High-efficiency liquid submerged fermentation culture medium of russula vinosa polysaccharide and fermentation method

Abstract

The invention relates to an efficient liquid submerged fermentation culture medium of russula vinosa polysaccharide and a fermentation method, and belongs to the technical field of fermentation. The formula of the efficient liquid submerged fermentation culture medium for russula vinosa polysaccharide comprises the following components: glucose, a russula vinosa stem water extract, a schizochytrium limacinum degreased powder water extract, a nannochloropsis oculata degreased powder protein enzymatic hydrolysate, an anoectochilus formosanus leaf water extract, a soybean cake powder protein enzymatic hydrolysate, wort, proteoglycan, monopotassium phosphate and magnesium sulfate heptahydrate. On the basis of the fermentation culture medium, a gradient fermentation technology is matched, efficient fermentation production of russula vinosa polysaccharide is achieved by adjusting the components and nutrient concentration of the culture medium in stages, the bottleneck of current russula vinosa polysaccharide resource acquisition is broken through, and industrial application of russula vinosa polysaccharide is promoted.

Description

Technical Field

[0001] The invention relates to a high-efficiency liquid submerged fermentation medium and a fermentation method for russula polysaccharide, belonging to the technical field of fermentation. Background Art

[0002] Red mushroom ( Vinous Russula ) As a rare edible and medicinal fungus, it is rich in polysaccharides, polyphenols, flavonoids and other bioactive ingredients, and its nutritional value and medicinal value have attracted much attention. Among them, Russula polysaccharide, as the core functional ingredient, has been proven to have multiple biological activities such as antioxidant, immunomodulatory, anti-tumor, hypoglycemic, and anti-inflammatory, and has great potential in the development of functional foods, health products and drugs. However, Russula is a typical ectomycorrhizal fungus that relies on specific host trees for symbiotic growth. It has not been possible to cultivate it artificially, resulting in an extreme scarcity of wild resources. In addition, due to over-picking and habitat destruction, its natural production has declined year by year, which has seriously restricted the development and large-scale application of products based on Russula polysaccharide. Therefore, exploring efficient and sustainable extraction technology for the active ingredients of Russula has become an urgent need for industrial development.

[0003] Submerged fermentation technology offers a breakthrough for the efficient utilization of rare edible mushroom resources. By simulating the natural growth environment of mycelium, this technology enables the large-scale production of mycelium and active ingredients in controlled fermentation tanks. This technology offers advantages such as short cycle times, high efficiency, and freedom from seasonal restrictions. For Russula edodes, studies have utilized liquid fermentation to obtain mycelium and exopolysaccharides, preliminarily demonstrating its feasibility as a viable alternative to wild fruiting bodies. However, existing technologies often utilize conventional culture media (such as sucrose and yeast extract as carbon and nitrogen sources) and standard, constant fermentation parameters. This leads to low mycelial biomass and limited polysaccharide yields, resulting in inefficient utilization of the already scarce Russula edodes and a waste of resources. Therefore, developing efficient fermentation processes tailored to the physiological characteristics of Russula edodes, and increasing polysaccharide yields through culture medium innovation and process control, has become a key approach to addressing resource bottlenecks and promoting industrial application. Summary of the Invention

[0004] In response to the existing problems, the present invention provides a high-efficiency liquid deep fermentation culture medium and fermentation method for Russula polysaccharide. The method can efficiently produce Russula polysaccharide through liquid deep fermentation, overcoming the problems of low mycelium biomass and limited polysaccharide yield in the existing Russula polysaccharide fermentation process, helping to break through the bottleneck of obtaining Russula polysaccharide resources and promote the industrial application of Russula polysaccharide.

