenolic substances) after centrifugation; 5) The supernatant is pretreated by using a microporous membrane to intercept macromolecular substances. The membrane has a molecular weight cutoff of 100 kDa. 6) Collect the permeate (polyphenolic substances) from the microporous membrane. 7) The permeate from the microporous membrane is subjected to molecular sieve treatment using an ultrafiltration device with a membrane molecular weight of 10 kDa to retain non-target macromolecules with a molecular weight of 10 kDa or higher. 8) Obtain the target product (polyphenolic substance) with a molecular weight below 10 kDa; 9) Concentration: The target product with a molecular weight of less than 10 kDa is concentrated using nanofiltration equipment to remove organic acids, inorganic salts and water with a molecular weight of less than 200 kDa, and obtain a concentrated solution. 10) The concentrate is treated by spray drying or freeze drying to obtain a high-purity product.

[0008] Compared with the prior art, the beneficial effects of the present invention are: (1) In this invention, Auricularia aurea is used for pure culture. Only Auricularia aurea participates in the entire fermentation process, and no Agaricus blazei appears. A large number of cells are proliferated around Auricularia aurea to synthesize polyphenols. A carbon source control mode is adopted to obtain Auricularia aurea polyphenols with good water solubility and high yield.

[0009] (2) Polyphenols in auricularia auricula are produced when the mycelium ages and a large amount of golden pigment appears on the cell surface. The amount of polyphenols released is large, and the concentration in the fermentation broth at the end of fermentation can reach 300 μg / mL (30000 μg / 100mL), which is much higher than the concentration extracted from the fruiting body.

[0010] (3) Polyphenols were synthesized by converting golden ear cells into polyphenols using a liquid fermentation tank. The fermentation cycle was 7-10 days, which is much shorter than the cultivation time of the fruiting body.

[0011] (4) Polyphenols are synthesized using auricularia auricula-judae mycelium. The resulting fermentation broth has low viscosity and good fluidity, making it easy to process later.

[0012] (5) Polyphenols have good thermal and acid stability, and have very strong antioxidant and free radical scavenging abilities. 1 μg of auricularia polyphenols can scavenge 5 μg of DPPH, with a scavenging rate of 90% and an antioxidant capacity of over 92%. Attached Figure Description

[0013] Figure 1 This is a flowchart of a method for preparing *Auricularia auricula-judae* polyphenols by fermentation of the chakra cells of *Auricularia auricula-judae* TA58.21 fungus according to the present invention; Figure 2 The free radical scavenging rate of the fermentation product in one embodiment of the present invention; Figure 3 This invention provides a comparison of the free radical scavenging effects of Ganoderma lucidum polyphenols, hydrogen peroxide, sorghum liquor, and Ganoderma lucidum polysaccharides in one embodiment of the invention. Figure 4 This invention illustrates the changes in Do, pH, protein, and polyphenolic substances during the liquid fermentation of *Auricularia auricula-judae* mycelium in one embodiment of the invention. Figure 4 Figure A shows the dissolved oxygen variation curve. Figure 4 In the graph, B represents the pH change curve. Figure 4 The graph in section C shows the changes in protein content during the fermentation process. Figure 4 D represents the linear growth of polyphenols; Figure 5 This is a comparison of the DPPH scavenging ability of polyphenolic substances from *Auricularia auricula-judae* and the positive control vitamin C in one embodiment of the present invention. Figure 6 This invention provides an analysis of the stability of polyphenolic substances in acidic and alkaline environments in one embodiment of the present invention. Figure 7 This is a stability analysis of the free radical scavenging ability of polyphenols from *Auricularia auricula-judae* under pH=2 conditions in one embodiment of the present invention. Figure 8 This demonstrates the thermal stability of the polyphenolic substance in *Auricularia auricula-judae* at T=80℃ in one embodiment of the present invention. Detailed Implementation

[0014] The present invention will be described in detail below in conjunction with the embodiments shown in the accompanying drawings. It should be noted that these embodiments are not limitations on the present invention, and any equivalent transformation or substitution in function, method, or structure made by those of ordinary skill in the art based on these embodiments falls within the protection scope of the present invention.

