CN105779313A - Method for preparing needle mushroom liquid strain by taking mushroom bran extracting solution as raw material - Google Patents
Method for preparing needle mushroom liquid strain by taking mushroom bran extracting solution as raw material Download PDFInfo
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Abstract
The invention discloses a method for preparing a needle mushroom liquid strain by taking mushroom bran extracting solution as a raw material. The method comprises the following steps: preparing mushroom bran extracting solution, preparing a shake-flask culture medium, inoculating, determining pellet form and hypha content, testing enzyme activity, expanding culture by virtue of a fermentation tank, and making a cultivation bottle, wherein a formula of the shake-flask culture medium comprises 36g of first-grade white granulated sugar, 5.8g of 140-mesh soybean meal, 1.24g of analytically pure KH2PO4, 1.28g of analytically pure MgSO4.7H2O and 2000mL of mushroom bran extracting solution. Mushroom bran left after needle mushrooms are harvested is taken as the raw material for preparing the needle mushroom liquid strain, the prepared liquid strain has the advantages of strong hypha activity, dense hyphae, high biological conversion rate and short production period, economic profit of an enterprise is effectively improved, and the problems of resource waste and environmental pollution, caused by arbitrary stacking of the mushroom bran, are alleviated.
Description
Technical field
The preparation method that the present invention relates to a kind of liquid spawn, particularly relates to a kind of method preparing flammulina velutipes liquid strains for raw material with mushroom bran extracting solution.
Background technology
Flammulina velutiper (Fr.) Sing (Flammulinavelutipes), formal name used at school hair handle money bacterium, also known as hair handle little fire mushroom, Flammulina velutipes (Fr.) Sing, Flammulina velutipes, freeze bacterium, Flammulina velutipes, intelligence mushroom etc..Flammulina velutiper (Fr.) Sing is nutritious, and its amino acid content is higher than general mushrooms, and especially the content of lysine is very high, has the function promoting children ' s intelligence development.Research shows, Flammulina velutiper (Fr.) Sing also has good antitumaous effect.Therefore, Flammulina velutiper (Fr.) Sing is not only a kind of ticbit, is again good health product, in recent years extremely people's favor especially, and its yield rises year by year.By the method for strain cultivation Flammulina velutiper (Fr.) Sing, there is advantage with short production cycle, that cell age consistent, sprouting fast, fruiting is neat, energetic, pollution rate is low, therefore the modernization commercial production of Flammulina velutiper (Fr.) Sing is many based on liquid strain cultivation pattern, thus being replaced gradually by traditional solid spawn cultivation mode.But the preparation method of current domestic flammulina velutipes liquid strains is comparatively single, culture medium prescription composition is all bought from market, not only expensive and component is single, and this limits the vigor room for promotion of Flammulina velutiper (Fr.) Sing liquid bacterial strain to a certain extent；Additionally, the unicity of culture medium prescription limits the re-using to the garbage produced in Flammulina velutiper (Fr.) Sing production process, cause the great wasting of resources and ambient pressure.
Summary of the invention
In order to solve the weak point existing for above-mentioned technology, the invention provides a kind of method preparing flammulina velutipes liquid strains for raw material with mushroom bran extracting solution.