[0005] The technical solutions of the present invention are as follows: The present invention provides a high-efficiency liquid submerged fermentation medium for Russula polysaccharide, the medium composition of which is as follows: Culture medium A (g / L): potato juice 150-220, Russula stem water extract 80-100, glucose 10-15, lactose 5-8, vitamin B1 0.001-0.005, agar powder 15-20; Medium B (g / L): glucose 15-25%, Russula edodes stem aqueous extract 80-100%, Schizochytrium defatted powder aqueous extract 12-16%, Nannochloropsis defatted powder protein hydrolyzate 16-18%, Anoectochilus roxburghii leaf aqueous extract 8-12%, soybean meal protein hydrolyzate 5-10%, malt extract 8-12%, protein 5-7%, potassium dihydrogen phosphate 3-3.5%, magnesium sulfate heptahydrate 2.2-3.3%, pH 5.5-7. Medium C (g / L): 24-28g glucose, 50-60g Russula stem aqueous extract, 20-24g Schizochytrium defatted powder aqueous extract, 33-36g Nannochloropsis defatted powder protein hydrolyzate, 36-45g Anoectochilus roxburghii leaf aqueous extract, 5-10g soybean meal protein hydrolyzate; 8-12g malt extract; 5-7g protein; 3-3.5g potassium dihydrogen phosphate, 2.2-3.3g magnesium sulfate heptahydrate, pH 5.5-7. Culture medium D (g / L): glucose 50-85, Russula edodes stipe aqueous extract 20-24, Schizochytrium defatted powder aqueous extract 26-28, Nannochloropsis defatted powder protein hydrolyzate 40-44, Anoectochilus roxburghii leaf aqueous extract 45-52, soybean meal protein hydrolyzate 15-20; malt extract 8-12; protein core 5-7; potassium dihydrogen phosphate 3-3.5, magnesium sulfate heptahydrate 2.2-3.3, pH 5.5-7.

[0006] The preparation processes of the water extract of Russula edulis stem, the water extract of Schizochytrium defatted powder, the protein hydrolyzate of Nannochloropsis defatted powder, the water extract of Anoectochilus roxburghii leaves, and the protein hydrolyzate of soybean cake powder are as follows: (1) Water extract of Russula stem: Add 3 to 5 times the mass ratio of fresh or frozen Russula stem to water, grind into a slurry, and filter the residue to obtain the water extract of Russula stem. (2) Water extract of defatted Schizochytrium powder: After extracting the oil, add 10-15 times the mass ratio of water to the remaining Schizochytrium powder, boil for 25-30 minutes, and filter the residue to obtain the water extract of defatted Schizochytrium powder; (3) Enzyme hydrolysate of defatted Nannochloropsis powder: Add 10-15 times the mass of water to the remaining Nannochloropsis powder after oil extraction, add neutral protease at a ratio of 0.5-0.75 g / L, heat in a water bath at 45-55 °C for 2.5-3 h, then heat to 95-100 °C, maintain for 8-15 min, and then cool to below 40 °C. After filtering the residue, the result is the enzyme hydrolysate of defatted Nannochloropsis powder. (4) Aqueous extract of roxburghii leaves: fresh leaves of roxburghii with a growth period of 8 to 9 months, add 3 to 5 times the amount of water by weight, grind into a pulp, and filter to obtain the aqueous extract of roxburghii leaves; (5) Soybean cake powder protein hydrolysate: Add 10-15 times the mass of water to defatted soybean cake powder, add trypsin at a ratio of 0.8-1.2 g / L, heat in a water bath at 35-40℃ for 2.5-3 h, then heat to 95-100℃, maintain for 8-15 min, then reduce to below 40℃, filter the residue, and the result is soybean cake powder protein hydrolysate.

[0007] The present invention also provides a high-efficiency liquid submerged fermentation method for Russula polysaccharide, comprising the following steps: (1) Inoculate the preserved Russula edulis strain onto solid culture medium A for activation at a temperature of 25-28°C for 5-8 days; (2) Then, pick a 0.5-1.5 cm bacterial block with high activity at the edge of the colony and transfer it to liquid culture medium B. Culture it at 29-31°C for 4-6 days with a shaker speed of 180-220 rpm. (3) Transfer the fermentation broth from medium B together with the mycelium to medium C at a ratio of 5-10%, and culture at 32-35°C for 4-6 days with a shaking speed of 250-300 rpm; (4) Transfer the fermentation broth from medium B together with the mycelium to medium C at a ratio of 5-10%, culture at 32-35°C for 4-6 days, and shake at 250-300 rpm; (5) Then, add 20-30 g / L of sugar, reduce the fermentation temperature to 22-24°C at a rate of 0.5°C / h, and reduce the shaker speed to 100-120 rpm at a rate of 10 rpm / h. Continue fermentation for 3-5 days until the glucose in the fermentation liquid is exhausted. (6) The mycelium in the fermentation liquid is separated and dried, crushed, water-extracted, precipitated with alcohol, deproteinized and purified to obtain the polysaccharide of mycelium of Russula, i.e., the intracellular polysaccharide; the fermentation liquid remaining after the mycelium is separated is concentrated, precipitated with alcohol, deproteinized and purified to obtain the polysaccharide of fermentation liquid of Russula, i.e., the extracellular polysaccharide.

