CN104737817B - Method for improving oyster mushroom laccase production capability - Google Patents
Method for improving oyster mushroom laccase production capability Download PDFInfo
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Abstract
The invention discloses a method for improving oyster mushroom laccase production capability. The method comprises the following steps of preparing drying materials for cultivating oyster mushrooms; adding copper salt solutions into the drying materials and uniformly mixing to prepare oyster mushroom culture mediums; sterilizing the oyster mushroom culture mediums, inoculating oyster mushroom seed solutions, and then improving the oyster mushroom laccase production capability by utilizing the copper salt solutions in the oyster mushroom culture mediums. According to the oyster mushroom culture mediums, the oyster mushroom laccase production capability can be greatly improved so that the oyster mushroom yield is increased, and a new idea for researching high-yield oyster mushroom culture mediums is provided.
Description
Technical field
The invention belongs to fungus growing technique field, more particularly to a kind of method for improving Pleurotus ostreatus laccase production ability.
Background technology
Pleurotus ostreatus scientific name rough bark is picked up the ears Pleurotus ostreatus (Jacq.) P.Kumm., belongs to basidiomycetes on taxonomy
Door Basidiomycota, gill fungi guiding principle Agaricomycetes, Agaricaless Agaricales, Pleurotaceae Pleurotaceae pick up the ears
Category Pleurotus, is one of most important edible fungi, while be also a kind of medicinal fungi, with treatment lumbago and skelalgia, Shou Zuma
Wood, grain are not dredged, the effect of inhibiting cancer.
Pleurotus ostreatus rely primarily on the laccase of its generation and lignocellulose are degraded, so as to obtain nutrient growth sporophore
(mushroom).Laccase (Lac) is a kind of extracellular glycoprotein containing 4 copper atoms, special according to spectrum and electron paramagnetic resonance (EPR)
Levy and can be divided into 3 classes：I types Cu and II types Cu are each 1, are single electricity receptors, in paramagnetism；Type III Cu has 2, is double electron acceptor,
In diamagnetism.Phenol structure unit in the presence of oxygen, in Lac primary challenge lignins：The core of phenol lose 1 electronics and
The free active group containing phenoxy group is oxidized to, with reference to enzyme active center --- I type copper atoms site, by Cys-His approach
Three core copper cluster sites are given by electron transmission, again electron transmission oxygen supply, oxygen occurs 4 electron reductions as electron acceptor in the site,
Generate the H of 2 molecules2O, simultaneous CαOxidation, Cα-CβThe series reaction such as cracking and alkyl-aryl cracking.Due to Lac
Oxidation-reduction potential it is relatively low, it is impossible to the non-phenolic construction unit being in the great majority in direct oxidation lignin degrading structure, therefore
The compound of some lower oxidation reduction positions need to be added as redox modulating agent.These regulators form height in the presence of enzyme
The stable intermediate of activity, then electron transmission is obtained from oxygen molecule to lignin molecule so as to lignin degrading.Due to laccase
The height of yield is directly affected to be applied to ligocellulose degradation, therefore, the yield of laccase has huge shadow to the yield of Pleurotus ostreatus
Ring.
Although China is Pleurotus ostreatus big producing country, it is not production power, current mushroom cultivation yet suffers from that per unit area yield is low, yield
The low problem of shakiness, poor quality, benefit.At present to oyster mushroom culture medium Quality Research mainly directly from the angle for improving Yield of Pleurotus Ostreatus
Spend the formula to oyster mushroom culture medium matter to be improved, and go to improve oyster mushroom culture medium from the angle for improving Pleurotus ostreatus laccase production ability
Matter, so improve Yield of Pleurotus Ostreatus method have not been reported.
The content of the invention
The purpose of the present invention is for a difficult problem of the prior art, there is provided a kind of method of raising Pleurotus ostreatus laccase production ability,
Wood flour after by being processed using steam explosion is used as major ingredient with corn cob, cotton seed hullss, and is aided with mantoquita to prepare Pleurotus ostreatus culture
Substrate so that Pleurotus ostreatus produce the ability of laccase and greatly improve, and then Yield of Pleurotus Ostreatus is improved, it is that Pleurotus ostreatus high-yield culture medium Quality Research is carried
New approaches are supplied.
To realize this purpose, the present invention provides a kind of method for improving Pleurotus ostreatus laccase production ability, including：
Prepare the siccative for mushroom cultivation；
Copper salt solution is added in the siccative, is uniformly mixed, obtain oyster mushroom culture medium matter；
The oyster mushroom culture medium matter is carried out after sterilization treatment, Pleurotus ostreatus seed liquor is inoculated with, using the oyster mushroom culture medium matter
In copper salt solution improve Pleurotus ostreatus laccase production ability.
