High-uronic-acid tremella polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microbial fermentation and biochemistry, in particular to high-uronic-acid tremella polysaccharide as well as a preparation method and application thereof. According to the method, a specific tremella fuciformis strain FTPS005.1 is adopted, and the high-molecular-weight (HU-HTPS) and low-molecular-weight (HU-LTPS) tremella fuciformis polysaccharides are synchronously prepared through one-step fermentation by optimizing the composition of a fermentation culture medium, controlling the temperature in a segmented manner, controlling the pH in a four-segment manner and adopting a material supplementing strategy. The uronic acid content is not less than 30%, the total sugar content is not less than 90%, and the protein content is not more than 0.1%. The obtained product has excellent oxidation resistance and moisturizing performance, and can be widely applied to the fields of food, health care products and cosmetics. The method is simple in process, high in yield and controllable in cost, and has good industrial popularization value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial fermentation and biochemistry, and particularly relates to a high uronic acid Tremella polysaccharide and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in the background of the present application is only intended to increase the understanding of the overall background of the present application and should not necessarily be regarded as acknowledging or implying in any form that the information constitutes prior art known to those skilled in the art.

[0003] Tremella fuciformis Berk, also known as white fungus, is a traditional medicinal and edible fungus in China, and is known as the "crown of fungi". It is mainly distributed in Fujian, Sichuan and other places, and is a unique edible fungus species in China. Tremella fuciformis Berk is rich in carbohydrates, proteins, various amino acids, vitamins and Tremella polysaccharide. Tremella polysaccharide is the main active substance of Tremella fuciformis Berk, and is currently extracted directly from the fruiting body. It has the effects of immune regulation, antioxidant, anti-tumor, blood glucose regulation, yin-nourishing and lung-moistening, and is widely used in food, health food and cosmetics.

[0004] Uronic acid, as an important acidic monosaccharide, is a key structural unit of various biological macromolecules. Its unique physicochemical properties and biological activities have a decisive influence on the function of polysaccharides. First, the carboxyl group (-COOH) in the uronic acid molecule acts as a strong hydrophilic group, which can bind a large number of water molecules through hydrogen bonding, significantly enhancing the water-holding capacity of polysaccharides. Second, the content of uronic acid directly affects the ability of polysaccharides to form specific types of gels, and is a key factor in regulating gel properties. In addition, uronic acid, as the functional center of various bioactive polysaccharides, is widely involved in physiological processes such as detoxification, anti-inflammatory, immune regulation, intestinal health maintenance, anticoagulation, and blood lipid, blood glucose and blood pressure reduction. At the same time, the negative charge carried by its carboxyl group can strongly bind to positively charged metal ions such as calcium, lead and cadmium, which not only helps to regulate mineral absorption and balance in the body, but also plays a role as a heavy metal detoxifier. Finally, uronic acid is also an important component of cell walls and extracellular matrix, providing necessary structural support for cells.

[0005] At present, the content of uronic acid in Tremella polysaccharide prepared by conventional methods is generally between 18% and 22%. Although some documents have reported that Tremella polysaccharide with uronic acid content exceeding 30% can be obtained by column chromatography purification technology, this process has problems such as low total sugar yield and high purification cost, which is difficult to meet the needs of large-scale production. Therefore, it is of important research value and application prospect to develop a new preparation process that can directly and efficiently synthesize Tremella polysaccharide with high uronic acid content through fermentation process. SUMMARY

[0006] In view of this, the present application provides a high uronic acid tremella polysaccharide and its preparation method and application. Through optimizing the composition of the fermentation medium, segmental temperature control, four-stage pH control and feeding strategy, the present application realizes the one-step fermentation for the simultaneous preparation of high molecular weight (HU-HTPS) and low molecular weight (HU-LTPS) tremella polysaccharides. The obtained products have high uronic acid content and high purity, and have excellent antioxidant, moisturizing properties and good biocompatibility, and can be widely used in the fields of functional food, health products and cosmetics (especially antioxidant and moisturizing skin care products).

[0007] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme: In a first aspect, the present application provides a preparation method of a high uronic acid tremella polysaccharide, comprising the following steps: (1) inoculating the activated strain into a seed culture medium to culture to obtain a first-stage seed liquid, and then inoculating the first-stage seed liquid into a second-stage seed culture medium to culture to obtain a second-stage seed liquid; (2) inoculating the activated strain into a seed culture medium to culture to obtain a first-stage seed liquid, and then inoculating the first-stage seed liquid into a second-stage seed culture medium to culture to obtain a second-stage seed liquid; (3) inoculating the second-stage seed liquid into a fermentation medium to culture to obtain a tremella fermentation liquid; (4) subjecting the tremella fermentation liquid to hot alkali hydrolysis, enzymolysis, centrifugation, then filtering, desalting and decolorizing the supernatant, alcohol precipitation, washing, dehydration and drying to obtain a high uronic acid high molecular weight tremella polysaccharide (HU-HTPS, tremella polysaccharide of high molecular weight of High uronic acid content); (5) subjecting the centrifuged bacterial cells to homogenization, acid hydrolysis, enzymolysis, filtration, desalting and decolorizing, and spray drying to obtain a high uronic acid low molecular weight tremella polysaccharide (HU-LTPS, tremella polysaccharide of low molecular weight of High uronic acid content).

[0008] Further, the tremella spore strain FTPS005.1 (described in CN119736169A) is a laboratory strain, which has been preserved in the China Center for Type Culture Collection on October 21, 2024, at the address of China. Wuhan. Wuhan University, with the preservation number of CCTCC M 20242283.

[0009] Further, in step (1), the activation culture parameters are: 24-28 ℃ culture for 2-4 days.

[0010] Further, in step (1), the activation culture medium comprises the following components: glucose 40-80 g / L, yeast powder 1-3 g / L, peptone 1-3 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, potassium dihydrogen phosphate 0.3-0.5 g / L, magnesium sulfate heptahydrate 0.5-2 g / L, vitamin B1 5-20 mg / L, agar powder 10-30 g / L, solvent is water, and pH is natural; preferably, the activation culture medium comprises the following components: glucose 60 g / L, yeast powder 2 g / L, peptone 2 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.46 g / L, magnesium sulfate heptahydrate 1 g / L, vitamin B1 10 mg / L, agar powder 20 g / L, solvent is water, and pH is natural.

[0011] Further, in step (2), the specific operation is as follows: the activated strain obtained in step (1) is inoculated into a seed culture medium, and the seed culture medium is cultured at 26-28 ℃ and a rotation speed of 160-220 r / min until OD 560 ≥5, and the first-stage seed liquid is inoculated into a second-stage seed culture medium at a volume ratio of 5%-15%, and the second-stage seed culture medium is cultured at 24-28 ℃ and a rotation speed of 160-220 r / min until OD 560 ≥15, to obtain a second-stage seed liquid.

[0012] Further, in step (2), the first-stage seed culture medium comprises the following components: glucose 40-80 g / L, yeast powder 1-3 g / L, peptone 1-3 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, potassium dihydrogen phosphate 0.3-0.5 g / L, magnesium sulfate heptahydrate 0.5-2 g / L, vitamin B1 5-20 mg / L, solvent is water, and pH is natural; preferably, the first-stage seed culture medium comprises the following components: glucose 60 g / L, yeast powder 2 g / L, peptone 2 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.46 g / L, magnesium sulfate heptahydrate 1 g / L, vitamin B1 10 mg / L, solvent is water, and pH is natural.

[0013] Further, in step (2), the second-stage seed culture medium comprises the following components: glucose 5-20 g / L, sucrose 5-20 g / L, yeast powder 1-3 g / L, peptone 2-6 g / L, dipotassium hydrogen phosphate 0.5-2 g / L, potassium dihydrogen phosphate 0.3-0.5 g / L, magnesium sulfate heptahydrate 0.5-2 g / L, solvent is water, and pH is natural; preferably, the second-stage seed culture medium comprises the following components: glucose 10 g / L, sucrose 10 g / L, yeast powder 2 g / L, peptone 4 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.46 g / L, magnesium sulfate heptahydrate 1 g / L, solvent is water, and pH is natural.

[0014] Further, in step (3), the secondary seed liquid is inoculated into the spore fermentation medium at a volume ratio of 8%-15%; the fermentation endpoint is reached when the pH rises to above 5.5 and the viscosity no longer increases.

[0015] Further, in step (3), the fermentation culture is operated in two temperature control sections and four pH control sections; the two temperature control sections are that the temperature is controlled at 28±0.5 ℃ for 0-96 h, and then reduced to 26±0.5 ℃; the four pH control sections are that the pH is natural for 0-24 h, controlled at 5.5-6.5 for 24-48 h, controlled at 4.5-5.0 for 48-120 h, and then natural.

[0016] Further, in step (3), the sugar is supplemented after 60 h of fermentation, and the sugar is supplemented in two batches with an interval of 2-4 h, and the total amount of the supplemented sugar is calculated according to the fructose content of 35-60 g / L in the fermentation system.

[0017] Further, in step (3), the sugar is supplemented with F42 fructose-glucose syrup (dry matter content greater than 71%, fructose content greater than 42%, and glucose content greater than 52%), F55 fructose-glucose syrup (dry matter content greater than 77%, fructose content greater than 55%, and glucose content greater than 40%), or F60 fructose-glucose syrup (dry matter content greater than 75%, fructose content greater than 60%, and glucose content greater than 35%); preferably, the sugar is supplemented with F55 fructose-glucose syrup (dry matter content greater than 77%, fructose content greater than 55%, and glucose content greater than 40%).

[0018] Further, in step (3), the pH regulation is performed using 10%-30% sodium hydroxide aqueous solution and 5%-10% sulfuric acid aqueous solution; preferably, the pH regulation is performed using 30% sodium hydroxide aqueous solution and 10% sulfuric acid aqueous solution.

[0019] Further, in step (3), the dissolved oxygen is 20%-30%, the aeration amount is 1-2 VVM, and the initial stirring speed is 200-500 r / min.

