WO2020134688A1 - Procédé de préparation de polysaccharide de hericium erinaceus de haute pureté par fermentation de hericium erinaceus, et milieu de fermentation correspondant - Google Patents

Procédé de préparation de polysaccharide de hericium erinaceus de haute pureté par fermentation de hericium erinaceus, et milieu de fermentation correspondant Download PDF

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WO2020134688A1
WO2020134688A1 PCT/CN2019/118947 CN2019118947W WO2020134688A1 WO 2020134688 A1 WO2020134688 A1 WO 2020134688A1 CN 2019118947 W CN2019118947 W CN 2019118947W WO 2020134688 A1 WO2020134688 A1 WO 2020134688A1
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fermentation
mass
hericium erinaceus
polysaccharide
purity
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PCT/CN2019/118947
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English (en)
Chinese (zh)
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贾玉倩
陆震
魏玉洁
杨旭
孙元军
石艳丽
郭学平
栾贻宏
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华熙生物科技股份有限公司
山东华熙海御生物医药有限公司
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Publication of WO2020134688A1 publication Critical patent/WO2020134688A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the invention relates to the field of microbial fermentation engineering, in particular to a method for preparing Hericium erinaceus polysaccharide by fermenting Hericium erinaceus and fermentation medium.
  • Hericium erinaceus also known as Hericium erinaceus, Ursinus edodes, and Hedgehog, belongs to the genus Hericium of the Polyphaga order Dentaceae, and is a well-known edible and medicinal bacteria in my country.
  • Xu Guangqi's "Full Agricultural Policy” records that it is delicious, fragrant and delicious. The folks often use it to treat indigestion and neurasthenia.
  • Hericium erinaceus has the effect of "assisting digestion and benefiting the five internal organs". Modern medicine has proved that Hericium erinaceus has a significant effect on digestive tract diseases and malignant tumors.
  • Hericium erinaceus polysaccharides have effects on immune regulation, cancer treatment, glucose and lipid reduction, antioxidant and anti-aging It has great efficacy and is a fungal polysaccharide with rich development potential and broad application prospects.
  • Japan and European and American countries began research on fungal polysaccharides in the 1930s, and accumulated a large amount of scientific research materials.
  • some polysaccharide drugs have rapidly entered the market and become new drugs and health products.
  • Hericium polysaccharides can be divided into intracellular polysaccharides and extracellular polysaccharides according to their locations. Intracellular polysaccharides exist in mycelial cells. Extracellular polysaccharides refer to polysaccharides secreted into the culture fluid under liquid culture conditions. Hericium erinaceus is extracted and isolated from the dried mycelium or obtained from the mycelium in the fermentation broth, and the extracellular polysaccharides in the fermentation broth are often ignored. For example, most active polysaccharides in various health products are processed from Hericium erinaceus fruiting body. Hericium erinaceus fruiting body takes about 2 to 3 months from seed production to harvest.
  • Patent Document 1 discloses "a hericium erinaceus polysaccharide and a preparation method thereof". First, the hericium erinaceus is alcohol-dried to prepare a crude polysaccharide. The crude polysaccharide is then subjected to adsorption treatment with an ion exchange resin to obtain the final hericium erinaceus polysaccharide. Although the steps in this process are relatively simple, the efficiency of the ion exchange method is low and it is difficult to scale up production, and the purity of polysaccharides has not been studied.
  • Patent Document 2 discloses "a method for extracting polysaccharides of Hericium erinaceus", which is obtained by crushing the fruit body parts of Hericium erinaceus. It is compared with microwave, hot water extraction, and ultrasonic extraction. Finally, it is concentrated, precipitated and dried The product can be made. Although this process can remove some small molecular impurities during alcohol precipitation, it does not involve the removal of large molecular impurities such as protein impurities, and the product purity is not high.
  • Patent Document 3 discloses "a hericium erinaceus active polysaccharide and a preparation method thereof". In this process, the hericium erinaceus polysaccharide is pretreated with raw materials, ultrasonically degraded, and finally freeze-dried to produce a hericium erinaceus high-activity polysaccharide product. Although the patented process is relatively simple, the freeze-drying process is not suitable for scale-up due to its high cost. In addition, the patent does not explain the source of the raw material of Hericium erinaceus polysaccharide.
  • Non-Patent Document 1 discloses an article "Optimization of Fermentation Production and Extraction Process of Hericium erinaceus Polysaccharide". The process is the fermentation of Hericium erinaceus hyphae, collecting the hyphae, the hyphae are extracted by hot water, and then Polysaccharides are prepared by precipitation with anhydrous ethanol. In this process, only the mycelium is collected and the extracellular polysaccharides present in the fermentation broth are discarded. At the same time, the purity of the prepared polysaccharides is not stated.
