CN106916861B - Method for simultaneously producing auricularia auricula polysaccharide and melanin - Google Patents

Method for simultaneously producing auricularia auricula polysaccharide and melanin Download PDF

Info

Publication number
CN106916861B
CN106916861B CN201611052845.0A CN201611052845A CN106916861B CN 106916861 B CN106916861 B CN 106916861B CN 201611052845 A CN201611052845 A CN 201611052845A CN 106916861 B CN106916861 B CN 106916861B
Authority
CN
China
Prior art keywords
parts
fermentation
polysaccharide
melanin
black fungus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201611052845.0A
Other languages
Chinese (zh)
Other versions
CN106916861A (en
Inventor
杨海龙
周化斌
杨欢东
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Wenzhou University
Original Assignee
Wenzhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wenzhou University filed Critical Wenzhou University
Priority to CN201611052845.0A priority Critical patent/CN106916861B/en
Publication of CN106916861A publication Critical patent/CN106916861A/en
Application granted granted Critical
Publication of CN106916861B publication Critical patent/CN106916861B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Botany (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)

Abstract

The invention provides a method for simultaneously producing auricularia auricula polysaccharide and melanin, which comprises the following specific steps: 1) activating strains; 2) seed culture in a shake flask; 3) fermenting and culturing; 4) separating and purifying black pigment of the agaric; 5) and (5) separating and purifying the agaric polysaccharide. The invention has the beneficial effects that: the Auricularia auricula can simultaneously produce Auricularia melanin and Auricularia polysaccharide, and directly extract melanin and Auricularia polysaccharide from fermentation broth; the fermentation medium can induce the black fungus to secrete melanin and agaric polysaccharide, so that the quantity of the melanin and the agaric polysaccharide secreted by the black fungus is increased, the secretion quantity of other substances irrelevant to the good quality of the product is reduced, the fermentation efficiency of the product is improved, and the preparation time of the product is shortened; the product obtained by separation and purification has high purity, strong activity and high extraction rate, and the structure of the product cannot be damaged.