In order to solve above technical problem, the technical solution used in the present invention is: a kind of method preparing flammulina velutipes liquid strains for raw material with mushroom bran extracting solution, comprises the following steps:
A, preparation mushroom bran extracting solution: being enriched with remaining mushroom bran after Flammulina velutiper (Fr.) Sing is gathered, prepare according to material-water ratio 1:25, standing at room temperature 24h, then with eight layers of gauze or filtration devices, filtrate is mushroom bran extracting solution；
B, preparation Shake flask medium: the formula of Shake flask medium is:
One-level white sugar 36g, 140 order bean cake powder 5.8g,
KH2PO4Analytical pure 1.24g, MgSO4·7H2O analytical pure 1.28g,
Mushroom bran extracting solution 2000mL；
After adjusting pH to 6.0～6.9, by above-mentioned culture medium subpackage to the conical flask containing rotor；With the tight conical flask bottleneck of tampon plug, and pack tightly bottleneck with aluminium-foil paper, autoclaving 30～60min at 121 DEG C；After sterilizing terminates, it is cooled to room temperature；
C, inoculation: in an aseptic environment, hook up Flammulina velutiper (Fr.) Sing mother with inoculating loop and plant truffle 8～10 pieces, be transferred in the Shake flask medium after cooling, shake-flask culture 7 days, shaking speed 150r/min under 20～25 DEG C of conditions；
D, pellet form and hyphae content measure: after shake-flask culture terminates, look first at pellet form and liquid clarity in culture bottle, then test the pH of bacterium solution with pH meter, it is judged that the upgrowth situation of strain；Randomly draw three shaking flask strains again, it is placed on magnetic stirring apparatus to be blended by strain, 10g bacterium solution is drawn in the centrifuge tube of known quality with syringe, it is centrifuged after trim and abandons supernatant, by weighing the quality of precipitation and centrifuge tube, calculate the average content of mycelia, and the strain hyphae content prepared with conventional method compares；
E, enzyme activity are tested: randomly draw nine shaking flask strains, be divided into three groups；First prepare crude enzyme liquid and draw glucose standard curve；Stirring three groups of shaking flask strains to crush on magnetic stirring apparatus and take the bacterium solution after 5mL crushes respectively in 10mL centrifuge tube, 3500r/min is centrifuged 10min, takes supernatant as crude enzyme liquid；The three groups of crude enzyme liquids obtained carry out the work of carboxymethylcelluloenzyme enzyme enzyme respectively, hemicellulase enzyme is lived, the test of laccase activity, and the crude enzyme liquid enzyme prepared with conventional method is lived and contrasted；
F, fermentation tank spread cultivation: carry out the preparation of fermentation medium according to formula as below:
One-level white sugar 12595g, 140 order bean cake powder 2025g,
KH2PO4Analytical pure 429g, MgSO4·7H2O analytical pure 443g,
Mushroom bran extracting solution 700L；
Autoclaving after tune pH to 6.0～6.9；Fermentation medium in fermentation tank is cooled to room temperature, in inoculation step C shake-flask culture terminate after bacterium solution；20～25 DEG C of condition bottom fermentations are cultivated 7 days, ventilation 0.1MP；
G, making culture bottle: weigh corn cob, Testa oryzae, wheat bran, cotton seed hulls, soybean cover, distiller's grains of beer, megasse, by above-mentioned raw material stirring mixing 25min, then limit adds mushroom bran extracting solution limit and continues stirring 40～50min, be modulated into water content be 65% ± 1, pH be the composite material of 6.3～6.9；Planting in culture bottle into above-mentioned composite material, making every bottle of indicated weight is 1020 ± 20g；Adopt five hole perforating methods, make charge level smooth distance culture bottle bottleneck 2.0cm, 121 DEG C of autoclaving 80～100min, be cooled to room temperature, last inoculation step F spread cultivation after fermentation liquid strain.
The method for drafting of glucose standard curve is as follows:
I, accurately weigh anhydrous glucose 250.0mg, be dissolved in distilled water and be settled to 250mL, obtain the Glucose standards solution of 1.0mg/mL；Glucose standards solution is diluted to 0mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL respectively；
II, measure the glucose solution of the above-mentioned variable concentrations absorbance at 540nm wavelength place by DNS method, draw glucose standard curve, draw regression equation.
The method of testing that carboxymethylcelluloenzyme enzyme enzyme is lived is: add the sodium carboxymethyl cellulose 1.5mL of 0.5% in 20ml tool plug test tube, add the crude enzyme liquid 0.5mL of dilution 10 times, water-bath 30min in 50 DEG C of thermostat water baths, DNS reagent 3mL is added immediately after taking-up, it is then placed in 5min in 100 DEG C of hot baths, taking-up is settled to 20mL with distilled water after being cooled to room temperature, mixing；Measure OD value with spectrophotometer at 540nm place, to boil 15min inactivator liquid for comparison, calculate enzyme activity.