  • Non-Patent Document 2 discloses a paper on "liquid fermentation of Hericium erinaceus and extraction and purification of polysaccharides". This paper prepared three components of pure polysaccharides from fermentation optimization to final purification. 84.31%, 78.47% and 72.16%, the purity of its products still needs to be improved.
  • Patent Literature 1 CN 102643359 A
  • Patent Literature 2 CN103044563A
  • Patent Literature 3 CN 106478829 A
  • Non-Patent Document 1 "The Fermentation Production and Extraction Process Optimization of Polysaccharide from Hericium erinaceum", Inner Mongolia
  • Non-Patent Literature 2 "Study on Liquid Fermentation of Hericium erinaceus and Extraction and Purification of Polysaccharide", Master Thesis of Jiangsu University, 2008.
  • the present invention aims to provide a method for preparing high-purity hericium polysaccharides by fermenting hericium erinaceus. To achieve the above purpose, the present invention adopts the following technical solutions:
  • a method for fermenting Hericium erinaceus to prepare high-purity Hericium erinaceus polysaccharides characterized in that it includes the following steps:
  • Step 1 Inoculate Hericium erinaceus strains into the seed culture medium, shake and cultivate the seeds to obtain the seed liquid;
  • Step 2 Inoculate the seed liquid in Step 1 into the fermentation medium containing glass beads;
  • Step 3 Perform temperature control and pH control during fermentation, and add carbon source at the same time;
  • Step 4 After the fermentation is completed, fungal enzyme is added for enzymolysis to obtain a fermentation broth containing broken cells;
  • Step 5 Treat the fermentation broth to obtain Hericium erinaceus polysaccharide.
  • step 1 the deposit number of Hericium erinaceus used is CCTCC NO: M2018567.
  • the seed culture medium includes the following components: 1-10 mass% carbon source, 1-5 mass% organic nitrogen source, 0.5-1 Inorganic nitrogen source by mass%, inorganic salt of 0.01 to 0.1% by mass.
  • the fermentation medium includes the following components: 1 to 5% by mass of carbon source, 1 to 5% by mass of organic nitrogen source, 0.5 to 1 Inorganic nitrogen source in mass%, inorganic salt in 0.01 to 1% by mass, trace elements in 0.0001 to 0.001% by mass, coenzyme in 0.0001 to 0.001% by mass, dispersant in 0.05 to 1% by mass, glass beads 10-50 particles/100mL
  • the dispersant is any one or more selected from Tween 80, EDTA and Tween 60.
  • step 3 the temperature control is such that the fermentation temperature is set to 24 to 30°C for 2 to 3 days of fermentation, and the fermentation temperature is set to 20 to 24 for 3 to 10 days of fermentation °C.
  • step 3 the pH control is such that the fermentation pH is natural when the fermentation is 2 to 3 days, and the pH is set to 5.0 to 5.5 when the fermentation is 3 to 10 days.
  • step 3 The method according to item 1, characterized in that in step 3, the carbon source is added at 4-6 days of fermentation, and the carbon source concentration of the fermentation broth is maintained at 1 to 1.5% by mass.
  • step 3 fermentation is stopped until 7 to 8 days of sugar supplementation, and the fermentation end point is that there is no residual sugar in the fermentation broth.
  • step 4 the enzymolysis process is performed on a shaker, and the rotation speed of the shaker is 200-300 rpm.
  • step 4 the fungal enzyme is one or more selected from cellulase, snail enzyme, protease, and fungal lysozyme, and the added amount is 0.1 ⁇ 1% by mass, and the enzymolysis time is 60 ⁇ 120min.
  • step 5 the treatment includes heating, decolorization, filtration, ultrafiltration, alcohol precipitation and drying.
  • the decolorization is adsorption decolorization of activated carbon
  • the activated carbon is one or more activated carbons selected from the group consisting of injection type, coconut shell type, wood powder type and coal columnar type.
  • the amount of activated carbon added to the fermentation broth is 0.1 to 1% by mass
  • the adsorption temperature is 30 to 80°C
  • the adsorption time is 30 to 120 min.
  • a fermentation medium characterized in that its composition is 1 to 5 mass% carbon source, 1 to 5 mass% organic nitrogen source, 0.5 to 1 mass% inorganic nitrogen source, 0.01 to 1 mass% Inorganic salt, 0.0001 to 0.001% by mass of trace elements, 0.0001 to 0.001% by mass of coenzyme, 0.05 to 1% by mass of dispersant, 10 to 50 glass beads per 100 mL, the rest is water.
  • a composition comprising Hericium erinaceus and a fermentation broth, the fermentation broth comprising Hericium erinaceus polysaccharide, wherein the concentration of Hericium erinaceus polysaccharide in the fermentation broth is 2 g/L or more, preferably It is 3 g/L or more, more preferably 4 g/L or more, and preferably 5 to 6 g/L.