Wherein, in the copper salt solution, the concentration of copper ion is 2.5-5.5mg/L.
Particularly, in the copper salt solution, the concentration of copper ion is 4.5-5.0mg/L.
Wherein, the ratio of the volume of the weight of the siccative and the copper salt solution is：1:(1.2-1.5), above-mentioned weight
Volume ratio meets the most conventional usage in this area, with g/mL or kg/L as correspondence unit dose, such as in the siccative of 1g
Add 1.2-1.5mL copper salt solutions or the copper salt solution of 1.2-1.5L is added in the siccative of 1kg.
Wherein, the mantoquita is soluble copper salt.
Particularly, the mantoquita be copper nitrate, copper chloride, one or more in Schweinfurt green, preferably copper nitrate.
Wherein, the siccative prepared for mushroom cultivation includes：
Wood flour is crushed to into 3-5cm；
The wood flour of 3-5mm is placed in blaster, saturated steam is passed through, individual composition is carried out；
The baiting valve of blaster is opened, explosion treatment is carried out to wood flour；
Wood flour after explosion treatment is dried into process；
Wood flour after dried is mixed with corn cob, cotton seed hullss, starch, wheat bran, Gypsum Fibrosum, Calx, carbamide, stirring is equal
It is even, obtain final product.
Wherein, the wood flour is poplar bits, pear wood bits, Fructus Mali pumilae wood flour, peach wood bits, the one kind in Fructus Pruni wood flour, Fructus Crataegi wood flour
Or various, preferably poplar bits.
Wherein, the relative pressure of the individual composition is 1.5-2Mpa, and the individual composition time is 5-10min.
Wherein, the temperature of described dried is 100-200 DEG C, and the time of dried is 45-90min.
Wherein, after the dried wood flour, corn cob, cotton seed hullss, starch, wheat bran, Gypsum Fibrosum, Calx, the weight of carbamide
Measuring part proportioning is：Wood flour 0.10-1.15, corn cob 0.10-1.15 after dried, cotton seed hullss 0.5-0.7, starch 0.05-
0.15th, wheat bran 0.05-0.15, Gypsum Fibrosum 0.015-0.025, Calx 0.015-0.025, carbamide 0.005-0.015.
Particularly, after the dried wood flour, corn cob, cotton seed hullss, starch, wheat bran, Gypsum Fibrosum, Calx, carbamide
Weight is：Wood flour 0.39, corn cob 0.76 after dried, cotton seed hullss 0.6, starch 0.1, wheat bran 0.1, Gypsum Fibrosum
0.02nd, Calx 0.02, carbamide 0.01.
Wherein, the temperature of the sterilization treatment is 115-125 DEG C, and preferably 121 DEG C, the pressure of sterilization treatment is 0.10-
0.13MPa, preferably 0.12Mpa, the time of sterilization treatment is 25-35min, preferably 30min.
Wherein, the Pleurotus ostreatus seed liquor is prepared as follows and forms：
Pleurotus ostreatus strains tested is seeded on solid medium, activation culture is carried out, is grown mycelia；
To be cultivated in mycelium inoculation to liquid seed culture medium, obtained primary seed solution；
Primary seed solution is seeded in liquid seed culture medium and is cultivated, obtained secondary seed solution and be described putting down
Mushroom seed liquor.
Wherein, the solid medium includes：Deionized water, glucose, fructus hordei germinatus leaching powder, agar powder, KH2PO4, wherein, often
Contain glucose 10.0g, fructus hordei germinatus leaching powder 20.0g, agar powder 18.0g, KH in 1000mL solid mediums2PO43.0g, remaining is
Deionized water.
Wherein, the condition of the activation culture is：Under closed dark condition, temperature is 26 ± 2 DEG C, and the time is 5-7 days.
Wherein, the liquid seed culture medium includes：Deionized water, glucose, yeast extract, MgSO4、VB1、KH2PO4,
Wherein, glucose 20.00g, yeast extract 5.00g, MgSO are contained in every 1000mL fluid mediums40.50g、VB10.01g、
KH2PO41g, remaining is deionized water.
Wherein, the condition of culture of the primary seed solution is：Temperature is 26 ± 2 DEG C, and rotating speed 150r/min is cultivated 7 days.
Wherein, the primary seed solution and the ratio of the volume of the liquid seed culture medium are 1:10.
