CN112593437A - Biodegradation method of lignin - Google Patents
Biodegradation method of lignin Download PDFInfo
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Abstract
The invention discloses a biodegradation method of lignin, and belongs to the technical field of lignin degradation. The operation steps are as follows: (1) raoultella ornithinolyticaRaoultella ornithinolyticaInoculating slant strains to an LB solid culture medium to obtain activated strains; (2) inoculating the activated strain to an LB liquid culture medium to obtain a fermentation liquid; fermenting the fermentation liquor again to obtain bacterial suspension; (3) drying and crushing the treated lignin; (4) adding the powder into a liquid inorganic salt culture medium for fermentation, adding the fermentation product into the bacterial suspension, and stirring for reaction to obtain a degradation product; wherein the lignin degradation rate reaches 20-25%, and the surface of the degradation product is in a loose and porous structure state. Compared with the prior art, the invention discloses Raoultella ornithinolyticaRaoultella ornithinolyticaThe strain has strong adaptability to the growth environment of bacteria, and peroxidase, manganese peroxidase and laccase generated by the bacteria have good stability, wider optimal pH range, better tolerance to chloride ions and other excellent characteristics. The method has mild reaction and easy operation.
Description
Technical Field
The invention belongs to the technical field of lignin degradation, and particularly relates to a biodegradation method of lignin.
Background
Lignocellulose is biomass consisting of lignin, cellulose and hemicellulose, and is a cell wall composition component of woody and herbaceous plants which is difficult to degrade, wherein the cellulose is used as a framework, the lignin and the cellulose are dispersed in or around the cellulose in a contained form, and the combination of the lignin and the cellulose is very stable, so that the lignocellulose is a main raw material for biomass research at present. But because of its compact structure, it is not easy to degrade and is greatly hindered in production and utilization. The lignin treatment method commonly used at present mainly comprises a physical method, a chemical method, a biological method or a combined treatment of a plurality of methods. Although these methods increase the utilization of biomass resources to some extent, they still have many drawbacks in the operation process.
The physical methods include an ultrasonic method, microwave radiation and gamma ray radiation, and mainly destroy the structural stability of lignin by generating transient local high temperature, high pressure and rapid temperature change in the lignin to generate chemical reaction, but the method has unstable effect and is difficult to industrially amplify and apply due to the fact that noise is unfavorable to the environment. The chemical method is to destroy some chemical bonds in lignin by some strong acid, strong base and the like to degrade the lignin, but the method is not environment-friendly, and has the problems of high cost, difficult recovery of reagents after use and the like. Compared with the two methods, the biological method is mild and has good treatment effect, and the existing biological treatment mainly comprises the steps of secreting ligninase and cellulase with strong activity by white rot fungi with strong lignin degradation capability to degrade lignin.
Research shows that when lignin peroxidase degrades lignin, an electron is obtained from a benzene ring to form a benzene free radical, and then other types of free radicals are generated through chain reaction to break some chemical bonds in the lignin; the manganese peroxidase has the main function of removing lignin methoxyl, so that a structure which is easy to oxidize is formed, and aromatic rings are broken; the laccase has the effect similar to that of the first two enzymes, namely, the laccase breaks some chemical bonds of lignin in the degradation process, so that the lignin degradation effect is achieved. However, biological methods have high culture conditions for strains and slow degradation process, so the research on biological treatment methods is mainly focused on finding a strain capable of degrading efficiently.
Nowadays, the aim of the high-efficiency biodegradation bacteria is to put on fungi, so most of the researches are on how the fungi can efficiently degrade lignin, and the researches on the bacteria are very rare. But more and more recent studies have shown that bacteria have advantages over fungi in certain aspects, such as environmental suitability and biodiversity. In fact, through a large number of experiments, bacteria have abundant and diverse metabolic pathways in the aspect of degrading aromatic compounds, namely simple phenolic compounds and high-aggregation complex lignin, and the high genetic variability of the bacteria becomes a potential advantage for degrading the lignin.
Disclosure of Invention
The invention provides a lignin biodegradation method in order to realize convenient, stable and environment-friendly efficient lignin degradation.
The strain used by the invention is Raoultella ornithinolyticaRaoultella ornithinolyticaPurchased from the strain preservation center of Guangdong province.