  • the fermentation medium used to produce the fermentation broth includes the following components: 1 to 5 mass% carbon source, 1 to 5 mass% organic nitrogen source, 0.5 to 1 mass% inorganic nitrogen source, 0.01 to 1 mass% Inorganic salts, 0.0001 to 0.001% by mass of trace elements, 0.0001 to 0.001% by mass of coenzyme, 0.05 to 1% by mass of dispersant, glass beads 10 to 50 particles/100mL, the dispersant is selected from Tween 80, EDTA and Any one or more of Tween 60.
  • the present invention provides a method for fermenting Hericium erinaceus to prepare high-purity Hericium erinaceus polysaccharides, characterized in that it includes the following steps:
  • Step 1 Inoculate Hericium erinaceus strains into the seed culture medium, shake and cultivate the seeds to obtain the seed liquid;
  • Step 2 Inoculate the seed liquid in Step 1 into the fermentation medium containing glass beads;
  • Step 3 Perform temperature control and pH control during fermentation, and add carbon source at the same time;
  • Step 4 After the fermentation is completed, fungal enzyme is added for enzymolysis to obtain a fermentation broth containing broken cells;
  • Step 5 Treat the fermentation broth to obtain Hericium erinaceus polysaccharide.
  • Hericium erinaceus is preferably the Hericium erinaceus with the deposit number CCTCC No: M2018567.
  • the bacterium is Hericium erinaceus mycelium deposited on the inclined surface of the test tube;
  • the above-mentioned glass beads are sterilized glass beads added to the seed medium before inoculation, the diameter of the glass beads is 1-10 mm, and the added amount is 10-50 particles/100 mL;
  • the dispersant may be any one selected from Tween 80, EDTA and Tween 60, preferably Tween 80, and the added amount is 0.05-0.1% by mass;
  • the above-mentioned seed culture medium includes the following components: a carbon source of 1 to 10% by mass, an organic nitrogen source of 1 to 5% by mass, an inorganic nitrogen source of 0.5 to 1% by mass, and an inorganic salt of 0.01 to 0.1% by mass.
  • step 2 specifically, the seed liquid is inoculated with 5 to 10% by volume and shaken with a shaker of 100 to 200 rpm;
  • the fermentation medium includes the following components: 1 to 5 mass% carbon source, 1 to 5 mass% organic nitrogen source, 0.5 to 1 mass% inorganic nitrogen source, 0.01 to 1 mass% inorganic salt, 0.0001 to 0.001 Trace elements by mass%, 0.0001 to 0.001 mass% of coenzyme, 0.05 to 1 mass% of dispersant, glass beads 10 to 50 particles/100mL;
  • the dispersant may be any one or more selected from Tween 80, EDTA and Tween 60, preferably Tween 80, the addition amount is 0.05-0.1% by mass, and the dispersant is added to the fermentation medium to improve the fermentation process Mycelium dispersion efficiency;
  • the diameter of the glass beads is 1-10 mm, and the glass beads are used to disperse the cells during the fermentation process.
  • both the seed medium and the fermentation medium contain a carbon source, a nitrogen source, and inorganic salts.
  • the above carbon source is a common carbon source for microorganisms, and may be one or more of glycerin, sucrose, fructose, glucose, and maltose.
  • the seed medium is preferably sucrose, and the fermentation medium is preferably glucose;
  • the above nitrogen source is a common nitrogen source for microbial culture, which may be one or more of beef extract, peptone, yeast powder and soybean cake powder.
  • the seed medium is preferably beef extract and the fermentation medium is preferably yeast powder;
  • the above-mentioned inorganic salts are commonly used inorganic salts for microbial culture, and can be one or more of sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride and sodium sulfate. Both the inorganic salts of the seed medium and the fermentation medium are Preferred are sodium dihydrogen phosphate and sodium sulfate;
  • the fermentation medium contains trace elements, which may be one or more of ferrous chloride and zinc chloride, preferably zinc chloride;
  • the fermentation medium contains coenzyme, which may be one or more of biotin, niacin, pyridoxal phosphate, betaine, vitamin B 12 and riboflavin, preferably pyridoxal phosphate.
  • coenzyme which may be one or more of biotin, niacin, pyridoxal phosphate, betaine, vitamin B 12 and riboflavin, preferably pyridoxal phosphate.
  • step 3 specifically, the above temperature control is: the fermentation temperature is set to 24 to 30°C for 2 to 3 days of fermentation, and the temperature is set to 20 to 24°C for 3 to 10 days of fermentation;
  • the above pH control is: the fermentation pH is natural when the fermentation is 2 to 3 days, and the pH is set to 5.0 to 5.5 when the fermentation is 3 to 10 days; the pH can be controlled to 5.0 to 5.5 by adding alkali, and the alkali can be NaOH, ammonia and urea. One or more, preferably NaOH solution.
  • the carbon source is added when the fermentation is for 4-6 days, and the carbon source concentration of the fermentation broth is maintained at 1 to 1.5% by mass;
  • Fermentation is stopped until 7-8 days, and the end point of fermentation is that there is no residual sugar in the fermentation broth.
