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] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for fermenting and preparing tremella polyphenols using the tremella aurantialba TA58.21 fungal chassis cells. Background Art

[0002] Tremella aurantialba belongs to the Fungi kingdom, Basidiomycota phylum, Tremellomycetes class, Tremellales order, Helicobasidiaceae family, Helicobasidium genus, and Tremella aurantialba species. The fruiting body of Tremella aurantialba is rich in nutrients such as protein, dietary fiber, fat, β-carotene, various amino acids, vitamins (VB, VC, VD), minerals (zinc, iron, potassium, magnesium), etc., as well as bioactive substances such as polysaccharides, polyphenols, flavonoids, and coumarins. It has effects such as hypoglycemic, hypolipidemic, antioxidant, anti-inflammatory, antithrombotic, anticoagulant, anti-radiation, antiviral, resolving phlegm and relieving cough, and enhancing immunity. It is a rare medicinal and edible fungus and a nourishing nutritional product, and is widely used in the fields of food, medicine, and cosmetics.

[0003] Tremella polyphenols are natural compounds present in Tremella aurantialba and have various biological activities, including antioxidant, hypoglycemic, hypolipidemic, enhancing immunity, anti-inflammatory, anticoagulant, and anti-tumor effects.

[0004] Currently, there is little research on fermenting polyphenolic substances using the mycelium of Tremella aurantialba, and there are also few research reports on extracting polyphenolic substances from the fruiting body. Tremella polyphenolic substances are mostly extracted from the fruiting body of Tremella aurantialba, and the fruiting body of Tremella aurantialba is a product formed by the symbiosis of Tremella aurantialba and Stereum hirsutum. The content of colloidal substances is very high and it is very viscous. The operation steps for extracting polyphenolic substances are cumbersome, and the extraction concentration is low and the yield is not high. The fruiting body is produced by symbiotic bacteria, and the hydrolysis products are complex mixtures. It is not advisable to extract polyphenolic substances from the fruiting body. Summary of the Invention

[0005] The purpose of the present invention is to provide a biological manufacturing method for fermenting and preparing tremella polyphenols using the tremella aurantialba TA58.21 fungal chassis cells, using pure culture of Tremella aurantialba, culturing the mycelium of Tremella aurantialba in a liquid deep fermentation mode, and synthesizing polyphenolic substances using the mycelial cells of Tremella aurantialba. This polyphenolic substance has high temperature resistance and acid resistance, and has obvious antioxidant and free radical scavenging effects. Every μg of tremella polyphenols can scavenge 5 μg of DPPH, and the DPPH free radical scavenging ability reaches more than 90%, and the antioxidant ability is more than 92%. In terms of thermal stability, it will not be damaged under the condition of 120 °C, and its biological activity is not easily inactivated under the condition of pH = 2.

[0006] The present invention provides a method for fermenting and preparing tremella polyphenols using the tremella aurantialba TA58.21 fungal chassis cells, comprising the following steps: 1) Add cooked soybean powder medium into a 200L fermenter. After high-temperature sterilization and cooling, inoculate Tremella aurantialba 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 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 aurantialba seed liquid is Tremella aurantialba TA58.21; 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 walls of Tremella aurantialba hyphae to break and release polyphenol substances; 3) Use a centrifuge to centrifuge the mycelium and remove the precipitate; 4) Collect the supernatant (polyphenol substances) after centrifugation; 5) Pretreat the supernatant by using a microporous membrane to intercept macromolecular substances, with the molecular weight cut-off of the membrane being 100 KDa; 6) Collect the permeate (polyphenol substances) of the microporous membrane; 7) Perform molecular sieve treatment on the permeate of the microporous membrane through an ultrafiltration device with a membrane molecular weight of 10 KDa, and intercept non-target macromolecular substances with a molecular weight above 10 KDa; 8) Obtain the target product (polyphenol substances) with a molecular weight below 10 KDa; 9) Concentrate, and perform concentration treatment on the target product with a molecular weight below 10 KDa through a nanofiltration device to remove organic acids, inorganic salts, and water with a molecular weight below 200 Da to obtain a concentrated solution; 10) Treat the concentrated solution by spray drying or freeze drying to obtain a high-purity product.