The method of testing that hemicellulase enzyme is lived is: add the wheat stalk hemicellulose solution 1.5mL of 0.5% in 20mL tool plug test tube；Wheat stalk hemicellulose solution pH is 4.8, and concentration is the acetate buffer preparation of 0.1mol/L；Add crude enzyme liquid 0.5mL, the water-bath 30min in 50 DEG C of thermostat water baths of dilution 10 times, after taking-up, add DNS reagent 3mL immediately, be then placed in 5min in 100 DEG C of hot baths, take out and be settled to 20mL with distilled water after being cooled to room temperature, mixing；Measure OD value with spectrophotometer at 540nm place, to boil 15min inactivator liquid for comparison, calculate enzyme activity.
The method of testing of laccase activity is: add the guaiacol of 0.5mL80mmol/L in 20mL tool plug test tube, add the acetate buffer solution 3mL that 0.1mol/L, pH are 4.8, add crude enzyme liquid 0.1mL, 30min is reacted at 28 DEG C, afterwards with spectrophotometer in 490nm wavelength place mensuration OD value, to boil the inactivator liquid of 15min as comparison, calculate enzyme and live.
The present invention with Flammulina velutiper (Fr.) Sing gather after remaining mushroom bran be prepared for flammulina velutipes liquid strains for raw material, it is energetic, dense that the liquid spawn prepared by the present invention has mycelia, the advantage that biological transformation ratio is high, with short production cycle, not only it is effectively increased the economic profit of enterprise, and alleviates mushroom bran and wantonly pile up the wasting of resources and problem of environmental pollution that cause.
Figure of description
Fig. 1 is the overall flow figure of the present invention.
Fig. 2 is glucose standard curve figure.
Detailed description of the invention
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
As it is shown in figure 1, the present invention comprises the following steps:
A, preparation mushroom bran extracting solution: being enriched with remaining mushroom bran after Flammulina velutiper (Fr.) Sing is gathered, prepare according to material-water ratio 1:25, prepared rear standing at room temperature 24h, then with eight layers of gauze or filtration devices, the filtrate after filtration is mushroom bran extracting solution；
B, preparation Shake flask medium: the formula of Shake flask medium is:
One-level white sugar 36g, 140 order bean cake powder 5.8g,
KH2PO4Analytical pure 1.24g, MgSO4·7H2O analytical pure 1.28g,
Mushroom bran extracting solution 2000mL；
After adjusting pH to 6.0～6.9, by above-mentioned culture medium subpackage to the conical flask containing rotor；With the tight conical flask bottleneck of tampon plug, and pack tightly bottleneck with aluminium-foil paper, autoclaving 30～60min at 121 DEG C；After sterilizing terminates, it is cooled to room temperature；
C, inoculation: in an aseptic environment, hook up Flammulina velutiper (Fr.) Sing mother with inoculating loop and plant truffle 8～10 pieces, be transferred in the Shake flask medium after cooling, shake-flask culture 7 days, shaking speed 150r/min under 20～25 DEG C of conditions；
D, pellet form and hyphae content measure: after shake-flask culture terminates, look first at pellet form and liquid clarity in culture bottle, then test the pH of bacterium solution with pH meter, it is judged that the upgrowth situation of strain；Randomly draw three shaking flask strains again, it is placed on magnetic stirring apparatus to be blended by strain, 10g bacterium solution is drawn in the centrifuge tube of known quality with syringe, it is centrifuged after trim and abandons supernatant, by weighing the quality of precipitation and centrifuge tube, calculate the average content of mycelia, and the strain hyphae content prepared with conventional method compares；
E, enzyme activity are tested: randomly draw nine shaking flask strains, be divided into three groups；First prepare crude enzyme liquid and draw glucose standard curve；The three groups of crude enzyme liquids obtained carry out the work of carboxymethylcelluloenzyme enzyme enzyme respectively, hemicellulase enzyme is lived, the test of laccase activity, and the crude enzyme liquid enzyme prepared with conventional method is lived and contrasted；
F, fermentation tank spread cultivation: carry out the preparation of fermentation medium according to formula as below:
One-level white sugar 12595g, 140 order bean cake powder 2025g,
KH2PO4Analytical pure 429g, MgSO4·7H2O analytical pure 443g,
Mushroom bran extracting solution 700L；
Autoclaving after tune pH to 6.0～6.9；Fermentation medium in fermentation tank is cooled to room temperature, in inoculation step C shake-flask culture terminate after bacterium solution；20～25 DEG C of condition bottom fermentations are cultivated 7 days, ventilation 0.1MP；
G, making culture bottle: weigh corn cob, Testa oryzae, wheat bran, cotton seed hulls, soybean cover, distiller's grains of beer, megasse, by above-mentioned raw material stirring mixing 25min, then limit adds mushroom bran extracting solution limit and continues stirring 40～50min, be modulated into water content be 65% ± 1, pH be the composite material of 6.3～6.9；Planting in culture bottle into above-mentioned composite material, making every bottle of indicated weight is 1020 ± 20g；Adopt five hole perforating methods, make charge level smooth distance culture bottle bottleneck 2.0cm, 121 DEG C of autoclaving 80～100min, be cooled to room temperature, last inoculation step F spread cultivation after fermentation liquid strain.
The concrete invention process of the present invention is as follows:
(1) screening of extracting solution composition
A, in order to the garbage that will produce in Flammulina velutiper (Fr.) Sing production process re-uses, selected three kinds of extracting solution compositions screen, namely after sterilizing after compost extracting solution, the full bottle of mycelia mycelia culture material complex extracting solution, Flammulina velutiper (Fr.) Sing gather after residue mushroom bran extracting solution；Mycelia culture material complex, mushroom bran after compost, the full bottle of mycelia after sterilizing are prepared by material-water ratio 1:15 respectively, standing at room temperature 24h, eight layers of gauze or filtration devices, the filtrate after filtration i.e. respectively above-mentioned three kinds of extracting solution, select distilled water to be contrast experiment simultaneously；
B, preparation contain the fluid medium of above-mentioned three kinds of extracting solution compositions respectively and contrast culture medium, and culture medium prescription is:
One-level white sugar 40g, 140 order bean cake powder 6.6g,
KH2PO4Analytical pure 1.32g, MgSO4·7H2O analytical pure 1.32g,
Extracting solution or water 2000mL；
Adjusting pH to 6.0～6.9, at 121 DEG C, autoclaving 30～60min, is cooled to room temperature；
C, Flammulina velutiper (Fr.) Sing mother is planted truffle it is transferred in above-mentioned four kinds of fluid mediums respectively, 22 DEG C of shake-flask culture 7～9 days；
After d, shake-flask culture terminate, observe the pellet form under four kinds of fluid mediums, liquid clarity；After the static 10min of shaking flask strain, if supernatant clear, surface without or have that a small amount of foam, fungus ball even particle size be consistent, fungus ball surface mycelia is divergent shape, then growth is good, it is possible to normally use；If supernatant is muddy, there is a large amount of foam on surface, fungus ball smooth surface, explanation shaking flask strain have polluted or aging, unusable；Hypha form fungus ball is little, bacterium thorn is plentiful, fungus ball is in the same size as well；PH, the pH of three kinds of extracting solution is measured between 6～7 better with pH meter；Centrifugal supernatant of abandoning, calculating hyphae content after smashing shaking flask strain；By pellet form, clarity, hyphae content, it is determined that going out the extracting solution being suitable for doing liquid seeds, comparative result is as shown in table 1；
Table 1, the morphological characteristic of four kind of liquid spawn, hyphae content
As shown in Table 1, three kinds of extracting solution compare, with common distilled water culture medium, the growth being suitable for flammulina velutipes liquid strains, consider from hypha form and hyphae content, the liquid spawn that mushroom bran extracting solution makes, fungus ball is in the same size, and hyphae content is moderate, therefore selecting mushroom bran extracting solution is that primary raw material prepares flammulina velutipes liquid strains.