Wherein, the condition of culture of the secondary seed solution is：Temperature is 26 ± 2 DEG C, and rotating speed 150r/min is cultivated 5 days.
The inventive method has the advantage that：
1st, oyster mushroom culture medium matter of the invention adopts wood flour that Steam explosion treatment crosses and corn cob, cotton seed hullss to match somebody with somebody for major ingredient
System is formed, and is aided with mantoquita, is greatly improved can the ability of the product laccase of Pleurotus ostreatus, and then be greatly improved the yield of Pleurotus ostreatus.
2nd, the present invention, from the angle of Pleurotus ostreatus laccase production ability is improved improveing oyster mushroom culture medium matter, is Pleurotus ostreatus high-yield culture medium
The development of matter provides new approaches.
3rd, oyster mushroom culture medium matter preparation method of the invention is simple, low cost.
4th, the Pleurotus ostreatus quality better cultivated using the oyster mushroom culture medium matter of the present invention, yield are high, copper content is in edible fungi state
In mark safety range.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not constitute any restriction to the scope of the present invention.People in the art
Member is it should be understood that can enter to the details of technical solution of the present invention and form without departing from the spirit and scope of the invention
Row modification is replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
The strains tested of the present invention is Eight Strains of Pleurotus Ostreatus Pleurotus ostreatus CCEF89, by the Chinese Academy of Agricultural Sciences's agricultural
Resource is provided with agricultural regionalization institute, and the bacterial strain is currently the kind of Chinese extensively cultivation.
The present invention solid culture based component be：Glucose 10.0g/L, fructus hordei germinatus leaching powder 20.0g/L, agar powder 18.0g/L,
KH2PO43.0g/L, pH nature.
The present invention liquid seed culture medium composition be：Glucose 20.00g/L, yeast extract 5.00g/L,
MgSO40.50g/L, VB10.01g/L, KH2PO41g/L, pH nature.
Embodiment 1
1st, prepare oyster mushroom culture medium matter
1) wood flour pretreatment
Wood flour is crushed to into 3-5cm；Then wood flour is placed in blaster, is passed through saturated steam, in relative pressure be
Under the conditions of 1.5Mpa, individual composition 10min is carried out；After individual composition terminates, the rapid baiting valve for opening blaster enters to wood flour
Row explosion treatment；Wood flour after explosion treatment is placed in the baking oven that temperature is 100 DEG C and is dried process, dried when
Between be 90min.
2) get the raw materials ready according to following weight proportion
By above-mentioned raw materials mix homogeneously, the siccative for mushroom cultivation is obtained, it is standby；
3) copper chloride and water are mixed, copper chloride solution of the copper ion concentration for 5mg/L is obtained；
4) under agitation, copper chloride solution is added in siccative, stirred, obtain oyster mushroom culture medium matter；
Wherein, the weight of the siccative and the ratio of the volume of copper chloride solution are 1：1.2, i.e., add in the siccative of 1g
1.2mL copper chloride solutions or in the siccative of 1kg add 1.2L copper chloride solution.
5) oyster mushroom culture medium matter is divided in conical flask, each conical flask loads 2g oyster mushroom culture medium matter, is subsequently placed in
In high-pressure steam sterilizing pan, it is 121 DEG C in temperature, pressure is the 30min that sterilizes under the conditions of 0.12MPa, takes out, in the cleaning of sterilization
Less than 25 DEG C are naturally cooled under environment, is obtained final product.
2nd, prepare Pleurotus ostreatus seed liquor
Pleurotus ostreatus strains tested is activated on solid medium, treats that mycelia covers with flat board, growing way is taken with card punch consistent, straight
5 pieces of the bacteria cake of footpath 6mm carries out shake-flask culture in being seeded to the triangular flask equipped with liquid seed culture medium, liquid seed culture medium
Liquid amount is the 2/5 of triangular flask volume, i.e., in 250mL triangular flasks load 100mL liquid seed culture mediums, temperature be 26 ±
2 DEG C, cultivate 7 days under the conditions of rotating speed 150r/min, obtain primary seed solution；
Primary seed solution is carried out after homogenized, is connected to equipped with liquid seed culture medium according to 10% (V/V) inoculum concentration
Triangular flask in carry out shake-flask culture, the liquid amount of liquid seed culture medium is the 2/5 of triangular flask volume, i.e., in 250mL triangles
Load 100mL liquid seed culture mediums in bottle, be 26 ± 2 DEG C in temperature, culture under the conditions of rotating speed 150r/min obtains two grades in 5 days
Seed liquor；
Secondary seed solution is preserved at 4 DEG C, it is standby.