The biodegradation operation of the lignin comprises the following steps:
(1) culturing the strains
Raoultella ornithinolyticaRaoultella ornithinolyticaInoculating the slant strains in a LB solid culture medium in a loop, and culturing at 28 ℃ for 24-72 h to obtain activated strains;
(2) preparation of the bacterial suspension
Inoculating the activated strain on an LB liquid culture medium, wherein the inoculation amount is 2-4%, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a fermentation liquid;
inoculating 2ml of fermentation liquor into 100ml of LB liquid culture medium, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a bacterial suspension;
(3) treatment of lignin materials
Drying and crushing the lignin material at the temperature of 45 ℃; sieving with 30-40 mesh sieve, and oven drying at 45 deg.C to obtain powder; the lignin material is crop straws or plant straws;
(4) degradation of
Adding 10g of powder into 20ml of liquid inorganic salt culture medium, uniformly stirring, sterilizing, and cooling to room temperature to obtain a fermentation product; adding 5% of the bacterial suspension in the step (2) into the fermentation product, uniformly stirring, and culturing at 28 ℃ for 2-3 days to obtain a degradation product; the degradation rate of lignin in the degradation product reaches 20-25%, and the surface of the degradation product is in a loose and porous structure state.
Measurement of Raoultella ornithinolytica by spectrophotometryRaoultella ornithinolyticaThe activity of the laccase, the peroxidase, the manganese peroxidase, the carboxymethyl cellulase and the activity of the filter paper enzyme are contained, and the activity of the manganese peroxidase is 219.49U/L.
The lignin material is corn straw or wheat straw or apple branches or rice straw or rice hull.
The operation of measuring the lignin degradation related enzyme activity is as follows:
1. preparation of crude enzyme solution
Taking 2-3 pieces of 0.25mm from the activated LB solid medium2Inoculating the large and small fungus blocks into a sterilized 100ml liquid alkali lignin culture medium, culturing for 2-3 days on a shaking table at 28 ℃ and 180r/min, taking 20ml, centrifuging in a centrifuge at room temperature of 8000rpm for 20min, and taking supernatant to obtain a crude enzyme solution for subsequent enzyme activity determination.
2. Determination of lignin-related enzyme activity
2.1 determination of peroxidase
1) Adding 250mM, pH4.5 tartaric acid-sodium tartrate buffer solution 2.7mL and veratryl alcohol solution 0.1mL of 10mmol/L into a test tube;
2) then 0.1mL of crude enzyme solution is added, and 0.1mL of 20mmol/L hydrogen peroxide solution is added at 28 ℃ to start the whole reaction;
3) after reacting for 2min, measuring the absorbance OD value at the wavelength of 310 nm;
4) performing an enzymatic reaction under the above experimental conditions with an enzyme activity unit (U) of 1.0. mu. mol veratryl alcohol per minute;
the enzyme activity of the peroxidase is determined to be 28.28U/L by a spectrophotometric method.
2.2 determination of manganese peroxidase
1) Adding 3.4mL of lactic acid-sodium lactate buffer solution with the pH value of 5.0 and 0.1mL of manganese sulfate solution with the mol/L of 0.4 into a test tube;
2) accurately adding 0.4mL of diluted crude enzyme solution to be detected, and adding 0.1mL of 3.2mmol/L hydrogen peroxide solution at 28 ℃ to start the whole reaction;
3) after reacting for 3min, measuring the absorbance OD value at the wavelength of 240 nm;
4) the enzymatic reaction was carried out under the above experimental conditions with an enzyme activity unit (U) of 1.0. mu. mol Mn produced per minute3+；
The enzyme activity of the manganese peroxidase is 219.49U/L by spectrophotometry.
2.3 determination of laccase
1) Adding 2.7mL of 50mM, pH5.0 acetic acid-sodium acetate buffer solution and 0.2mL of 1.0mmol/L ABTS solution into a test tube;
2) then accurately adding 0.1mL of crude enzyme solution to be detected, and adding 0.1mL of 20mmol/L hydrogen peroxide solution at 28 ℃ to start the whole reaction;
3) after reacting for 2min, measuring the absorbance OD value under the wavelength of 430 nm;
4) carrying out an enzymatic reaction under the experimental conditions described above, one unit of enzyme activity (U) producing 1.0. mu. mol of ABTS per minute;
the enzyme activity of the laccase is determined to be 1.31U/L by a spectrophotometric method.