[0020] Further, in step (4), after the fermentation is completed, water is added according to a fermentation liquid:water volume ratio of 1:1-3, then the fermentation liquid is directly heated to 60-80 ℃, the pH is controlled at 8-10, and the alkaline treatment is performed for 1-3 h.

[0021] Further, in step (4), the enzymolysis is performed by reducing the temperature to 40-50 ℃, controlling the pH at 7-8, adding 0.5‰-1.5‰ trypsin, and enzymolysis for 0.5-1.5 h.

[0022] Further, in step (4), centrifugation is performed by adding 2%-4% sodium chloride to the pretreated fermentation broth and centrifuging it in a tube-type centrifuge with a centrifugal force controlled at 12000-14000 g, and collecting the supernatant and bacterial cells respectively.

[0023] Further, in step (4), filtration includes coarse filtration and fine filtration; coarse filtration involves adjusting the pH of the supernatant to 8.5-9.0, selecting 3-5 μm diatomaceous earth filter paper, using perlite as a filter aid, and filtering with a plate and frame filter; fine filtration involves adjusting the pH of the filtrate to 5.0-6.0, selecting 1-3 μm diatomaceous earth filter paper + 1-4 μm activated carbon deep filter paper, using diatomaceous earth as a filter aid, and circulating the filtrate twice with a plate and frame filter; after passing through 0.45 μm and 0.22 μm filter membranes, a clear filtrate is obtained.

[0024] Further, in step (4), desalting, decolorizing and alcohol precipitation involves transferring the clarified filtrate into an empty container, turning on the stirrer, slowly adding 95% ethanol to an alcohol content of 62°-65°, continuing to stir for 15-20 min, and then letting it stand.

[0025] Further, in step (4), the washing process involves discarding the supernatant, adding 75° ethanol to the precipitate, stirring for 15-20 min, and letting it stand; discarding the supernatant again, adding 80° ethanol to the precipitate, stirring for 15-20 min, and letting it stand; discarding the supernatant again, adding 85° ethanol to the precipitate, stirring for 15-20 min, and letting it stand.

[0026] Further, in step (4), the supernatant is discarded during dehydration, 95° ethanol is added to the precipitate, stirring is continued for 15-20 minutes, and then the mixture is allowed to stand.

[0027] Further, in step (4), the drying is carried out by negative pressure filtration in a Buchner funnel. After the ethanol is removed, the product is dried in a vacuum drying oven at 50-60 ℃ for 4-6 h. After drying, the product is pulverized evenly in a pulverizer and passed through a 200-mesh sieve to obtain a pure white HU-HTPS product.

[0028] Further, in step (5), homogenization involves re-dissolving the bacterial cells in water at 1 / 3 to 1 / 2 of the original fermentation liquid volume, homogenizing them twice in a high-pressure homogenizer at 800-1000 bar, and then adding water to the original fermentation liquid volume.

[0029] Further, in step (5), acid hydrolysis involves transferring the homogenized liquid to a glass reactor, starting the stirrer, controlling the temperature at 65-80 ℃, and after the temperature stabilizes, adding sulfuric acid solution to the system, controlling the final concentration of sulfuric acid in the solution to reach 0.21-0.3 M, and hydrolyzing for 2-3 h.

[0030] Further, in step (5), the enzymatic hydrolysis involves adjusting the temperature to 30-45 °C, adjusting the pH to 7.5-8 with NaOH, adding 0.5‰-1.5‰ trypsin, 0.5‰-1.5‰ esterase, and 0.5‰-1‰ snailase to the system, and reacting for 1-2 h.

[0031] Further, in step (5), filtration includes coarse filtration and fine filtration; coarse filtration involves adjusting the pH of the supernatant to 8.5-9.0, selecting 3-5 μm diatomaceous earth filter paper, using perlite as a filter aid, and filtering with a plate and frame filter; fine filtration involves adjusting the pH of the filtrate to 5.0-6.0, selecting 1-3 μm diatomaceous earth filter paper + 1-4 μm activated carbon deep filter paper, using diatomaceous earth as a filter aid, and circulating filtration with a plate and frame filter to obtain fine filtrate; the fine filtrate is then filtered through 0.45 μm and 0.22 μm filter membranes to obtain a clear filtrate.

[0032] Further, in step (5), the desalted and decolorized filtrate is obtained by using a combination of activated carbon deep filter paper filtration + 1-3KD organic membrane ultrafiltration. During the fine filtration stage, the pH is adjusted to 5.0-6.0. 1-3 μm diatomaceous earth filter paper + 1-4 μm activated carbon deep filter paper are selected. Diatomaceous earth is used as a filter aid. After circulating filtration by the plate and frame filter, the solution is filtered through 0.45 μm and 0.22 μm filter membranes. Then, the solution is concentrated by 1-3 KD organic membrane ultrafiltration. The ultrafiltration concentrate is recovered. Finally, the solution is filtered through 0.45 μm, 0.22 μm and 0.1 μm filter membranes to obtain a colorless HU-LTPS solution with low conductivity.

[0033] Further, in step (5), spray drying involves the filtrate entering a spray dryer and spray drying at 190-210 ℃ to obtain the HU-LTPS finished product.

[0034] Further, the fermentation medium comprises the following components: glucose 20-100 g / L, yeast extract 2-10 g / L, peptone 1-5 g / L, *Clerodendrum inerme* extract 20-100 g / L, monosodium glutamate 0.1-1 g / L, glutamine 0.1-1 g / L, serine 0.1-0.5 g / L, histidine 0.1-0.5 g / L, calcium sulfate 0.1-1 g / L, manganese sulfate 0.1-1 g / L, magnesium sulfate 0.1-1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and water as the solvent; preferably, the fermentation medium comprises the following components: glucose 40 g / L, yeast extract 4 g / L, peptone 2 g / L, *Clerodendrum inerme* extract 60 g / L, monosodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L. The following ingredients are present in the following concentrations: g / L calcium sulfate, 0.5 g / L manganese sulfate, 1 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, and water as the solvent.

[0035] Furthermore, the preparation method of the *Amanita muscaria* extract is as follows: (a) After activating the 'fragrant gray fungus', inoculate it into a sawdust culture medium and culture it at 24-28 ℃ until the sawdust culture medium is covered with white 'fragrant gray fungus' mycelia and the sawdust culture medium turns brown to black, thus obtaining the 'fragrant gray fungus' sawdust culture. (b) Adjust the moisture content of the sawdust culture of *Aureobasidium sarmentosum* obtained in step (a) to 60%-70%, weigh 50-100 g, add water, boil in a water bath for 20-60 min, filter, retain the filtrate, wash the solid residue with water 2-3 times, retain the washing liquid, discard the solid residue, mix all the liquids, filter through a 3-5 μm pore size to remove large insoluble particles, pass through a 100 nm ceramic membrane, and control the final volume to be the same as the volume of water added; Further, in step (a), the ash fungus is inoculated onto PDA enriched medium and cultured at 24-28 ℃ until it covers the entire plate before being used directly; or it is transferred to PDA enriched medium and cultured at 24-28 ℃ until it covers the entire plate, then the fungal blocks are prepared and inoculated onto sawdust medium.

[0036] Further, the PDA-enriched culture medium comprises the following components: potato 100-300 g / L, glucose 40-80 g / L, yeast extract 2-5 g / L, peptone 2-5 g / L, potassium dihydrogen phosphate 1-2 g / L, magnesium sulfate heptahydrate 1-2 g / L, agar powder 10-30 g / L, and water as the solvent; the sawdust culture medium comprises the following components by weight: sawdust 60-70 parts, wheat bran 20-40 parts, glucose 1-3 parts, gypsum 1-2 parts, with a material-to-water ratio of 1:1-1.5; preferably, the PDA-enriched culture medium comprises the following components: potato 200 g / L, glucose 60 g / L, yeast extract 3 g / L, peptone 3 g / L, potassium dihydrogen phosphate 1.5 g / L, magnesium sulfate heptahydrate 1.5 g / L, and agar powder 20 g / L. The solvent is water; the sawdust culture medium comprises the following components by weight: 67 parts sawdust, 30 parts wheat bran, 2 parts glucose, 1 part gypsum, with a material-to-water ratio of 1:1.3.

[0037] Furthermore, in step (b), the volume of water added is 0.8-2 L.

[0038] Secondly, the present invention provides a high-uronic acid Tremella polysaccharide prepared by the preparation method described in the first aspect, wherein the uronic acid content is not less than 30%, the total sugar content is ≥90%, and the protein content is ≤0.1%.

[0039] Furthermore, the high-molecular-weight thyme polysaccharide includes the high-molecular-weight product HU-HTPS, with a molecular weight of 600,000 to 1,000,000 Daltons; and the low-molecular-weight product HU-LTPS, with a molecular weight of 20,000 to 50,000 Daltons.

[0040] Thirdly, the present invention provides the application of the high-uronic acid Tremella polysaccharide prepared in the first aspect or the high-uronic acid Tremella polysaccharide described in the second aspect in the preparation of food, health products or cosmetics.

[0041] Furthermore, the cosmetic product is a skincare product with antioxidant or moisturizing functions.

[0042] Compared with the prior art, the present invention has achieved the following beneficial effects: This invention optimizes the fermentation medium and fermentation conditions to produce two products, HU-LTPS and HU-HTPS, with high uronic acid content. The fermentation yield of HU-LTPS reaches 20.43 g / L and that of HU-HTPS reaches 24.3 g / L, demonstrating high yield and stability. The fermentation cycle is short, the conditions are easier to control, and the fermentation process is more advantageous. Both products can be extracted with just one fermentation step. HU-LTPS has a molecular weight of 20,000-50,000 Daltons, and HU-HTPS has a molecular weight of 600,000-1,000,000 Daltons. Both products have a uronic acid content of ≥30%, and the finished products are pure white powders. Compared with traditional Tremella polysaccharides, they have better water retention and antioxidant capacity. Attached Figure Description

[0043] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0044] Figure 1 This is a growth curve of strain FTPS005.1 under conditions of 26 ℃ and 28 ℃; Figure 2 These are OD graphs of bacterial growth under different pH conditions; Figure 3 This is a graph showing the DPPH free radical scavenging rate; Figure 4 This is a comparison chart of water loss rates under different concentration conditions; Figure 5 This is a graph showing cell viability under different concentrations of HU-HTPS and HU-LTPS. Figure 6 This is a diagram of a cell scratch assay at 8 mg / mL. Figure 7 This is a comparison chart of cell migration rates under the 8 mg / mL condition. Detailed Implementation

[0045] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention.