  • step 4 specifically, the enzymolysis process is performed on a shaker, and the rotation speed of the shaker is 200-300 rpm;
  • the above fungal enzyme is one or more selected from the group consisting of cellulase, snail enzyme, protease, and fungal lysozyme, preferably fungal lysozyme, and the amount of fungal enzyme added is 0.1 to 1% by mass, preferably 0.5% by mass %, the enzymolysis time is 60 to 120 min, and the temperature is 25 to 35°C, preferably 30°C.
  • the above treatment may include heating, decolorization, filtration, ultrafiltration, alcohol precipitation and drying;
  • the above heating temperature is 50 ⁇ 100°C, and the heating time is 10 ⁇ 60min to extract polysaccharides and denature the precipitated protein;
  • the above decolorization is adsorption decolorization of activated carbon.
  • the activated carbon is one or more activated carbons selected from the group consisting of injection type, coconut shell type, wood powder type and coal columnar type.
  • the amount of activated carbon added in the fermentation broth is 0.1 to 1% by mass. It is preferably 0.5% by mass, the adsorption temperature is 30 to 80°C, preferably 50°C, and the adsorption time is 30 to 120 min, preferably 40 min;
  • the pore size of the filter membrane used in the above filtration is 0.22 ⁇ m to remove macromolecular impurities and microorganisms.
  • the molecular weight cutoff of the filter membrane used in the above ultrafiltration may be 1000 to 5000 Da, preferably 1000 Da, to remove small molecular impurities.
  • the above alcohol precipitation is precipitation using ethanol, the volume of ethanol used is 2 to 5 times of the filtrate, preferably 4 times, and the number of ethanol precipitations is 2 or more times.
  • the above drying is vacuum drying, the drying temperature is 20-50°C, preferably 25°C, and the drying time is 15-30h, preferably 20h.
  • the content of the polysaccharide of Hericium erinaceus obtained by the method of the present invention is 5-6 g/L, and the purity is 90% or more.
  • the invention also provides a fermentation medium, which is composed of a carbon source of 1 to 5% by mass, an organic nitrogen source of 1 to 5% by mass, an inorganic nitrogen source of 0.5 to 1% by mass, and an inorganic salt of 0.01 to 1% by mass , 0.0001 to 0.001% by mass of trace elements, 0.0001 to 0.001% by mass of coenzyme, 0.05 to 1% by mass of dispersant, glass beads 10 to 50 particles/100mL, the rest is water.
  • a fermentation medium which is composed of a carbon source of 1 to 5% by mass, an organic nitrogen source of 1 to 5% by mass, an inorganic nitrogen source of 0.5 to 1% by mass, and an inorganic salt of 0.01 to 1% by mass , 0.0001 to 0.001% by mass of trace elements, 0.0001 to 0.001% by mass of coenzyme, 0.05 to 1% by mass of dispersant, glass beads 10 to 50 particles/100mL, the rest is water.
  • the dispersant is any one or more selected from Tween 80, EDTA and Tween 60.
  • the preparation method of the invention uses the fermentation metabolism control theory to control the fermentation process, and can increase the fermentation yield of the polysaccharide of Hericium erinaceus.
  • the introduction of fermentation and dispersion technology, combined with high-efficiency fungal enzyme digestion technology can produce broken wall protoplast cells, and the total intracellular and extracellular polysaccharides can be obtained in a higher yield with less impurities and the content is about It is 5 ⁇ 6g/L, the purity of the final preparation of Hericium erinaceus polysaccharides can reach more than 90%, the process is simple and feasible, and it is easy to scale up production, and has great potential for industrial production.
  • the method for preparing high-purity hericium erinaceus polysaccharide by fermenting hericium erodes includes the following steps:
  • Step 1 Inoculate Hericium erinaceus strains into the seed culture medium, shake and cultivate the seeds to obtain the seed liquid;
  • Step 2 Inoculate the seed liquid in Step 1 into the fermentation medium containing glass beads;
  • Step 3 Perform temperature control and pH control during fermentation, and add carbon source at the same time;
  • Step 4 After the fermentation is completed, fungal enzyme is added for enzymolysis to obtain a fermentation broth containing broken cells;
  • Step 5 Treat the fermentation broth to obtain Hericium erinaceus polysaccharide.
  • the preparation method of the hericium erinaceus polysaccharide of the present invention introduces fermentation and dispersion technology, combined with high-efficiency fungal enzyme digestion technology, can prepare broken wall protoplast cells, and the total intracellular and extracellular polysaccharides can be purified under the premise of introducing less impurities. Higher yields are obtained.
  • Step 1 Inoculate the Hericium erinaceus strains into the seed culture medium, shake and cultivate the seeds to obtain a seed solution.
  • Hericium erinaceus is preferably the Hericium erinaceus with the deposit number CCTCC No: M2018567.