3rd, inoculation and culture
Under aseptic technique, 2mL secondary seed solutions are seeded to the cone of the oyster mushroom culture medium matter prepared equipped with step 1
In shape bottle, carry out sending out bacterium culture 5 days under the dark airtight condition that temperature is 26 DEG C.
4th, extract laccase crude enzyme liquid
60mL 0.1mmol/L sodium-acetate buffers (pH 4.6) are added in the conical flask after inoculated and cultured, by conical flask
Be placed in shaking table, temperature be 26 DEG C, rotating speed be 150r/min under the conditions of, extract 4h, after taking-up rotating speed be 12000r/min
Under the conditions of 10min is centrifuged, take supernatant, as crude enzyme liquid.
5. laccase activity is determined
Reaction system：0.2mol/L 4.0 disodium hydrogen phosphates of pH-citrate buffer solution 2mL, 0.5mmol/L ABTS1mL,
Crude enzyme liquid 0.5mL, compares the crude enzyme liquid for the inactivation of boiling water bath, at ultraviolet-visible double beam spectrophotometer measurement 420nm
Optical density value (OD), and per 1min record OD420.Experiment is all provided with three groups of repetitions, averages.
Enzyme activity computing formula is as follows：
U/L=A × V × 106/ (e × L × t × v),
Wherein, A-light absorption value；V-reaction system cumulative volume, mL；E-molar absorption coefficient, laccase are 3.6 × 104
(mol/L)-1cm-1；L-cuvette diameter, cm；T-response time, min；V-sample volume, mL.
Enzyme activity measurement result is shown in Table 1.
Embodiment 2
1st, prepare oyster mushroom culture medium matter
1) wood flour pretreatment
Wood flour is crushed to into 3-5cm；Then wood flour is placed in blaster, is passed through saturated steam, in relative pressure be
Under the conditions of 2.0Mpa, individual composition 5min is carried out；After individual composition terminates, the rapid baiting valve for opening blaster enters to wood flour
Row explosion treatment；Wood flour after explosion treatment is placed in the baking oven that temperature is 150 DEG C and is dried process, dried when
Between be 60min.
2) get the raw materials ready according to following weight proportion
Above-mentioned raw materials mix homogeneously is obtained for cultivating the siccative of Pleurotus ostreatus, it is standby；
3) copper nitrate and water are mixed, copper nitrate solution of the copper ion concentration for 2.5mg/L is obtained；
4) under agitation, copper nitrate solution is added in siccative, stirred, obtain oyster mushroom culture medium matter；
Wherein, the weight of the siccative and the ratio of the volume of copper nitrate solution are 1：1.3, i.e., add in the siccative of 1g
1.3mL copper nitrate solutions or in the siccative of 1kg add 1.3L copper nitrate solution.
5) oyster mushroom culture medium matter is divided in conical flask, loads 2g oyster mushroom culture medium matter in each conical flask, then put
In high-pressure steam sterilizing pan, it is 115 DEG C in temperature, after pressure is for the 35min that sterilizes under the conditions of 0.1Mpa, takes out, in sterilization
Less than 25 DEG C are naturally cooled under clean environment, is obtained final product.
2nd, prepare Pleurotus ostreatus seed liquor
Pleurotus ostreatus strains tested is activated on solid medium, treats that mycelia covers with flat board, growing way is taken with card punch consistent, straight
5 pieces of the bacteria cake of footpath 6mm carries out shake-flask culture in being seeded to the triangular flask equipped with liquid seed culture medium, liquid seed culture medium
Liquid amount is the 2/5 of triangular flask volume, i.e., in 250mL triangular flasks load 100mL liquid seed culture mediums, temperature be 26 ±
2 DEG C, 7d is cultivated under the conditions of rotating speed 150r/min, obtain primary seed solution；
Primary seed solution is carried out after homogenized, is connected to equipped with liquid seed culture medium according to 10% (V/V) inoculum concentration
Triangular flask in carry out shake-flask culture, the liquid amount of liquid seed culture medium is the 2/5 of triangular flask volume, i.e., in 250mL triangles
Load 100mL liquid seed culture mediums in bottle, be 26 ± 2 DEG C in temperature, 5d cultivated under the conditions of rotating speed 150r/min and obtains two
Level seed liquor；
Secondary seed solution is preserved at 4 DEG C, it is standby.
3rd, inoculation and culture
Under aseptic technique, 2mL secondary seed solutions are seeded to the cone of the oyster mushroom culture medium matter prepared equipped with step 1
In shape bottle, carry out sending out bacterium under the dark airtight condition of 26 DEG C of temperature and cultivate 4 days.