3. Determination of cellulose-related enzyme Activity
3.1 glucose Standard Curve determination
1) Weighing 1g of dry glucose, dissolving in 1000mL of distilled water, and preparing a glucose standard solution with the concentration of 1 mg/mL;
2) taking 6 clean and dried 25mL test tubes with plug scales, respectively adding 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL glucose standard solutions to a constant volume of 1mL, diluting into diluents with the concentrations of 0mg/mL, 0.2mg/mL, 0.4mg/mL, 0.6mg/mL, 0.8mg/mL and 1.0mg/mL, and numbering;
3) add 3mLDNS solution to each tube (three samples in parallel per tube);
4) shaking, boiling in water bath for 10min, taking out, rapidly cooling, adding distilled water to constant volume of 25mL, and mixing;
5) zeroing the test tube without adding glucose, and measuring the absorbance of the solution in other tubes at the wavelength of 540 nm;
a glucose standard curve is plotted with glucose concentration as the x-axis and the measured absorbance as the y-axis in FIG. 1.
3.2 determination of the Activity of the Filter paper enzyme (FPase)
1) Preparation of a sample: uniformly mixing the crude enzyme solution in a test tube to be detected;
2) preparing a filter paper strip: making filter paper into a size of 1.0 multiplied by 6.0cm, and putting the filter paper into an oven to be dried, wherein the mass of the filter paper is 0.5g for later use;
3) the operation process is as follows:
taking 4 cleaned and dried 25mL test tubes with scales and plugs (one blank tube and three sample tubes), and numbering;
sample tube: respectively adding 0.5g of filter paper strip, 1mL of crude enzyme solution and 1mL of acetic acid buffer solution into three test tubes;
blank tube: adding 1mL of crude enzyme solution and 1mL of acetic acid buffer solution;
and (3) simultaneously preserving the heat of 4 test tubes in a water bath at 50 ℃ for 1h, taking out the test tubes, immediately adding a 3mL of the solution of the zinc sulfate to each test tube to stop the enzyme reaction, cooling the test tubes, then adding distilled water to a constant volume of 25mL, and fully and uniformly mixing. The absorbance of the tubes was measured at 540nm wavelength and recorded, with blank tubes being zeroed. Finding out the corresponding glucose content on the standard curve, and calculating the enzyme activity of the filter paper. Under the above conditions, 1mL of the enzyme solution consumed the substrate per hour to produce reducing sugars corresponding to 1umol of glucose, defined as one enzyme activity unit (U);
the enzyme activity of the filter paper enzyme measured by spectrophotometry is 1.03U/L.
3.3 determination of the Activity of carboxymethyl cellulase (CMCase)
1) Preparation of a sample: uniformly mixing the crude enzyme solution in a test tube to be detected;
2) preparation of sodium carboxymethylcellulose-acetic acid buffer: weighing 2g of sodium carboxymethylcellulose, heating and dissolving the sodium carboxymethylcellulose in 50mM acetic acid-sodium acetate buffer solution with pH of 5.0, cooling and then supplementing to 100 mL;
3) the operation process is as follows:
taking 4 cleaned and dried 25mL test tubes with scales and plugs (one blank tube and three sample tubes), and numbering;
sample tube: 1mL of crude enzyme solution and 1mL of sodium carboxymethylcellulose-acetic acid buffer solution are respectively added into three test tubes;
blank tube: adding 1mL of distilled water and 1mL of sodium carboxymethylcellulose-acetic acid buffer solution;
and (3) simultaneously keeping the temperature of 4 test tubes in a water bath at 50 ℃ for 30min, taking out the test tubes, immediately adding a 3mL of the solution of the zinc sulfate to each test tube to stop the enzyme reaction, cooling the test tubes, then adding distilled water to a constant volume of 25mL, and fully and uniformly mixing. The absorbance of the tubes was measured at 540nm wavelength and recorded, with blank tubes being zeroed. Finding out the corresponding glucose content on the standard curve, under the conditions, consuming the substrate by 1mL of enzyme liquid per hour to generate reducing sugar equivalent to 1umol of glucose, and defining the reducing sugar as one enzyme activity unit (U);
the enzyme activity of the carboxymethyl cellulose measured by a spectrophotometry method is 0.89U/L.