  • Mushroom mushroom is a fungus of the basidiomycete polypore order Hydatidaceae Hericium (Rullex F.) Pers. It is a saprophyte and a well-known edible fungus. It looks like a hedgehog or a monkey head, so it is commonly known as hericium erinaceus or monkey mushroom.
  • Hericium erinaceus is preferably Hericium erinaceus CCTCC No: M 2018567, which was deposited on August 23, 2018 at the Chinese Type Culture Collection (CCTCC). This strain is a new one discovered by the applicant. Hericium erinaceus species, and found that it can be used for ergothioneine biosynthesis.
  • the Hericium erinaceus species are Hericium erinaceus mycelium deposited on the slope of the test tube.
  • glass beads and dispersant are added to the seed medium, and the seeds are cultured by shaking; the glass beads are sterilized glass beads added to the seed medium before inoculation, and the glass beads have a diameter of 1-10 mm ,
  • the amount of addition is 10-50 particles/100mL; glass beads are used to disperse the cells; the dispersant can be any one selected from Tween 80, EDTA and Tween 60, preferably Tween 80, the amount of addition is 0.05 ⁇ 0.1% by mass, the dispersion efficiency of mycelium can be improved by adding a dispersant.
  • the shaking culture is performed at 20-25° C. and shaking at 100-200 rpm for 5-10 days. Under this condition, Hericium erinaceus can grow well.
  • the above-mentioned seed culture medium includes the following components: a carbon source of 1 to 10% by mass, an organic nitrogen source of 1 to 5% by mass, an inorganic nitrogen source of 0.5 to 1% by mass, and an inorganic salt of 0.01 to 0.1% by mass.
  • the carbon source may be a carbon source commonly used by microorganisms, and is not particularly limited.
  • it may be glycerin, sucrose, fructose, glucose, maltose, etc., preferably sucrose.
  • the nitrogen source may be a nitrogen source commonly used for microbial culture, and is not particularly limited.
  • it may be beef extract, peptone, yeast powder, bean cake powder, etc., preferably beef extract.
  • the inorganic salt may be an inorganic salt commonly used for microorganism cultivation, and is not particularly limited.
  • it may be sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium sulfate, etc., preferably sodium dihydrogen phosphate and sodium sulfate.
  • Step 2 Inoculate the seed liquid from Step 1 into the fermentation medium containing glass beads.
  • glass beads By adding glass beads to the fermentation medium, fermentation and dispersion techniques can be utilized.
  • the seed liquid is cultured with shaking at a shaker of 100 to 200 rpm at an inoculation amount of 5 to 10% by volume.
  • the fermentation medium includes the following components: 1-5% by mass carbon source, 1-5% by mass organic nitrogen source, 0.5-1% by mass inorganic nitrogen source, 0.01-1% by mass % Inorganic salts, 0.0001 to 0.001% by mass of trace elements, 0.0001 to 0.001% by mass of coenzyme, 0.05 to 1% by mass of dispersant, 10 to 50 glass beads per 100 mL.
  • the dispersant may be any one or more selected from Tween 80, EDTA and Tween 60, preferably Tween 80, the addition amount is 0.05-0.1% by mass, and the dispersant is added to the fermentation medium to improve the fermentation process
  • Tween 80 the addition amount is 0.05-0.1% by mass
  • the carbon source may be a carbon source commonly used by microorganisms, and is not particularly limited.
  • it may be glycerin, sucrose, fructose, glucose, maltose, etc., preferably glucose.
  • the nitrogen source may be a commonly used nitrogen source for microorganism cultivation, and is not particularly limited.
  • it may be beef extract, peptone, yeast powder, bean cake powder, etc., preferably yeast powder.
  • the inorganic salt may be an inorganic salt commonly used for microorganism cultivation, and is not particularly limited.
  • the trace element is also not particularly limited, and for example, it may be ferrous chloride, zinc chloride, etc., preferably zinc chloride.
  • the coenzyme may be, for example, biotin, niacin, pyridoxal phosphate, betaine, vitamin B 12 , riboflavin, etc., preferably pyridoxal phosphate.
  • Step 3 Temperature control and pH control are carried out during the fermentation process, and the carbon source is added at the same time.
  • the use of fermentation metabolism control theory to control the fermentation process can improve the fermentation yield of Hericium erinaceus polysaccharides.
  • the temperature control is: the fermentation temperature is set to 24 to 30°C for 2 to 3 days of fermentation, and the temperature is set to 20 to 24°C for 3 to 10 days of fermentation; the pH is controlled to be fermented for 2 to 3 days
  • the pH is natural, and the pH is set to 5.0 to 5.5 during 3 to 10 days of fermentation; the pH can be controlled to 5.0 to 5.5 by adding alkali.
  • the alkali can be one or more of NaOH, ammonia, and urea, preferably a NaOH solution.