[0007] Compared with the prior art, the beneficial effects of the present invention are: (1) The present invention adopts pure culture of Tremella aurantialba. Only Tremella aurantialba participates in the whole fermentation process, and there is no appearance of Stereum hirsutum. A large number of cells are proliferated around Tremella aurantialba to synthesize polyphenol substances, and a carbon source control mode is adopted to obtain Tremella aurantialba polyphenols with good water solubility and high yield.

[0008] (2) Tremella aurantialba polyphenols are produced when the mycelium ages and a large amount of golden pigments appear on the cell surface. The release amount of Tremella aurantialba polyphenols is large, and the concentration in the fermentation broth at the end of fermentation can reach 300 μg / mL (30000 μg / 100 mL), which is much higher than the extraction concentration from fruiting bodies.

[0009] (3) Use a liquid fermenter to transform and synthesize polyphenol substances from Tremella aurantialba cells. The fermentation cycle is 7 - 10 days, which is much lower than the cultivation time of fruiting bodies.

[0010] (4)Using Tremella aurantialba mycelium to synthesize polyphenolic substances, the formed fermentation broth has a low viscosity and good fluidity, which is convenient for post-treatment.

[0011] (5)The polyphenolic substances have good thermal stability and acid stability, and their antioxidant and free radical scavenging abilities are very strong. 1 μg of Tremella aurantialba polyphenols can scavenge 5 μg of DPPH, with a scavenging rate of 90% and an antioxidant ability of over 92%. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a flow chart of a method for fermenting and preparing Tremella aurantialba polyphenols using the fungal chassis cell of Tremella aurantialba TA58.21 of the present invention; Figure 2 It is the scavenging rate of the fermentation product on free radicals in an embodiment of the present invention; Figure 3 It is a comparison of the free radical scavenging of Tremella aurantialba polyphenols, hydrogen peroxide, sorghum wine, and Ganoderma lucidum polysaccharide in an embodiment of the present invention; Figure 4 It is the change of the liquid fermentation parameters Do, pH, protein, and polyphenolic substances of Tremella aurantialba mycelium in an embodiment of the present invention; Figure 5 It is a comparison of the DPPH scavenging abilities of Tremella aurantialba polyphenolic substances and the positive control Vc in an embodiment of the present invention; Figure 6 It is the stability analysis of the polyphenolic substances of Tremella aurantialba in an acidic and alkaline environment in an embodiment of the present invention; Figure 7 It is the stability analysis of the free radical scavenging ability of the polyphenolic substances of Tremella aurantialba under the condition of pH = 2 in an embodiment of the present invention; Figure 8 It is the thermal stability of the polyphenolic substances of Tremella aurantialba under the condition of T = 80 °C in an embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0013] The present invention will be described in detail below with reference to the embodiments shown in the drawings. However, it should be noted that these embodiments are not intended to limit the present invention. 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.

[0014] 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 ( Tremella aurantialba ), with the product number Bio-52358, purchased from Zhili Zhongte (Wuhan) Biotechnology Co., Ltd. After repeated screening for many times, the strain TA58.21 suitable for liquid fermentation was obtained (the TA58.21 is a self-numbered strain).

[0015] Taking a 200L fermenter as an example, with a liquid filling volume of 150L, the specific steps are as follows: 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 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 culturing for 24 h; the strain in the Tremella aurantialba seed liquid is Tremella aurantialba TA58.21.

[0016] 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 breakdown of the cell wall of Tremella aurantialba mycelium and release polyphenolic substances. This process is to release polyphenolic substances and inactivate the Tremella aurantialba at the same time.

[0017] 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.

[0018] 4) Collect the supernatant after centrifugation. This supernatant contains a large amount of polyphenolic substances.

[0019] 5) Pretreat the supernatant by using a microporous membrane to intercept macromolecular substances. The molecular weight cut-off of the membrane is 100KDa; 6) Collect the permeate of the microporous membrane (polyphenolic substance solution); 7) Perform molecular sieve treatment on the permeate of the microporous membrane through an ultrafiltration device with a membrane molecular weight of 10KDa to intercept non-target macromolecular substances with a molecular weight above 10KDa.