[0046] Example 1: Preparation of Tremella polysaccharide by fermentation of microbial strains Fermentation strain: The laboratory-owned strain FTPS005.1 was deposited at the China Center for Type Culture Collection on October 21, 2024, at Wuhan University, Wuhan, China, with accession number CCTCC M 20242283.

[0047] Activation of Tremella fuciformis spores: Spores of strain FTPS005.1 were inoculated into activation medium and cultured at 26 ℃ for 3 days.

[0048] The activated culture medium consists of: 60 g / L glucose, 2 g / L yeast extract, 2 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 0.46 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 10 mg / L vitamin B1, 20 g / L agar powder, and water as the solvent, with a natural pH.

[0049] The activated bacterial strain is inoculated into seed culture medium for cultivation, as follows: Select the above-mentioned activated bacterial strains and inoculate them into seed culture medium. Incubate at 26°C and 220 r / min until OD reaches [value missing]. 560 When the OD value reaches 5 or higher, a primary seed culture is obtained. This primary seed culture is then inoculated into a secondary seed culture medium at a volume ratio of 10%, and cultured at 26°C and a rotation speed of 160 r / min. 560 When the value reaches 15, a secondary seed solution is obtained.

[0050] The prepared seed liquid was fermented to prepare Tremella polysaccharide, as detailed below: The secondary seed culture was inoculated into the above-mentioned Tremella fuciformis spore fermentation medium at a volume ratio of 10%. The parameters were controlled as shown in Table 1. Sugar was added after fermentation for 60 h, in two batches, with an interval of 2 h between the two batches.

[0051] Table 1 Fermentation Parameter Control

[0052] The fermentation ended when the pH rose above 5.5 and the viscosity no longer increased. The fermentation cycle lasted for 6 days.

[0053] The primary seed culture medium consisted of: 60 g / L glucose, 2 g / L yeast extract, 2 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 0.46 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, and 10 mg / L vitamin B1, with water as the solvent and natural pH.

[0054] The secondary seed culture medium consisted of: 10 g / L glucose, 10 g / L sucrose, 2 g / L yeast extract, 4 g / L peptone, 1 g / L dipotassium hydrogen phosphate, 0.46 g / L potassium dihydrogen phosphate, and 1 g / L magnesium sulfate heptahydrate, with water as the solvent and natural pH.

[0055] The fermentation medium comprises the following components: 40 g / L glucose, 4 g / L yeast extract, 2 g / L peptone, 60 g / L *Alternaria alternata* extract, 0.5 g / L monosodium glutamate, 0.5 g / L glutamine, 0.5 g / L serine, 0.5 g / L histidine, 0.5 g / L calcium sulfate, 0.5 g / L manganese sulfate, 1 g / L magnesium sulfate, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, and water as the solvent.

[0056] The preparation method of *Amanita muscaria* extract is as follows: *Amanita muscaria* is inoculated onto PDA-enriched medium and cultured at 24 ℃ until it covers the entire plate. Then, it is prepared into mycelial blocks and inoculated onto sawdust medium. It is cultured at 24 ℃ until the sawdust medium is covered with white *Amanita muscaria* mycelia and the sawdust medium turns brown to black, thus obtaining *Amanita muscaria* sawdust culture. The moisture content of the obtained *Amanita muscaria* sawdust culture is adjusted to 70%. 100 g of the culture is weighed, 1 L of water is added, and the mixture is boiled in a water bath for 30 min. The mixture is filtered, and the filtrate is retained. The solid residue is washed three times with water, and the washing liquid is retained. The solid residue is discarded. All liquids are mixed and filtered through a 3-5 μm pore size filter to remove large insoluble particles. The mixture is then passed through a 100 nm ceramic membrane, and the final volume is controlled to be the same as the volume of water added.

[0057] The sugar replenishment flow is F55 fructose syrup (dry matter content greater than 77%, fructose content greater than 55%, glucose content greater than 40%). The total amount of F55 fructose syrup added is calculated based on a fructose concentration of 40 g / L, with a total sugar replenishment of 10.91 kg.

[0058] Acid-base regulation is achieved using a 10%-30% sodium hydroxide aqueous solution and a 5%-10% sulfuric acid aqueous solution.

[0059] Preparation of high-uronic acid content Tremella polysaccharide from Tremella spore strain fermentation broth: Preparation of HU-HTPS: After fermentation, the fermentation broth was diluted with water at a volume ratio of 1:3, heated to 70℃, and the pH was controlled at 9. Alkali treatment was then performed for 2 hours. The temperature was lowered to 50℃, the pH was controlled at 7.5, and 0.5‰ (w / vg / mL) trypsin (enzyme activity 250 NF U / mg, purchased from Beijing Solarbio Science & Technology Co., Ltd.) was added for enzymatic hydrolysis for 1 hour to obtain the pretreatment solution. The pretreatment solution was dissolved in 2% sodium chloride and then directly centrifuged in a tubular centrifuge at a centrifugal force of 14000 g. The supernatant was collected. The pH of the supernatant was adjusted to 9.0, and 10 sheets of 3-5 μm diatomaceous earth filter paper were used as a filter aid and filtered using a plate and frame filter press to obtain the coarse filtrate. The pH of the coarse filtrate was further adjusted to 5.5, and 1-4 sheets of 1-3 μm diatomaceous earth filter paper were stacked together. Two sheets of μm activated carbon deep filter paper were used, with diatomaceous earth as a filter aid. The mixture was filtered twice using a plate and frame filter press to obtain a fine filtrate. This fine filtrate was then passed through 0.45 μm and 0.22 μm filter membranes to obtain a clear filtrate. The filtrate was transferred to an empty container, stirred, and 95% ethanol was slowly added until the alcohol content reached 62°. Stirring was continued for 15 min. The supernatant was discarded, and three volumes of 75° ethanol were added to the precipitate. Stirring was continued for 15 min, and the mixture was allowed to stand. The supernatant was discarded again, and three volumes of 80° ethanol were added to the precipitate. Stirring was continued for 15 min, and the mixture was allowed to stand. The supernatant was discarded again, and three volumes of 85° ethanol were added to the precipitate. Stirring was continued for 15 min, and the mixture was allowed to stand. The supernatant was discarded again, and the precipitate was transferred to a Buchner funnel for negative pressure filtration. After removing the ethanol, the precipitate was transferred to a 55° vacuum drying oven and dried for 6 hours. h; The dried product is pulverized evenly in a pulverizer, passed through a 200-mesh sieve, and the pulverizing and sieving process is repeated until no particles are present, resulting in pure white HU-HTPS.

[0060] Preparation of HU-LTPS: The bacterial cells were reconstituted with water at half the volume of the original fermentation broth, and then homogenized twice using a high-pressure homogenizer at 1000 bar to obtain a homogenized solution. The homogenized solution was transferred to a glass reactor and stirred. The temperature was controlled at 70 °C, and sulfuric acid was added to control the final concentration at 0.3 M. Acid hydrolysis was carried out for 2 h. The temperature was then lowered to 30 °C, and the pH was adjusted to 7.5. 0.5‰ (w / vg / mL) trypsin (enzyme activity 250 NF U / mg, purchased from Beijing Solarbio Science & Technology Co., Ltd.), 0.5‰ (w / vg / mL) esterase (enzyme activity 250 NFU / mg, purchased from Beijing Solarbio Science & Technology Co., Ltd.), and 0.1‰ (w / vg / mL) snailase (10 mg / mL, purchased from Shanghai Yuanye Biotechnology Co., Ltd.) were added, and the reaction was carried out for 1 h. The pH of the solution was adjusted to 9.0, and the reaction was carried out at 3-5°C. Ten sheets of 1-3 μm diatomaceous earth filter paper were used as a filter aid and filtered using a plate and frame filter press to obtain a coarse filtrate. The pH of the coarse filtrate was adjusted to 5.5, and eight sheets of 1-3 μm diatomaceous earth filter paper were stacked on top of two sheets of 1-4 μm activated carbon deep filter paper. Using diatomaceous earth as a filter aid, the filter paper was circulated and filtered twice using a plate and frame filter press to obtain a fine filtrate. The fine filtrate was then filtered through 0.45 μm and 0.22 μm filter membranes to obtain a clear filtrate. The clear filtrate was then subjected to ultrafiltration desalination and decolorization using a 1KD organic membrane. The ultrafiltration concentrate was recovered and then filtered through 0.45 μm, 0.22 μm, and 0.1 μm filter membranes before being directly spray-dried at 190 °C to obtain pure white HU-LTPS.

[0061] Experimental Example 1 Dissolved oxygen profile, dynamic viscosity, and OD were measured in the fermentation broth of Example 1. 560 The yield of the final obtained Tremella polysaccharide, as well as the content and molecular weight of each component in the Tremella polysaccharide, were determined and calculated. The specific determination methods are as follows, and the test results are shown in Table 2.

[0062] (1) The dissolved oxygen data of the fermentation broth is automatically detected online by the fermenter. The dissolved oxygen data represents the growth of the Tremella strain. The secretion of Tremella polysaccharide is mainly concentrated in the stable growth period of Tremella spores. At this time, the strain has a high oxygen demand and the dissolved oxygen in the fermentation broth will drop rapidly. When the dissolved oxygen rises rapidly, it indicates that the strain has begun to die and has entered the decline period, which indicates the end of fermentation. (2) Dynamic viscosity determination: 20 mL of fermentation broth was directly measured by a Bollerfeld DV2TLVTJ0 rotational viscometer. The unit of dynamic viscosity is mPa·s. (3) OD 560 Determination: The fermentation broth was diluted appropriately to control the OD value within the range of 0.2-0.8. Using water as a blank, the absorbance was directly measured at 560 nm using a Beijing Purkinje General Instrument Co., Ltd. T6 New Century UV spectrophotometer. 560 =A560 ×N In the formula: A 560 : Absorbance of the solution at 560 mm; N: Dilution factor.