  • the supplementary carbon source is as follows: the supplementary carbon source is started when fermentation takes 4 to 6 days, and the carbon source concentration of the fermentation broth is maintained at 1 to 1.5% by mass; the supplementation of sugar is stopped until 7 to 8 days, and the fermentation end point is that there is no residual sugar in the fermentation broth.
  • the fermentation yield of hericium erinaceus polysaccharide can be improved.
  • Step 4 After the fermentation is completed, fungal enzymes are added for enzymolysis to obtain a fermentation broth containing broken cells.
  • fungal enzymatic hydrolysis technology broken wall protoplast cells can be prepared, and the total intracellular and extracellular polysaccharides can be obtained in a higher yield under the premise of introducing less impurities after purification.
  • the enzymatic hydrolysis process is performed on a shaker, and the rotation speed of the shaker is 200-300 rpm.
  • the fungal enzyme may be one or more selected from the group consisting of cellulase, snail enzyme, protease, and fungal lysolytic enzyme, preferably a fungal lysolytic enzyme, and the amount of fungal enzyme added is 0.1 to 1% by mass, preferably 0.5% by mass %, the enzymolysis time is 60 to 120 min, and the temperature is 25 to 35°C, preferably 30°C, so that the protoplast cells can be more thoroughly broken, and the intracellular polysaccharide can be released outside the cells.
  • Step 5 Treat the fermentation broth to obtain Hericium erinaceus polysaccharide.
  • the above treatment is not particularly limited as long as it can obtain Hericium erinaceus polysaccharide in high yield, and may include heating, decolorization, filtration, ultrafiltration, alcohol precipitation, and drying.
  • the heating temperature is 50 to 100° C.
  • the heating time is 10 to 60 min.
  • the above-mentioned decolorization is not particularly limited as long as it can decolor the fermentation broth, and it is preferably activated carbon adsorption decolorization.
  • the activated carbon may be one or more types of activated carbon selected from the group consisting of injection type, coconut shell type, wood powder type and coal columnar type.
  • the amount of activated carbon added to the fermentation broth is 0.1 to 1% by mass, preferably 0.5% by mass, the adsorption temperature is 30 to 80 °C, preferably 50 °C, the adsorption time is 30 to 120 min, preferably 40 min, decolorization under this condition , Can obtain the hericium polysaccharide with better hue.
  • Filtration is not particularly limited, and the pore size of the filter membrane used is preferably 0.22 ⁇ m, whereby macromolecular impurities and microorganisms can be better removed.
  • Ultrafiltration is not particularly limited, and the used filter membrane may have a molecular weight cut-off of 1,000 to 5,000 Da, preferably 1,000 Da, so that small molecular impurities can be better removed.
  • the alcohol precipitation is not particularly limited, and may be ethanol precipitation.
  • the volume of ethanol used is 2 to 5 times that of the filtrate, preferably 4 times, and the number of ethanol precipitations is 2 or more times, thereby extracting polysaccharides of Hericium erinaceus with higher yield. .
  • the drying is not particularly limited, and it may be vacuum drying.
  • the drying temperature is 20 to 50°C, preferably 25°C, and the drying time is 15 to 30h, preferably 20h, thereby better retaining the activity of hericium erinaceus polysaccharide.
  • the content of the polysaccharide of Hericium erinaceus obtained by the preparation method of the present invention is 5-6 g/L, and the purity is more than 90%.
  • Medium preparation Weigh 20 parts of sucrose, 10 parts of beef extract, 1 part of sodium dihydrogen phosphate, 0.5 part of sodium sulfate, 1 part of Tween 80 according to the mass fraction, add water to 1000 parts, the pH is natural.
  • Seed culture Fill 250mL Erlenmeyer flask with 100mL of the above-mentioned seed medium, and add about 20 glass beads with a diameter of 3mm to the seed medium. After sterilization, inoculate 3 cm 2 of the fungus from the inclined surface of Hericium erinaceus into the seed culture medium, place it in a shaker at 200 rpm, and shake at 25°C for 7 days to synthesize a large amount of mycelium to prepare mycelium-dispersed seeds. liquid.
  • Preparation of culture medium Weigh 35 parts of glucose, 10 parts of yeast powder, 0.5 parts of sodium dihydrogen phosphate, 0.5 parts of sodium sulfate, 1 part of Tween 80, 0.1 parts of zinc chloride, 0.005 parts of pyridoxal phosphate according to the mass fraction , Add water to 1000 parts.
  • Fermentation culture fill 1mL Erlenmeyer flask with 300mL of the above fermentation medium, add about 50 glass beads with a diameter of 3mm to the fermentation medium, after sterilization, inoculate the seed liquid into the fermentation culture according to the inoculation amount of 5% by volume
  • the base put it in a shaker at 200 rpm, shake at 25°C, and set the pH to be natural, pH 4.5 to 5, pH 5 to 5.5, pH 5.5 to 6.0, pH segment control, that is, to control the pH to 5 to 5.5 after 3 days of fermentation
  • the phenol sulfuric acid method was used to determine the polysaccharide content in the fermentation broth.