[0008] Different from the prior art, the present invention has the following beneficial effects: 1. The present invention adds five active ingredients, including water extract of Russula stem, water extract of Schizochytrium defatted powder, protein hydrolyzate of Nannochloropsis defatted powder, water extract of Anoectochilus roxburghii leaves, and protein hydrolyzate of soybean cake powder, to the liquid fermentation medium of Russula, which can effectively promote the rapid growth of Russula and improve the yield of polysaccharides.

[0009] 2. The present invention also achieves efficient production of Russula polysaccharide through a liquid deep fermentation method. The present invention combines gradient fermentation technology with the fermentation medium and adjusts the medium components and nutrient concentration in stages, so that the Russula experiences a gradient-changing growth environment at different stages, optimizes the growth environment and metabolic level of the mycelium, and greatly improves the yield of high-value-added Russula polysaccharide. DETAILED DESCRIPTION

[0010] The present invention will be further described below with reference to the examples. The examples are provided only to illustrate the present invention, but not to limit the scope of the invention.

[0011] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. The methods in the following embodiments are all conventional methods unless otherwise specified.

[0012] The Russula strain involved in the following embodiments is from the China Forestry Microorganism Culture Collection Center, and the strain number is cfcc 88186.

[0013] Example 1 This embodiment provides a high-efficiency liquid submerged culture medium and fermentation method for Russula polysaccharide, comprising the following steps: (1) Raw material preparation: The following steps were used to prepare the water extract of Russula stem, the water extract of Schizochytrium defatted powder, the protein hydrolyzate of Nannochloropsis defatted powder, the water extract of Anoectochilus roxburghii leaves, and the protein hydrolyzate of soybean cake powder: 1) Russula stem aqueous extract: fresh or frozen Russula stems, add 4 times the mass ratio of water, grind into a slurry, filter the residue to obtain Russula stem aqueous extract; 2) Defatted Schizochytrium powder aqueous extract: After oil extraction, the remaining Schizochytrium powder is added with 12 times the mass of water, boiled for 28 minutes, and the residue is filtered to obtain the defatted Schizochytrium powder aqueous extract. 3) Enzyme hydrolysate of defatted Nannochloropsis powder: Add 13 times the mass of water to the remaining Nannochloropsis powder after oil extraction, add 0.6 g / L of neutral protease, and heat in a 50°C water bath for 2.8 h. Then, heat to 98°C and maintain for 11 min. Then, cool to 35°C. Filter the residue to obtain the enzyme hydrolysate of defatted Nannochloropsis powder. 4) Aqueous extract of roxburghii leaves: Fresh roxburghii leaves from a 9-month-old plant are ground into a slurry with 4 times the amount of water. The slurry is filtered to obtain the aqueous extract. 5) Soybean cake meal protein hydrolysate: Add 125 times the mass of water to defatted soybean cake meal, add trypsin at a ratio of 1.0 g / L, and heat in a 38°C water bath for 2.8 hours. Then, heat to 98°C and maintain for 10 minutes. Then, cool to 32°C. Filter the residue to obtain the soybean cake meal protein hydrolysate.

[0014] (2) Culture medium preparation: Activation medium A was prepared according to the following formula: potato juice 200 g / L, Russula stem water extract 90 g / L, glucose 13 g / L, lactose 6.5 g / L, vitamin B1 0.003 g / L, agar powder 18 g / L; Fermentation medium B was prepared according to the following recipe: 20 g / L glucose, 90 g / L aqueous extract of Russula oleracea stems, 14 g / L aqueous extract of Schizochytrium defatted powder, 17 g / L protein hydrolyzate of Nannochloropsis defatted powder, 10 g / L aqueous extract of Anoectochilus roxburghii leaves, 8 g / L protein hydrolyzate of soybean meal; 10 g / L malt extract; 6 g / L protein; 3.2 g / L potassium dihydrogen phosphate, and 2.8 g / L magnesium sulfate heptahydrate. The initial pH of fermentation medium B was controlled at 6. Fermentation medium C was prepared according to the following recipe: glucose 26 g / L, Russula oleifera stem aqueous extract 55 g / L, Schizochytrium defatted powder aqueous extract 22 g / L, Nannochloropsis defatted powder protein hydrolyzate 34 g / L, Anoectochilus roxburghii leaf aqueous extract 40 g / L, soybean meal protein hydrolyzate 8 g / L; malt extract 10 g / L; protein 6 g / L; potassium dihydrogen phosphate 3.2 g / L, magnesium sulfate heptahydrate 2.8 g / L, and the initial pH of fermentation medium C was controlled at 6. Fermentation medium D was prepared according to the following recipe: glucose 70 g / L, Russula edodes stipe aqueous extract 22 g / L, Schizochytrium defatted powder aqueous extract 27 g / L, Nannochloropsis defatted powder protein hydrolyzate 42 g / L, Anoectochilus roxburghii leaf aqueous extract 48 g / L, soybean meal protein hydrolyzate 17 g / L; malt extract 10 g / L; protein 6 g / L; potassium dihydrogen phosphate 3.2 g / L, magnesium sulfate heptahydrate 2.8; and the initial pH value of fermentation medium D was controlled at 6.