[0015] As shown Figure 1 in the present embodiment, a method for fermenting and preparing tremella polyphenols using tremella TA58.21 fungal chassis cells is provided. The strain used in this method is tremella TA58.21, and the preservation number of the tremella TA58.21 strain is CGMCC No. 42090, and the microbial classification and naming is tremella Naematelia aurantialba , which is preserved in the General Microbiological Center of the China Microbial Culture Collection Center. The preservation address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation time is August 28, 2025.

[0016] Taking a 200L fermenter as an example, the liquid filling volume is 150L, and the specific steps are as follows: 1) Add cooked soybean powder medium into the fermenter. After high-temperature sterilization and cooling, inoculate the tremella mycelium seed liquid, introduce sterile oxygen, the aeration ratio is 0.5:1, control the initial stirring speed of the fermenter at 150 rpm, the culture temperature is 25 - 30 °C, and continuously culture for 5 - 8 days. When the fermentation broth turns red and a large amount of foam is generated, close the exhaust valve, maintain the pressure in the tank above 0.1 MPa, raise the fermentation temperature to 50 °C, and continue to culture for 24 h; the strain in the tremella seed liquid is tremella TA58.21.

[0017] 2) Increase the stirring speed of the fermenter to 350 rpm, heat the fermentation broth to 80 - 90 °C, hold for 4 - 8 h, stop stirring, continue to heat up to 120 °C, hold for 30 min, causing the tremella mycelium cell wall to break and release polyphenol substances. This process is to release polyphenol substances and inactivate the tremella fungus at the same time.

[0018] 3) Use a centrifuge to centrifuge the mycelium to remove the precipitate, that is, centrifuge the fermented broth treated at high temperature to remove the precipitate and mycelium.

[0019] 4) Collect the supernatant after centrifugation, and this supernatant contains a large amount of polyphenol substances.

[0020] 5) The supernatant is pretreated by using a microporous filter membrane to intercept macromolecular substances, and the molecular weight cutoff of the filter membrane is 100KDa; 6) Collect the permeate of the microporous filter membrane (polyphenol substance solution); 7) Perform molecular sieve treatment on the permeate of the microporous filter membrane through an ultrafiltration device with a membrane molecular weight of 10KDa to intercept non-target macromolecular substances above 10KDa.

[0021] 8) Obtain the target product (polyphenolic substance) with a molecular weight below 10 kDa; 9) Concentration: The target product with a molecular weight of less than 10 kDa is concentrated using nanofiltration equipment to remove organic acids, inorganic salts and water with a molecular weight of less than 200 kDa, and obtain a concentrated solution. 10) The concentrate is treated by spray drying or freeze drying to obtain a high-purity product.

[0022] This method can be used to obtain auricularia polyphenol products with good water solubility, heat resistance, and acid resistance, and a molecular weight of less than 10,000 Da.

[0023] Figure 2 The figure shows the free radical scavenging rate of the fermentation products in the example (analysis of free radical scavenging by polyphenols produced at different fermentation stages of Auricularia auricula-judae). The figure shows that after 120 hours of fermentation, a large amount of Auricularia auricula-judae polyphenols began to be secreted and produced.

