CN112042466B - Tremella cultivation method for extracting high-molecular tremella polysaccharides - Google Patents
Tremella cultivation method for extracting high-molecular tremella polysaccharides Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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Abstract
A tremella cultivation method for extracting high-molecular tremella polysaccharide relates to the technical field of tremella cultivation, solves the problem of low molecular weight of tremella polysaccharide obtained by the existing cultivation technology, and comprises the steps of obtaining tremella spores through low-temperature domestication, preparing a novel culture medium, dressing seeds, uniformly mixing lower-layer incense ash hypha and upper-layer tremella hypha in a clean area at a ratio of 1: 2, and then inoculating within 12 hours; optimizing the tremella cultivation process, including tremella strain domestication, novel culture medium preparation, sterilization, inoculation and optimization of culture conditions; after the tremella strain is domesticated at low temperature, the tremella strain is adaptive to the reduction of growth temperature, and cellulase, hemicellulase and polyphenol oxidase secreted by associated fungi cinerea are well controlled in the growth and development process of tremella; in order to optimize the initial growth of the tremella, important raw materials, namely tremella polysaccharide extraction waste residues and bagasse are added into a culture medium, so that the normal growth and development of the tremella at the initial stage are ensured; the tremella polysaccharide extraction waste residue in the culture medium is recycled, and the environmental protection problem of waste residue treatment is solved.
Description
Technical Field
The invention relates to a tremella cultivation method, in particular to a tremella cultivation method for extracting high-molecular tremella polysaccharide.
Background
Tremella, also known as Tremella fuciformis and Tremella fuciformis, has high nutritive value and medicinal efficacy, is a very precious edible fungus and is a name of 'crown in fungus'. The research and cultivation of white fungus in China is in the leading position all over the world, areas such as the Tongjiang Sichuan and the ancient field of Fujian are used as the genuine producing areas of white fungus, and cut-log (green periploca) cultivation and substitute (bottle cultivation and bag cultivation) cultivation are respectively used as representatives; the yield and quality of the tremella fuciformis planted in the Tongjiang section are the first in China, and 90% of the tremella fuciformis planted in China is used as a substitute for tremella fuciformis planted in a ancient field. The research on tremella mainly focuses on the aspects of biological characteristics, production and cultivation, pest control, processing technology, separation and purification, medicinal functions, economic value, genetic breeding and the like.
The tremella polysaccharide is a novel plant-derived high-efficiency humectant extracted and refined from tremella, is also considered as a plant-derived hyaluronic acid substance, has the molecular weight of 100-150 ten thousand, is the only plant-derived humectant with the molecular weight of more than 100 ten thousand in the cosmetic raw material world so far, and is particularly suitable for cosmetics such as essence, facial masks, cream, emulsion, skin lotion and the like.
In the cosmetic market, the demand of tremella polysaccharide for high molecules is gradually increased, and the situation that the molecular weight is high in innovation continuously appears in the market. At present, 160 ten thousand Da molecular weight tremella polysaccharide appears in the market, the demands of many customers are more than 180 ten thousand Da, and the high molecular weight tremella polysaccharide has a large application market space. However, when the existing tremella raw materials are extracted, the molecular weight of the extracted products is different from 90 to 160 ten thousand Da under the same conditions, and the fact that the raw materials play a very critical role in extracting tremella polysaccharides can be seen.
The analysis shows that the associated bacteria, namely cellulase, hemicellulase and polyphenol oxidase secreted by the cinerea virens in the tremella cultivation process have important influence on the molecular weight of tremella polysaccharides, so that the control of the content of secretion of the cinerea virens becomes a key factor for controlling the molecular weight of the tremella polysaccharides.