Compared with the prior art, the beneficial technical effects of the invention are embodied in the following aspects:
(1) the invention utilizes a biological method to decompose the Raoultella ornithiiRaoultella ornithinolyticaThe lignin degradation is carried out, the aromatic ring chemical bond between lignin is destroyed by enzyme, mainly manganese peroxidase, generated by microorganisms in the fermentation process, so that the lignin degradation effect is achieved, and the activity of the manganese peroxidase generated by the strain is relatively higher than that of other lignin degradation bacteria, and reaches 219.49U/L. Raoultella ornithinolyticaRaoultella ornithinolyticaWidely storedIn water, soil and plants, bioinformatics research results recently show that the strains have important enzyme coding genes in a lignin degradation pathway, such as peroxidase, Fe-Mn type superoxide dismutase, catechol 1, 2-dioxygenase, protocatechuic acid-3, 4-dioxygenase and the like, and the genes are expressed to different degrees under different culture conditions. The method has important significance for promoting the development of the lignocellulose application industry.
(2) The invention utilizes a biological method to decompose the Raoultella ornithiiRaoultella ornithinolyticaCompared with physical and chemical methods, the method for treating the lignin has the advantages of mildness, good stability, easiness in operation and good lignin degradation effect, and as shown in fig. 2 and fig. 3, the untreated corn straw has the characteristics of smooth and complete surface, regular arrangement, compactness and order and typical fibrous state, and the compact structure of the treated corn straw is damaged to form coarse, loose and porous and disordered fine fibers. The comparison shows that the Raoultella ornithinolytica is decomposedRaoultella ornithinolyticaCan damage the lignin structure in the growth process, thereby achieving a good degradation effect.
(3) The invention relates to a Raoultella ornithinolytica bacteriumRaoultella ornithinolyticaCompared with the prior treatment mode of degrading lignin by fungi, the method for degrading lignin has the advantages that the culture condition is easy to achieve, the degradation effect is ideal, the period of bacterial growth is short, and compared with fungi, peroxidase, manganese peroxidase and laccase generated by the bacteria have the excellent characteristics of better stability, wider optimal pH range, better tolerance to chloride ions and the like.
(4) The invention discovers that the lignin degrading bacteria have the function of fixing nitrogen, so that the lignin degrading bacteria can grow without adding an additional nitrogen source in the growth process, as shown in figure 4.
Drawings
FIG. 1 is a graph of a glucose standard curve.
FIG. 2 is a structural diagram of an electron microscope before corn stalks are not degraded.
FIG. 3 is the electron microscope structure diagram after the corn stalk is fermented for 3 days.
FIG. 4 is a graph of nitrogen-free medium growth.
Detailed Description
The invention will now be further described with reference to specific examples.
The following examples illustrate the sources of the raw materials used as follows:
the strain is Raoultella ornithinolyticaRaoultella ornithinolyticaPurchased from Guangdong province strain preservation center, the enzyme activity of the manganese peroxidase reaches 219.49U/L; LB solid medium purchased from Aladdin Biochemical agent shop; LB liquid medium purchased from Aladdin Biochemical agent shop; the inorganic salt liquid culture medium was purchased from the alatin biochemical reagent store.
Example 1
The biodegradation operation of the lignin comprises the following steps:
(1) culturing the strains
Raoultella ornithinolyticaRaoultella ornithinolyticaInoculating the slant strains in a LB solid culture medium in a loop, and culturing at 28 ℃ for 24-72 h to obtain activated strains;
(2) preparation of the bacterial suspension
Inoculating the activated strain on an LB liquid culture medium, wherein the inoculation amount is 2%, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a fermentation liquid;
inoculating 2ml of fermentation liquor into 100ml of LB liquid culture medium, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a bacterial suspension;
(3) treatment of lignin materials
The lignin material of the embodiment is corn stalks;
drying and crushing corn straws at the temperature of 45 ℃; sieving with 30-40 mesh sieve, and oven drying at 45 deg.C to obtain corn stalk powder;
(4) degradation of
Adding 10g of corn straw powder into 20ml of liquid inorganic salt culture medium, uniformly stirring, sterilizing, and cooling to room temperature to obtain a fermentation product; adding 5% of the bacterial suspension in the step (2) into the fermentation product, uniformly stirring, and culturing at 28 ℃ for 2-3 days to obtain the degradation product.