Description

Method for simultaneously producing auricularia auricula polysaccharide and melanin
Technical Field
The invention belongs to the field of biological fermentation, and particularly relates to a method for simultaneously producing auricularia auricula polysaccharide and melanin.
Background
Auricularia (Auricularia auricula) also called Tremella, Chuan ear, and black fungus, also called Hecai, Wood moth, Tree chicken, wood fungus, Auricularia, Ulmus pumila, and Morus alba, etc., because it grows on the side of tree, it is similar to human ear in shape. Is a traditional fungus used as both medicine and food in China, has rich nutrition, has the efficacies of tonifying qi and strengthening the body, invigorating qi and blood, moistening lung, stopping bleeding, relieving pain, relaxing the bowels and the like, and is an important Chinese medicine resource in China.
Researches show that main active ingredients in the agaric fungus comprise melanin, agaric polysaccharide, amino acid, trace elements and the like. The melanin has biological activities of resisting oxidation, scavenging free radicals, stimulating immunity, inducing synthesis of tumor necrosis factor TNF-a and interleukin IL-6, protecting liver, and preventing ultraviolet radiation. The Auricularia polysaccharide has effects of enhancing immunity, reducing blood lipid, resisting thrombi, resisting aging, reducing blood sugar, resisting blood coagulation, resisting oxidation, resisting radiation and stopping bleeding.
In recent years, the fermentation method for producing the agaric mycelium, polysaccharide and melanin by using agaric as a starting bacterium has been researched and reported, and the prior art such as the Chinese patent with the publication number of CN102766658B discloses a production process for preparing the agaric melanin by the fermentation method. The black fungus melanin prepared by the method has the characteristics of short production period, low cost, high yield, convenience for subsequent processing and the like, and the black fungus melanin product is powdery, has the purity of 70-85% and the yield of 900-1100 mg/L. In the prior art, for example, a Chinese patent with an authorization publication number of CN103641924B discloses a method for extracting auricularia auricula polysaccharide from auricularia auricula, which comprises the steps of selecting dry auricularia auricula as a raw material, soaking, cleaning, mashing, adding cellulase, and controlling enzymolysis conditions to carry out enzymolysis so as to remove cell walls; leaching the agaric residues subjected to enzymolysis with hot water; mixing the Auricularia enzymolysis solution and Auricularia leaching solution, filtering, adding tannic acid solution to remove protein in the filtrate, and precipitating the centrifugally collected polysaccharide solution with ethanol; finally, the polysaccharide product is obtained through vacuum drying. The extraction method has the advantages of reasonable process, simple operation, comprehensive component retention, high polysaccharide yield and purity, and good product safety.
The existing research only relates to the fermentation method for producing the auricularia auricula polysaccharide or the melanin independently, because the auricularia auricula polysaccharide and the melanin are respectively a primary metabolite and a secondary metabolite, strains produced by the fermentation of the auricularia auricula polysaccharide and the melanin are different in fermentation conditions, strains capable of simultaneously producing the auricularia auricula polysaccharide and the melanin at high yield need to be screened, and the production conditions of the auricularia auricula polysaccharide and the melanin are comprehensively considered and optimized. The agaric polysaccharide and the melanin are bioactive substances with good application prospects, and the agaric polysaccharide and the melanin can be obtained simultaneously by producing the agaric polysaccharide and the melanin simultaneously through a liquid fermentation method, but no research report about the simultaneous production of the agaric polysaccharide and the melanin through the liquid fermentation method exists at present.
Disclosure of Invention
The invention aims to provide a method for simultaneously extracting melanin and auricularia auricula polysaccharide, and producing the melanin and the auricularia auricula polysaccharide with high yield and high purity.
Aiming at the problems mentioned in the background technology, the invention adopts the technical scheme that: the method comprises the steps of taking black fungus as an initial strain, activating slant strains, carrying out liquid shake flask culture and fermentation culture to obtain black fungus fermentation liquor, and respectively obtaining the agaric polysaccharide and melanin from the obtained fermentation liquor after separation and purification.
In particular, the present inventionThe invention relates to a process for simultaneously producing auricularia auricula polysaccharide and melanin by a liquid fermentation method, wherein the adopted strain of auricularia auricula (A) and A (B) is auricularia nigraAuriculariaauricula) In 2014, 9 and 29 days, the culture is preserved in the China general microbiological culture Collection center (CGMCC), and the preservation address is as follows: the number of the collection is CGMCC No.9711 in Xilu No. 1 Beijing, Chaoyang.