[0020] 8) Obtain the target product (polyphenolic substances) with a molecular weight below 10KDa; 9) Concentrate. Concentrate the target product with a molecular weight below 10KDa through a nanofiltration device to remove organic acids, inorganic salts, and water with a molecular weight below 200Da to obtain a concentrated solution; 10) Treat the concentrated solution by spray drying or freeze drying to obtain a high-purity product.

[0021] Through this method, it is possible to obtain a Tremella aurantialba polyphenol product with good water solubility, heat resistance, acid resistance, and a molecular weight less than 10,000Da.

[0022] Figure 2 For the scavenging rate of free radicals by the fermentation product in the example (analysis of the scavenging of free radicals by polyphenolic substances produced during different periods of Tremella aurantialba fermentation), this figure shows that after 120 h of fermentation time, a large amount of Tremella aurantialba polyphenols begin to be secreted and produced.

[0023] Figure 3 For the comparison of free radical scavenging effects of golden ear polyphenols and hydrogen peroxide, sorghum wine and ganoderma lucidum polysaccharide in the embodiment (free radical scavenging analysis of golden ear polyphenols and hydrogen peroxide, sorghum wine and ganoderma lucidum), Figure 3 It can be seen that golden ear and Ganoderma lucidum polysaccharides both have the function of scavenging free radicals, while hydrogen peroxide and sorghum wine cannot scavenge free radicals and themselves contain a certain concentration of free radicals; adding golden ear polyphenols to hydrogen peroxide can eliminate free radicals.

[0024] Figure 4 The changes of Do, pH, protein and polyphenols in the liquid fermentation of Tremella fuciformis mycelium in the embodiment (analysis of the changes of various parameters of Tremella fuciformis polyphenols in different time periods) are shown in FIG. Figure 4 It can be seen that A and B are the curves of dissolved oxygen and pH changes. Tremella fuciformis has a low demand for oxygen, and the pH drops and remains at around 4.0; C is the change in protein content during the fermentation process, and the increase is basically small; D is the linear growth of polyphenols.

[0025] Figure 5 This is a comparison of the DPPH scavenging ability of golden ear polyphenols and the positive control Vc in the examples (comparison of the anti-free radical activity of golden ear polyphenols and Vc). The comparison of the free radical scavenging ability of golden ear polyphenols with VC shows that the free radicals scavenged by 40μg of Vc can be replaced by 100μg of golden ear polyphenols.

[0026] Figure 6 The stability analysis of Tremella fuciformis polyphenols in acid and alkaline environments in the examples is shown in the figure. Figure 6 It shows that the optimal pH range for the free radical scavenging effect of the golden ear polyphenols is 4-8.0.

[0027] Figure 7 This is the stability analysis of the free radical scavenging ability of Tremella fuciformis polyphenols under the condition of pH=2 in the examples. Figure 7 The results showed that golden ear polyphenols were acid-resistant at pH 2.0, and their free radical scavenging ability did not decrease as the treatment time was extended to 5 h, indicating good stability.

[0028] Figure 8 The thermal stability of the golden ear polyphenols in the embodiment at T = 80 ° C is obtained by Figure 8 It can be seen that the DPPH radical scavenging ability of golden ear polyphenol did not decrease after being treated at 80℃ for 5h, and remained at a high point, indicating that the golden ear polyphenol has good thermal stability; while the control Vc is not heat-resistant and has poor stability. As the insulation time increases, the free radical scavenging activity continues to decrease.