[0063] (4) Loss on drying: directly measured using a Mettler Toledo HS153 halogen moisture analyzer.

[0064] (5) Determination of total sugar content: An optimized phenol-sulfuric acid method was used, with the following specific steps: 0, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 mL of mannose standard stock solution were placed in 25 mL stoppered test tubes, and each tube was filled to 2.0 mL with distilled water. 1.0 mL of 6% phenol solution was added to each tube, and the mixture was shaken well. At room temperature, 6.0 mL of concentrated sulfuric acid was added vertically while the tube was suspended in the air. The mixture was allowed to stand for 10 min, then mixed thoroughly with a shaker. The mixture was then placed in a boiling water bath for 30 min. After the reaction, the mixture was cooled to room temperature in an ice-water bath. The absorbance A value was measured at a wavelength of 490 nm. A standard curve was plotted with the absorbance value on the ordinate and the amount of mannose (µg) on ​​the abscissa.

[0065] Weigh approximately 0.03 g of sample (accurate to 0.0001 g) and place it in a 500 mL volumetric flask. Add distilled water to the mark, mix well, and let stand for 2 hours. Pipette 2.0 mL of the above solution into a 25 mL stoppered test tube, add 1.0 mL of 6% phenol solution, mix well, and vertically add 6.0 mL of concentrated sulfuric acid while the pipette is suspended at room temperature. Let stand for 10 minutes, mix thoroughly with a shaker, and react in a boiling water bath for 30 minutes. After the reaction, cool to room temperature in an ice-water bath. Use 2.0 mL of water as a blank control and measure the absorbance at a wavelength of 490 nm. The formula for calculating the total sugar content is as follows:

[0066] In the formula: X: total sugar content, % C 1: Mannitol content on the standard curve corresponding to the absorbance measured by the sample, μg; m: sample mass, g; h: percentage of weight loss after drying, %; 1.25: conversion factor for total sugar to mannose.

[0067] (6) Determination of glucuronic acid: The sulfuric acid-carbazole method was adopted. The specific steps are as follows: Accurately weigh 100 mg of glucuronic acid standard dried to constant weight at 105 °C, put it into a 100 mL volumetric flask, add water to dissolve and dilute to the mark, shake well to obtain glucuronic acid standard stock solution; use a pipette to take 5 mL of glucuronic acid standard stock solution into a 100 mL volumetric flask, add purified water to dilute to the mark, and obtain a glucuronic acid standard solution of about 50 μg / mL; accurately measure 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of glucuronic acid standard solution, put them into 25 mL stoppered test tubes respectively, add purified water to 1.0 mL of each, mix well, cool in an ice bath, slowly add 5.0 mL of borax sulfuric acid solution that has been cooled in an ice bath, stopper tightly, mix well, heat in a boiling water bath for 10 min, and cool to room temperature in an ice bath. Accurately add 0.2 mL of carbazole test solution, mix well, heat in a boiling water bath for 15 min, and cool to room temperature in an ice bath. Measure the absorbance at 530 nm using a UV-Vis spectrophotometer, and calculate the regression equation using the concentration of glucuronic acid against the corresponding absorbance.

[0068] Weigh 0.1 g (W1, accurate to 0.001 g) of the test sample and place it in a 100 mL volumetric flask. Dissolve and dilute to volume with water. Weigh 5 g (W2, accurate to 0.01 g) of the sample and place it in a 50 mL volumetric flask. Dilute to the mark with purified water and shake well to obtain the test solution. Take two stoppered test tubes and add 1 mL of the test solution to each. Take another 25 mL stoppered test tube and add 1 mL of purified water as a blank sample. Mix well and cool in an ice bath. Slowly add 5.0 mL of borax-sulfuric acid solution that has been cooled in an ice bath. Stopper tightly, mix well, heat in a boiling water bath for 10 min, and cool to room temperature in an ice bath. Accurately add 0.2 mL of carbazole test solution, mix well, heat in a boiling water bath for 15 min, and cool to room temperature in an ice bath. Measure the absorbance at a wavelength of 530 nm using a UV-Vis spectrophotometer. The formula for calculating the glucuronic acid content is as follows:

[0069] In the formula: X G : Glucuronic acid content, %; C1: Glucuronic acid concentration in the test solution calculated by regression equation, μg / mL; W1: Mass of the test sample weighed, g; W2: Amount of the test solution weighed in the second step of dilution, g; h: Percentage of weight loss after drying of the sample, %.

[0070] (7) Molecular weight determination: Ubbelohde viscometer method was used.

[0071] Table 2 Indicator Measurement

[0072] OD 560This reflects the relative number of Tremella fuciformis spores, OD 560 The larger the viscosity, the higher the growth activity of the strain. The dynamic viscosity of the fermentation broth reflects the molecular weight of the Tremella polysaccharide; a higher viscosity indicates a higher molecular weight and also reflects an increase in product content. Total sugar, glucuronic acid, and protein content are all quality control indicators for Tremella polysaccharide. Total sugar indicates the purity of the prepared Tremella polysaccharide, glucuronic acid is the main indicator of this invention, and protein is the main impurity in Tremella polysaccharide. Tremella polysaccharides of different molecular weights have different effects. Medium and high molecular weight Tremella polysaccharides have superior water retention capacity, while low molecular weight Tremella polysaccharides have excellent transdermal absorption capacity. HU-HTPS and HU-LTPS obtained by fermentation of strain FTPS005.1 are both white powders with a total sugar content ≥90%, a glucuronic acid content >30%, and a protein content ≤0.1%, meeting the enterprise standard Q / FCG 1001S-2024. The glucuronic acid content of the HU-HTPS and HU-LTPS prepared in this way is >30%, which is more than 15% higher than the glucuronic acid content of Tremella polysaccharides currently sold on the market.

[0073] The process of this invention successfully prepared two types of high-uronic acid Tremella polysaccharides in one step. The uronic acid content of both exceeded 30%, and the purity (total sugar ≥90%, protein ≤0.1%) was high, with a clear molecular weight distribution.

[0074] Experimental Example 2: Effects of different culture media on HU-HTPS and HU-LTPS, with other conditions the same as in Example 1.

[0075] Control culture medium: 50 g / L granulated sugar, 10 g / L sugarcane juice, 2 g / L yeast powder, 4 g / L corn steep liquor, 0.5 g / L monosodium glutamate, 0.5 g / L glutamine, 0.5 g / L serine, 0.5 g / L histidine, 60 g / L ash extract, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, with water as the solvent.

[0076] Experimental Group 1: Glucose 60 g / L, yeast powder 2 g / L, corn steep liquor 4 g / L, ash extract 60 g / L, monosodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, solvent: water.

[0077] Experimental Group 2: F55 fructose syrup 78 g / L, yeast powder 2 g / L, corn steep liquor 4 g / L, styrax extract 60 g / L, monosodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, solvent: water.

[0078] Experimental Group 3: F55 fructose syrup 78 g / L, yeast powder 4 g / L, peptone 2 g / L, *Alternaria alternata* extract 60 g / L, monosodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, solvent: water.

[0079] Experimental Group 4: F55 fructose syrup 78 g / L, yeast powder 4 g / L, peptone 2 g / L, *Alternaria alternata* extract 60 g / L, monosodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L, calcium sulfate 0.5 g / L, manganese sulfate 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, solvent: water.

[0080] Fermentation: The secondary seed culture was inoculated into the fermentation medium at a volume ratio of 10%. The culture conditions were: 26℃, aeration rate of 1 VVM, rotation speed of 300-800 r / min, dissolved oxygen (with dissolved oxygen-related stirring) maintained at 20%-30%, and pH controlled at 3.5-5.0. The fermentation endpoint was defined as the fermentation broth viscosity no longer changing and pH > 5.5. The effects of different culture media on HU-HTPS and HU-LTPS are shown in Table 3.

[0081] Table 3 Effects of different culture media on HU-HTPS and HU-LTPS

[0082] Comparing the effects of different culture medium compositions on the yield and uronic acid content of HU-HTPS and HU-LTPS, the results showed that using glucose as the sole carbon source (experimental group 1) was beneficial for cell growth (OD). 560The polysaccharide yield was 67.43 g / L, but the polysaccharide yield was the lowest (HU-HTPS only 5.58 g / L), and the uronic acid content was less than 20%. Using F55 fructose syrup and peptone as a nitrogen source (experimental group 3), the polysaccharide yield was improved, but the uronic acid content was still in the range of 20-21%. Supplementing experimental group 3 with calcium sulfate and manganese sulfate (experimental group 4) significantly increased the uronic acid content, with HU-HTPS and HU-LTPS reaching 25.46% and 26.52% respectively, while the molecular weight of the products remained stable.

[0083] Adding appropriate amounts of calcium sulfate and manganese sulfate to the culture medium can significantly improve the synthesis efficiency of uronic acid in Tremella fuciformis polysaccharide, which is one of the key factors in achieving a high uronic acid content (≥25%). Using F55 fructose syrup in combination with peptone as a nitrogen source can increase yield. The preferred culture medium formulation of this invention has a clear advantage in improving product quality.

[0084] Experimental Example 3: The effect of two-stage temperature control on the yield and uronic acid content of HU-HTPS and HU-LTPS. The remaining operations are the same as in Example 1.

[0085] Fermentation medium: F55 fructose syrup 78 g / L, yeast extract 4 g / L, peptone 2 g / L, *Alternaria alternata* extract 60 g / L, monosodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L, calcium sulfate 0.5 g / L, manganese sulfate 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, solvent: water.

[0086] Fermentation: The secondary seed culture was inoculated into the fermentation medium at a volume ratio of 10%. The culture conditions were: 300-800 r / min, dissolved oxygen (with dissolved oxygen-related stirring) maintained at 20%-30%, pH 4.5-5.0. Fermentation ended when the viscosity of the fermentation broth no longer changed and the pH > 5.5. Temperature control methods are shown in Table 4; the effects of different temperature control methods on HU-HTPS and HU-LTPS are shown in Table 5; the growth curves of strain FTPS005.1 at 26 ℃ and 28 ℃ are shown in Table 5. Figure 1 As shown.