Disclosure of Invention
In order to solve the problems, the invention provides a tremella cultivation method for extracting high-molecular tremella polysaccharide, and provides a tremella cultivation method for cultivating high-molecular tremella polysaccharide. In order to achieve the purpose, the invention adopts the technical scheme that: a tremella cultivation method for extracting high-molecular tremella polysaccharide comprises the following steps:
1) domesticating Tremella fuciformis strains at low temperature under aseptic condition, culturing at the temperature of 23 ℃, performing spore ejection separation on Tremella fuciformis strains with strong growth after eighty percent maturity, performing hypha culture and strain cultivation on the separated spores, changing the culture temperature to 22 ℃, and repeatedly screening Tremella fuciformis spores capable of thriving at the temperature of 18-20 ℃;
2) preparing a culture medium: 30-50% of wood chips, 10-20% of bran, 10-20% of corncobs, 10-20% of tremella polysaccharide extraction waste residues, 5-10% of bagasse, 1-5% of yeast peptone, 1-2% of gypsum and 1-2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) seed dressing, namely uniformly mixing the lower layer incense ash hypha and the upper layer tremella hypha at a ratio of 1: 2 in a clean area, and inoculating within 12 hours;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
Further, the domesticated strain in the step 1) grows at 18-20 ℃, the temperature is lower than 23 ℃ of the growth environment of the strain before domestication, the temperature of the growth environment of the domesticated strain is lower than 3-5 ℃ of the growth environment of the strain before domestication, the temperature is controlled in the cultivation stage to adjust the growth state of the incense ash hyphae, so that the cellulase, hemicellulase and polyphenol oxidase secreted by the incense ash hyphae are controlled, and the situation that the molecular weight of polysaccharide in tremella is reduced due to excessive secretion of the cellulase, hemicellulase and polyphenol oxidase is avoided.
Further, the domestication method in the step 1) adopts a constant-temperature incubator for cultivation.
Further, the growth cycle, the leaf diameter, the fresh weight and the dry weight of a single tremella and the molecular weight of extracted tremella polysaccharide are controlled by tremella polysaccharide extraction waste residues, bagasse and yeast peptone in the culture medium in the step 2).
Further, the step 3) is carried out by using a high-pressure high-temperature sterilization pot, so that the sterilization time is effectively shortened, and the production efficiency is improved.
Further, the tremella fuciformis strain is prepared in the step 4), seed dressing is carried out, tremella fuciformis is inoculated on a culture medium in a clean area, a small amount of incense ash hyphae are inoculated after the tremella fuciformis is cultured for 10 days, the tremella fuciformis is cultured for 7-10 days at the same temperature, and the tremella fuciformis is uniformly stirred when the incense ash hyphae spread in a whole test tube, so that the original seeds are obtained.
Compared with the prior art, the invention has the following beneficial effects: optimizing the tremella cultivation process, including tremella strain domestication, culture medium preparation, sterilization, inoculation and optimized culture conditions; after the tremella strain is domesticated at low temperature, the tremella strain is adaptive to the reduction of growth temperature, and cellulase, hemicellulase and polyphenol oxidase secreted by associated fungi cinerea are well controlled in the growth and development process of tremella; in order to optimize the initial growth of the tremella, important raw materials, namely tremella polysaccharide extraction waste residues and bagasse are added into a culture medium, so that the normal growth and development of the tremella at the initial stage are ensured; the tremella polysaccharide extraction waste residues in the culture medium are recycled, so that the environmental protection problem of waste residue treatment is solved; the method can improve the molecular weight of mucopolysaccharide in the tremella and solve the environmental protection problem of tremella polysaccharide waste residue treatment, and is low in cost and suitable for industrial production.
Detailed Description
In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention is further described below with reference to specific examples.