  • Standard solution Accurately weigh 100 mg of dry and constant weight glucose (analytical purity) into a volumetric flask, add water to bring the volume to 250 mL, shake up and accurately draw 10 mL of the solution, and add water to bring the volume to 100 mL.
  • Preparation of 80% phenol solution Accurately remove 80mL of re-distilled phenol (re-distilled phenol), add distilled water to 100mL, and then obtain 80% phenol solution. Store in a dark bottle protected from light.
  • Sample preparation Weigh 0.2g or 0.2mL of the sample to be tested in a 50mL volumetric flask, add appropriate amount of water, dissolve completely and add water to the mark to the mark as a stock solution, shake well. Measure 5mL of the stock solution before use, put it in a 50mL volumetric flask, add water to the mark, and then dilute 10 times in the same way. Take 2mL in a test tube with a stopper, start from the above "adding 6% phenol solution 1.0mL", and operate in the same way. The polysaccharide concentration of the sample to be measured is obtained from the standard curve, and the polysaccharide content is calculated according to the dilution factor.
  • the optimal fermentation pH is to control the pH at 5.0 to 5.5 after 3 days of fermentation, at which time the polysaccharide content is highest.
  • seed liquid was prepared according to Example 1.
  • the culture medium was prepared according to Example 1.
  • Fermentation culture Fill 1mL Erlenmeyer flask with 300mL of the above fermentation medium, add about 50 glass beads with a diameter of 3mm to the fermentation medium, after sterilization, inoculate the seed liquid into the fermentation medium according to the inoculation amount of 5%
  • shake culture at 200 rpm in a shaker pH control of fermentation after 3 days of fermentation, control the pH value at 5 ⁇ 5.5, set the fermentation temperature of 22 °C, 25 °C, 28 °C, respectively, the temperature is controlled by stage, that is, the temperature is reduced to 22 after 3 days of fermentation
  • the fermentation was performed by shaking culture until the glucose was completely exhausted, and the fermentation was completed.
  • seed liquid was prepared according to Example 1.
  • Preparation of culture medium configure the culture medium according to Example 1.
  • Fermentation culture Fill 1mL Erlenmeyer flask with 300mL of the above fermentation medium, add about 50 glass beads with a diameter of 3mm to the fermentation medium, and after sterilization, inoculate the seed liquid into the fermentation culture according to the inoculation amount of 5%
  • the base put it in a shaker at 200 rpm, shake at 25°C for 3 days, and then start to add NaOH to control the pH value at 5.0 to 5.5.
  • the temperature is reduced to 22°C and continue to ferment to the fifth day.
  • the fermentation is completed.
  • the polysaccharide content in the fermentation broth is measured according to Example 1.
  • the polysaccharide content of sugar supplement is higher than that of polysaccharide without sugar supplement.
  • Example 4 Comparison of enzymolysis conditions of Hericium erinaceus fermentation broth
  • Snail enzyme 2.5% addition, enzymolysis temperature 35°C, enzymolysis time 4h, enzymolysis pH 6.5;
  • Cellulase, snail enzyme compound enzyme cellulase addition amount 1.5%, snail enzyme addition amount 2.5%, enzymolysis temperature 35 °C, enzymolysis time 5h, enzymolysis pH 6.5;
  • Snail enzyme, collapsing enzyme compound enzyme snail enzyme added amount 2.5%, collapsing enzyme added amount 0.005%, enzymolysis temperature 38°C, enzymolysis time 4h, enzymolysis pH 6.5;
  • Fungal wall-solving enzyme 0.5% addition amount, enzymolysis temperature 30°C, enzymolysis time 2h, enzymolysis pH 6.0;
  • Ultrasound Ultrasonic power 1200w, ultrasonic time 30min;
  • Homogenization spindle speed is 3000r/min, homogenization time is 10 ⁇ 30min;
  • Enzymatic hydrolysis Polysaccharide content in enzymatic hydrolysis treatment solution (g/L) Cellulase 4.61 Snail enzyme 4.64 Cellulase, snail enzyme complex enzyme 4.98 Snail enzyme, breakdown enzyme complex enzyme 5.17 Fungal lysozyme 5.97 Ultrasound 4.52 Homogeneous 4.48
  • the best extraction process is the enzymolysis conditions: adding 0.5% fungal wall-solving enzyme, enzymolysis temperature 30°C, enzymolysis time 2h, enzymolysis pH 6.0.