[0087] Table 4 Temperature Control Methods

[0088] according to Figure 1 It can be seen that under the conditions of 26 ℃ and 28 ℃, 0-24 h is the lag period, 24 h-96 h is the logarithmic growth period, 96 h-144 h is the stationary period, and after 144 h is the decline period.

[0089] Table 5. Effects of different temperature control methods on HU-HTPS and HU-LTPS

[0090] By comparing the effects of constant temperature throughout the fermentation process and segmented temperature control, it was found that constant temperature throughout the fermentation process (28 ℃ or 26 ℃) could not balance cell growth and product synthesis: 28 ℃ throughout the process was conducive to rapid cell growth, but the polysaccharide yield was low (HU-HTPS 10.19 g / L); 26 ℃ throughout the process increased the yield, but prolonged the fermentation cycle.

[0091] A segmented temperature control strategy (28 ℃ in the early stage, then reduced to 26 ℃ in the middle and late stages of the logarithmic growth phase) resulted in an increased HU-HTPS yield of 14.90 g / L and a uronic acid content of 27.32% in experimental group 1, superior to all isothermal control groups. This two-stage temperature control strategy can promote cell growth while simultaneously enhancing polysaccharide synthesis and uronic acid accumulation, effectively balancing fermentation efficiency and product quality, and demonstrating good process controllability and scale-up potential.

[0092] Experimental Example 4: Effect of four-stage pH control on the yield of HU-HTPS and HU-LTPS and the content of uronic acid. The remaining operations are the same as in Example 1.

[0093] Fermentation medium: F55 fructose syrup 78 g / L, yeast extract 4 g / L, peptone 2 g / L, *Alternaria alternata* extract 60 g / L, monosodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L, calcium sulfate 0.5 g / L, manganese sulfate 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, solvent: water.

[0094] Fermentation: The secondary seed culture was inoculated into the fermentation medium at a volume ratio of 10%. The culture conditions were: 28 ℃ for 0-60 h, 26 ℃ for 60-144 h; rotation speed 300-800 r / min; dissolved oxygen (with dissolved oxygen-related stirring) maintained at 20%-30%; fermentation endpoint was defined as no further change in viscosity and pH > 5.5. The pH control strategy is shown in Table 6. The effects of different pH control methods on HU-HTPS and HU-LTPS are shown in Table 7. The OD values ​​of the strains under different pH conditions are shown in Table 8. Figure 2 As shown.

[0095] Table 6 pH Control Strategy

[0096] Table 7. Effects of different pH control methods on HU-HTPS and HU-LTPS

[0097] Experiment 4 systematically compared the effects of different pH control strategies on the fermentation process and products. Neither natural pH throughout the process nor a single pH control method could simultaneously achieve high cell density and high uronic acid content. By adopting a four-stage pH control strategy (natural pH for 0-24 h, pH controlled at 5.5-6.5 for 24-48 h, pH controlled at 4.5-5.0 for 48-120 h, and natural pH after 120 h), the HU-HTPS yield in Experiment 4 reached 16.81 g / L, the uronic acid content was 28.60%, and the molecular weight increased to 642,800 Dals. This strategy effectively alleviated the problem of pH regulation lag in the later stage of high-viscosity fermentation, which is conducive to accurate determination of fermentation endpoint and stable product structure.

[0098] The four-stage pH control strategy can effectively coordinate cell growth and product synthesis, significantly increase polysaccharide yield and uronic acid content, and improve the controllability and reproducibility of the fermentation process, making it suitable for industrial application.

[0099] Experimental Example 5: Effect of feed addition on the yield of HU-HTPS and HU-LTPS and the content of uronic acid. The remaining operations are the same as in Example 1.

[0100] Fermentation medium: glucose 60 g / L, yeast extract 4 g / L, peptone 2 g / L, *Alternaria alternata* extract 60 g / L, monosodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L, calcium sulfate 0.5 g / L, manganese sulfate 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, solvent: water.

[0101] Fermentation: The secondary seed culture was inoculated into the fermentation medium at a volume ratio of 10%. The culture conditions are shown in Table 8, the sugar supplementation strategy is shown in Table 9, and the effects of different feeding methods on HU-HTPS and HU-LTPS are shown in Table 10.

[0102] Table 8 Cultivation Conditions

[0103] After the residual sugar concentration in the fermentation broth was measured to be 0, sugar was replenished according to the sugar replenishment strategy in Table 9.

[0104] Table 9 Sugar Supplementation Strategies

[0105] The fermentation endpoint was defined as the fermentation broth viscosity no longer changing and pH > 5.5.

[0106] Table 10. Effects of different feeding methods on HU-HTPS and HU-LTPS

[0107] Experiment 5 systematically investigated the effects of different fed-batch strategies on the yield, uronic acid content, and molecular weight of high-uronic acid Tremella fuciformis polysaccharides (HU-HTPS and HU-LTPS). The results showed that the fed-batch method significantly regulated the product accumulation and structural characteristics during fermentation: In the unfed control group, under the initial culture medium conditions, the HU-HTPS yield was 16.67 g / L, the uronic acid content was 28.71%, and the molecular weight was 624,400 DaL, demonstrating a certain basic synthetic capacity, but the overall yield and structural indicators still had room for improvement; In the single-fed groups (experimental groups 1-3), as the proportion of fructose in the added fructose syrup increased (F42→F55→F60), the HU-HTPS yield gradually increased from 18.70 g / L to 20.79 g / L, the uronic acid content also increased from 29.38% to 30.69%, and the molecular weight increased simultaneously. This indicates that fed-batch feeding effectively promotes polysaccharide synthesis, and increasing the fructose ratio helps increase uronic acid content. However, compared to the F55 fructose syrup group (Experiment 2), the F60 fructose syrup group (Experiment 3) showed only a slight increase in polysaccharide content and virtually no increase in uronic acid content, while the price increased significantly, resulting in a lower overall cost-effectiveness. The batch-feeding group (Experiment 4) adopted a two-batch feeding strategy with F55 fructose syrup, further increasing HU-HTPS yield to 22.17 g / L, uronic acid content to 30.87%, and molecular weight to 840,800 Dals. Simultaneously, HU-LTPS yield reached 18.61 g / L, with uronic acid content at 34.09%. This strategy significantly improved polysaccharide yield and molecular weight while maintaining a high uronic acid level, demonstrating good process coordination.

[0108] Although the continuously fed-batch group (experimental group 5) was slightly better than experimental group 4 in terms of HU-HTPS yield (23.81 g / L) and uronic acid content (31.28%), it had a large total sugar supplementation, high operational complexity, and a lower polysaccharide increment per unit sugar consumption (Δyield / Δsugar supplementation). Considering industrialization costs and operational feasibility, it was less economical than experimental group 4. In summary, experimental group 4 (fed-batch F55 fructose syrup in two batches) achieved a better balance between yield, uronic acid content, molecular weight, and process complexity, and is a feasible strategy for achieving efficient and economical production of high-uronic acid Tremella polysaccharide.

[0109] This invention, through optimized feeding process, demonstrates that a batch feeding strategy can effectively increase the yield and uronic acid content of Tremella fuciformis polysaccharides, while also improving the molecular structure of the product. Experimental group 4, employing a two-batch feeding method with F55 fructose syrup, achieved excellent results with a HU-HTPS yield of 22.17 g / L and a uronic acid content of 30.87%, and a HU-LTPS yield of 18.61 g / L and a uronic acid content of 34.09%, respectively, without excessively increasing raw material costs or operational complexity. Furthermore, the molecular weight distribution met the requirements for high-quality polysaccharides. This strategy offers advantages such as ease of operation, high sugar consumption efficiency, and ease of scale-up, making it suitable for continuous industrial production and providing reliable process support for the large-scale preparation of high-uronic acid Tremella fuciformis polysaccharides.

[0110] Experimental Example 62: Scale-up Experiment of a Tank Activation of Tremella fuciformis spores: Spores of strain FTPS005.1 were inoculated into activation medium and cultured at 26 ℃ for 3 days.

[0111] Primary seed culture: Pick the above-mentioned activated bacterial strain and inoculate it into 5 L of seed culture medium. Incubate at 26 °C and 220 r / min until OD reaches 100%. 560 If the value is 5 or higher, a first-grade seed solution is obtained.

[0112] Secondary seed culture: The primary seed culture was inoculated into a 500 L seed tank (128 L sample volume) at a volume ratio of 10% (12.8 L). Culture conditions: temperature 26 ℃, aeration rate 1 VVM, stirring speed 147 r / min, pH at rest, cultured for 2.5 days (OD). 560 (Up to 15), secondary seed solution is obtained; Fermentation culture: The total fermentation volume was calculated based on 80% of the sample loading (1.6 tons). The secondary seed liquid was inoculated into the Tremella fuciformis spore fermentation medium at a volume ratio of 8%. The parameters were controlled as shown in Table 11. Sugar was added after 60 h of fermentation, and F55 fructose syrup was added in two batches (the total fructose concentration was controlled at 40 g / L).

[0113] Table 11 Fermentation Parameter Control

[0114] The fermentation ended when the pH rose above 5.5 and the viscosity no longer increased. The fermentation cycle lasted for 6 days.