(2) orthogonal test
The selected liquid culture based formulas being raw material with mushroom bran extracting solution carries out the orthogonal test of five factor four levels, and five factors are respectively as follows: white sugar, MgSO4·7H2O、KH2PO4, bean cake powder, material-water ratio；It is 40g, 36g, 32g, 28g that the addition of each factor is respectively set to white sugar；MgSO4·7H2O is 1.32g, 1.28g, 1.24g, 1.20g；KH2PO4For 1.32g, 1.28g, 1.24g, 1.20g；Bean cake powder is 6.6g, 6.2g, 5.8g, 5.4g；Material-water ratio is 1:15,1:20,1:25,1:30；According to the orthogonal test formula of five factor four levels, prepare liquid spawn respectively；Orthogonal test table is as shown in table 2；
Table 2, orthogonal test table
After shake-flask culture terminates, the 16 kinds of liquid spawns obtained are carried out the mensuration of strain form, hyphae content；Determine best orthogonal test formula；Orthogonal experiments is as shown in table 3；
Table 3, orthogonal experiments
By orthogonal test table it can be seen that white sugar has the greatest impact, next to that bean cake powder, followed by KH2PO4、MgSO4·7H2O and material-water ratio, the optimum combination drawn in orthogonal test is: A2B2C3D3E3, it may be assumed that
One-level white sugar 36g, 140 order bean cake powder 5.8g,
KH2PO4Analytical pure 1.24g, MgSO4·7H2O analytical pure 1.28g,
Material-water ratio is 1:25.
(3) enzyme is lived and is tested
The best medium formula that orthogonal test is drawn carries out the work of carboxymethylcelluloenzyme enzyme enzyme, hemicellulase enzyme is lived and the mensuration of laccase activity；First prepare crude enzyme liquid and draw glucose standard curve；Being stirred by three groups of shaking flask strains broken on magnetic stirring apparatus, take the bacterium solution after 5mL crushes in 10mL centrifuge tube, 3500r/min is centrifuged 10min, takes supernatant as crude enzyme liquid, 4 DEG C of low-temperature preservations；The method for drafting of glucose standard curve is as follows:
Accurately weigh anhydrous glucose 250.0mg, be dissolved in distilled water and be settled to 250mL, obtain the Glucose standards solution of 1.0mg/mL；Glucose standards solution is diluted to 0mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL respectively；Measure the glucose solution of the above-mentioned variable concentrations absorbance at 540nm wavelength place by DNS method, draw glucose standard curve, as shown in Figure 2；Draw regression equation: y=1.766x-0.0002, R2=0.997；
The three groups of crude enzyme liquids obtained carry out the work of carboxymethylcelluloenzyme enzyme enzyme respectively, hemicellulase enzyme is lived, the test of laccase activity, and the crude enzyme liquid enzyme prepared with cellar culture based formulas is lived and contrasted；Definition OD value change 0.001 per minute is an enzyme activity unit.Cellar culture based formulas is:
One-level white sugar 40g, 140 order bean cake powder 6.6g,
KH2PO4Analytical pure 1.32g, MgSO4·7H2O analytical pure 1.32g,
Distilled water 2000mL；
The method of testing that carboxymethylcelluloenzyme enzyme enzyme is lived is: add the sodium carboxymethyl cellulose 1.5mL of 0.5% in 20ml tool plug test tube, add the crude enzyme liquid 0.5mL of dilution 10 times, water-bath 30min in 50 DEG C of thermostat water baths, DNS reagent 3mL is added immediately after taking-up, it is then placed in 5min in 100 DEG C of hot baths, taking-up is settled to 20mL with distilled water after being cooled to room temperature, mixing；Measure OD value with spectrophotometer at 540nm place, to boil 15min inactivator liquid for comparison, calculate enzyme activity.