Detecting degradation products by using a Van Soest PJ lignocellulose quantitative method, referring to fig. 2 and fig. 3, comparing that a plurality of pores are generated on the surface of the degraded corn straw, and the degradation rate of the corn straw lignin reaches 22.76%.
Example 2
The biodegradation operation of the lignin comprises the following steps:
(1) culturing the strains
Raoultella ornithinolyticaRaoultella ornithinolyticaInoculating the slant strains in a LB solid culture medium in a loop, and culturing at 28 ℃ for 24-72 h to obtain activated strains;
(2) preparation of the bacterial suspension
Inoculating the activated strain on an LB liquid culture medium, wherein the inoculation amount is 2%, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a fermentation liquid;
inoculating 2ml of fermentation liquor into 100ml of LB liquid culture medium, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a bacterial suspension;
(3) treatment of lignin materials
The lignin material of the embodiment is wheat straw;
drying wheat straw at 45 deg.C, and pulverizing; sieving with 30-40 mesh sieve, and oven drying at 45 deg.C to obtain wheat straw powder;
(4) degradation of
Adding 10g of wheat straw powder into 20ml of liquid inorganic salt culture medium, uniformly stirring, sterilizing, and cooling to room temperature to obtain a fermented product; adding 5% of the bacterial suspension in the step (2) into the fermentation product, uniformly stirring, and culturing at 28 ℃ for 2-3 days to obtain the degradation product.
The degradation products are detected by a Van Soest PJ lignocellulose quantitative method, and the degradation rate of the lignin in the wheat straws reaches 23.58%.
Example 3
The biodegradation operation of the lignin comprises the following steps:
(1) culturing the strains
Raoultella ornithinolyticaRaoultella ornithinolyticaSlant strain is inoculated on LB in a loopCulturing the solid culture medium at 28 ℃ for 24-72 h to obtain an activated strain;
(2) preparation of the bacterial suspension
Inoculating the activated strain on an LB liquid culture medium, wherein the inoculation amount is 2%, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a fermentation liquid;
inoculating 2ml of fermentation liquor into 100ml of LB liquid culture medium, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a bacterial suspension;
(3) treatment of lignin materials
The lignin material of this example was apple shoots;
drying apple branches at 45 ℃, and crushing; sieving with 30-40 mesh sieve, and oven drying at 45 deg.C to obtain apple branch powder;
(4) degradation of
Adding 10g of apple branch powder into 20ml of liquid inorganic salt culture medium, uniformly stirring, sterilizing, and cooling to room temperature to obtain a fermented product; adding 5% of the bacterial suspension in the step (2) into the fermentation product, uniformly stirring, and culturing at 28 ℃ for 2-3 days to obtain the degradation product.
The degradation product is detected by a quantitative method of lignocellulose of Van Soest PJ, and the degradation rate of the lignin of the apple branches reaches 20.06 percent.
Example 4
The biodegradation operation of the lignin comprises the following steps:
(1) culturing the strains
Raoultella ornithinolyticaRaoultella ornithinolyticaInoculating the slant strains in a LB solid culture medium in a loop, and culturing at 28 ℃ for 24-72 h to obtain activated strains;
(2) preparation of the bacterial suspension
Inoculating the activated strain on an LB liquid culture medium, wherein the inoculation amount is 2%, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a fermentation liquid;
inoculating 2ml of fermentation liquor into 100ml of LB liquid culture medium, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a bacterial suspension;
(3) treatment of lignin materials
The lignin material of the embodiment is rice straw;
drying and crushing rice straws at the temperature of 45 ℃; sieving with 30-40 mesh sieve, and oven drying at 45 deg.C to obtain rice straw powder;
(4) degradation of
Adding 10g of rice straw powder into 20ml of liquid inorganic salt culture medium, uniformly stirring, sterilizing, and cooling to room temperature to obtain a fermentation product; adding 5% of the bacterial suspension in the step (2) into the fermentation product, uniformly stirring, and culturing at 28 ℃ for 2-3 days to obtain the degradation product.