4th, extract laccase crude enzyme liquid
60mL 0.1mmol/L sodium-acetate buffers (pH 4.6) are added in the conical flask after inoculated and cultured, by conical flask
Be placed in shaking table, temperature be 26 DEG C, rotating speed be 150r/min under the conditions of, extract 4h, after taking-up rotating speed be 12000r/min
Under the conditions of 10min is centrifuged, take supernatant, as crude enzyme liquid.
5. laccase activity is determined
Reaction system：0.2mol/L 4.0 disodium hydrogen phosphates of pH-citrate buffer solution 2mL, 0.5mmol/L ABTS1mL,
Crude enzyme liquid 0.5mL, compares the crude enzyme liquid for the inactivation of boiling water bath, at ultraviolet-visible double beam spectrophotometer measurement 420nm
Optical density value (OD), and per 1min record OD420.Experiment is all provided with three groups of repetitions, averages.
Enzyme activity computing formula is as follows：
U/L=A × V × 106/ (e × L × t × v),
Wherein, A-light absorption value；V-reaction system cumulative volume, mL；E-molar absorption coefficient, laccase are 3.6 × 104
(mol/L)-1cm-1；L-cuvette diameter, cm；T-response time, min；V-sample volume, mL.
Enzyme activity measurement result is shown in Table 1.
Embodiment 3
1st, prepare oyster mushroom culture medium matter
1) wood flour pretreatment
Wood flour is crushed to into 3-5cm；Then wood flour is placed in blaster, is passed through saturated steam, in relative pressure be
Under the conditions of 1.8Mpa, individual composition 8min is carried out；After individual composition terminates, the rapid baiting valve for opening blaster enters to wood flour
Row explosion treatment；Wood flour after explosion treatment is placed in the baking oven that temperature is 200 DEG C and is dried process, dried when
Between be 45min.
2) get the raw materials ready according to following weight proportion
Above-mentioned raw materials mix homogeneously is obtained for cultivating the siccative of Pleurotus ostreatus, it is standby；
3) Schweinfurt green and water are mixed, acetic acid copper solution of the copper ion concentration for 3mg/L is obtained；
4) under agitation, acetic acid copper solution is added in siccative, stir, obtain oyster mushroom culture medium matter；
Wherein, the weight of the siccative and the ratio of the volume of acetic acid copper solution are 1：1.4, i.e., add in the siccative of 1g
1.4mL acetic acid copper solution or in the siccative of 1kg add 1.4L acetic acid copper solution.
5) oyster mushroom culture medium matter is divided in conical flask, loads 2g oyster mushroom culture medium matter in each conical flask, then put
In high-pressure steam sterilizing pan, it is 125 DEG C in temperature, pressure is 0.13MPa, after high temperature sterilize 25min, takes out, in sterilization
Less than 25 DEG C are naturally cooled under clean environment, is obtained final product.
2nd, prepare Pleurotus ostreatus seed liquor
Pleurotus ostreatus strains tested is activated on solid medium, treats that mycelia covers with flat board, growing way is taken with card punch consistent, straight
5 pieces of the bacteria cake of footpath 6mm carries out shake-flask culture in being seeded to the triangular flask equipped with liquid seed culture medium, liquid seed culture medium
Liquid amount is the 2/5 of triangular flask volume, i.e., in 250mL triangular flasks load 100mL liquid seed culture mediums, temperature be 26 ±
2 DEG C, 7d is cultivated under the conditions of rotating speed 150r/min, obtain primary seed solution；
Primary seed solution is carried out after homogenized, is connected to equipped with liquid seed culture medium according to 10% (V/V) inoculum concentration
Triangular flask in carry out shake-flask culture, the liquid amount of liquid seed culture medium is the 2/5 of triangular flask volume, i.e., in 250mL triangles
Load 100mL liquid seed culture mediums in bottle, be 26 ± 2 DEG C in temperature, 5d cultivated under the conditions of rotating speed 150r/min and obtains two
Level seed liquor；
Secondary seed solution is preserved at 4 DEG C, it is standby.
3rd, inoculation and culture
Under aseptic technique, 2mL secondary seed solutions are seeded to the cone of the oyster mushroom culture medium matter prepared equipped with step 1
In shape bottle, carry out sending out bacterium culture 7 days under the conditions of dark closed, temperature is for 26 DEG C.