[0015] (3) Fermentation process: S1. Inoculate the preserved Russula spp. onto activation medium A for activation at 26°C for 6 days; S2. Then, pick a 1 cm bacterial block with high activity at the edge of the colony and transfer it to fermentation medium B. Incubate at 30°C for 5 days with a shaker speed of 200 rpm. S3, then transfer the fermentation broth of fermentation medium B together with the mycelium at a ratio of 8% to fermentation medium C, and culture at 33°C for 5 days with a shaking speed of 280 rpm; S4, then the fermentation broth of fermentation medium C together with the mycelium was transferred to fermentation medium D at a ratio of 8%, and cultured at 33°C for 5 days with a shaking speed of 260 rpm; S5. Finally, glucose is added to the fermentation medium D fermentation broth obtained in step S4 until the glucose concentration reaches 25 g / L, and the fermentation temperature is lowered to 23° C. at a rate of 0.5° C. / h, and the shaker speed is lowered to 110 rpm at a rate of 10 rpm / h. Fermentation is continued for 4 days until the glucose in the fermentation broth is exhausted; the mycelium in the fermentation broth is then separated and subjected to drying, crushing, water extraction, alcohol precipitation, deproteinization, and purification to obtain Russula polysaccharide, which is a Russula mycelium polysaccharide, i.e., an intracellular polysaccharide; the fermentation broth remaining after separation of the mycelium is concentrated, precipitated with alcohol, deproteinized, and purified to obtain Russula polysaccharide, which is a Russula fermentation broth polysaccharide, i.e., an extracellular polysaccharide.

[0016] Example 2 This embodiment provides a high-efficiency liquid submerged culture medium and fermentation method for Russula polysaccharide, comprising the following steps: (1) Raw material preparation: The following steps were used to prepare the water extract of Russula stem, the water extract of Schizochytrium defatted powder, the protein hydrolyzate of Nannochloropsis defatted powder, the water extract of Anoectochilus roxburghii leaves, and the protein hydrolyzate of soybean cake powder: 1) Russula stem aqueous extract: Add 5 times the mass ratio of water to fresh or frozen Russula stems, grind into a slurry, and filter the residue to obtain Russula stem aqueous extract. 2) Defatted Schizochytrium powder aqueous extract: After extracting the oil, add 15 times the mass of water to the remaining Schizochytrium powder, boil for 30 minutes, and filter the residue to obtain the defatted Schizochytrium powder aqueous extract. 3) Enzyme hydrolysate of defatted Nannochloropsis powder: Add 15 times the mass of water to the remaining Nannochloropsis powder after oil extraction, add 0.75 g / L of neutral protease, and heat in a 55°C water bath for 3 h. Then, heat to 98°C and maintain for 8 min. Then, cool to 28°C. Filter the residue to obtain the enzyme hydrolysate of defatted Nannochloropsis powder. 4) Aqueous extract of roxburghii leaves: Fresh roxburghii leaves from a 9-month-old plant are ground into a slurry with 5 times the amount of water. The slurry is filtered to obtain the aqueous extract. 5) Soybean cake meal protein hydrolysate: Add 15 times the amount of water by weight to defatted soybean cake meal, add trypsin at a ratio of 1.2 g / L, heat in a 40°C water bath for 3 h, then heat to 95°C, maintain for 15 min, and then cool to 28°C. Filter the residue to obtain the soybean cake meal protein hydrolysate.