The degradation products are detected by a Van Soest PJ lignocellulose quantitative method, and the degradation rate of the rice straw lignin reaches 23.01 percent.
Example 5
The biodegradation operation of the lignin comprises the following steps:
(1) culturing the strains
Raoultella ornithinolyticaRaoultella ornithinolyticaInoculating the slant strains in a LB solid culture medium in a loop, and culturing at 28 ℃ for 24-72 h to obtain activated strains;
(2) preparation of the bacterial suspension
Inoculating the activated strain on an LB liquid culture medium, wherein the inoculation amount is 2%, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a fermentation liquid;
inoculating 2ml of fermentation liquor into 100ml of LB liquid culture medium, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a bacterial suspension;
(3) treatment of lignin materials
The lignin material of the embodiment is rice hulls;
drying and crushing rice hulls at the temperature of 45 ℃; sieving with 30-40 mesh sieve, and oven drying at 45 deg.C to obtain rice hull powder;
(4) degradation of
Adding 10g of rice hull powder into 20ml of liquid inorganic salt culture medium, uniformly stirring, sterilizing, and cooling to room temperature to obtain a fermented product; adding 5% of the bacterial suspension in the step (2) into the fermentation product, uniformly stirring, and culturing at 28 ℃ for 2-3 days to obtain the degradation product.
The degradation products are detected by a quantitative method of lignocellulose of Van Soest PJ, and the degradation rate of the rice hull lignin reaches 21.59 percent.
Claims (3)
1. A method for biodegradation of lignin, characterized by the following operating steps:
(1) culturing the strains
Raoultella ornithinolyticaRaoultella ornithinolyticaInoculating the slant strains in a LB solid culture medium in a loop, and culturing at 28 ℃ for 24-72 h to obtain activated strains;
(2) preparation of the bacterial suspension
Inoculating the activated strain on an LB liquid culture medium, wherein the inoculation amount is 2-4%, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a fermentation liquid;
inoculating 2ml of fermentation liquor into 100ml of LB liquid culture medium, and culturing for 2-3 days on a shaking table at the temperature of 28 ℃ and 180r/min to obtain a bacterial suspension;
(3) treatment of lignin materials
Drying and crushing the lignin material at the temperature of 45 ℃; sieving with 30-40 mesh sieve, and oven drying at 45 deg.C to obtain powder; the lignin material is crop straws or plant straws;
(4) degradation of
Adding 10g of powder into 20ml of liquid inorganic salt culture medium, uniformly stirring, sterilizing, and cooling to room temperature to obtain a fermentation product; adding 5% of the bacterial suspension in the step (2) into the fermentation product, uniformly stirring, and culturing at 28 ℃ for 2-3 days to obtain a degradation product; the degradation rate of lignin in the degradation product reaches 20% -25%, and the surface of the degradation product after degradation is in a loose and porous structure state.
2. A method of biodegradation of lignin according to claim 1, characterized in that: the Raoultella ornithinolyticaRaoultella ornithinolyticaThe enzyme activity of the manganese peroxidase is 219.49U/L.
3. A method of biodegradation of lignin according to claim 1, characterized in that: the lignin material is corn straw or wheat straw or apple branches or rice straw or rice hull.
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| CN116285405A (en) * | 2022-10-20 | 2023-06-23 | 南京高新工大生物技术研究院有限公司 | Edible fungus fermentation process for secreting laccase and application thereof |
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| CN113777064A (en) * | 2021-09-10 | 2021-12-10 | 合肥工业大学 | Kit and method for detecting in-vitro antioxidant activity of xylaria intracellular polysaccharide |
| CN113777064B (en) * | 2021-09-10 | 2022-04-01 | 合肥工业大学 | Kit and method for detecting in-vitro antioxidant activity of xylaria intracellular polysaccharide |
| CN116285405A (en) * | 2022-10-20 | 2023-06-23 | 南京高新工大生物技术研究院有限公司 | Edible fungus fermentation process for secreting laccase and application thereof |
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