[0029] In this embodiment, Tremella aurantialba is used for pure culture, and the liquid submerged fermentation mode is utilized to culture the mycelium of Tremella aurantialba, and polyphenolic substances are synthesized by the cells of Tremella aurantialba. During the fermentation process of Tremella aurantialba, by increasing the dissolved oxygen and agitation, a large amount of carbon source in the culture medium is consumed, and after the starch polysaccharide in the fermentation source is degraded, the concentration of polysaccharide substances in the fermentation broth is lower than 3 mg / mL, the reducing sugar is lower than 2 mg / mL, and the concentration of the produced polyphenolic substances is higher than 300 μg / mL. Specifically, the following advantages are included: 1) In the present invention, Tremella aurantialba is used for pure culture, and polyphenolic substances are synthesized by Tremella aurantialba to obtain Tremella aurantialba polyphenolic substances with good water solubility and high yield. The average concentration of the fermentation broth can reach 300 μg / mL, which is much higher than the extraction concentration from the fruiting body. The fruiting body of Tremella aurantialba is accompanied by the growth of Tremella aurantialba ( Naematelia aurantialba (Bandoni&M. Zang) Millanes&Wedin ), and Stereum hirsutum ( Stereum hirsutum(Willd.)Fr ). The mycelia of the two grow together, and their common tissue develops into the fruiting body of Tremella aurantialba. The formed products are quite different, and the concentration of polyphenolic substances extracted from the fruiting body is low.

[0030] 2) In the present invention, a liquid fermentation tank is used to transform and synthesize polyphenolic substances from the cells of Tremella aurantialba, which has made a major breakthrough in terms of time and space. The fermentation period is 7 days, and the mycelium of Tremella aurantialba grows well on the entire fermentation tank without being restricted by space.

[0031] 3) In the present invention, polyphenolic substances are synthesized by the mycelium of Tremella aurantialba, and the viscosity of the formed fermentation broth is not high, and the fluidity is relatively good, which is convenient for post-treatment.

[0032] 4) The antioxidant and free radical scavenging abilities of the polyphenolic substances in the present invention are very powerful. Every μg of Tremella aurantialba polyphenols can scavenge 5 μg of DPPH, and the scavenging rate is 90%, and the antioxidant ability is more than 92%. DPPH (2,2-diphenyl-1-picrylhydrazyl) is a commonly used method for free radical scavenging experiments. It is a stable free radical that can react with antioxidants to evaluate the activity of antioxidants. The principle of the DPPH free radical scavenging experiment is that when the DPPH molecule encounters an antioxidant, the antioxidant will capture the unpaired electron of DPPH, causing the DPPH molecule to change from purple to colorless. This change can be quantitatively measured by the change in absorbance at a wavelength of 517 nm using a spectrophotometer. The mechanisms of action of free radical scavengers include directly scavenging free radicals, inhibiting the generation of free radicals, and enhancing the body's antioxidant defense system, etc. Through these mechanisms, free radical scavengers contribute to maintaining human health, delaying the aging process, and preventing the occurrence of various diseases.

[0033] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in all respects, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Accordingly, all changes that fall within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

Claims

1. A method for fermenting and preparing tremella aurantialba polyphenols using tremella aurantialba TA58.21 fungal chassis cells, characterized in that, It includes the following steps: 1) Add cooked soybean powder medium into a 200L fermenter. After high-temperature sterilization and cooling, inoculate Tremella aurantialba 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 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 aurantialba seed liquid is Tremella aurantialba TA58.21; 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 heat up to 120 °C, and keep it warm for 30 min to cause the cell wall of Tremella aurantialba mycelium to break and release polyphenolic substances; 3) Centrifuge the mycelium using a centrifuge to remove the precipitate; 4) Collect the supernatant after centrifugation; 5) Pretreat the supernatant by using a microporous membrane to intercept macromolecular substances, with the molecular weight cut-off of the membrane being 100KDa; 6) Collect the permeate of the microporous membrane; 7) Perform molecular sieve treatment on the permeate of the microporous membrane through an ultrafiltration device with a membrane molecular weight of 10KDa to intercept non-target macromolecular substances with a molecular weight above 10KDa; 8) Obtain the target product with a molecular weight below 10KDa; 9) Concentrate, perform concentration treatment on the target product with a molecular weight below 10KDa through a nanofiltration device to remove organic acids, inorganic salts, and water with a molecular weight below 200Da to obtain a concentrated solution; 10) Treat the concentrated solution by spray drying or freeze drying to obtain a high-purity product.

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