[0017] (2) Culture medium preparation: Activation medium A was prepared according to the following formula: potato juice 220 g / L, Russula stem water extract 100 g / L, glucose 15 g / L, lactose 8 g / L, vitamin B1 0.005 g / L, agar powder 20 g / L; Fermentation medium B was prepared according to the following recipe: 25 g / L glucose, 100 g / L aqueous extract of Russula oleracea stems, 16 g / L aqueous extract of Schizochytrium defatted powder, 18 g / L protein hydrolyzate of Nannochloropsis defatted powder, 12 g / L aqueous extract of Anoectochilus roxburghii leaves, 10 g / L protein hydrolyzate of soybean meal; 12 g / L malt extract; 7 g / L protein; 3.5 g / L potassium dihydrogen phosphate, and 3.3 g / L magnesium sulfate heptahydrate. The initial pH of fermentation medium B was controlled at 7. Fermentation medium C was prepared according to the following recipe: 28 g / L glucose, 60 g / L aqueous extract of Russula oleracea stems, 24 g / L aqueous extract of Schizochytrium defatted powder, 36 g / L protein hydrolyzate of Nannochloropsis defatted powder, 45 g / L aqueous extract of Anoectochilus roxburghii leaves, 10 g / L protein hydrolyzate of soybean meal; 12 g / L malt extract; 7 g / L protein; 3.5 g / L potassium dihydrogen phosphate; and 3.3 g / L magnesium sulfate heptahydrate. The initial pH of fermentation medium C was controlled at 7. Fermentation medium D was prepared according to the following recipe: glucose 85 g / L, Russula oleifera stem aqueous extract 24 g / L, Schizochytrium defatted powder aqueous extract 28 g / L, Nannochloropsis defatted powder protein hydrolyzate 44 g / L, Anoectochilus roxburghii leaf aqueous extract 52 g / L, soybean meal protein hydrolyzate 20 g / L; malt extract 12 g / L; protein 7 g / L; potassium dihydrogen phosphate 3.5 g / L, magnesium sulfate heptahydrate 3.3 g / L, and the initial pH value of fermentation medium D was controlled at 7.

[0018] (3) Fermentation process: S1. Inoculate the preserved Russula spp. onto solid culture medium A for activation at 28°C for 5 days; S2. Then, pick a 1.5 cm bacterial block with high activity at the edge of the colony and transfer it to liquid medium B. Incubate at 31°C for 4 days with a shaker speed of 220 rpm. S3. Transfer the fermentation broth from medium B together with the mycelium to medium C at a ratio of 10%, and culture at 35°C for 4 days with a shaker speed of 300 rpm. S4. Transfer the fermentation broth from medium C together with the mycelium to medium D at a ratio of 10%, and culture at 35°C for 6 days with a shaker speed of 300 rpm. S5. Finally, glucose is added to the fermentation broth of the fermentation medium D obtained in step S4 until the glucose concentration reaches 30 g / L, and the fermentation temperature is lowered to 24° C. at a rate of 0.5° C. / h, and the shaker speed is lowered to 120 rpm at a rate of 10 rpm / h. Fermentation is continued for 3 days until the glucose in the fermentation broth is exhausted; the mycelium in the fermentation broth is then separated and subjected to drying, crushing, water extraction, alcohol precipitation, deproteinization, and purification to obtain Russula polysaccharide, which is a Russula mycelium polysaccharide, i.e., an intracellular polysaccharide; the fermentation broth remaining after separation of the mycelium is concentrated, precipitated with alcohol, deproteinized, and purified to obtain Russula polysaccharide, which is a Russula fermentation broth polysaccharide, i.e., an extracellular polysaccharide.