The method of testing that hemicellulase enzyme is lived is: add the wheat stalk hemicellulose solution 1.5mL of 0.5% in 20mL tool plug test tube；Wheat stalk hemicellulose solution pH is 4.8, and concentration is the acetate buffer preparation of 0.1mol/L；Add crude enzyme liquid 0.5mL, the water-bath 30min in 50 DEG C of thermostat water baths of dilution 10 times, after taking-up, add DNS reagent 3mL immediately, be then placed in 5min in 100 DEG C of hot baths, take out and be settled to 20mL with distilled water after being cooled to room temperature, mixing；Measure OD value with spectrophotometer at 540nm place, to boil 15min inactivator liquid for comparison, calculate enzyme activity.
The method of testing of laccase activity is: add the guaiacol of 0.5mL80mmol/L in 20mL tool plug test tube, add the acetate buffer solution 3mL that 0.1mol/L, pH are 4.8, add crude enzyme liquid 0.1mL, 30min is reacted at 28 DEG C, afterwards with spectrophotometer in 490nm wavelength place mensuration OD value, to boil the inactivator liquid of 15min as comparison, calculate enzyme and live.The result that each enzyme is lived is as shown in table 4.
Table 4, enzyme test result alive
As shown in Table 4, with the flammulina velutipes liquid strains that mushroom bran extracting solution is prepared for primary raw material, compared with the flammulina velutipes liquid strains that conventional formulation prepares, carboxymethylcelluloenzyme enzyme enzyme is lived, hemicellulase enzyme is lived and laccase activity vigor all effectively improves.
(4) spread cultivation and make culture bottle
Carry out the preparation of fermentation medium with reference to orthogonal test optimal result, cultivate 7 days in inoculation after fermentation tank；Preparation culture bottle material, sterilizing, be cooled to room temperature after inoculate the strain after above-mentioned fermentation spreads cultivation.Fruiting experiment result such as table 5 represents.
Table 5, mycelium growth vigor and fruiting situation
+++ represent mycelium length dense.
As shown in Table 5, the culture bottle fruiting yield that prepared by mushroom bran extracting solution strain is higher, the production cycle is shorter.
Producing the factory of 200,000 bottles of culture bottles for every day, the invention have the benefit that every bottle of net weight after needle mushroom fruiting is increased to 454g by 447g, yield of flammulina velutipes increases and cost savings every days is nearly 10,000 yuan of the direct profit that company brings；Additionally, use shake-flask culture cycle of preparing of the present invention and fermentation cycle that spreads cultivation to foreshorten to 7d by 9d；After inoculation culture bottle, full bottle was foreshortened to 20 days by original 22 days, and the fruiting time was foreshortened to 45 days by original 48 days.
Due to nutrient substance such as sugary in mushroom bran extracting solution, aminoacid, nitrogen, the present invention is made full use of the addition that can reduce other nutrition, thus reducing production cost, meets the strategy of sustainable development of agricultural economy；Additionally, by the effect of microorganism, mushroom bran is likely to produce some secondary metabolites improving fungus ball vigor, makes shaking flask cycle, fermentation period all substantially shorten；Use mushroom bran extracting solution make culture bottle, it is possible to make Flammulina velutiper (Fr.) Sing as early as possible be converted to reproductive stage from vegetative growth phase, thus shortening the fruiting phase, improve yield.
Above-mentioned embodiment is not limitation of the present invention, and the present invention is also not limited to the example above, the change made within the scope of technical scheme of those skilled in the art, remodeling, interpolation or replacement, also belongs to protection scope of the present invention.