[0024] Figure 3 This example compares the free radical scavenging effects of *Ganoderma lucidum* polyphenols, hydrogen peroxide, sorghum liquor, and *Ganoderma lucidum* polysaccharides (free radical scavenging analysis of *Ganoderma lucidum* polyphenols, hydrogen peroxide, sorghum liquor, and *Ganoderma lucidum*). Figure 3 The hydrogen peroxide used was 3% concentrated, and the sorghum liquor was freshly distilled with an alcohol concentration of 50% (V / V). Both contain free radicals. After these two substances react with the DPPH reagent, the solution color is much deeper than the DPPH reagent background color. Figure 3 A negative DPPH radical scavenging rate indicates that the product has no ability to scavenge free radicals. Figure 3 The results of experiments on the scavenging of free radicals in hydrogen peroxide by dermal growth factor polyphenols (i.e., hydrogen peroxide + dermal growth factor polyphenols) show that dermal growth factor polyphenols can scavenge free radicals in hydrogen peroxide. This indicates that dermal growth factor polyphenols can be applied in the food, beverage, and alcohol industries containing free radicals, effectively reducing the concentration of free radicals in the system. Figure 3 It can be seen that golden ear fungus (i.e., golden ear fungus polyphenols) and Ganoderma lucidum polysaccharides (i.e.,...) Figure 3 Both Ganoderma lucidum (a type of mushroom) and other similar substances have free radical scavenging effects. Hydrogen peroxide and sorghum liquor (specifically, freshly distilled sorghum liquor) do not have free radical scavenging capabilities. However, adding polyphenols from Ganoderma lucidum to hydrogen peroxide can eliminate free radicals. Figure 3 It is known that both golden ear fungus (i.e., golden ear fungus polyphenols) and Ganoderma lucidum polysaccharides have the effect of scavenging free radicals. Hydrogen peroxide and sorghum liquor cannot scavenge free radicals and contain a certain concentration of free radicals themselves. Adding golden ear fungus polyphenols to hydrogen peroxide can eliminate free radicals.

[0025] Figure 4 The changes in Do, pH, protein, and polyphenols during the liquid fermentation of Auricularia auricula-judae mycelium in this example (analysis of changes in various parameters of Auricularia auricula-judae polyphenols at different time points) were analyzed by... Figure 4As can be seen, A and B are the curves showing the changes in dissolved oxygen and pH. The demand for oxygen in golden ear fungus is not high, and the pH decreases and remains around 4.0. C shows the change in protein content during fermentation, with a relatively small increase. D shows the linear increase in polyphenol content.

[0026] Figure 5 In the example, the DPPH scavenging ability of *Auricularia auricularia* polyphenol and the positive control vitamin C was compared (comparison of the free radical scavenging activities of *Auricularia auricularia* polyphenol and vitamin C). The comparison of the free radical scavenging ability of *Auricularia auricularia* polyphenol with that of vitamin C showed that 100 μg of *Auricularia auricularia* polyphenol could replace 40 μg of vitamin C in scavenging free radicals.

[0027] Figure 6 For the stability analysis of polyphenols in acidic and alkaline environments in the examples, Figure 6 The optimal pH range for this polyphenol to scavenge free radicals is 4-8.0.

[0028] Figure 7 This study analyzed the stability of the free radical scavenging ability of the polyphenols from *Auricularia auricula-judae* at pH 2 in the examples. Figure 7 The results showed that the polyphenols in the ear of the ear exhibited good acid resistance at pH 2.0, and their free radical scavenging ability did not decrease as the treatment time was extended to 5 hours, demonstrating good stability.

[0029] Figure 8 To ensure the thermal stability of the polyphenolic substance in the example at T=80℃, it was prepared by... Figure 8 It can be seen that when treated at 80℃ for 5 hours, the DPPH free radical scavenging ability of the auricularia polyphenol did not decrease and remained at a high level, indicating that the auricularia polyphenol has good thermal stability; while the control vitamin C is not heat-resistant and has poor stability, and its free radical scavenging activity continued to decrease with the extension of the heat preservation time.

[0030] This embodiment uses *Auricularia auricula-judae* for pure culture, employing a liquid submerged fermentation mode to cultivate *Auricularia auricula-judae* mycelium, and utilizing the cells of *Auricularia auricula-judae* to synthesize polyphenols. During fermentation, by increasing dissolved oxygen and agitation, *Auricularia auricula-judae* consumes a large amount of carbon source in the culture medium, degrading the starch polysaccharides in the fermentation source, resulting in a polysaccharide concentration in the fermentation broth below 3 mg / mL, a reducing sugar concentration below 2 mg / mL, and a polyphenol concentration above 300 μg / mL. Specifically, this includes the following advantages: 1) This invention uses *Auricularia auricula-judae* for pure culture, utilizing *Auricularia auricula-judae* to synthesize polyphenolic substances, obtaining *Auricularia auricula-judae* polyphenols with good water solubility and high yield. The average fermentation broth concentration can reach 300 μg / mL, far exceeding the concentration extracted from the fruiting body. The fruiting body of *Auricularia auricula-judae* is obtained from *Auricularia auricula-judae* (… Naematelia aurantialba (Bandoni&M. Zang) Millanes&Wedin ) and Leatherbacterium ( Stereum hirsutum (Willd.) Fr They grow together, and their mycelia develop together into the fruiting body of *Auricularia auricula-judae*. The products formed are quite different, and the concentration of polyphenols extracted from the fruiting body is low.