[0019] Example 3 This embodiment provides a high-efficiency liquid submerged culture medium and fermentation method for Russula polysaccharide, comprising the following steps: (1) Raw material preparation: The following steps were used to prepare the water extract of Russula stem, the water extract of Schizochytrium defatted powder, the protein hydrolyzate of Nannochloropsis defatted powder, the water extract of Anoectochilus roxburghii leaves, and the protein hydrolyzate of soybean cake powder: 1) Russula stem aqueous extract: fresh or frozen Russula stems, add 3 times the mass ratio of water, grind into a slurry, filter the residue to obtain Russula stem aqueous extract; 2) Defatted Schizochytrium powder aqueous extract: After oil extraction, add 10 times the mass of water to the remaining Schizochytrium powder, boil for 25 minutes, and filter the residue to obtain the defatted Schizochytrium powder aqueous extract. 3) Enzyme hydrolysate of defatted Nannochloropsis powder: Add 10 times the mass of water to the remaining Nannochloropsis powder after oil extraction, add 0.5 g / L of neutral protease, and heat in a 45°C water bath for 2.5 h. Then, heat to 95°C and maintain for 15 min. Then, cool to 36°C. Filter the residue to obtain the enzyme hydrolysate of defatted Nannochloropsis powder. 4) Aqueous extract of roxburghii leaves: Fresh roxburghii leaves from an 8-month-old plant are ground into a pulp with 3 times the amount of water. The pulp is filtered to obtain the aqueous extract. 5) Soybean cake meal protein hydrolysate: Add 10 times the amount of water by weight to defatted soybean cake meal, add trypsin at a ratio of 0.8 g / L, heat in a 35°C water bath for 2.5 h, then heat to 95°C, maintain for 15 min, and then cool to 30°C. Filter the residue to obtain the soybean cake meal protein hydrolysate.

[0020] (2) Culture medium preparation: Activation medium A was prepared according to the following formula: potato juice 150 g / L, Russula stem water extract 80 g / L, glucose 10 g / L, lactose 5 g / L, vitamin B1 0.001 g / L, agar powder 15 g / L; Fermentation medium B was prepared according to the following recipe: 15 g / L glucose, 80 g / L aqueous extract of Russula oleracea stems, 12 g / L aqueous extract of Schizochytrium defatted powder, 16 g / L protein hydrolyzate of Nannochloropsis defatted powder, 8 g / L aqueous extract of Anoectochilus roxburghii leaves, 5 g / L protein hydrolyzate of soybean meal; 8 g / L malt extract; 5 g / L protein stalks; 3 g / L potassium dihydrogen phosphate, and 2.2 g / L magnesium sulfate heptahydrate. The initial pH of fermentation medium B was controlled at 5.5. Fermentation medium C was prepared according to the following recipe: 24 g / L glucose, 50 g / L aqueous extract of Russula oleracea stems, 20 g / L aqueous extract of Schizochytrium defatted powder, 33 g / L protein hydrolyzate of Nannochloropsis defatted powder, 36 g / L aqueous extract of Anoectochilus roxburghii leaves, 5 g / L protein hydrolyzate of soybean meal; 8 g / L malt extract; 5 g / L protein stalks; 3 g / L potassium dihydrogen phosphate; and 2.2 g / L magnesium sulfate heptahydrate. The initial pH of fermentation medium C was controlled at 5.5. Fermentation medium D was prepared according to the following recipe: glucose 50 g / L, Russula edodes stipe aqueous extract 20 g / L, Schizochytrium defatted powder aqueous extract 26 g / L, Nannochloropsis defatted powder protein hydrolyzate 40 g / L, Anoectochilus roxburghii leaf aqueous extract 45 g / L, soybean meal protein hydrolyzate 15 g / L; malt extract 8 g / L; protein 5 g / L; potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 2.2 g / L, and the initial pH of fermentation medium D was controlled at pH 5.5.

[0021] (3) Fermentation process: S1. Inoculate the preserved Russula spp. onto solid culture medium A for activation at 25°C for 8 days; S2. Then, pick a 0.5 cm bacterial block with high activity at the edge of the colony and transfer it to liquid medium B. Incubate at 29°C for 6 days with a shaker speed of 180 rpm. S3. Transfer the fermentation broth from medium B together with the mycelium to medium C at a ratio of 5%, and culture at 32°C for 6 days with a shaker speed of 250 rpm. S4. Transfer the fermentation broth from medium C together with the mycelium to medium D at a ratio of 5%, and culture at 32°C for 4 days with a shaker speed of 250 rpm. S5. Finally, glucose is added to the fermentation broth of the fermentation medium D obtained in step S4 until the glucose concentration reaches 20 g / L, and the fermentation temperature is lowered to 22° C. at a rate of 0.5° C. / h, and the shaker speed is lowered to 100 rpm at a rate of 10 rpm / h. Fermentation is continued for 5 days until the glucose in the fermentation broth is exhausted; the mycelium in the fermentation broth is then separated and subjected to drying, crushing, water extraction, alcohol precipitation, deproteinization, and purification to obtain Russula polysaccharide, which is a Russula mycelium polysaccharide, i.e., an intracellular polysaccharide; the fermentation broth remaining after separation of the mycelium is concentrated, precipitated with alcohol, deproteinized, and purified to obtain Russula polysaccharide, which is a Russula fermentation broth polysaccharide, i.e., an extracellular polysaccharide.