The specific operation steps are as follows:
1) activating strains: inoculating a black fungus (Auricularia auricula) strain with the preservation number of CGMCC No.9711 to a slant culture medium for activation, controlling the temperature at 25-35 ℃, the pH at 5.5-6.5, and the activation time at 50-100 h, and finishing the activation when hyphae grow over the slant culture medium. The strain activation is to change the black fungus strain which is in a preservation state and has low activity into a strain which has vigorous activity and strong reproductive capacity and metabolic capacity, and to lay a cushion for the next step of strain expansion culture;
2) and (3) seed culture in a shaking flask: inoculating the test tube slant strains into a shake flask filled with a liquid culture medium for amplification culture, wherein the liquid filling coefficient is 0.15-0.25, carrying out rotary culture, the rotary culture speed is 150-200 r/min, the culture temperature is 24-30 ℃, the pH is 5.7-6.5, and the culture time is 72-120 h. The black fungus strains are uniformly distributed in the shake flask culture medium, the propagation speed is accelerated, the strain quantity rapidly rises, and target strains with enough quantity are obtained;
3) fermentation culture: the volume of the fermentation tank is generally 30L-1000L, no matter the volume is 80% of the volume of the tank, the fermentation tank is subjected to air elimination before adding the culture medium, the air elimination temperature is 100-130 ℃, the time is 20-40 min, then the prepared fermentation medium is added when the temperature of the tank is reduced to 50-60 ℃, the fermentation medium is added and then is subjected to actual elimination, the actual elimination temperature is 110-115 ℃, and the time is 20-40 min. The fermentation tank is subjected to high-temperature sterilization in both air sterilization and actual sterilization, so that harmful bacteria are prevented from having adverse effects on the fermentation of the soybean milk by the black fungus strains. After the actual digestion is finished, inoculating the strain obtained after the expanded culture to ferment when the temperature of the culture medium is reduced to 20-30 ℃. The inoculation amount of the strain is 5-10% of the volume of the fermentation medium, the fermentation temperature is 32-45 ℃, the pressure of a fermentation tank is 0.03-0.06 MPa, the stirring speed is 100-150 r/min, and the ventilation rate is 1: 0.5-1: 1v/v/m, and the fermentation time is 96-144 h. The black fungus is aerobic, and oxygen is introduced during fermentation to keep vigorous activity, so that the fermentation efficiency is high, and the capacity of producing melanin and agaric polysaccharide is strong. The prepared fermentation medium is suitable for fermenting the black fungus strains, the fermentation efficiency is high, the secretion of black fungus melanin is large, the fermentation time is shortened, and the contents of black fungus melanin and black fungus polysaccharide are improved;
4) and (3) separating and purifying black fungus melanin: and (3) centrifuging the fermentation culture obtained in the step (3) at the rotating speed of 10000-20000 r/min for 20-40 min, and separating the agaricus bisporus mycelium from the fermentation clear liquid. And (3) concentrating the fermented clear liquid in vacuum at low temperature to 1/3-1/2 of the original volume, adjusting the pH to 1.8-2.4 by using 2-4 mol/L HCl, standing for 0.8-1.2 h, centrifuging, collecting precipitates, washing the precipitates by using distilled water and 0.005-0.015 mol/L HCl respectively, and freeze-drying to obtain the agaric melanin. The melanin obtained by separation has high purity, no damage to planar structure and spatial structure, and strong oxidation resistance. Freeze drying can protect melanin activity and keep it for a long time;
5) and (3) separating and purifying the agaric polysaccharide: and (3) concentrating the supernatant obtained in the step (3) at low temperature in vacuum to 1/4-1/3 of the original volume, adding 25-35% of ethanol, and precipitating protein and amino acid alcohol secreted by the agaric strain to improve the purity of the agaric polysaccharide. Centrifuging to obtain a supernatant, adding 65-80% ethanol containing 0.002-0.004% of ferric chloride and 0.004-0.008% of magnesium chloride into the supernatant, centrifuging to obtain a precipitate, and freeze-drying the precipitate to obtain the auricularia auricula polysaccharide. The addition of trace ferric chloride and magnesium chloride in ethanol can increase the precipitation rate of Auricularia polysaccharide in the supernatant, and improve the extraction rate of Auricularia polysaccharide.
The slant culture medium comprises the following components in parts by weight: 40-70 parts of potato juice, 20-40 parts of agar, 10-15 parts of glucose, 2-5 parts of liquorice extract, 1-4 parts of curcumin, 0.05-0.15 part of sodium acetate and 0.01-0.1 part of dodecyl dimethyl betaine (preferably 0.01 part of dodecyl dimethyl betaine). The slant culture medium can fully provide carbon sources, nitrogen sources, trace elements and growth factors required during the activation of black fungus strains, can accelerate the activation of black fungus, shorten the preparation time of the high-oxidation-resistance soybean milk, reduce the cost and improve the economic benefit.