4th, extract laccase crude enzyme liquid
60mL 0.1mmol/L sodium-acetate buffers (pH 4.6) are added in the conical flask after inoculated and cultured, by conical flask
Be placed in shaking table, temperature be 26 DEG C, rotating speed be 150r/min under the conditions of, extract 4h, after taking-up rotating speed be 12000r/min
Under the conditions of 10min is centrifuged, take supernatant, as crude enzyme liquid.
5. laccase activity is determined
Reaction system：0.2mol/L 4.0 disodium hydrogen phosphates of pH-citrate buffer solution 2mL, 0.5mmol/L ABTS1mL,
Crude enzyme liquid 0.5mL, compares the crude enzyme liquid for the inactivation of boiling water bath, at ultraviolet-visible double beam spectrophotometer measurement 420nm
Optical density value (OD), and per 1min record OD420.Experiment is all provided with three groups of repetitions, averages.
Enzyme activity computing formula is as follows：
U/L=A × V × 106/ (e × L × t × v),
Wherein, A-light absorption value；V-reaction system cumulative volume, mL；E-molar absorption coefficient, laccase are 3.6 × 104
(mol/L)-1cm-1；L-cuvette diameter, cm；T-response time, min；V-sample volume, mL.
Enzyme activity measurement result is shown in Table 1.
Embodiment 4
1st, prepare oyster mushroom culture medium matter
1) wood flour pretreatment
Wood flour is crushed to into 3-5cm；Then wood flour is placed in blaster, is passed through saturated steam, in relative pressure be
Under the conditions of 1.6Mpa, individual composition 6min is carried out；After individual composition terminates, the rapid baiting valve for opening blaster enters to wood flour
Row explosion treatment；Wood flour after explosion treatment is placed in the baking oven that temperature is 170 DEG C and is dried process, dried when
Between be 80min.
2) get the raw materials ready according to following weight proportion
Above-mentioned raw materials mix homogeneously is obtained for cultivating the siccative of Pleurotus ostreatus, it is standby；
3) copper chloride and water are mixed, copper chloride solution of the copper ion concentration for 5.5mg/L is obtained；
4) under agitation, copper chloride solution is added in siccative, stirred, obtain oyster mushroom culture medium matter；
Wherein, the weight of the siccative and the ratio of the volume of copper chloride solution are 1：1.5, i.e., add in the siccative of 1g
1.5mL copper chloride solutions or in the siccative of 1kg add 1.5L copper chloride solution.
5) oyster mushroom culture medium matter is divided in conical flask, loads 2g oyster mushroom culture medium matter in each conical flask, be placed in height
In pressure steam sterilization pan, it is 121 DEG C in temperature, pressure is 0.12MPa, after high temperature sterilize 30min, takes out, in the cleaning of sterilization
Less than 25 DEG C are naturally cooled under environment, is obtained final product.
2nd, prepare Pleurotus ostreatus seed liquor
Pleurotus ostreatus strains tested is activated on solid medium, treats that mycelia covers with flat board, growing way is taken with card punch consistent, straight
5 pieces of the bacteria cake of footpath 6mm carries out shake-flask culture in being seeded to the triangular flask equipped with liquid seed culture medium, liquid seed culture medium
Liquid amount is the 2/5 of triangular flask volume, i.e., in 250mL triangular flasks load 100mL liquid seed culture mediums, temperature be 26 ±
2 DEG C, 7d is cultivated under the conditions of rotating speed 150r/min, obtain primary seed solution；
Primary seed solution is carried out after homogenized, is connected to equipped with liquid seed culture medium according to 10% (V/V) inoculum concentration
Triangular flask in carry out shake-flask culture, the liquid amount of liquid seed culture medium is the 2/5 of triangular flask volume, i.e., in 250mL triangles
Load 100mL liquid seed culture mediums in bottle, be 26 ± 2 DEG C in temperature, 5d cultivated under the conditions of rotating speed 150r/min and obtains two
Level seed liquor；
Secondary seed solution is preserved at 4 DEG C, it is standby.
3rd, inoculation and culture
Under aseptic technique, 2mL secondary seed solutions are seeded to the cone of the oyster mushroom culture medium matter prepared equipped with step 1
In shape bottle, carry out sending out bacterium culture 6 days under the conditions of dark closed, temperature is for 26 DEG C.
4th, extract laccase crude enzyme liquid
60mL 0.1mmol/L sodium-acetate buffers (pH 4.6) are added in the conical flask after inoculated and cultured, by conical flask
Be placed in shaking table, temperature be 26 DEG C, rotating speed be 150r/min under the conditions of, extract 4h, after taking-up rotating speed be 12000r/min
Under the conditions of 10min is centrifuged, take supernatant, as crude enzyme liquid.