Claims (5)
1. the method preparing flammulina velutipes liquid strains for raw material with mushroom bran extracting solution, it is characterised in that: said method comprising the steps of:
A, preparation mushroom bran extracting solution: being enriched with remaining mushroom bran after Flammulina velutiper (Fr.) Sing is gathered, prepare according to material-water ratio 1:25, prepared rear standing at room temperature 24h, then with eight layers of gauze or filtration devices, the filtrate after filtration is mushroom bran extracting solution；
B, preparation Shake flask medium: the formula of Shake flask medium is:
One-level white sugar 36g, 140 order bean cake powder 5.8g,
KH2PO4Analytical pure 1.24g, MgSO4·7H2O analytical pure 1.28g,
Mushroom bran extracting solution 2000mL；
After adjusting pH to 6.0～6.9, by above-mentioned culture medium subpackage to the conical flask containing rotor；With the tight conical flask bottleneck of tampon plug, and pack tightly bottleneck with aluminium-foil paper, autoclaving 30～60min at 121 DEG C；After sterilizing terminates, it is cooled to room temperature；
C, inoculation: in an aseptic environment, hook up Flammulina velutiper (Fr.) Sing mother with inoculating loop and plant truffle 8～10 pieces, be transferred in the Shake flask medium after cooling, shake-flask culture 7 days, shaking speed 150r/min under 20～25 DEG C of conditions；
D, pellet form and hyphae content measure: after shake-flask culture terminates, look first at pellet form and liquid clarity in culture bottle, then test the pH of bacterium solution with pH meter, it is judged that the upgrowth situation of strain；Randomly draw three shaking flask strains again, it is placed on magnetic stirring apparatus to be blended by strain, 10g bacterium solution is drawn in the centrifuge tube of known quality with syringe, it is centrifuged after trim and abandons supernatant, by weighing the quality of precipitation and centrifuge tube, calculate the average content of mycelia, and the strain hyphae content prepared with conventional method compares；
E, enzyme activity are tested: randomly draw nine shaking flask strains, be divided into three groups；First prepare crude enzyme liquid and draw glucose standard curve；Being stirred by three groups of shaking flask strains broken on magnetic stirring apparatus, take the bacterium solution after 5mL crushes respectively in 10mL centrifuge tube, 3500r/min is centrifuged 10min, takes supernatant as crude enzyme liquid；The three groups of crude enzyme liquids obtained carry out the work of carboxymethylcelluloenzyme enzyme enzyme respectively, hemicellulase enzyme is lived, the test of laccase activity, and the crude enzyme liquid enzyme prepared with conventional method is lived and contrasted；
F, fermentation tank spread cultivation: carry out the preparation of fermentation medium according to formula as below:
One-level white sugar 12595g, 140 order bean cake powder 2025g,
KH2PO4Analytical pure 429g, MgSO4·7H2O analytical pure 443g,
Mushroom bran extracting solution 700L；
Autoclaving after tune pH to 6.0～6.9；Fermentation medium in fermentation tank is cooled to room temperature, in inoculation step C shake-flask culture terminate after bacterium solution；20～25 DEG C of condition bottom fermentations are cultivated 7 days, ventilation 0.1MP；
G, making culture bottle: weigh corn cob, Testa oryzae, wheat bran, cotton seed hulls, soybean cover, distiller's grains of beer, megasse, by above-mentioned raw material stirring mixing 25min, then limit adds mushroom bran extracting solution limit and continues stirring 40～50min, be modulated into water content be 65% ± 1, pH be the composite material of 6.3～6.9；Planting in culture bottle into above-mentioned composite material, making every bottle of indicated weight is 1020 ± 20g；Adopt five hole perforating methods, make charge level smooth distance culture bottle bottleneck 2.0cm, 121 DEG C of autoclaving 80～100min, be cooled to room temperature, last inoculation step F spread cultivation after fermentation liquid strain.