Example one
1) Culturing Tremella fuciformis strains under aseptic conditions, culturing at the temperature of 23 ℃ accurately, and not performing any domestication treatment on original Tremella fuciformis strains;
2) preparing a culture medium: 40% of wood chips, 15% of bran, 15% of corncobs, 15% of tremella polysaccharide extraction waste residues, 8% of bagasse, 3% of yeast peptone, 2% of gypsum and 2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) seed dressing, namely uniformly mixing the lower layer incense ash hypha and the upper layer tremella hypha at a ratio of 1: 2 in a clean area, and inoculating within 12 hours;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
Example two
1) Domesticating Tremella fuciformis strains at low temperature under aseptic condition, culturing at the temperature of 23 ℃, performing spore ejection separation on Tremella fuciformis strains with strong growth after eighty percent maturity, performing hypha culture and strain cultivation on the separated spores, changing the culture temperature to 20 ℃, and repeatedly screening Tremella fuciformis spores capable of thriving at the temperature of 15-17 ℃;
2) preparing a culture medium: 40% of wood chips, 15% of bran, 15% of corncobs, 15% of tremella polysaccharide extraction waste residues, 8% of bagasse, 3% of yeast peptone, 2% of gypsum and 2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) seed dressing, namely uniformly mixing the lower layer incense ash hypha and the upper layer tremella hypha at a ratio of 1: 2 in a clean area, and inoculating within 12 hours;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
EXAMPLE III
1) Domesticating Tremella fuciformis strains at low temperature under aseptic condition, culturing at the temperature of 23 ℃, performing spore ejection separation on Tremella fuciformis strains with strong growth after eighty percent maturity, performing hypha culture and strain cultivation on the separated spores, changing the culture temperature to 22 ℃, and repeatedly screening Tremella fuciformis spores capable of thriving at the temperature of 18-20 ℃;
2) preparing a culture medium: 40% of wood chips, 15% of bran, 15% of corncobs, 15% of tremella polysaccharide extraction waste residues, 8% of bagasse, 3% of yeast peptone, 2% of gypsum and 2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) seed dressing, namely uniformly mixing the lower layer incense ash hypha and the upper layer tremella hypha at a ratio of 1: 2 in a clean area, and inoculating within 12 hours;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
Example four
1) Domesticating Tremella fuciformis strains at low temperature under aseptic condition, culturing at the temperature of 23 ℃, performing spore ejection separation on Tremella fuciformis strains with strong growth after eighty percent maturity, performing hypha culture and strain cultivation on the separated spores, changing the culture temperature to 22 ℃, and repeatedly screening Tremella fuciformis spores capable of thriving at the temperature of 18-20 ℃;
2) preparing a culture medium: 40% of wood chips, 15% of bran, 15% of corncobs, 23% of bagasse, 3% of yeast peptone, 2% of gypsum and 2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) seed dressing, namely uniformly mixing the lower layer incense ash hypha and the upper layer tremella hypha at a ratio of 1: 2 in a clean area, and inoculating within 12 hours;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
EXAMPLE five
1) Domesticating Tremella fuciformis strains at low temperature under aseptic condition, culturing at the temperature of 23 ℃, performing spore ejection separation on Tremella fuciformis strains with strong growth after eighty percent maturity, performing hypha culture and strain cultivation on the separated spores, changing the culture temperature to 22 ℃, and repeatedly screening Tremella fuciformis spores capable of thriving at the temperature of 18-20 ℃;
2) preparing a culture medium: 40% of wood chips, 15% of bran, 15% of corncobs, 15% of tremella polysaccharide extraction waste residues, 8% of bagasse, 3% of yeast peptone, 2% of gypsum and 2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) dressing seeds, namely inoculating tremella on a culture medium in a clean area, culturing for 10 days, inoculating a little incense ash hypha, culturing for 7-10 days at the same temperature, and uniformly stirring when the incense ash hypha spreads the whole test tube to obtain stock seeds;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
EXAMPLE six
1) Domesticating Tremella fuciformis strains at low temperature under aseptic condition, culturing at the temperature of 23 ℃, performing spore ejection separation on Tremella fuciformis strains with strong growth after eighty percent maturity, performing hypha culture and strain cultivation on the separated spores, changing the culture temperature to 22 ℃, and repeatedly screening Tremella fuciformis spores capable of thriving at the temperature of 18-20 ℃;