5. laccase activity is determined
Reaction system：0.2mol/L 4.0 disodium hydrogen phosphates of pH-citrate buffer solution 2mL, 0.5mmol/L ABTS1mL,
Crude enzyme liquid 0.5mL, compares the crude enzyme liquid for the inactivation of boiling water bath, at ultraviolet-visible double beam spectrophotometer measurement 420nm
Optical density value (OD), and per 1min record OD420.Experiment is all provided with three groups of repetitions, averages.
Enzyme activity computing formula is as follows：
U/L=A × V × 106/ (e × L × t × v),
Wherein, A-light absorption value；V-reaction system cumulative volume, mL；E-molar absorption coefficient, laccase are 3.6 × 104
(mol/L)-1cm-1；L-cuvette diameter, cm；T-response time, min；V-sample volume, mL.
Enzyme activity measurement result is shown in Table 1.
Reference examples 1
In addition to replacing copper salt solution with isopyknic water in the step of preparing oyster mushroom culture medium matter, remaining and embodiment
1 is identical.Enzyme activity measurement result is shown in Table 1.
Reference examples 2
Get the raw materials ready according to following weight proportion in the step of oyster mushroom culture medium matter is prepared except step 1：
Outside, remaining is same as Example 1,
Enzyme activity measurement result is shown in Table 1.
Reference examples 3
Get the raw materials ready according to following weight proportion in oyster mushroom culture medium matter except prepared by step 1：
Outside, remaining is same as Example 1,
Enzyme activity measurement result is shown in Table 1.
Reference examples 4
Get the raw materials ready according to following weight proportion in oyster mushroom culture medium matter except prepared by step 1：
Outside, remaining is same as Example 1,
Enzyme activity measurement result is shown in Table 1.
Reference examples 5
In addition to oyster mushroom culture medium matter is prepared according to the formula of traditional oyster mushroom culture medium matter A, remaining and embodiment 1
It is identical, wherein, the formula of conventional oyster mushroom culture medium matter A is as follows：
1.78 parts of wood flour, 0.2 part of wheat bran, 0.02 part of Calx, material-water ratio 1:1.3.
Wherein, wood flour is not through the common wood flour of Steam explosion treatment.
Enzyme activity measurement result is shown in Table 1.
Reference examples 6
In addition to oyster mushroom culture medium matter is prepared according to the formula of traditional oyster mushroom culture medium matter B, remaining and embodiment 1
It is identical, wherein, the formula of conventional oyster mushroom culture medium matter B is as follows：
1 part of corn cob, 0.94 part of beanstalk, 0.04 part of wheat bran, 0.02 part of Calx, material-water ratio 1:1.3.
Enzyme activity measurement result is shown in Table 1.
The Pleurotus ostreatus laccase activity measurement result of 1. embodiment 1-4 of table and reference examples 1-6
| Laccase activity U/L | |
| Embodiment 1 | 651.94 |
| Embodiment 2 | 638.23 |
| Embodiment 3 | 645.36 |
| Embodiment 4 | 640.17 |
| Reference examples 1 | 209.15 |
| Reference examples 2 | 284.91 |
| Reference examples 3 | 110.21 |
| Reference examples 4 | 109.08 |
| Reference examples 5 | 71.65 |
| Reference examples 6 | 62.43 |
As it can be seen from table 1 the oyster mushroom culture medium matter of the present invention can significantly improve the ability that Pleurotus ostreatus produce laccase, laccase is lived
Power >=638.23U/L, significantly larger than using single major ingredient as oyster mushroom culture medium matter or the oyster mushroom culture medium matter without mantoquita
And traditional oyster mushroom culture medium matter.
Test example 1
Using the oyster mushroom culture medium matter of the embodiment of the present invention 1 and the preparation of reference examples 1-6 in identical flat mushroom strain, culture bar
Cultivated under part, harvested 6 batches of Pleurotus ostreatus altogether, statistics is adopted the Pleurotus ostreatus biological transformation ratio of seven kinds of different oyster mushroom culture medium matter, as a result
It is shown in Table 2.
Biological transformation ratio, refers to that per bag of product fresh mushroom gross weight is multiplied by 100% again with the ratio of packed oyster mushroom culture medium matter weight,
It is the Common Parameters that mushroom yield is weighed in this area.
2. every batch of mushroom body yield of table and biological transformation ratio statistical result
As seen from the results in Table 2, using the present invention oyster mushroom culture medium matter cultivate Pleurotus ostreatus, its biological transformation ratio apparently higher than
The Pleurotus ostreatus cultivated using the oyster mushroom culture medium matter of reference examples 1-6.