[0115] Preparation of HU-HTPS: After fermentation, the fermentation broth was transferred to a pretreatment tank and diluted with water at a fermentation broth:water volume ratio of 1:3. The temperature was raised to 70 ℃, the pH was controlled at 9.0, and alkali treatment was performed for 2 h. The temperature was then lowered to 50 ℃, the pH was controlled at 7.5, and 0.5‰ (w / vg / mL) trypsin was added for enzymatic hydrolysis for 1 h to obtain the pretreatment solution. After dissolving the pretreatment solution with 2% sodium chloride, it was directly centrifuged in a tube bundle centrifuge at a centrifugal force of 14000 g. The supernatant was collected and transferred to an intermediate tank. The pH of the supernatant was adjusted to 9.0, and a plate and frame filter press was used with 3-5 μm diatomaceous earth filter paper and perlite as a filter aid to obtain the coarse filtrate, which was transferred to the coarse filtrate storage tank. The pH of the coarse filtrate was further adjusted to 5.5, and 1-3 μm diatomaceous earth filter paper was used with 1-4 μm perlite as a filter aid. A 1000 μm activated carbon deep filter paper, using diatomaceous earth as a filter aid, was used for two circulation filtrations using a plate and frame filter press to obtain a fine filtrate. The fine filtrate was then further filtered through 0.45 μm and 0.22 μm filter membranes to obtain a clear, purified filtrate, which was transferred to a fine filtration tank. Stirring was started, and 5% ethanol and purified filtrate were added to a sedimentation tank at a ratio of 1:0.532 (95% ethanol: purified filtrate), and stirring was continued for 15 min. The supernatant was discarded, and 3 times the volume of 75° ethanol was added to the sediment, stirred for 15 min, and allowed to stand. The supernatant was discarded, and 3 times the volume of 80° ethanol was added to the sediment, stirred for 15 min, and allowed to stand. The supernatant was discarded, and 3 times the volume of 85° ethanol was added to the sediment, stirred for 15 min, and allowed to stand. The supernatant was discarded, and 3 times the volume of 95° ethanol was added to the sediment, stirred for 15 min, and allowed to stand. min, let stand; discard the supernatant, transfer the precipitate to a four-in-one dryer for drying; pulverize the dried product evenly in a pulverizer, pass it through a 200-mesh sieve, repeat the pulverizing and sieving process until no particles are found, and obtain pure white HU-HTPS.

[0116] Preparation of HU-LTPS: After centrifugation, the bacterial cells were reconstituted with water at half the original fermentation broth volume, and then homogenized twice at 1000 bar using a high-pressure homogenizer to obtain a homogenized solution. The homogenized solution was transferred to an enamel jar and stirred. The temperature was controlled at 70 °C, and sulfuric acid was added to control the final concentration at 0.3 M. Acid hydrolysis was carried out for 2 h. The temperature was lowered to 30 °C, and the pH was adjusted to 7.5. 0.5‰ (w / vg / mL) trypsin, 0.5‰ (w / vg / mL) esterase, and 0.1‰ (w / vg / mL) snailase were added, and the reaction was carried out for 1 h. The pH of the solution was adjusted to 9.0, and 10 sheets of 3-5 μm diatomaceous earth filter paper were used as filter aids and filtered using a plate and frame filter press to obtain a coarse filtrate, which was transferred to a coarse filtrate storage tank. The pH of the coarse filtrate was further adjusted to 5.5, and 1-4 sheets of 1-3 μm diatomaceous earth filter paper were stacked together. Two sheets of μm activated carbon deep filter paper were used, with diatomaceous earth as a filter aid, and the filter was circulated twice using a plate and frame filter press to obtain a fine filtrate. The fine filtrate was then filtered through 0.45 μm, 0.22 μm, and 0.1 μm filter membranes to obtain a purified filtrate, which was then transferred to a purified filtrate storage tank. The purified filtrate was then subjected to ultrafiltration desalination and decolorization using a 1 KD organic membrane. The ultrafiltration concentrate was recovered and directly spray-dried at 190℃ to obtain pure white HU-LTPS. The index measurements are shown in Table 12.

[0117] Table 12 Measurement of Indicators for 2-ton Tanks

[0118] Scale-up experiments conducted in a 2-ton fermenter demonstrated that the high-uronic acid Tremella fuciformis polysaccharide preparation process described in this invention exhibits good industrial adaptability and stability. (Fermentation broth OD) 560 The value reached 127.68, significantly higher than the laboratory scale, indicating more vigorous cell growth during scale-up, which is attributed to the optimized dissolved oxygen control and nutrient supply system. The dynamic viscosity of the fermentation broth reached 918,300 mPa·s, reflecting the sufficient accumulation of high molecular weight polysaccharides. This also demonstrates that the high-viscosity environment did not adversely affect process control, showcasing the effectiveness of the segmented temperature control and four-stage pH control strategy in large-scale production.

[0119] Product extraction results showed that the yield of high molecular weight Tremella fuciformis polysaccharide (HU-HTPS) reached 24.30 g / L, and the yield of low molecular weight product (HU-LTPS) was 20.43 g / L, both significantly higher than the pilot-scale level, confirming that the process has a good scale-up effect. The obtained products have excellent purity, with total sugar contents of 95.18% and 94.73% for HU-HTPS and HU-LTPS, respectively, and uronic acid contents of 32.47% and 34.23%, respectively. Protein residue was less than 0.01% for both, and all indicators met the standards for high-quality polysaccharides. Molecular weight determination showed that HU-HTPS was 896,600 Da and HU-LTPS was 31,900 Da, indicating that the scale-up process did not cause significant changes in the product structure, and the process reproducibility was good.

[0120] This scale-up experiment employed a segmented temperature control and four-stage pH control strategy, combined with a batch feeding method using F55 fructose syrup, enabling accurate determination of the fermentation endpoint and process control. Even when scaled up to a 2-ton scale, the entire process remained simple to operate and stable in control, with a high polysaccharide yield per unit of sugar consumption, demonstrating good economic efficiency and industrial feasibility. In summary, the process of this invention exhibits excellent repeatability and stability in scaled-up production, and the prepared high-uronic acid Tremella fuciformis polysaccharide meets ideal indicators in terms of yield, purity, and functional properties, possessing significant value for industrial application.

[0121] Experiment Example 7: Comparison of the antioxidant properties of Tremella polysaccharides prepared by different processes The method for determining the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radical scavenging ability of Tremella fuciformis polysaccharide is as follows: (1) Accurately weigh 1.97 mg DPPH and dilute to 50 mL with anhydrous ethanol to obtain a DPPH solution; (2) Control group 1: Take 10 mg of Tremella fuciformis polysaccharide from control group 1 (Treme HA polysaccharide product (prepared by extraction method, trade name: Treme HA)). ® Tremella polysaccharide (product manufacturer: Shandong Focus Freda Biotechnology Co., Ltd.) was dissolved in ultrapure water to prepare sample solutions with concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.07815, and 0.039075 mg / mL, respectively. (3) Control group 2: Take 10 mg of Tremella polysaccharide (Tremella polysaccharide finished product (prepared by fermentation method), patent publication number CN119736169A) from control group 2 and dissolve it in ultrapure water. Prepare a series of sample solutions according to the preparation method in step (2). (3) Experimental group 1: Dissolve 10mg of HU-HTPS prepared in Experimental Example 6 in ultrapure water and prepare a series of sample solutions according to the preparation method in step (2); (4) Experimental group 2: Dissolve 10 mg of HU-LTPS prepared in Experimental Example 6 in ultrapure water and prepare a series of sample solutions according to the preparation method in step (2); (4) Dissolve 500 mg of vitamin C in ultrapure water to prepare vitamin C solutions with concentrations of 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, and 0.001563 g / mL as positive control solutions; (5) Use anhydrous ethanol as the ethanol control solution; (6) Add 100 μL of the test sample to each well in a 96-well plate. The test solution includes the sample solution and the vitamin C control solution. Set up 3 replicates for each reaction. The sample addition is shown in Table 13.

[0122] Table 13 Solution Preparation

[0123] (7) React at room temperature in the dark for 30 min. Measure the absorbance at 517 nm using an ELISA reader. Calculate the DPPH scavenging rate for different concentrations of samples according to the formula: DPPH scavenging rate = [1 - (absorbance value of Ai - absorbance value of Aj) / absorbance value of Ac] * 100%. The DPPH scavenging rate of the vitamin C control solution is shown in Table 14, and the DPPH scavenging rate of the sample solutions is shown in Table 15. The DPPH free radical scavenging rate curves for different groups are shown in... Figure 3 As shown.

[0124] Table 14 DPPH clearance rate of vitamin C control solution

[0125] Table 15 DPPH scavenging rate of sample solutions

[0126] This experiment evaluated the antioxidant capacity of Tremella fuciformis polysaccharides from different sources by measuring DPPH free radical scavenging rate. The results showed that experimental group 1 (HU-HTPS) and experimental group 2 (HU-LTPS) exhibited superior DPPH scavenging rates compared to the two control groups (commercially available Tremella fuciformis polysaccharides prepared by extraction and fermentation methods) at all tested concentrations. Especially at higher concentrations (10 mg / mL), experimental group 2 (HU-LTPS) achieved a scavenging rate of 86.81%, close to the scavenging rate of the positive control vitamin C at 0.1 mg / mL (88.21%), demonstrating extremely strong antioxidant activity. As the concentration decreased, the scavenging rate of all samples decreased, but the experimental groups consistently outperformed the control groups, indicating that the high-uronic acid Tremella fuciformis polysaccharides prepared in this invention have antioxidant advantages over a wider concentration range.

[0127] Experiment Example 8: Comparison of the moisturizing properties of Tremella polysaccharides prepared by different processes Experimental Group 1: Tremella polysaccharide finished product (prepared by extraction method, trade name: Treme HA® Tremella polysaccharide), product manufacturer: Shandong Focus Freda Biotechnology Co., Ltd.; Experimental Group 2: Tremella polysaccharide finished product (prepared by fermentation method), patent publication number CN119736169A; Experimental Group 3: HU-HTPS prepared in Experimental Example 6.

[0128] Water loss rate experiment: (1) Take the sample powders from experimental group 1, experimental group 2 and experimental group 3 and prepare solutions with three concentration gradients of 0.1%, 0.5% and 1% (w / v) respectively. Store them in a refrigerator at 4 ℃ for later use.

[0129] (2) Negative control group: ultrapure water.

[0130] (3) Equilibration: The instrument and sample were equilibrated for 2 h at a temperature of 25±1 ℃ and a relative humidity of 45±5%.

