A method for preparing *Auricularia auricula-judae* polyphenols by fermentation of the chakra cells of *Auricularia auricula-judae* TA58.21 fungus.

Abstract

This invention belongs to the field of biopharmaceutical technology and relates to a method for preparing *Auricularia auricula-judae* polyphenols by fermentation of the basal cells of *Auricularia auricula-judae* TA58.21 fungus. The method includes: adding cooked soybean powder culture medium and *Auricularia auricula-judae* seed liquid to a fermenter; introducing sterile oxygen; controlling the stirring speed of the fermenter; maintaining the culture temperature at 25-30℃; continuously culturing for 5-8 days; when the fermentation broth turns red and produces a large amount of foam; closing the exhaust valve; maintaining the tank pressure at 0.1 MPa; increasing the fermentation temperature to 50℃; continuing culture for 24 hours; increasing the stirring speed of the fermenter; raising the temperature of the fermentation broth to 80-90℃; maintaining this temperature for 4-8 hours; stopping stirring; and continuing to raise the temperature to 120℃; maintaining this temperature for 30 minutes; centrifuging the fermentation broth to remove precipitates and mycelia; collecting the supernatant for pretreatment of macromolecules and fungal fragments; collecting the permeate and passing it through a nanofiltration membrane to obtain a concentrated solution; and spray-drying or freeze-drying the concentrated solution to obtain a high-purity product. This invention enables the production of auricularia polyphenol products with good water solubility, heat resistance, and acid resistance, and a molecular weight of less than 10,000 Da.

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 chakra cells of *Auricularia auricula-judae* TA58.21 fungus, comprising the following steps:

[0008] 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.

[0009] 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.

[0010] 3) Centrifuge the mycelium to remove precipitates;

[0011] 4) Collect the supernatant (polyphenolic substances) after centrifugation;

[0012] 5) The supernatant is pretreated by using a microporous membrane to intercept macromolecular substances. The membrane has a molecular weight cutoff of 100 kDa.

[0013] 6) Collect the permeate (polyphenolic substances) from the microporous membrane.

[0014] 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.

[0015] 8) Obtain the target product (polyphenolic substance) with a molecular weight below 10 kDa;

[0016] 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.

[0017] 10) The concentrate is treated by spray drying or freeze drying to obtain a high-purity product.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] (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.

[0020] (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.

[0021] (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.

[0022] (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.

[0023] (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

[0024] 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;

[0025] Figure 2 The free radical scavenging rate of the fermentation product in one embodiment of the present invention;

[0026] 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.

[0027] 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 change 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;

[0028] 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.

[0029] 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.

[0030] Figure 7 Stability analysis of the free radical scavenging ability of Tremella aurantialba polyphenols under the condition of pH = 2 in an embodiment of the present invention;

[0031] Figure 8 Thermal stability of Tremella aurantialba polyphenols at T = 80 °C in an embodiment of the present invention. Detailed implementation manners

[0032] The present invention will be described in detail below in conjunction with the embodiments shown in the drawings. However, it should be noted that these embodiments are not limitations of the present invention, and any equivalent transformation or substitution in terms of function, method, or structure made by those of ordinary skill in the art based on these embodiments shall fall within the protection scope of the present invention.

[0033] Refer Figure 1 As shown, this embodiment provides a method for fermenting and preparing Tremella aurantialba polyphenols using the fungal chassis cell of Tremella aurantialba TA58.21. The strain used in this method is Tremella aurantialba TA58.21, and the preservation number of the Tremella aurantialba TA58.21 strain is CGMCC No. 42090, and the microbial classification name is Tremella aurantialba Naematelia aurantialba , which is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation time is August 28, 2025.

[0034] Taking a 200 L fermenter as an example, with a liquid loading volume of 150 L, the specific steps are as follows:

[0035] 1) Add cooked soybean powder medium into the fermenter. After high-temperature sterilization and cooling, inoculate the Tremella aurantialba mycelium seed liquid, introduce sterile oxygen, with an aeration ratio of 0.5:1, control the initial stirring speed of the fermenter at 150 rpm, the culture temperature at 25 - 30 °C, and continuously culture for 5 - 8 days. When the fermentation broth turns red and a large amount of foam appears, 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 aurantialba seed liquid is Tremella aurantialba TA58.21.

[0036] 2) Increase the stirring speed of the fermenter to 350 rpm, heat the fermentation broth to 80 - 90 °C, keep it warm for 4 - 8 h, stop stirring, continue to raise the temperature to 120 °C, and keep it warm for 30 min to cause the cell wall of the Tremella aurantialba mycelium to break and release polyphenols. This process is to release polyphenols and inactivate the Tremella aurantialba fungus at the same time.

[0037] 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.

[0038] 4) Collect the supernatant after centrifugation, which contains a large amount of polyphenols.

[0039] 5) The supernatant is pretreated by using a microporous membrane to intercept macromolecular substances. The membrane has a molecular weight cutoff of 100 kDa.

[0040] 6) Collect the permeate (polyphenol solution) from the microporous membrane.

[0041] 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.

[0042] 8) Obtain the target product (polyphenolic substance) with a molecular weight below 10 kDa;

[0043] 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.

[0044] 10) The concentrate is treated by spray drying or freeze drying to obtain a high-purity product.

[0045] 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.

[0046] 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.

[0047] 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 3Both 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.

[0048] Figure 4 The changes in Do, pH, protein, and polyphenolic substances 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 4 As 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.

[0049] Figure 5 In this 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.

[0050] 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.

[0051] Figure 7 This study analyzed the stability of the free radical scavenging ability of the polyphenols from *Auricularia auricula-judae* under pH=2 conditions 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.

[0052] 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 the DPPH free radical scavenging ability of the polyphenols treated at 80℃ for 5 hours did not decrease and remained at a high level, indicating that the polyphenols have 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 treatment time.

[0053] 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:

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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 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 culture. 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 broth turns red and produces a large amount of foam, close the exhaust valve, maintain the pressure inside the tank above 0.1 MPa, raise the fermentation temperature to 50℃, and continue culture for 24 hours. The strain in the *Auricularia auricula-judae* seed culture is *Auricularia auricula-judae* TA58.21, with preservation number CGMCC No. 42090, and microbiologically named *Auricularia auricula-judae*. Naematelia aurantialba It 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. 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 a microporous membrane to retain 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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