  • Example 5 Preparation of high-purity Hericium erinaceus polysaccharides by fermentation of Hericium erinaceus
  • Fermentation culture Fill 1mL Erlenmeyer flask with 300mL of the above fermentation medium, add about 50 glass beads with a diameter of 3mm to the fermentation medium, and after sterilization, take the example according to 5% of the inoculation amount (1 )
  • Medium seed solution was inoculated into the fermentation medium, placed in a shaker at 200 rpm, shaking culture at 25 °C for 3 days, began to add NaOH to control the pH value at 5.0 ⁇ 5.5, while the temperature was reduced to 22 °C continue to ferment to the fifth day, start to supplement Add glucose to maintain the sugar concentration in the shake flask at 1-1.5%, stop the sugar supplement on the 8th day of fermentation, and ferment until the 10th day after the glucose is completely exhausted.
  • Example 6 50L fermentation scale test of Hericium erinaceus
  • Fermentation culture After the above-mentioned medium is sterilized, the seed liquid in Example (1) is inoculated into the fermentation medium according to the inoculation amount of 5%, and the fermentor is stirred by a flat-leaf turbine agitating blade to facilitate mycelium dispersion ,
  • the fermentation conditions are:
  • Fermentation temperature Sampling at 3 to 4 days at 25°C to detect sugar concentration. When the sugar concentration is about 2% to 2.5%, the temperature drops to 22°C and fermentation continues to the end.
  • Fermentation pH The fermentation pH is natural when the initial temperature is 25°C. After the temperature drops to 22°C, NaOH is added to control the pH at 5.0-5.5 to the end of fermentation.
  • Sugar supplement method take 5-5 days of fermentation to test the sugar concentration. When the sugar concentration drops below 1%, start to add glucose to maintain the sugar concentration in the fermentation broth at 1 to 1.5%. Fermentation stops on the 8th day. On day 10, glucose was completely depleted.
  • Fermentation culture Take 1L Erlenmeyer flask and fill it with 300mL of the above fermentation medium. After sterilization, inoculate the fermentation medium from the seed solution in Example (1) according to 5% inoculation volume and place it on a shaker at 200rpm At 25°C, the fermentation was carried out with shaking until the glucose was completely consumed on the sixth day.
  • Precipitation and drying The filtrate is precipitated with 4 volumes of 95% ethanol, and the supernatant is discarded to obtain a precipitate.
  • the polysaccharide can be obtained after the precipitation is dried in vacuum for 20h, weighing a total of 0.78g, and the purity of the polysaccharide measured by phenol sulfuric acid method is 74.6%.
  • Example 5 Except that the dispersant Tween 80 was not added to the fermentation medium, the operation of Example 5 was followed to obtain the hericium erinaceus polysaccharide, which weighed a total of 1.34 g, and the purity of the polysaccharide measured by the phenol sulfate method was 90.7%.
  • Example 5 Except that no glass beads were added to the fermentation medium, the operations of Example 5 were followed to obtain Hericium erinaceus polysaccharide, which weighed a total of 1.40 g, and the purity of the polysaccharide measured by the phenol sulfate method was 91.0%.
  • Comparative Example 1 In the fermentation medium of Comparative Example 1, glass beads and dispersant Tween 80 were not added, temperature and pH control was not performed, carbon source was not supplemented, and enzymatic hydrolysis was not performed, so high-purity hericium polysaccharides could not be obtained.
  • Comparative Example 2 since the dispersant Tween 80 was not added to the fermentation medium, the purity and yield of hericium erinaceus polysaccharide were relatively high, but still lower than in Example 5.
  • Comparative Example 3 since glass beads were not added to the fermentation medium, the purity and yield of hericium erinaceus polysaccharide were relatively high, but they were still lower than in Example 5. Comparative Example 4 lacks key enzymatic hydrolysis, so the purity and yield of Hericium erinaceus polysaccharide are lower than in Example 5, and the purity of Hericium erinaceus polysaccharide is less than 90%.

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Abstract

Procédé de préparation d'un polysaccharide d'Hericium erinaceus de haute pureté par fermentation d'Hericium erinaceus. Le procédé comprend les étapes suivantes : inoculation d'un milieu de graine liquide avec une souche de Hericium erinaceus et sa la culture dans une phase logarithmique; transfert des graines vers un milieu de fermentation et ajout de billes de verre pour disperser le thallus; simultanément, ajout d'une coenzyme pour favoriser la transformation et la régulation de la valeur de pH, la température et la concentration de source de carbone; sa la mise en place sur un agitateur pour la fermentation sous agitation; soumettre le bouillon fermenté à une enzymolyse pour permettre au mycélium de former des cellules protoplastes à rupture de paroi uniques; et effectuer un chauffage, une décoloration, un filtrage, une ultrafiltration, des précipités d'alcool répétés et des précipités de séchage pour obtenir le polysaccharide d'Hericium erinaceus de haute pureté, la pureté du polysaccharide pouvant atteindre au moins 90 %, et sa teneur étant d'environ 5 À 6 g/L
PCT/CN2019/118947 2018-12-26 2019-11-15 Procédé de préparation de polysaccharide de hericium erinaceus de haute pureté par fermentation de hericium erinaceus, et milieu de fermentation correspondant WO2020134688A1 (fr)

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