The shake flask seed culture medium comprises the following components in parts by weight: 10-30 parts of glucose, 20-35 parts of beef extract and 0.75-1.5 parts of K2HPO40.75 to 1.5 parts of KH2PO40.5-1 part of MgSO41 to 3 parts of CaCO31-4 parts of NaCl, 5-10 parts of yeast extract, 2-3 parts of sodium citrate, 1-4 parts of propolis extract, 5-8 parts of soybean protein isolate and 0.01-0.1 part of sorbitan trioleate (preferably, 0.02 part of sorbitan trioleate). The culture medium can not only provide nutrient substances required by the expanded culture of the black fungus strain, but also stimulate the growth of the strain, improve the capability of secreting melanin and black fungus polysaccharide and improve the production efficiency.
The fermentation medium comprises the following components in parts by weight: 30-60 parts of glucose, 35-70 parts of corn starch, 2-5 parts of tyrosine, 0.06-1.5 parts of GP defoaming agent, 1-3 parts of curcumin, 5-10 parts of algal polysaccharide, 1-5 parts of cowberry fruit extract and 0.001-0.01 part of arachidonic acid (preferably, 0.001 part of arachidonic acid). The fermentation culture agent can induce the black fungus to secrete melanin and agaric polysaccharide, so that the quantity of the black fungus to secrete melanin and agaric polysaccharide is increased, the purity of the melanin and agaric polysaccharide is relatively improved by arachidonic acid, and meanwhile, the secretion quantity of other substances irrelevant to the good quality of the product is reduced, the fermentation efficiency of the product is improved, and the preparation time of the product is shortened.
Compared with the prior art, the invention has the advantages that: the method can produce the black fungus melanin and the black fungus polysaccharide simultaneously, and has the advantages of simple operation, short time consumption and high economic benefit; the prepared slant culture medium can fully provide carbon sources, nitrogen sources, trace elements and growth factors required during the activation of black fungus strains, can accelerate the activation of black fungus, shorten the preparation time of the high-oxidation-resistance soybean milk, reduce the cost and improve the economic benefit; the shake flask culture medium can not only provide nutrients required by the expanded culture of the black fungus strains, but also stimulate the growth of the strains, improve the capacity of secreting melanin and black fungus polysaccharide and improve the production efficiency; the fermentation medium can induce the black fungus to secrete melanin and agaric polysaccharide, so that the quantity of the black fungus and the agaric polysaccharide secreted by the black fungus is increased, the secretion of other substances irrelevant to the good quality of the product is reduced, the fermentation efficiency of the product is improved, and the preparation time of the product is shortened. The product obtained by separation and purification has high purity, strong activity and high extraction rate, and the structure of the product cannot be damaged.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The scheme of the invention is further illustrated by the following examples:
example 1:
a method for simultaneously producing auricularia auricula polysaccharide and melanin comprises the following specific steps:
1) activating strains: inoculating a black fungus (Auricularia auricula) strain with the preservation number of CGMCC No.9711 to a slant culture medium for activation, controlling the temperature at 25-35 ℃, the pH at 5.5-6.5, and the activation time at 50-100 h, and finishing the activation when hyphae grow over the slant culture medium. The strain activation is to change the black fungus strain which is in a preservation state and has low activity into a strain which has vigorous activity and strong reproductive capacity and metabolic capacity, and to lay a cushion for the next step of strain expansion culture;
2) and (3) seed culture in a shaking flask: inoculating the test tube slant strains into a shake flask filled with a liquid culture medium for amplification culture, wherein the liquid filling coefficient is 0.15-0.25, carrying out rotary culture, the rotary culture speed is 150-200 r/min, the culture temperature is 24-30 ℃, the pH is 5.7-6.5, and the culture time is 72-120 h. The black fungus strains are uniformly distributed in the shake flask culture medium, the propagation speed is accelerated, the strain quantity rapidly rises, and target strains with enough quantity are obtained;