[0131] (4) Sample weighing and addition: Take a series of clean weighing bottles, number them and record the numbers. Use an analytical balance to accurately weigh the mass (W0) of each empty container. Then, add 2.0 mL of test samples from different groups, ensuring that the liquid surface is flat and the thickness is uniform. Set up 3 parallel samples for each group of experiments, weigh and record the total mass (W1) again. (5) Initial weight record: Calculate the initial mass (W) of each sample. 初始 = W1 - W0), and record it in the data table. The sample mass at this point serves as the baseline for subsequent calculations; (6) Drying process and timed weighing: Place all samples in a constant temperature and humidity chamber and start timing. Remove samples and weigh them quickly at the following time points: 0.5, 1, 2, 4, 6, 8, 12, and 24 hours. The weighing process should be rapid to avoid the samples absorbing or losing moisture during weighing. Record the data (W) after each weighing. t Then immediately return it to its original environment; (7) Endpoint treatment: After 12 hours of measurement, all samples were placed in a 105℃ oven to dry to constant weight, and the dry weight of the samples (W) was measured. 干 This step aims to remove all moisture from the sample to facilitate subsequent calculations.

[0132] (8) According to the formula, water loss rate (%) = [(W 初始 - W t ) / (W 初始 - W 干 The water loss rate of the sample was calculated by multiplying the result by 100%. The water loss rate data is shown in Table 16. A comparison of the water loss rates of different samples under different concentration conditions is shown below. Figure 4As shown.

[0133] Table 16 Water Loss Rate Data Table

[0134] The moisturizing properties of different Tremella fuciformis polysaccharide samples were evaluated through a water loss rate experiment. The results are as follows: Experimental group 3 (HU-HTPS) showed the lowest water loss rate at all time points and concentrations, indicating its optimal moisturizing performance. At a concentration of 0.5%, HU-HTPS exhibited the best moisturizing effect, with a significantly lower water loss rate than other groups. Experimental groups 1 (extraction method) and 2 (fermentation method) showed higher water loss rates and relatively poorer moisturizing performance. Even after extending the time to 24 hours, the water loss rate of experimental group 1 remained low, indicating its long-lasting moisturizing ability. The HU-HTPS prepared in this invention exhibits excellent moisturizing properties, especially at a concentration of 0.5%, with significantly better moisturizing ability than Tremella fuciformis polysaccharides prepared by traditional extraction and fermentation methods. This suggests that HU-HTPS has broad application prospects as a highly effective moisturizing ingredient in cosmetics, skincare products, and other fields.

[0135] Experiment Example 9: Cytotoxicity Assay: This experiment uses the CCK-8 assay to quantitatively evaluate the cytotoxicity of HU-HTPS and HU-LTPS samples on L-929 mouse fibroblasts in order to assess their biocompatibility.

[0136] 1. Experimental materials Cell line (L-929 mouse fibroblast cell line); test samples (HU-HTPS, HU-LTPS, sterilized by filtration through a 0.22 μm microporous membrane before use); CCK-8 kit; DMEM complete medium containing 10% fetal bovine serum; PBS buffer; trypsin-EDTA digestion solution.

[0137] 2. Sample solution preparation The HU-HTPS and HU-LTPS samples were diluted with sterile PBS to the following eight concentration gradients: 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 5 mg / mL, 8 mg / mL, and 10 mg / mL.

[0138] 3. Cell seeding and culture L-929 cells in logarithmic growth phase were harvested, digested, resuspended, and counted. The cell density was adjusted to 1×10⁻⁶. 5 cells / mL. Seed the cell suspension at 100 μL per well in a 96-well plate. Pre-culture the plate at 37 ℃ in a 5% CO2 incubator for 24 h to allow for full cell adhesion.

[0139] 4. Sample processing After the cells adhere to the culture medium, the original culture medium is aspirated and discarded.

[0140] Experimental group: 100 μL of HU-HTPS or HU-LTPS sample solution of each concentration was added, with 6 replicates for each concentration. Negative control group: 100 μL of fresh complete culture medium was added. Blank control group: 100 μL of cell-free culture medium was added.

[0141] Return the culture plate to the incubator and continue culturing for 24 hours.

[0142] 5. CCK-8 assay for cell viability After 24 h of sample treatment, 10 μL of CCK-8 solution was added to each well; the culture plate was placed in a 37 ℃ incubator and incubated in the dark for 2 h; after incubation, the absorbance of each well was immediately measured at 450 nm using a microplate reader.

[0143] 6. The relative cell viability is calculated using the following formula: Cell viability (%) = (OD experimental group - OD blank group) / (OD negative control group - OD blank group) × 100% Cell scratch assay: This experiment uses a cell scratch assay to quantitatively evaluate the inhibitory or promoting effect of the test sample on the migration behavior of L-929 mouse fibroblasts. 1. Experimental Materials Cell line (L-929 mouse fibroblast cell line); Test samples: (HU-HTPS, HU-LTPS, prepared to the required working concentration with sterile PBS or culture medium before use); DMEM complete medium; serum-free DMEM medium; PBS buffer; trypsin-EDTA digestion solution; paraformaldehyde fixative (4%).

[0144] 2. Cell plating and culture Take L-929 cells in the logarithmic growth phase, digest and resuspend them, and adjust the cell density to 5 × 10⁶ cells / year using complete culture medium. 5 cells / mL. Add 1 mL of cell suspension to each well of a 12-well plate and gently agitate to distribute the cells evenly. Incubate the plate at 37°C with 5% CO2 for 24 h, until the cells grow to a monolayer with 90%-100% confluence.

[0145] 3. Cell scratch technique Using the tip of a sterile 200 μL pipette (or a dedicated cell scratcher), perpendicular to the bottom of the well plate, make a uniform and straight stroke across the cell layer along the diameter of the well plate. Discard the old culture medium and gently rinse the well plate twice with PBS buffer to remove any suspended cell debris. Add 1 mL of serum-free culture medium (or serum-free culture medium containing different concentrations of test samples) to each well. Experimental group: serum-free culture medium containing 8 mg / mL HU-HTPS or HU-LTPS samples was added; Negative control group: Serum-free culture medium containing no sample was added.

[0146] 4. Image Acquisition and Data Analysis 0 h image acquisition: Immediately after sample addition (recorded as 0 h), at least 3 pre-selected fields of view images are taken at the same location of each well scratch under an inverted microscope (10× objective lens); Culture and subsequent data collection: The culture plate was returned to the incubator for further culture. Images were taken again at the same location after 24 hours. Image analysis: Use image analysis software such as ImageJ to measure the width (or area) of scratches in all images; Calculate cell migration rate: Cell migration rate (%) = (1 - average scratch width T) h / Average scratch width T0) ×100%; where T0 is 0 h, T h For training time.

[0147] The cell viability data under different concentrations of HU-HTPS and HU-LTPS are shown in Table 17. Figure 5 As shown in the figure. Cell scratch assay at 8 mg / mL is illustrated below. Figure 6 As shown in Table 18, cell migration rate data under the 8 mg / mL condition are presented. A comparison graph of cell migration rates under the 8 mg / mL condition is also shown. Figure 7 As shown.

[0148] Table 17 Cell viability data under different concentrations of HU-HTPS and HU-LTPS

[0149] Table 18. Cell migration rate data at 8 mg / mL.

[0150] Experiment 9 evaluated the effects of HU-HTPS and HU-LTPS on the cytotoxicity and migration behavior of L-929 mouse fibroblasts using the CCK-8 assay and cell scratch assay. Cytotoxicity results showed that within the concentration range of 0.25–8 mg / mL, the relative cell viability of both polysaccharide treatments remained above 100%. HU-HTPS showed the highest viability at 0.5–2 mg / mL (121.56%), while HU-LTPS reached 114.78% in the same range, indicating that it not only had no cytotoxicity but also a certain degree of cell proliferation promotion. When the concentration was increased to 10 mg / mL, the cell viability remained at 97.62% (HU-HTPS) and 91.29% (HU-LTPS), respectively, with no significant toxic reaction, indicating that both polysaccharides possess good biocompatibility over a wide concentration range.

[0151] In the cell scratch assay, after treatment with 8 mg / mL HU-HTPS and HU-LTPS for 24 h, the cell migration rates were 18% and 15%, respectively, significantly higher than the 9% in the negative control group, indicating that both effectively promoted the migration behavior of L-929 cells. Combined with cytotoxicity data, this migration-promoting effect can be attributed not to cell proliferation, but rather to the regulation of cell motility by the polysaccharides themselves. Overall, both HU-HTPS and HU-LTPS exhibited excellent biocompatibility at the experimental concentrations, without causing cell damage, and possessed certain cell migration-promoting activity, providing reliable in vitro experimental evidence for their application as restorative functional ingredients in skincare products.

[0152] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A preparation method of high glycuronate tremella polysaccharide, characterized in that, Comprise the following steps: (1) The strain of tremella sporae is coated on the activated culture medium to activate the culture to obtain the activated strain; (2) The activated strain is inoculated into the seed culture medium to culture to obtain the first-stage seed liquid, and the first-stage seed liquid is inoculated into the second-stage seed culture medium to culture to obtain the second-stage seed liquid; (3) The second-stage seed liquid is inoculated into the fermentation culture medium to ferment and culture to obtain the tremella fermentation liquor; (4) The tremella fermentation liquor is hydrolyzed by hot alkali, enzymatically hydrolyzed, centrifuged, and then the supernatant is filtered, desalted and decolorized, alcohol precipitated, washed, dehydrated and dried to obtain high-sugar uronic acid high-molecular tremella polysaccharide HU-HTPS; (5) The bacteria after centrifugation are homogenized, acid hydrolyzed, enzymatically hydrolyzed, filtered, desalted and decolorized, and then spray dried to obtain high-sugar uronic acid low-molecular tremella polysaccharide HU-LTPS; In step (3), the fermentation culture is operated in two temperature control sections and four pH control sections; the two temperature control sections are 0-96 h of fermentation at 28 DEG C, and 96 h later, the temperature is reduced to 26 DEG C; the four pH control sections are 0-24 h, the pH is natural; 24-48 h, the pH is controlled to be 5.5-6.5; 48-120 h, the pH is controlled to be 4.5-5.0; 120 h later, the pH is natural.