The Pleurotus ostreatus sampling cultivated to the oyster mushroom culture medium matter using embodiment 1, determines the content of its copper, copper in new fresh flat mushroom
Content≤10.000mg/kg, in dry Pleurotus ostreatus, copper content≤10.000mg/kg, exists《GB7096-2003 edible fungi sanitary standards》
The safety range of regulation.
Claims (9)
1. it is a kind of improve Pleurotus ostreatus laccase production ability method, it is characterised in that comprise the steps：
Prepare the siccative containing the wood flour after explosion treatment for mushroom cultivation；
Copper salt solution is added in the siccative containing the wood flour after explosion treatment, is uniformly mixed, is obtained containing explosion
The siccative and Cu of the wood flour after process2+The oyster mushroom culture medium matter of mix homogeneously；
By the siccative and Cu of the wood flour containing after explosion treatment2+The oyster mushroom culture medium matter of mix homogeneously carries out sterilization treatment
Afterwards, Pleurotus ostreatus seed liquor is inoculated with, Pleurotus ostreatus laccase production ability is improved using the copper salt solution in the oyster mushroom culture medium matter；
Wherein, in the copper salt solution, the concentration of copper ion is 2.5-5.5mg/L.
2. method according to claim 1, it is characterised in that the concentration of copper ion is 4.5- in the copper salt solution
5.0mg/L。
3. method according to claim 1, it is characterised in that the volume of the weight of the siccative and the copper salt solution it
Than for 1:(1.2-1.5).
4. method according to claim 3, it is characterised in that the mantoquita is copper nitrate, copper chloride, in Schweinfurt green
Plant or various.
5. method according to claim 3, it is characterised in that the preparation be used for mushroom cultivation containing explosion treatment after
The siccative of wood flour include：
Wood flour is crushed to into 3-5cm；
The wood flour of 3-5cm is placed in blaster, saturated steam is passed through, individual composition is carried out；
The baiting valve of blaster is opened, explosion treatment is carried out to wood flour；
Wood flour after explosion treatment is dried into process；
Wood flour after dried is mixed with corn cob, cotton seed hullss, starch, wheat bran, Gypsum Fibrosum, Calx, carbamide, is stirred,
Obtain final product.
6. method according to claim 5, it is characterised in that the relative pressure of the individual composition is 1.5-2MPa, vapour
Steaming process time is 5-10min.
7. method according to claim 5, it is characterised in that the temperature of described dried is 100-200 DEG C, is dried
The time of process is 45-90min.
8. method according to claim 5, it is characterised in that wood flour, corn cob, cotton seed hullss after the dried,
Starch, wheat bran, Gypsum Fibrosum, Calx, the weight of carbamide are：Wood flour 0.10-1.15, corn cob 0.10- after dried
1.15th, cotton seed hullss 0.5-0.7, starch 0.05-0.15, wheat bran 0.05-0.15, Gypsum Fibrosum 0.015-0.025, Calx 0.015-
0.025th, carbamide 0.005-0.015.
9. method according to claim 1, it is characterised in that the temperature of the sterilization treatment is 115-125 DEG C, at sterilizing
The pressure of reason is 0.10-0.13MPa, and the time of sterilization treatment is 25-35min.
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| CN108929865A (en) * | 2017-05-27 | 2018-12-04 | 北京林业大学 | A method of induction Produced from Pleurotus ostreatus accelerates to produce laccase |
| CN108395332A (en) * | 2018-05-07 | 2018-08-14 | 山东省农业科学院农业资源与环境研究所 | A kind of culture medium of edible fungus and preparation method thereof |
| CN112126632B (en) * | 2020-09-14 | 2023-05-30 | 廊坊师范学院 | Method for improving laccase production activity of Pleurotus ostreatus |
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| CN102150564A (en) * | 2011-03-29 | 2011-08-17 | 福建绿宝食品集团有限公司 | Cultivation method for oyster mushroom |
| CN102498937B (en) * | 2011-10-24 | 2014-04-09 | 榆树市腾龙食用菌种植专业合作社 | Method for culturing oyster mushroom |
| CN102617240B (en) * | 2012-04-10 | 2014-07-02 | 绥化学院 | Novel edible fungus culture medium and preparation method thereof |
| CN103314774A (en) * | 2013-05-24 | 2013-09-25 | 苏州市经纬农产品有限公司 | Oyster mushroom cultivation method |
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