3) fermentation culture: the volume of the fermentation tank is generally 30L-1000L, no matter the volume is 80% of the volume of the tank, the fermentation tank is subjected to air elimination before adding the culture medium, the air elimination temperature is 100-130 ℃, the time is 20-40 min, then the prepared fermentation medium is added when the temperature of the tank is reduced to 50-60 ℃, the fermentation medium is added and then is subjected to actual elimination, the actual elimination temperature is 110-115 ℃, and the time is 20-40 min. The fermentation tank is subjected to high-temperature sterilization in both air sterilization and actual sterilization, so that harmful bacteria are prevented from having adverse effects on the fermentation of the soybean milk by the black fungus strains. After the actual digestion is finished, inoculating the strain obtained after the expanded culture to ferment when the temperature of the culture medium is reduced to 20-30 ℃. The inoculation amount of the strain is 5-10% of the volume of the fermentation medium, the fermentation temperature is 32-45 ℃, the pressure of a fermentation tank is 0.03-0.06 MPa, the stirring speed is 100-150 r/min, and the ventilation rate is 1: 0.5-1: 1v/v/m, and the fermentation time is 96-144 h. The black fungus is aerobic, and oxygen is introduced during fermentation to keep vigorous activity, so that the fermentation efficiency is high, and the capacity of producing melanin and agaric polysaccharide is strong. The prepared fermentation medium is suitable for fermenting the black fungus strains, the fermentation efficiency is high, the secretion of black fungus melanin is large, the fermentation time is shortened, and the contents of black fungus melanin and black fungus polysaccharide are improved;
4) and (3) separating and purifying black fungus melanin: and (3) centrifuging the fermentation culture obtained in the step (3) at the rotating speed of 10000-20000 r/min for 20-40 min, and separating the agaricus bisporus mycelium from the fermentation clear liquid. And (3) concentrating the fermented clear liquid in vacuum at low temperature to 1/3-1/2 of the original volume, adjusting the pH to 1.8-2.4 by using 2-4 mol/L HCl, standing for 0.8-1.2 h, centrifuging, collecting precipitates, washing the precipitates by using distilled water and 0.005-0.015 mol/L HCl respectively, and freeze-drying to obtain the agaric melanin. The melanin obtained by separation has high purity, no damage to planar structure and spatial structure, and strong oxidation resistance. Freeze drying can protect melanin activity and keep it for a long time;
5) and (3) separating and purifying the agaric polysaccharide: and (3) concentrating the supernatant obtained in the step (3) at low temperature in vacuum to 1/4-1/3 of the original volume, adding 25-35% of ethanol, and precipitating protein and amino acid alcohol secreted by the agaric strain to improve the purity of the agaric polysaccharide. Centrifuging to obtain a supernatant, adding 65-80% ethanol containing 0.002-0.004% of ferric chloride and 0.004-0.008% of magnesium chloride into the supernatant, centrifuging to obtain a precipitate, and freeze-drying the precipitate to obtain the auricularia auricula polysaccharide. The addition of trace ferric chloride and magnesium chloride in ethanol can increase the precipitation rate of Auricularia polysaccharide in the supernatant, and improve the extraction rate of Auricularia polysaccharide.
The slant culture medium comprises the following components in parts by weight: 40-70 parts of potato juice, 20-40 parts of agar, 10-15 parts of glucose, 2-5 parts of a licorice extract, 1-4 parts of curcumin, 0.05-0.15 part of sodium acetate and 0.01-0.1 part of dodecyl dimethyl betaine. The slant culture medium can fully provide carbon sources, nitrogen sources, trace elements and growth factors required during the activation of black fungus strains, can accelerate the activation of black fungus, shorten the preparation time of the high-oxidation-resistance soybean milk, reduce the cost and improve the economic benefit.
The shake flask seed culture medium comprises the following components in parts by weight: 10-30 parts of glucose, 20-35 parts of beef extract and 0.75-1.5 parts of K2HPO40.75 to 1.5 parts of KH2PO40.5-1 part of MgSO41 to 3 parts of CaCO31-4 parts of NaCl, 5-10 parts of yeast extract, 2-3 parts of sodium citrate, 1-4 parts of propolis extract, 5-8 parts of soybean protein isolate and 0.01-0.1 part of sorbitan trioleate. The culture medium can not only provide nutrient substances required by the expanded culture of the black fungus strain, but also stimulate the growth of the strain, improve the capability of secreting melanin and black fungus polysaccharide and improve the production efficiency.
The fermentation medium comprises the following components in parts by weight: 30-60 parts of glucose, 35-70 parts of corn starch, 2-5 parts of tyrosine, 0.06-1.5 parts of GP defoaming agent, 1-3 parts of curcumin, 5-10 parts of algal polysaccharide, 1-5 parts of cowberry fruit extract and 0.001-0.01 part of arachidonic acid. The fermentation culture agent can induce the black fungus to secrete melanin and agaric polysaccharide, so that the quantity of the black fungus to secrete melanin and agaric polysaccharide is increased, wherein the purity of the melanin and the agaric polysaccharide is relatively improved by arachidonic acid, and the secretion quantity of other substances irrelevant to the good quality of the product is reduced, so that the fermentation efficiency of the product is improved, and the preparation time of the product is shortened.
Example 2:
a method for simultaneously producing auricularia auricula polysaccharide and melanin comprises the most preferable steps of:
1) activating strains: inoculating Auricularia auricula (Auricularia auricula) strain with preservation number of CGMCC No.9711 to slant culture medium, activating at 30 deg.C and pH of 6 for 80 hr, and finishing activation when the slant culture medium is full of mycelia. The strain activation is to change the black fungus strain which is in a preservation state and has low activity into a strain which has vigorous activity and strong reproductive capacity and metabolic capacity, and to lay a cushion for the next step of strain expansion culture;