[0022] Comparative Example 1 This comparative example uses conventional fermentation technology to ferment Russula polysaccharide, including the following steps: (1) Culture medium composition Solid activated medium (g / L): potato juice 200, glucose 20, peptone 3, agar 18, potassium dihydrogen phosphate 1, magnesium sulfate heptahydrate 3, natural pH; Seed culture medium (g / L): potato juice 200, glucose 20, potassium dihydrogen phosphate 2, magnesium sulfate 1, natural pH; Fermentation medium (g / L): sucrose 20, yeast extract 2, potassium dihydrogen phosphate 2, magnesium sulfate 1.

[0023] (2) Fermentation method Activation of the fungus: Cut a 1 cm mycelium block from the mother tube of Russula and inoculate it in the middle of the slope, then culture it at 30°C for 6 days. Seed culture: Cut the activated slant into 1 cm mycelial blocks and inoculate them into liquid culture medium at 30°C and 200 rpm for 6 days. Fermentation culture: liquid culture inoculation rate is 10%, 30℃, 200 r / min, and culture is carried out for 10 days.

[0024] Comparative Example 2 This comparative example uses conventional fermentation technology to ferment Russula polysaccharide, including the following steps: (1) Culture medium Solid activated medium (g / L): glucose 20, potato juice 20, potassium dihydrogen phosphate 3, magnesium sulfate heptahydrate 3, VB10.1, agar 20; pH natural; Liquid seed culture medium (g / L): glucose 20, peptone 2, yeast extract 1, magnesium sulfate heptahydrate 1, potassium dihydrogen phosphate 0.46, potassium hydrogen phosphate 1; natural pH value; Fermentation medium (g / L): glucose 40, peptone 5, magnesium sulfate heptahydrate 0.75, potassium dihydrogen phosphate 1.5; natural pH.

[0025] (2) Pick two small pieces of colonies from the activated slant and inoculate them into liquid seed culture medium. Place them on a shaker at 25°C and shake at 100 rpm for 4 days to obtain the first-level seed liquid. At a 10% inoculation rate, inoculate the first-level seed liquid into the liquid seed culture medium and place them on a shaker at 26°C and shake at 100 rpm for 4 days to obtain the second-level seed liquid. At a 10% inoculation rate, inoculate the second-level strain into the fermentation medium and place them on a shaker at 26°C and shake at 100 rpm for 8 days.

[0026] Table 1 compares the biomass, intracellular polysaccharide, and exopolysaccharide yields of Russula mycelium obtained by fermentation of Examples 1-3 and Comparative Examples 1-2. Examples 1-3 are applications of the technology of the present invention. Comparative Examples 1-2 are conventional fermentation technologies, serving as controls. The data show that the biomass of Russula mycelium fermented using the present invention is 3-4 times that of conventional fermentation technology, and the yields of intracellular polysaccharides and exopolysaccharides are 2-3 times that of conventional fermentation technology. This method has strong application value and plays an important role in promoting the industrial application of Russula polysaccharides.

[0027] Table 1 Biomass and polysaccharide yields of Russula mycelium obtained by fermentation

Claims

1. A high-efficiency liquid submerged fermentation medium for Russula polysaccharide, characterized in that: The culture medium composition is as follows: Culture medium A (g / L): potato juice 150-220, Russula stem water extract 80-100, glucose 10-15, lactose 5-8, vitamin B1 0.001-0.005, agar powder 15-20; Medium B (g / L): glucose 15-25%, Russula edodes stem aqueous extract 80-100%, Schizochytrium defatted powder aqueous extract 12-16%, Nannochloropsis defatted powder protein hydrolyzate 16-18%, Anoectochilus roxburghii leaf aqueous extract 8-12%, soybean meal protein hydrolyzate 5-10%, malt extract 8-12%, protein 5-7%, potassium dihydrogen phosphate 3-3.5%, magnesium sulfate heptahydrate 2.2-3.3%, pH 5.5-7. Medium C (g / L): 24-28g glucose, 50-60g Russula stem aqueous extract, 20-24g Schizochytrium defatted powder aqueous extract, 33-36g Nannochloropsis defatted powder protein hydrolyzate, 36-45g Anoectochilus roxburghii leaf aqueous extract, 5-10g soybean meal protein hydrolyzate; 8-12g malt extract; 5-7g protein; 3-3.5g potassium dihydrogen phosphate, 2.2-3.3g magnesium sulfate heptahydrate, pH 5.5-7. Culture medium D (g / L): glucose 50-85, Russula edodes stipe aqueous extract 20-24, Schizochytrium defatted powder aqueous extract 26-28, Nannochloropsis defatted powder protein hydrolyzate 40-44, Anoectochilus roxburghii leaf aqueous extract 45-52, soybean meal protein hydrolyzate 15-20; malt extract 8-12; protein core 5-7; potassium dihydrogen phosphate 3-3.5, magnesium sulfate heptahydrate 2.2-3.3, pH 5.5-7.