2) preparing a culture medium: 40% of wood chips, 15% of bran, 15% of corncobs, 15% of tremella polysaccharide extraction waste residues, 8% of bagasse, 3% of yeast peptone, 2% of gypsum and 2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) seed dressing, namely inoculating tremella on a culture medium in a clean area, culturing for 4-6 days, inoculating a proper amount of incense ash hypha, culturing for 7-10 days at the same temperature, and uniformly stirring the lower-layer incense ash hypha and the upper-layer tremella hypha in a ratio of 1: 1 to obtain stock seeds when the incense ash hypha spreads over a whole test tube;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
Results and analysis of the experiments
In each embodiment, 50 groups of tremella is picked randomly, the picked tremella is measured, and the average ear emergence rate, the average fresh weight of each tremella, the average dry weight of each tremella, the molecular weight and the growth condition of each tremella are obtained through statistics. Experimental results data and analysis were as follows:
table one: data on the results of the experiments in the examples
| Group of | Average ear growth (%) | Average fresh weight per flower (g) | Average single dry weight (g) | Molecular weight (kDa) | Problem of growth |
| Example one | 98% | 165 | 41 | 1657 | Is free of |
| Example two | 55% | 125 | 35 | 1337 | Low ear emergence rate |
| EXAMPLE III | 98% | 205 | 45 | 1964 | Is free of |
| Example four | 86% | 170 | 41 | 1766 | Low ear emergence rate |
| EXAMPLE five | 90% | 186 | 42 | 1923 | After dressing seedsUnstable Tremella hypha |
| EXAMPLE six | 98% | 152 | 48 | 1821 | Is free of |
Table two: influence of different domestication temperatures on molecular weight of tremella polysaccharide
| Group of | Incubation temperature (. degree.C.) | Acclimation temperature (. degree.C.) | Molecular weight (kDa) |
| Example one | 23 | No acclimation | 1657 |
| Example two | 23 | 15-17℃ | 1337 |
| EXAMPLE III | 23 | 18-20℃ | 1964 |
And (4) analyzing results: the independent variables of the first, second and third examples are different domestication temperatures, under the condition that the culture medium condition and the seed dressing condition are kept the same, the molecular weight of the tremella polysaccharide obtained by the tremella mycelia domesticated at 18-20 ℃ is obviously increased after the tremella mycelia are domesticated at low temperature, the molecular weight of the tremella polysaccharide obtained by the tremella mycelia domesticated at 15-17 ℃ is lower, and the conditions that the tremella is not suitable for growth and the yield is low under the condition that the tremella mycelia domesticated at 15-17 ℃ are obtained by combining the average earning rate and the growth condition of the first table.
Table three: influence of different seed dressing modes on molecular weight of tremella polysaccharide
| Group of | Seed dressing mode | Average ear growth (%) | Molecular weight (kDa) |
| EXAMPLE III | In a clean area, the lower layer incense ash hypha and the upper layer tremella hypha are evenly mixed at a ratio of 1: 2 and inoculated within 12 hours. | 98% | 1964 |
| EXAMPLE five | Inoculating Tremella in clean region on culture medium, culturing for 10 days, and inoculating small amount of incenseGrey hypha at the same temperature And (5) culturing for 7-10 days at the temperature, and obtaining the stock when the incense ash hyphae spread the whole test tube. | 90% | 1923 |
| EXAMPLE six | Inoculating Tremella in clean region on culture medium, culturing for 4-6 days, inoculating appropriate amount of incense ash mycelium, and culturing at the same temperature Culturing for 7-10 days, and allowing the lower layer of incense ash hyphae and the upper layer of tremella hyphae to be 1: 1 when the incense ash hyphae spread over the whole test tube And mixing to obtain the original seed. | 98% | 1821 |
And (4) analyzing results: the independent variables of the third, fifth and sixth examples are different in seed dressing mode, and therefore, the molecular weight of the tremella polysaccharide obtained in the third and fifth examples is obviously higher than that of the tremella polysaccharide obtained in the sixth example, and the third and fifth examples can both produce and prepare tremella polysaccharide with high molecular weight. However, the tremella fuciformis obtained in the fifth embodiment has poor ear emergence rate and is concentrated, which shows that the seed dressing mode has poor stability and has certain influence on tremella fuciformis planting industrialization.