2) and (3) seed culture in a shaking flask: inoculating the test tube slant strain into a shake flask filled with liquid culture medium for amplification culture, wherein the liquid filling coefficient is 0.22, performing rotary culture at a rotation speed of 150r/min, a culture temperature of 28 ℃, a pH value of 6, and a culture time of 100 h. The black fungus strains are uniformly distributed in the shake flask culture medium, the propagation speed is accelerated, the strain quantity rapidly rises, and target strains with enough quantity are obtained;
3) fermentation culture: fermenting with 100L fermentation tank, performing air digestion at 121 deg.C for 30min before adding culture medium, adding fermentation culture medium 80% of the tank volume when the tank temperature is reduced to 50 deg.C, and performing actual digestion at 110 deg.C for 40 min. The fermentation tank is subjected to high-temperature sterilization in both air sterilization and actual sterilization, so that harmful bacteria are prevented from having adverse effects on the fermentation of the soybean milk by the black fungus strains. After the culture medium is cooled to 25 ℃, inoculating the strain obtained after the expanded culture to ferment. The strain inoculation amount is 8 percent of the volume of the fermentation medium, the fermentation temperature is 40 ℃, the pressure of a fermentation tank is 0.004MPa, the stirring speed is 120 r/min, the ventilation rate is 1:1v/v/m, and the fermentation time is 110 h. The black fungus is aerobic, and oxygen is introduced during fermentation to keep vigorous activity, so that the fermentation efficiency is high, and the capacity of producing melanin and agaric polysaccharide is strong. The prepared fermentation medium is suitable for fermenting the black fungus strains, the fermentation efficiency is high, the secretion of black fungus melanin is large, the fermentation time is shortened, and the contents of black fungus melanin and black fungus polysaccharide are improved;
4) and (3) separating and purifying black fungus melanin: and (3) centrifugally separating the fermentation culture obtained in the step (3), wherein the rotating speed is 10000 r/min, centrifuging for 30min, and separating the agaricus mycelium from the fermentation clear liquid. And (3) concentrating the fermentation clear liquid at low temperature in vacuum to 1/3 of the original volume, adjusting the pH to 2 by using 3mol/L HCl, standing for 1h, centrifuging to collect precipitates, washing the precipitates by using distilled water and 0.01mol/L HCl respectively, and freeze-drying to obtain the agaric melanin. The melanin obtained by separation has high purity, no damage to planar structure and spatial structure, and strong oxidation resistance. Freeze drying can protect melanin activity and keep it for a long time;
5) and (3) separating and purifying the agaric polysaccharide: and (3) concentrating the supernatant obtained in the step (3) at low temperature in vacuum to 1/4 of the original volume, adding 30% ethanol, and precipitating the protein and amino acid alcohol secreted by the agaric strain to improve the purity of the agaric polysaccharide. Centrifuging to obtain supernatant, adding 70% ethanol containing 0.002% ferric chloride and 0.006% magnesium chloride into the supernatant, centrifuging to obtain precipitate, and freeze drying the precipitate to obtain Auricularia polysaccharide. The addition of trace ferric chloride and magnesium chloride in ethanol can increase the precipitation rate of Auricularia polysaccharide in the supernatant, and improve the extraction rate of Auricularia polysaccharide.
The slant culture medium comprises the following components in parts by weight: 60 parts of potato juice, 30 parts of agar, 15 parts of glucose, 4 parts of liquorice extract, 2 parts of curcumin, 0.05 part of sodium acetate and 0.01 part of dodecyl dimethyl betaine. The slant culture medium can fully provide carbon sources, nitrogen sources, trace elements and growth factors required during the activation of black fungus strains, can accelerate the activation of black fungus, shorten the preparation time of the high-oxidation-resistance soybean milk, reduce the cost and improve the economic benefit.
The shake flask seed culture medium comprises the following components in parts by weight: 25 parts of glucose, 30 parts of beef extract and 0.75 part of K2HPO40.75 part of KH2PO40.8 part of MgSO42 parts of CaCO33 parts of NaCl, 7 parts of yeast extract, 2 parts of sodium citrate, 3 parts of propolis extract, 6 parts of soy protein isolate and 0.02 part of sorbitan trioleate. The culture medium can not only provide nutrient substances required by the expanded culture of the black fungus strain, but also stimulate the growth of the strain, improve the capability of secreting melanin and black fungus polysaccharide and improve the production efficiency.