2. The production method according to claim 1, wherein In step (1), the activation culture parameters are: 24-28 DEG C culture for 2-4 days; and / or, in step (1), the activation culture medium comprises the following components: glucose 40-80 g / L, yeast powder 1-3 g / L, peptone 1-3 g / L, potassium phosphate dibasic 0.5-2 g / L, potassium phosphate monobasic 0.3-0.5 g / L, magnesium sulfate heptahydrate 0.5-2 g / L, vitamin B1 5-20 mg / L, agar powder 10-30 g / L, solvent is water, and the pH is natural.

3. The production method according to claim 1, wherein In step (2), the specific operation is: the activated strain obtained in step (1) is inoculated into a seed culture medium, and cultured at 26-28 ℃ and a rotation speed of 160-220 r / min until OD 560 ≥5, and the first-stage seed liquid is inoculated into a second-stage seed culture medium at a volume ratio of 5%-15%, and cultured at 24-28 ℃ and a rotation speed of 160-220 r / min until OD 560 ≥15, and the second-stage seed liquid is obtained. And / or, in step (2), the first-stage seed culture medium comprises the following components: glucose 40-80 g / L, yeast powder 1-3 g / L, peptone 1-3 g / L, potassium phosphate dibasic 0.5-2 g / L, potassium phosphate monobasic 0.3-0.5 g / L, magnesium sulfate heptahydrate 0.5-2 g / L, vitamin B1 5-20 mg / L, solvent is water, and the pH is natural; And / or, in step (2), the second-stage seed culture medium comprises the following components: glucose 5-20 g / L, sucrose 5-20 g / L, yeast powder 1-3 g / L, peptone 2-6 g / L, potassium phosphate dibasic 0.5-2 g / L, potassium phosphate monobasic 0.3-0.5 g / L, magnesium sulfate heptahydrate 0.5-2 g / L, solvent is water, and the pH is natural.

4. The production method according to claim 1, wherein In step (3), the second-stage seed liquid is inoculated into the spore fermentation culture medium at a volume ratio of 8%-15%; the pH is raised to above 5.5, and the viscosity no longer increases as the fermentation end point; And / or, in step (3), the fermentation is supplemented with sugar after 60 h, and the total amount of sugar supplementation is calculated according to the fructose content of 35-60 g / L in the fermentation system; And / or, in step (3), the sugar supplementation is F42 fructose syrup, F55 fructose syrup or F60 fructose syrup; And / or, in step (3), the sugar supplementation is F42 fructose syrup, F55 fructose syrup or F60 fructose syrup; And / or, in step (3), pH control uses 10%-30% sodium hydroxide aqueous solution, 5%-10% sulfuric acid aqueous solution; And / or, in step (3), the dissolved oxygen is 20%-30%, the aeration volume is 1-2 VVM, and the initial stirring speed is 200-500 r / min.

5. The production method according to claim 1, wherein In step (4), after the fermentation is completed, the hot alkaline hydrolysis is carried out by adding water to the fermentation broth in a volume ratio of 1:1-3, directly heating the fermentation broth to 60-80 ℃, controlling the pH to be 8-10, and alkali treatment for 1-3 h; And / or, in step (4), the enzymolysis is carried out by reducing the temperature to 40-50 ℃, controlling the pH to be 7-8, adding 0.5‰-1.5‰ trypsin, and enzymolysis for 0.5-1.5 h; And / or, in step (4), the centrifugation is carried out by adding 2%-4% sodium chloride to the pretreated fermentation broth, and then centrifuging in a tube bundle centrifuge, controlling the centrifugal force to be 12000-14000 g, and collecting the supernatant and the bacteria respectively; And / or, in step (4), the filtration includes coarse filtration and fine filtration; the coarse filtration is to adjust the pH of the supernatant to 8.5-9.0, select 3-5 μm diatomite filter paper board, use perlite as filter aid, and use plate and frame filter to filter; the fine filtration is to adjust the pH of the filtrate to 5.0-6.0, select 1-3 μm diatomite filter paper board+1-4 μm activated carbon deep filter paper board, use diatomite as filter aid, and use plate and frame filter to filter twice; after further filtering through 0.45 μm and 0.22 μm filter membranes, clear filtrate is obtained; And / or, in step (4), the desalination and decolorization and alcohol precipitation are carried out by transferring the clear filtrate into an empty barrel, opening the stirring, slowly adding 95% ethanol to the alcohol degree of 62°-65°, continuing to stir for 15-20 min, and standing; And / or, in step (4), the washing is carried out by discarding the supernatant, adding 75° ethanol to the precipitate, continuing to stir for 15-20 min, standing; continuing to discard the supernatant, adding 80° ethanol to the precipitate, continuing to stir for 15-20 min, standing; continuing to discard the supernatant, adding 85° ethanol to the precipitate, continuing to stir for 15-20 min, standing; And / or, in step (4), the dehydration is carried out by discarding the supernatant, adding 95° ethanol to the precipitate, continuing to stir for 15-20 min, and standing; And / or, in step (4), the drying is carried out by using a Buchner funnel to perform negative pressure filtration, after the ethanol is dried, drying in a vacuum drying oven at 50-60 ℃ for 4-6 h, after drying, uniformly crushing in a pulverizer, passing through a 200 mesh sieve, and obtaining pure white HU-HTPS finished product.

6. The production method according to claim 1, wherein In step (5), the homogenization is carried out by resuspending the bacteria with water in a volume of 1 / 3-1 / 2 of the original fermentation broth, homogenizing twice in a high-pressure homogenizer at 800-1000 bar, and supplementing water to the volume of the original fermentation broth; And / or, in step (5), the acidolysis is carried out by transferring the homogenized liquid to a glass reaction kettle, starting stirring, controlling the temperature to be 65-80 ℃, after the temperature is stable, adding sulfuric acid solution to the system, controlling the final concentration of sulfuric acid in the solution to be 0.21-0.3 M, and acidolysis for 2-3 h; And / or, in step (5), the enzymolysis is carried out after the temperature is adjusted to 30-45 DEG C and the pH is adjusted to 7.5-8 with NaOH, 0.5‰-1.5‰ trypsin, 0.5‰-1.5‰ esterase and 0.5‰-1‰ snailase are added into the system, and the reaction is carried out for 1-2 hours; And / or, in step (5), the filtration includes coarse filtration and fine filtration; the coarse filtration is carried out by adjusting the pH of the supernatant to 8.5-9.0, selecting 3-5 μm diatomite filter paper board, taking perlite as filter aid, and adopting plate and frame filter to filter; the fine filtration is carried out by adjusting the pH of the filtrate to 5.0-6.0, selecting 1-3 μm diatomite filter paper board+1-4 μm activated carbon deep filter paper board, taking diatomite as filter aid, and adopting plate and frame filter to filter, to obtain fine filtrate; the fine filtrate is further filtered through 0.45 μm and 0.22 μm filter membranes, to obtain clear filtrate; And / or, in step (5), the desalination and decolorization is carried out by adopting the combination mode of activated carbon deep filter paper board filtration+1-3 KD organic membrane ultrafiltration for the clear filtrate, adjusting the pH to 5.0-6.0 in the fine filtration stage, selecting 1-3 μm diatomite filter paper board+1-4 μm activated carbon deep filter paper board, taking diatomite as filter aid, adopting plate and frame filter to filter, and then further filtering through 0.45 μm and 0.22 μm filter membranes, and then further carrying out 1-3 KD organic membrane ultrafiltration and concentration, recovering the ultrafiltration and concentration liquid, and finally filtering through 0.45 μm, 0.22 μm and 0.1 μm filter membranes, to obtain colorless and low-conductivity HU-LTPS solution; And / or, in step (5), the spray drying is carried out by feeding the filtrate into a spray dryer, and carrying out spray drying at 190-210 DEG C, to obtain HU-LTPS product.

7. The production method according to claim 1, wherein The fermentation medium comprises the following components: glucose 20-100 g / L, yeast powder 2-10 g / L, proteose peptone 1-5 g / L, Lentinus tigrinus extract 20-100 g / L, sodium glutamate 0.1-1 g / L, glutamine 0.1-1 g / L, serine 0.1-0.5 g / L, histidine 0.1-0.5 g / L, calcium sulfate 0.1-1 g / L, manganese sulfate 0.1-1 g / L, magnesium sulfate 0.1-1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and solvent is water; preferably, the fermentation medium comprises the following components: glucose 40 g / L, yeast powder 4 g / L, proteose peptone 2 g / L, Lentinus tigrinus extract 60 g / L, sodium glutamate 0.5 g / L, glutamine 0.5 g / L, serine 0.5 g / L, histidine 0.5 g / L, calcium sulfate 0.5 g / L, manganese sulfate 0.5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 0.5 g / L, and solvent is water.

8. The production method according to claim 7, wherein The preparation method of the Lentinus tigrinus extract is as follows: (a) after the Lentinus tigrinus is activated, it is inoculated into wood chip medium, and cultured at 24-28 DEG C until the white Lentinus tigrinus mycelium is covered on the wood chip medium, and the wood chip medium is brown to black, to obtain Lentinus tigrinus wood chip culture; (b) Adjusting the water content of the culture of the step (a) to 60%-70%, taking 50-100 g, adding water, boiling in a water bath for 20-60 min, filtering, washing the solid residue with water for 2-3 times, retaining the washing liquid and discarding the solid residue, mixing all the liquids, removing the large insoluble particles by filtering through a 3-5 μm aperture, and passing through a 100 nm ceramic membrane, and finally controlling the volume to be the same as the volume of the added water.

9. The high glycuronosyl tremella polysaccharide prepared by the preparation method according to any one of claims 1-8, characterized in that, The high uronic acid Tremella polysaccharide has a uronic acid content of not less than 30%, a total sugar content of ≥90%, and a protein content of ≤0.1%; preferably, the high uronic acid Tremella polysaccharide includes a high molecular weight product HU-HTPS having a molecular weight of 60-100 million Daltons, and a low molecular weight product HU-LTPS having a molecular weight of 20-50 thousand Daltons.

10. Use of the high uronic acid Tremella polysaccharide prepared by the preparation method of any one of claims 1-8 or the high uronic acid Tremella polysaccharide of claim 9 in the preparation of food, health care products or cosmetics.

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