Table four: influence of different culture media on molecular weight of Tremella polysaccharides
| Group of | Media composition | Molecular weight （kDa） |
| EXAMPLE III | 40% of wood chips, 15% of bran, 15% of corncobs, 15% of tremella polysaccharide extraction waste residues, 8% of bagasse, 3% of yeast peptone, 2% of gypsum and 2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and packaging. | 1964 |
| Example four | 40% of wood chips, 15% of bran, 15% of corncobs, 23% of bagasse, 3% of yeast peptone, 2% of gypsum and 2% of dipotassium hydrogen phosphate; mixing, pulverizing, and blending Adding water to make the humidity of the culture medium reach 50-60%, mixing, and bagging. | 1766 |
And (4) analyzing results: the difference between the example four and the example three is that the bagasse replaces the white fungus polysaccharide extraction waste residue, but the molecular weight of the white fungus polysaccharide in the example four is smaller than that in the example three, which indicates that the addition of the white fungus polysaccharide extraction waste residue has a positive effect on increasing the molecular weight of the white fungus polysaccharide.
The technical solutions of the present invention or similar technical solutions designed by those skilled in the art based on the teachings of the technical solutions of the present invention are all within the scope of the present invention.
Claims (5)
1. A tremella cultivation method for extracting high-molecular tremella polysaccharide is characterized by comprising the following steps:
1) domesticating Tremella fuciformis strains at low temperature under aseptic condition, culturing at the temperature of 23 ℃, performing spore ejection separation on Tremella fuciformis strains with strong growth after eighty percent maturity, performing hypha culture and strain cultivation on the separated spores, changing the culture temperature to 22 ℃, and repeatedly screening Tremella fuciformis spores capable of thriving at the temperature of 18-20 ℃;
2) preparing a culture medium: 30-50% of wood chips, 10-20% of bran, 10-20% of corncobs, 10-20% of tremella polysaccharide extraction waste residues, 5-10% of bagasse, 1-5% of yeast peptone, 1-2% of gypsum and 1-2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) seed dressing, namely uniformly mixing the lower layer incense ash hypha and the upper layer tremella hypha at a ratio of 1: 2 in a clean area, and inoculating within 12 hours;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
2. The method for cultivating tremella fuciformis from which polymeric tremella polysaccharide is extracted as claimed in claim 1, wherein the temperature of the domesticated strain in step 1) is 18-20 ℃ lower than 23 ℃ of the growth environment of the pre-domesticated strain, and the temperature of the growth environment of the domesticated strain is 3-5 ℃ lower than the temperature of the growth environment of the pre-domesticated strain.
3. The method for cultivating white fungus by extracting high molecular weight white fungus polysaccharide according to claim 1, wherein the domestication method in the step 1) adopts a constant temperature incubator for cultivation.
4. The method for cultivating tremella fuciformis from which high-molecular tremella polysaccharides are extracted according to claim 1, wherein the tremella fuciformis is sterilized in a high-pressure high-temperature sterilizer in the step 3).