2. The high-efficiency liquid submerged fermentation medium of Russula polysaccharide according to claim 1, characterized in that: The Russula stem water extract is obtained by grinding fresh or frozen Russula stems into pulp in water and filtering the residue.

3. The high-efficiency liquid submerged fermentation medium of Russula polysaccharide according to claim 1, characterized in that: The Schizochytrium defatted powder water extract is obtained by boiling the Schizochytrium powder in water and filtering the residue.

4. The high-efficiency liquid submerged fermentation medium for Russula polysaccharide according to claim 1, characterized in that: The enzymatic hydrolysate of the defatted Nannochloropsis algae powder is prepared according to the following steps: adding 10 to 15 times the mass of water to the Nannochloropsis algae powder, adding neutral protease at a ratio of 0.5 to 0.75 g / L, heating in a water bath at 45 to 55° C. for 2.5 to 3 hours, then heating to 95 to 100° C., maintaining for 8 to 15 minutes, then cooling to room temperature, and filtering to obtain the enzymatic hydrolysate of the defatted Nannochloropsis algae powder.

5. The high-efficiency liquid submerged fermentation medium of Russula polysaccharide according to claim 1, characterized in that: The roxburghii leaf water extract is obtained by grinding fresh roxburghii leaves in water into a slurry and filtering the slurry.

6. The high-efficiency liquid submerged fermentation medium of Russula polysaccharide according to claim 1, characterized in that: The soybean meal protein hydrolysate is prepared according to the following steps: adding 10 to 15 times the mass of water to defatted soybean meal, adding trypsin at a ratio of 0.8 g / L, heating in a 35°C water bath for 2.5 hours, then heating to 95°C, maintaining for 15 minutes, and then cooling to 30°C. After filtering the residue, the soybean meal protein hydrolysate is obtained.

7. Using the high-efficiency liquid submerged fermentation medium of Russula polysaccharide according to any one of claims 1 to 6 for high-efficiency liquid submerged fermentation of Russula polysaccharide.

8. A high-efficiency submerged liquid fermentation method for producing Russula polysaccharide according to claim 7, characterized in that: The steps include: (1) Inoculate the preserved Russula edulis strain onto solid culture medium A for activation at a temperature of 25-28°C for 5-8 days; (2) Then, pick a 0.5-1.5 cm bacterial block with high activity at the edge of the colony and transfer it to liquid culture medium B. Culture it at 29-31°C for 4-6 days with a shaker speed of 180-220 rpm. (3) Transfer the fermentation broth from medium B together with the mycelium to medium C at a ratio of 5-10%, and culture at 32-35°C for 4-6 days with a shaking speed of 250-300 rpm; (4) Transfer the fermentation broth from medium B together with the mycelium to medium C at a ratio of 5-10%, culture at 32-35°C for 4-6 days, and shake at 250-300 rpm; (5) Then, add 20-30 g / L of sugar, reduce the fermentation temperature to 22-24°C at a rate of 0.5°C / h, and reduce the shaker speed to 100-120 rpm at a rate of 10 rpm / h. Continue fermentation for 3-5 days until the glucose in the fermentation liquid is exhausted. (6) The mycelium in the fermentation liquid is separated and dried, crushed, water-extracted, precipitated with alcohol, deproteinized and purified to obtain the polysaccharide of mycelium of Russula, i.e., the intracellular polysaccharide; the fermentation liquid remaining after the mycelium is separated is concentrated, precipitated with alcohol, deproteinized and purified to obtain the polysaccharide of fermentation liquid of Russula, i.e., the extracellular polysaccharide.

Images