The fermentation medium comprises the following components in parts by weight: 50 parts of glucose, 60 parts of corn starch, 3 parts of tyrosine, 1 part of GP defoaming agent, 2 parts of curcumin, 9 parts of algal polysaccharide, 4 parts of cowberry fruit extract and 0.001 part of arachidonic acid. The fermentation culture agent can induce the black fungus to secrete melanin and agaric polysaccharide, so that the quantity of the black fungus and the agaric polysaccharide secreted by the black fungus is increased, the secretion quantity of other substances irrelevant to the good quality of the product is reduced, the fermentation efficiency of the product is improved, and the preparation time of the product is shortened. The experimental data of the invention are in nonlinear change, and the action mechanism is not yet determined, so that the experimental result of the invention is to be verified.
The embodiments described above are intended to illustrate the technical solutions of the present invention in detail, and it should be understood that the above-mentioned embodiments are only specific embodiments of the present invention, and are not intended to limit the present invention, and any modification, supplement or similar substitution made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. A method for simultaneously producing auricularia auricula polysaccharide and melanin is characterized by comprising the following steps:
1) activating strains: activating the black fungus strain in the preservation state on a slant culture medium;
2) and (3) seed culture in a shaking flask: carrying out enlarged culture on the activated black fungus strain in a shake flask seed culture medium, wherein the liquid filling coefficient is 0.15-0.25, and carrying out rotary culture;
3) fermentation culture: performing air digestion on the fermentation tank, adding a fermentation culture medium, performing actual digestion, inoculating the strain obtained in the step 2) after the actual digestion is finished, fermenting, and centrifugally separating the agaricus mycelium and the fermentation clear liquid after the fermentation is finished;
4) and (3) separating and purifying black fungus melanin: concentrating the fermented clear liquid obtained in the step 3), adjusting the pH of the fermented clear liquid, centrifuging, collecting the precipitate, and freeze-drying the obtained precipitate;
5) and (3) separating and purifying the agaric polysaccharide: concentrating the supernatant obtained in the step 4), adding ethanol for primary precipitation, centrifuging to obtain a supernatant, precipitating with ethanol again, and freeze-drying the obtained precipitate;
the fermentation medium comprises the following components in parts by weight: 30-60 parts of glucose, 35-70 parts of corn starch, 2-5 parts of tyrosine, 0.06-1.5 parts of GP defoaming agent, 1-3 parts of curcumin, 5-10 parts of algal polysaccharide, 1-5 parts of cowberry fruit extract and 0.001-0.01 part of arachidonic acid;
the inoculation amount of the strains in the step 3) is 5-10% of the volume of the fermentation medium, the fermentation temperature is 32-45 ℃, the pressure of a fermentation tank is 0.03-0.06 MPa, the stirring speed is 100-150 r/min, and the ventilation amount is 1: 0.5-1: 1v/v/m, and the fermentation time is 96-144 h;
the shake flask seed culture medium comprises the following components in parts by weight: 10-30 parts of glucose, 20-35 parts of beef extract and 0.75-1.5 parts of K2HPO40.75 to 1.5 parts of KH2PO40.5-1 part of MgSO41 to 3 parts of CaCO31 to 4 parts of NaCl, 5 to 10 parts of yeast extract,2-3 parts of sodium citrate, 1-4 parts of propolis extract, 5-8 parts of isolated soy protein and 0.01-0.1 part of sorbitan trioleate;
the concentration of ethanol used for primary precipitation in the step 5) is 25-35%, the concentration of ethanol used for secondary precipitation is 65-80%, and 0.002-0.004% of ferric chloride and 0.004-0.008% of magnesium chloride are dissolved in 65-80% of ethanol;
the black fungus strain is an Auricularia auricula (Auricularia auricula) strain with the preservation number of CGMCC No. 9711.
2. The method for simultaneously producing auricularia auricula polysaccharide and melanin according to claim 1, wherein the method comprises the following steps: the slant culture medium comprises the following components in parts by weight: 40-70 parts of potato juice, 20-40 parts of agar, 10-15 parts of glucose, 2-5 parts of a licorice extract, 1-4 parts of curcumin, 0.05-0.15 part of sodium acetate and 0.01-0.1 part of dodecyl dimethyl betaine.
3. The method for simultaneously producing auricularia auricula polysaccharide and melanin according to claim 1, wherein the method comprises the following steps: the rotating speed of the rotary culture in the step 2) is 150-200 r/min, the culture temperature is 24-30 ℃, the pH value is 5.7-6.5, and the culture time is 72-120 h.
4. The method for simultaneously producing auricularia auricula polysaccharide and melanin according to claim 1, wherein the method comprises the following steps: and 3) performing air elimination at the temperature of 100-130 ℃ for 20-40 min, then adding the fermentation liquor when the temperature of the tank is reduced to 50-60 ℃, and performing actual elimination after adding, wherein the actual elimination temperature is 110-115 ℃ for 20-40 min.
CN201611052845.0A 2016-11-25 2016-11-25 Method for simultaneously producing auricularia auricula polysaccharide and melanin Active CN106916861B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201611052845.0A CN106916861B (en) 2016-11-25 2016-11-25 Method for simultaneously producing auricularia auricula polysaccharide and melanin