5. A tremella cultivation method for extracting high-molecular tremella polysaccharide is characterized by comprising the following steps:
1) domesticating Tremella fuciformis strains at low temperature under aseptic condition, culturing at the temperature of 23 ℃, performing spore ejection separation on Tremella fuciformis strains with strong growth after eighty percent maturity, performing hypha culture and strain cultivation on the separated spores, changing the culture temperature to 22 ℃, and repeatedly screening Tremella fuciformis spores capable of thriving at the temperature of 18-20 ℃;
2) preparing a culture medium: 30-50% of wood chips, 10-20% of bran, 10-20% of corncobs, 10-20% of tremella polysaccharide extraction waste residues, 5-10% of bagasse, 1-5% of yeast peptone, 1-2% of gypsum and 1-2% of dipotassium hydrogen phosphate; mixing, pulverizing, adding water to make culture medium humidity reach 50-60%, mixing, and bagging;
3) performing high-temperature high-pressure sterilization, wherein the sterilization conditions are as follows: increasing pressure, sterilizing at 121 deg.C for 2-3 hr, and cooling to 22-26 deg.C in inoculation chamber for inoculation;
4) dressing seeds, namely inoculating tremella on a culture medium in a clean area, culturing for 10 days, inoculating a little incense ash hypha, culturing for 7-10 days at the same temperature, and uniformly stirring when the incense ash hypha spreads the whole test tube to obtain stock seeds;
5) inoculating 2-4g of tremella strain into the inoculating hole by using the inoculating spoon, and culturing the fungus bag in a sterile environment for 4-8 days at 22-26 ℃;
6) transferring to a fungus room for cultivation, sealing the fungus bag with small holes of 6 x 6cm to make the bacterial colony contact with air sufficiently, controlling the temperature at 20-24 deg.C and humidity at 80-90%, and culturing for 5-10 days; after the bud base differentiates the ear bud, controlling the temperature at 18-22 ℃ and the humidity at 90-95%, and cultivating for 10-15 days;
7) stopping spraying water when the diameter of the leaves is larger than 18cm, maintaining natural humidity, increasing ventilation, and picking after the fungus bags are obviously contracted, wrinkled and lightened at 18-22 deg.C for 5-10 days;
8) after the tremella is dried under vacuum and low pressure at low temperature, high molecular weight tremella polysaccharide is extracted according to the following steps:
superfine grinding: selecting clean and disease-free dried tremella fuciformis, preparing into coarse powder, and continuously crushing by using a superfine crusher to obtain tremella fuciformis superfine powder;
decoloring and removing impurities: adding 70 wt% -100 wt% of ethanol into the obtained tremella fuciformis superfine powder, leaching, filtering, and removing small molecular impurities such as polyphenol, fat and pigment to obtain decolored and impurity-removed tremella fuciformis superfine powder;
low-temperature leaching: adding deionized water into the obtained decolorized and impurity-removed Tremella superfine powder, leaching at low temperature of less than or equal to 40 deg.C, centrifuging to obtain cold water soluble polysaccharide leaching solution and cold water insoluble Tremella residue;
deproteinization: concentrating the obtained polysaccharide leaching solution, adding a sevage reagent into the concentrated solution, fully stirring, standing for layering, and collecting an upper-layer polysaccharide solution;
purification and refining: adding the obtained polysaccharide solution into quaternary ammonium salt solution until complex precipitation is achieved, dissolving the precipitate in sodium chloride solution, removing insoluble substances, concentrating the supernatant until polysaccharide is nearly saturated, and performing alcohol precipitation, centrifugation, washing and drying to obtain water-soluble high-molecular-weight tremella polysaccharide;
utilization of the residue: drying and pulverizing the obtained cold water insoluble Tremella residues to obtain powdery Tremella residues; the molecular weight of the tremella polysaccharide was then determined by Gel Permeation Chromatography (GPC).
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| CN102037849B (en) * | 2009-10-12 | 2012-08-08 | 江寿根 | Tremella fuciformis strain culture method |
| CN102174615A (en) * | 2011-03-16 | 2011-09-07 | 浙江省农业科学院 | Method for producing fungal polysaccharide with residues after extraction of polysaccharide using needle mushroom |
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