2. the method preparing flammulina velutipes liquid strains for raw material with mushroom bran extracting solution according to claim 1, it is characterised in that: the method for drafting of described glucose standard curve is as follows:
I, accurately weigh anhydrous glucose 250.0mg, be dissolved in distilled water and be settled to 250mL, obtain the Glucose standards solution of 1.0mg/mL；Glucose standards solution is diluted to 0mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL, 1.0mg/mL respectively；
II, measure the glucose solution of the above-mentioned variable concentrations absorbance at 540nm wavelength place by DNS method, draw glucose standard curve, draw regression equation.
3. the method preparing flammulina velutipes liquid strains for raw material with mushroom bran extracting solution according to claim 1, it is characterized in that: the method for testing that described carboxymethylcelluloenzyme enzyme enzyme is lived is: in 20ml tool plug test tube, add the sodium carboxymethyl cellulose 1.5mL of 0.5%, add the crude enzyme liquid 0.5mL of dilution 10 times, water-bath 30min in 50 DEG C of thermostat water baths, DNS reagent 3mL is added immediately after taking-up, it is then placed in 5min in 100 DEG C of hot baths, taking-up is settled to 20mL with distilled water after being cooled to room temperature, mixing；Measure OD value with spectrophotometer at 540nm place, to boil 15min inactivator liquid for comparison, calculate enzyme activity.
4. the method preparing flammulina velutipes liquid strains for raw material with mushroom bran extracting solution according to claim 1, it is characterised in that: the method for testing that described hemicellulase enzyme is lived is: add the wheat stalk hemicellulose solution 1.5mL of 0.5% in 20mL tool plug test tube；Described wheat stalk hemicellulose solution pH is 4.8, and concentration is the acetate buffer preparation of 0.1mol/L；Add crude enzyme liquid 0.5mL, the water-bath 30min in 50 DEG C of thermostat water baths of dilution 10 times, after taking-up, add DNS reagent 3mL immediately, be then placed in 5min in 100 DEG C of hot baths, take out and be settled to 20mL with distilled water after being cooled to room temperature, mixing；Measure OD value with spectrophotometer at 540nm place, to boil 15min inactivator liquid for comparison, calculate enzyme activity.
5. the method preparing flammulina velutipes liquid strains for raw material with mushroom bran extracting solution according to claim 1, it is characterized in that: the method for testing of described laccase activity is: in 20mL tool plug test tube, add the guaiacol of 0.5mL80mmol/L, add the acetate buffer solution 3mL that 0.1mol/L, pH are 4.8, add crude enzyme liquid 0.1mL, 30min is reacted at 28 DEG C, measure OD value with spectrophotometer at 490nm wavelength place afterwards, to boil the inactivator liquid of 15min as comparison, calculate enzyme work.
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| CN111066574A (en) * | 2020-01-15 | 2020-04-28 | 青岛农业大学 | Method for preparing Lepista sordida cultivars by using mushroom dregs |
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| CN113317118A (en) * | 2021-06-22 | 2021-08-31 | 上海光明森源生物科技有限公司 | Industrial needle mushroom bottle cultivation method with good flower type and preservation performance |
| CN113317130A (en) * | 2021-06-08 | 2021-08-31 | 上海光明森源生物科技有限公司 | Method for preparing liquid strain of needle mushroom and culture medium used by method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108207492A (en) * | 2018-02-09 | 2018-06-29 | 山东省农业科学院农业资源与环境研究所 | A kind of Wood rot type edible fungus liquid culture growth medium, preparation method and application |
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| CN112210547A (en) * | 2019-07-12 | 2021-01-12 | 湖南农业大学 | Mushroom bran crude enzyme preparation and preparation method thereof |
| CN111066574A (en) * | 2020-01-15 | 2020-04-28 | 青岛农业大学 | Method for preparing Lepista sordida cultivars by using mushroom dregs |
| CN113317130A (en) * | 2021-06-08 | 2021-08-31 | 上海光明森源生物科技有限公司 | Method for preparing liquid strain of needle mushroom and culture medium used by method |
| CN113317118A (en) * | 2021-06-22 | 2021-08-31 | 上海光明森源生物科技有限公司 | Industrial needle mushroom bottle cultivation method with good flower type and preservation performance |
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