[0031] 2) This invention utilizes a liquid fermenter to transform and synthesize polyphenolic substances from Auricularia auricula-judae cells, representing a significant breakthrough in both time and space. The fermentation cycle is 7 days, and the Auricularia auricula-judae mycelium grows well throughout the entire fermenter without spatial limitations.

[0032] 3) This invention utilizes auricularia auricula mycelium to synthesize polyphenolic substances, resulting in a fermentation broth with low viscosity and good fluidity, which facilitates post-processing.

[0033] 4) The polyphenols of this invention exhibit extremely strong antioxidant and free radical scavenging capabilities. Each μg of *Auricularia auricula-judae* polyphenols scavenges 5 μg of DPPH, achieving a scavenging rate of 90% and an antioxidant capacity exceeding 92%. DPPH (2,2-diphenyl-1-picrylhydrazine) is a commonly used free radical scavenging assay. It is a stable free radical that can react with antioxidants to assess their activity. The principle of the DPPH free radical scavenging experiment is that when DPPH molecules encounter antioxidants, the antioxidants capture the unpaired electrons of DPPH, causing the DPPH molecule to change from purple to colorless. This change can be quantitatively measured by measuring the absorbance change at a wavelength of 517 nm using a spectrophotometer. The mechanisms of action of free radical scavengers include directly scavenging free radicals, inhibiting free radical generation, and enhancing the body's antioxidant defense system. Through these mechanisms, free radical scavengers help maintain human health, delay the aging process, and prevent the occurrence of various diseases.

[0034] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A strain of *Auricularia auricula-judae* TA58.21, characterized in that, The preservation number is CGMCC No. 42090, and the microbial classification name is Auricularia auricula-judae. Naematelia aurantialba It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on August 28, 2025.

2. A method for preparing *Auricularia auricula-judae* polyphenols by fermentation of the basal cells of *Auricularia auricula-judae* TA58.21 fungus, characterized in that, Includes the following steps: 1) Add cooked soybean powder culture medium to a 200L fermenter. After high-temperature sterilization and cooling, inoculate with Auricularia auricula-judae seed liquid. Introduce sterile oxygen at an aeration ratio of 0.5:

1. Initially control the stirring speed of the fermenter at 150 rpm and the culture temperature at 25-30℃. Culture continuously for 5-8 days. When the fermentation liquid turns red and a large amount of foam is produced, close the exhaust valve, maintain the pressure inside the tank above 0.1MPa, raise the fermentation temperature to 50℃, and continue to culture for 24 hours. The strain in the Auricularia auricula-judae seed liquid is Auricularia auricula-judae TA58.

21. 2) Increase the stirring speed of the fermenter to 350 rpm, heat the fermentation liquid to 80-90℃, keep it at this temperature for 4-8 hours, stop stirring, continue to raise the temperature to 120℃, keep it at this temperature for 30 minutes, causing the cell walls of the auricularia auricula mycelium to break down and release polyphenols. 3) Centrifuge the mycelium to remove precipitates; 4) Collect the supernatant after centrifugation; 5) The supernatant is pretreated by using a microporous membrane to intercept macromolecular substances. The membrane has a molecular weight cutoff of 100 kDa. 6) Collect the permeate from the microporous membrane; 7) The permeate from the microporous membrane is subjected to molecular sieve treatment using an ultrafiltration device with a membrane molecular weight of 10 kDa to retain non-target macromolecules with a molecular weight of 10 kDa or higher. 8) Obtain the target product with a molecular weight below 10 kDa; 9) Concentration: The target product with a molecular weight of less than 10 kDa is concentrated using nanofiltration equipment to remove organic acids, inorganic salts and water with a molecular weight of less than 200 kDa, and obtain a concentrated solution. 10) The concentrate is treated by spray drying or freeze drying to obtain a high-purity product.

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