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201611052845.0A CN106916861B (en) 2016-11-25 2016-11-25 Method for simultaneously producing auricularia auricula polysaccharide and melanin

Publications (2)

Publication Number Publication Date
CN106916861A CN106916861A (en) 2017-07-04
CN106916861B true CN106916861B (en) 2021-03-16

Family

ID=59453239

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201611052845.0A Active CN106916861B (en) 2016-11-25 2016-11-25 Method for simultaneously producing auricularia auricula polysaccharide and melanin

Country Status (1)

Country Link
CN (1) CN106916861B (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019148419A1 (en) * 2018-02-01 2019-08-08 江南大学 Method for improving yield of edible and medicinal fungus exopolysaccharide by adding tyrosine
CN108384820B (en) * 2018-02-01 2020-11-06 江南大学 Method for increasing yield of edible and medicinal fungus exopolysaccharide by adding tyrosine
CN108410751B (en) * 2018-02-05 2021-01-26 温州大学 Alcaligenes faecalis and application thereof in degradation and decoloration of azo dyes
US11655490B2 (en) 2018-04-09 2023-05-23 Sanko Tekstil Isletmeleri San. Ve Tic. A.S. Process for preparing a dyed biopolymer and products thereof
CN108841200B (en) * 2018-05-22 2019-11-15 福建农林大学 A kind of preparation method of water solubility agaric melanin
CN109258296B (en) * 2018-12-05 2020-11-06 江西天元药业有限公司 Semi-continuous submerged fermentation process for high-yield auricularia auricula polysaccharide

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101186932B (en) * 2007-11-11 2012-01-04 温州大学 Method for synchronously producing hypocrellin and shiraia bambusicola polysaccharides
CN102766658B (en) * 2012-07-30 2014-05-21 南京农业大学 Production process of Jew's ear melanin by fermentation and products of Jew's ear melanin

Also Published As

Publication number Publication date
CN106916861A (en) 2017-07-04

Similar Documents

Publication Publication Date Title
CN106916861B (en) Method for simultaneously producing auricularia auricula polysaccharide and melanin
CN103549428B (en) Active SOD enzyme preparation method in a kind of raising fruit and vegetable edible mushroom ferment
CN108504621B (en) Culture medium for paecilomyces hepiali Cs-4 and preparation method thereof
CN104187947A (en) Hericium erinaceus liquid fermented beverage and preparation method thereof
CN104293680B (en) The preparation method and product of red yeast rice strain, high active ingredient functional Monascus powder
CN107299063B (en) Preparation method of black-skin termitomyces liquid strain
CN106434373A (en) High-density fermentation medium formula of sparassis crispa and pharmaceutical grade glucan preparation method of high-density fermentation medium formula
CN103305396A (en) Method for producing cordyceps vinegar by use of cordyceps taishanensis fermentation liquor
CN105961021B (en) White cordyceps militaris production process
CN105309750A (en) Comprehensive utilization and production technology of high-content antrodia camphorate fungal active polysaccharide, triterpene fine powder, and byproducts thereof
CN116064685B (en) Preparation process and application of eurotium cristatum fermented edible traditional Chinese medicine
CN107410799B (en) Preparation method of multi-strain composite fermented persimmon beverage
CN111493247A (en) Large-fruit hawthorn and hericium erinaceus fermented beverage and preparation method thereof
CN111334440A (en) Rhizopus oryzae CH5 and application thereof in extraction of ganoderma sinensis polysaccharide
CN109504725B (en) Method for preparing high-purity hericium erinaceus polysaccharide by fermenting hericium erinaceus and fermentation culture medium
CN109258296B (en) Semi-continuous submerged fermentation process for high-yield auricularia auricula polysaccharide
CN106360614A (en) Method of preparing boletus edulis essential powder from boletus edulis deeply fermented mycelia
CN106491661B (en) Method for extracting fresh cordyceps sinensis raw pulp
CN111394258B (en) Rhizopus stolonifer FL-3 and application thereof in extracting pachyman
CN114591847A (en) Cordyceps militaris bacterium culture medium composition for increasing cordycepin content, liquid fermentation method and cordycepin preparation method
CN110607332B (en) Culture medium for improving content of functional red yeast rice Monacolin K and fermentation method
CN109076881B (en) Culture method and application of selenium-rich hericium erinaceus mycelium
CN113940421A (en) Method for preparing solid ginseng ferment by variable light, variable temperature, variable pressure and pulsation
CN101134940B (en) Fermentation production method for Chinese caterpillar fungus fungus-bat moth hirsutella sinensis
CN115074256B (en) Hericium erinaceus liquid fermentation medium and method for preparing triterpenes

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant