CN109797107B - Preparation method of fungus mycelium fine powder - Google Patents
Preparation method of fungus mycelium fine powder Download PDFInfo
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Abstract
The invention provides a preparation method of fungal mycelium fine powder, which solves the problems of treatment and storage of mycelium obtained by liquid culture. The method is carried out according to the following steps: 1) obtaining mycelium through liquid amplification culture; 2) carrying out suction filtration treatment on the liquid mycelia to obtain a fungus cake; 3) drying the fungus cake in vacuum drying equipment or microwave equipment; 4) drying the fungus cake, pulverizing, sieving, and storing. The fungus hypha fine powder prepared by the method has bright color and no peculiar smell, and the operation method is simple and easy to implement and has low production cost.
Description
Technical Field
The invention relates to a preparation method of fungal mycelium fine powder.
Background
The mycelium of fungus is made into fine mycelium powder, which is an effective method for increasing the preservation time. The expanding culture of fungi is a basic link, the separation and drying of mycelium and a culture system are the final output link, and the link design is closely related to the culture system.
In the conventional liquid culture method, the fungal sclerotia is mostly inoculated directly into the liquid culture medium, although disinfection and sterilization are carried out by using a 1% sodium hypochlorite solution before inoculation. However, if the concentration of sodium hypochlorite is too low, the sterilization and disinfection are not thorough, and the pollution is easy to happen, and if the concentration is too high, the activity of the fungus fruiting body is reduced, and even the biological activity is lost. Therefore, although the growth rate of fungi is superior to that of the solid culture method in the conventional liquid culture method, the culture of some strains is not ideal for practical industrial use because of insufficient stability.
For the separation of mycelium from fermentation broth, there are several laboratory practices: centrifugation, filtration, suction filtration, and comparative experiments in the laboratory on several methods, yielded the following results: although the centrifugal method can achieve solid-liquid separation, the mycelium still contains a large amount of liquid at the moment, and the vacuum drying time is increased; the filtering method is to separate the hypha fermentation liquor by using gauze, and can also achieve the purpose of solid-liquid separation, but the fermentation liquor is sticky, so that more time is consumed, and the drying time is increased; the suction filtration method is characterized in that the pressure generated by a vacuum pump is utilized to separate mycelium fermentation liquor, the separation speed is high, and the water content of the mycelium is low, so that the method is an ideal method.
Disclosure of Invention
The invention provides a preparation method of fungal hypha fine powder, which is used for meeting the use requirement of raw materials required by further in-depth research on fungi.
The preparation method of the fungus hypha fine powder comprises the following steps:
1) obtaining mycelium by a liquid culture method; the formula of the liquid culture medium for inoculating the hypha mother liquor is as follows: 35-45g/L of corn grits, 6-10g/L of peptone, 10-20g/L of glucose and 2-4g/L of monopotassium phosphate;
2) carrying out suction filtration treatment on the liquid mycelia to obtain a fungus cake;
3) drying the fungus cake in vacuum drying equipment or microwave drying equipment at 45-50 deg.C;
4) oven drying the fungus cake, pulverizing, sieving, and packaging.
Further, the step 1) specifically comprises the following steps:
1.1) inoculating sclerotium on a slant culture medium for activation to grow hypha;
1.2) inoculating activated sclerotium hyphae on a PDA liquid culture medium under the aseptic condition, and culturing for 3-4 days to obtain hypha mother liquor;
1.3) inoculating the hypha mother liquor on a liquid culture medium, and culturing for 7 days by a shaking table to obtain the mycelium.
Further, the optimal formula of the liquid culture medium is as follows: 35g/L of corn grits, 10g/L of peptone, 15g/L of glucose and 3g/L of monopotassium phosphate.
Further, in the step 1.3), the inoculation amount of the hypha mother liquor is 5%; the shaking culture is carried out under the aerobic condition, the temperature is 24 ℃, the rotating speed is 160r/min, and the pH value is 5.5 of the original value of the culture medium.
Further, in step 1.1), the culture temperature for activating the bacterial strain is 24-27 ℃.
Further, in step 2), the suction filtration treatment specifically comprises: and connecting the vacuum pump with a Buchner funnel, and performing suction filtration on the mycelia to obtain the fungus cakes.
Further, in the step 4), the dried fungus cakes are crushed, screened by a 40-mesh sieve, bagged, sealed and stored.
The preparation method is particularly suitable for a new strain Fibulizoctoria sp.
The new strain is collected in termite nest, identified as Fibulizoctonia sp.
The ITS molecular sequence is determined by molecular biology, and the ITS segment sequence with the number of DQ276871 Fibulizoctonia sp in the database is found to be most similar to the corresponding sequence of the novel fungal sclerotia by alignment, wherein the similarity reaches 595/597 (99%). The novel fungus was thus confirmed to be of the genus Fibulizotoxin. It has the following morphological characteristics: the diameter of the bacterial colony is 68-70mm, the bacterial colony is white to light brown, the middle part is compact and felty, the edge is loose, the bacterial colony has obvious concentric ring lines and radial wrinkles, a large amount of light brown sclerotia are generated on the surface, the back of the bacterial colony is white to light brown, and no water-soluble pigment exists. The vegetative hypha has smooth wall, thin wall, locked combination, multiple branches, width of 0.9-4.0 μm, and no spore production in liquid culture state.
The preservation number of the fungus strain is as follows: CCTCC M2018446, depository: china Center for Type Culture Collection (CCTCC), address: eight-path Lojia mountain in Wuchang district, Wuhan city, Hubei province, preservation date: year 2018, month 07, 04.
The invention has the following beneficial effects:
1. the mycelium fine powder can be preserved for a long time. The invention adopts the method that the liquid culture solution is filtered to obtain the fungus cake, and the fungus cake is dried in vacuum at the temperature of 45-50 ℃ to ensure that the hyphae are completely dehydrated, thereby not influencing the preparation of the hyphae into fungus powder and not causing the loss of effective active ingredients in the hyphae. Because in the dehydration state, the hypha in the fine powder state can not go mouldy, the prepared hypha fine powder has bright color and luster, no peculiar smell, is convenient to carry, has low production cost and simple and convenient operation, and when in subsequent pharmacological, toxicological and efficacy experiments, the hypha fine powder can be dissolved in water as a preparation or a solution and directly gavage to act on mice, can be completely practically applied in factory production, has good practicability and can generate good economic benefit and social effect.
2. The mycelium is prepared by liquid culture of corn grits and other basic nutritional formulas, has few chemical formulas, does not generate toxic or side effects on human bodies, and has low price.
3. The liquid culture method of the strain obviously shortens the production period, and the strain has consistent shape and size in a triangular flask, low production cost and simple and convenient inoculation. The hypha yield is high and can reach 19 g/L.
4. The used culture medium raw materials and the suction filtration drying equipment are relatively simple and easy to obtain, and the culture medium has the advantages of low preparation cost, easiness in storage, good color, no peculiar smell, high nutritional ingredients and the like.
5. The invention has universality.
Detailed Description
The preparation method of the fungal hypha fine powder is described in detail below by taking a new strain Fibulizoctonia sp, which is discovered by the applicant as an example:
(1) activation of strain and preparation of hypha mother liquor
Washing the collected fungus sclerotium with 1% (v/v) NaClO solution for several times, soaking for 2-3min, pouring off the waste liquid, washing off the residual liquid on the surface with sterile water, inoculating on the prepared slant culture medium, culturing at 24-27 deg.C for 3-4 days, finishing the culture, selecting the strain with good growth vigor, and storing at 4 deg.C for use. Wherein, the slant culture medium can be prepared by a conventional method.
Inoculating the stored hypha strain to PDA liquid culture medium (optionally agar) with sterile inoculating needle in sterile operation table, and shake culturing at 24-27 deg.C for 3-4 days to obtain hypha mother liquor.
(2) Preparation of liquid Medium (optimum formulation)
Weighing 35g of corn grits, boiling for 20min to make the corn grits be cooked but not rotten, filtering, removing residues, adding 15g of glucose, 10g of peptone and 3g of monopotassium phosphate, dissolving in the filtrate, and finally fixing the volume of the filtrate to 1000 mL. A250 mL Erlenmeyer flask was charged with more than 100mL of liquid medium. Sterilizing at 121 deg.C for 20 min.
(3) Inoculation of
In an aseptic operation table, a sterile test tube is used for sucking 5mL of hypha mother liquor to be inoculated into a triangular flask, and after inoculation, the opening of the triangular flask must be sealed by a plastic packaging film so as to avoid bacterial contamination.
(4) Culturing
Placing the inoculated triangular flask in a constant-temperature shaking incubator for culture. The temperature is controlled at 24-27 ℃, the rotating speed is 160r/min, and the culture is carried out for about one week. Thus, a large amount of hyphae were observed in the flask.
(5) Preparing fungus hypha cake
Taking out the triangular flask filled with the mycelium culture solution from the shaking table incubator, pouring the flask into a Buchner funnel, performing suction filtration on the mycelium culture solution by using a vacuum pump so as to achieve the purpose of separating the culture solution from the mycelium, and separating the mycelium from the culture solution after the suction filtration is completed to form a circular fungus cake.
(6) Preparing mycelium fine powder
Preparing all fermentation liquor with fungal mycelia in the above method into fungus cake, placing in a vacuum drying oven, and drying at 45-50 deg.C. And after the fungus cake is completely dried, crushing, sieving by a 40-mesh sieve, bagging, sealing and storing to obtain the mycelium fine powder.
In the liquid culture step (2) to (4) of the above method for producing fine powder of fungal hyphae, the fungal sclerotia is not directly inoculated into the liquid culture medium, but the activation of the fungal species is carried out before the liquid culture is carried out, and the optimum formula of the liquid culture medium is obtained by screening.
Screening experiments to obtain optimal liquid media are briefly described below:
control group 1:
screening of carbon sources: changing the source of a carbon source in a liquid culture medium, sequentially selecting 2% corn grit, glucose, starch, sucrose, lactose and dextrin as the carbon source, setting the content of peptone as the nitrogen source to be 1%, the content of inorganic salt to be 0.2% and the pH value to be a natural value.
Control group 2:
and (3) screening of nitrogen sources: changing the source of nitrogen source in the liquid culture medium, respectively selecting peptone, yeast extract, ammonium nitrate, soybean powder, urea and bran with concentration of 1% as nitrogen source, 2% glucose as carbon source, inorganic salt with concentration of 0.2%, and pH.
Control group 3:
screening inorganic salts: changing the source of inorganic salt in liquid culture medium, and selecting 0.2% KH2PO4，MgSO4，FeSO3，NaH2PO4，ZnSO4As the inorganic salt source, 1% of optimum nitrogen source, 2% of optimum carbon source and pH are selected.
The invention comprises the following steps:
after the liquid medium components are determined, the optimal screening of the distribution ratio of the medium components is performed through an orthogonal experiment (see the following table).
Orthogonal experiment factor level meter
Growth conditions were as follows:
control group 1:
the hypha growth rate in each group of culture bottles is slow, the volume of hypha balls is small, when the corn grits are used as a carbon source, the growth rate is higher than that of other groups of culture bottles, and the hypha yield is 1.41 g.
Control group 2:
the growth rate of hyphae in each group of culture bottles is slow, the volume of hyphae balls is small and small, when peptone is used as a nitrogen source, the growth rate is high, and the yield of the hyphae is 0.81 g.
Control group 3:
the growth speed of hyphae in each group of culture bottles is slow, the volume of hyphae balls is small, the growth speed of the hyphae is high when potassium dihydrogen phosphate is used as inorganic salt, and the yield of the hyphae is 1.24 g.
The invention comprises the following steps:
the growth speed of hyphae in each group of culture bottles of the orthogonal experiment is high, the time for the hyphae in the strain bottle to reach a saturated state is 7 days at the fastest, the strains are consistent in shape and size in triangular bottles, the size of hypha balls is uniform, the number of the hypha balls is large, and the maximum growth quantity of the hypha balls can reach 1.91 g. The optimal culture formula is determined as follows: 35g of corn grits, 10g of peptone, 15g of glucose and 3g of monopotassium phosphate.
The preparation method of the fungal mycelium fine powder has the advantages of low production cost and simple operation, and the prepared fungal mycelium fine powder has bright color, no peculiar smell and is convenient to carry.
The preparation method is particularly suitable for the new strain Fibulizoctoria sp (preservation number: CCTCC M2018446) discovered by the applicant. Experiments prove that the preparation method is also suitable for other fungi of the same type (particularly Altaibacteriaceae), and can obtain better fine powder products.
The applicant examined the composition of the fungal mycelium: the fungal mycelium contains various bioactive effective components such as polysaccharide, triterpenes, protein, etc., wherein the content of polysaccharide and triterpenes is high, wherein the content of triterpenes is 144.23mg/g, and the content of polysaccharide is 124 mg/g. At present, it is known that polysaccharides have the effects of resisting tumor, improving immunity, reducing blood sugar, resisting aging and the like, and triterpenoids have strong activity in the aspects of hemolysis, resisting cancer, resisting inflammation, resisting bacteria, resisting virus, reducing cholesterol, killing mollusks and the like.
The prepared hypha fine powder can be dissolved in water as a preparation or a solution and directly gavage to act on mice, can be completely practically applied in factory production, has good practicability and can generate good economic benefit and social effect.
Claims (7)
1. The preparation method of the fungus hypha fine powder is characterized in that the classification and the name of the fungus strains areFibulorhizoctoniasp. with the deposit number: CCTCC M2018446; the preparation method comprises the following steps:
1) obtaining mycelium by a liquid culture method; the formula of the liquid culture medium for inoculating the hypha mother liquor is as follows: 35-45g/L of corn grits, 6-10g/L of peptone, 10-20g/L of glucose and 2-4g/L of monopotassium phosphate;
2) carrying out suction filtration treatment on the liquid mycelia to obtain a fungus cake;
3) drying the fungus cake in vacuum drying equipment or microwave drying equipment at 45-50 deg.C;
4) oven drying the fungus cake, pulverizing, sieving, and packaging.
2. The preparation method of the fungal mycelium fine powder according to claim 1, wherein the step 1) specifically comprises the following steps:
1.1) inoculating sclerotium on a slant culture medium for activation to grow hypha;
1.2) inoculating activated sclerotium hyphae on a PDA liquid culture medium under the aseptic condition, and culturing for 3-4 days to obtain hypha mother liquor;
1.3) inoculating the hypha mother liquor on a liquid culture medium, and culturing for 7 days by a shaking table to obtain the mycelium.
3. The method for preparing the fungal mycelium fine powder according to claim 2, wherein in the step 1.3), the composition of the liquid culture medium is as follows: 35g/L of corn grits, 10g/L of peptone, 15g/L of glucose and 3g/L of monopotassium phosphate.
4. The method for preparing the fungal hypha fine powder according to claim 2, wherein in the step 1.3), the inoculation amount of the hypha mother liquor is 5%; the shaking culture is carried out under the aerobic condition, the temperature is 24 ℃, the rotating speed is 160r/min, and the pH value is 5.5 of the original value of the culture medium.
5. The method for preparing fungal hypha fine powder according to claim 2, wherein the culture temperature for activating the strain in step 1.1) is 24-27 ℃.
6. The preparation method of the fungus hypha fine powder according to claim 1, wherein in the step 2), the suction filtration treatment specifically comprises the following steps: and connecting the vacuum pump with a Buchner funnel, and performing suction filtration on the mycelia to obtain the fungus cakes.
7. The preparation method of the fungal mycelium fine powder as claimed in claim 1, wherein in the step 4), the dried fungal cake is crushed, sieved by a 40-mesh sieve, bagged, sealed and stored.
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