CN112831480A - Method for producing laccase by solid-state fermentation of pseudo-ginseng residue by lucid ganoderma - Google Patents
Method for producing laccase by solid-state fermentation of pseudo-ginseng residue by lucid ganoderma Download PDFInfo
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Abstract
The invention discloses a method for producing laccase by utilizing solid state fermentation of pseudo-ginseng residue. The invention is realized by the following steps: (1) drying, crushing and screening the pseudo-ginseng residue; (2) uniformly mixing the pretreated pseudo-ginseng residue, a proper amount of nitrogen source, water, copper sulfate, magnesium sulfate, monopotassium phosphate, tween-80 and veratryl alcohol according to a certain proportion; (3) carrying out steam sterilization treatment on the uniformly mixed materials; (4) inoculating at least one ganoderma lucidum strain on the sterilized material for solid state fermentation; (5) drying the fermentation culture to obtain the ganoderma laccase. By adopting the method, the pseudo-ginseng residue can be converted into laccase. The method utilizes the pseudo-ginseng residue as a resource, changes waste into valuable, reduces the pollution to the environment, and provides the laccase with low cost for the environmental protection industry.
Description
Technical Field
The invention belongs to the field of industrial waste recycling, and relates to a method for producing laccase by using pseudo-ginseng residue.
Background
With the rapid development of the traditional Chinese medicine industry in China, the waste amount of the traditional Chinese medicine residues in various large traditional Chinese medicine pharmaceutical factories in China is increasing day by day. The traditional treatment methods such as incineration, landfill and fixed area stacking are often adopted for the treatment of the traditional Chinese medicine residues, which not only needs to invest a large amount of capital, but also causes resource waste and serious environmental pollution. Therefore, how to reasonably dispose and utilize the herb residue is an important problem which cannot be avoided in the process of modernizing the Chinese herbs. At present, the approaches for recycling Chinese herb residues mainly comprise fermentation production of protein feed, use as a feed additive, preparation of organic fertilizer, preparation of active carbon, pyrolysis for gas production and the like.
Pseudo-ginseng is a traditional rare Chinese medicinal material in China and is also a plant used as both medicine and food, the root and the stem of the pseudo-ginseng can be used as a medicine, the main medicinal component of the pseudo-ginseng is saponin, and pseudo-ginseng slag after extracting effective components is generally treated as waste. The panax notoginseng residue after the total saponin extraction still contains nutrient substances such as starch, cellulose, protein, amino acid, various trace elements and the like, and the panax notoginseng residue is further developed and utilized as a fermentation substrate, so that waste can be changed into valuable, and the utilization efficiency of resources is improved. The Chinese invention patent with the application number of 201010232573.9 discloses 'a white spirit prepared by fermenting and distilling pseudo-ginseng residue and a method thereof'; the Chinese invention patent with the application number of 201110119291.2 discloses a method for producing protein feed by fermenting pseudo-ginseng residue; the Chinese invention patent with the application number of 201210196021.6 discloses a method for producing a trichoderma biocontrol agent by utilizing the fermentation of pseudo-ginseng residue; the Chinese invention patent with the application number of 201310437721.4 discloses a method for producing medicinal mycoplasm by using mixed fermentation of pseudo-ginseng residue; the Chinese patent with application number of 201710559473.9 discloses a method for producing red pigment by using pseudo-ginseng slag.
Laccase is a copper-containing polyphenol redox protein. Laccase can take molecular oxygen as a unique cofactor, and oxidize refractory compounds into water and other small molecular substances, so that the laccase is an environment-friendly biocatalyst. Laccase is found in various fungi and plant secretions at present, few bacteria and insects also produce laccase, and other laccases are also found in the blood plasma of some animals. Laccase has a wide application prospect in the field of environmental protection: the laccase can be used for manufacturing biosensors for environmental monitoring or for decolorizing dye wastewater, and can also be used for repairing organically polluted soil due to the strong degradation capability of the laccase to pesticides, herbicides and petroleum hydrocarbons. The invention takes the pseudo-ginseng residue as the fermentation substrate to carry out solid state fermentation to produce laccase, thereby realizing the resource utilization of the pseudo-ginseng residue and reducing the production cost of the laccase.
Disclosure of Invention
The invention aims to provide a method for producing laccase by using pseudo-ginseng residue as a raw material through a ganoderma lucidum solid state fermentation process. The invention relates to a method for producing laccase by solid-state fermentation of pseudo-ginseng residue by utilizing lucid ganoderma, which sequentially comprises the following process steps:
(1) pretreatment of pseudo-ginseng residue
Drying wet pseudo-ginseng residue, pulverizing, sieving, and selecting pseudo-ginseng residue with particle size of 0.1-0.3mm for subsequent process;
(2) compounding and compounding
Uniformly mixing the pretreated pseudo-ginseng residue, a proper amount of nitrogen source, water, copper sulfate, magnesium sulfate, monopotassium phosphate, tween-80 and veratryl alcohol, wherein the specific batching mode is as follows:
100 parts of pretreated pseudo-ginseng residue, 2-5 parts of nitrogen source, 0.02-0.04 part of copper sulfate, 0.1-0.2 part of monopotassium phosphate, 0.1-0.2 part of magnesium sulfate, 0.02-0.04 part of tween-80, 0.03-0.06 part of veratryl alcohol, wherein the mass ratio of solid material to water is 1:1.0-1: 2.0; when in material preparation, adding the nitrogen source, the copper sulfate, the monopotassium phosphate, the magnesium sulfate, the Tween-80 and the veratryl alcohol into water, uniformly mixing, and then uniformly mixing the prepared solution and the pseudo-ginseng residue;
(3) sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
(4) fermentation of
Performing solid state fermentation on the sterilized mixture, inoculating strains according to 20-25% of inoculation amount, and performing fermentation conditions: the pressure is normal pressure, the temperature is 27-29 ℃, and the fermentation period is 11-13 days;
(5) drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
In the method, the nitrogen source used in the material preparation is one or more of yeast powder, corn protein powder and peptone;
in the method, the strains used in the step 4 comprise ganoderma lucidum (Ganoderma lucidum (Fr.))Ganoderma lucidum5.896), Ganoderma Applanatum (Ganoderma applanatum)Ganoderma applanatum 3.15579), tropical Ganoderma (Ganoderma lucidum (Fr.) karst: (Ganoderma lucidum karst)Ganoderma tropicum3.7533), Ganoderma weberianum (Fr.) karst.)Ganoderma weberianum 5.1572) is used.
The invention has the following beneficial effects:
(1) by adopting the method, the pseudo-ginseng residue is converted into the laccase product, so that the resource utilization of the pseudo-ginseng residue can be realized, and the production cost of the laccase is reduced;
(2) compared with liquid fermentation, the solid fermentation process adopted by the invention has the advantages of simple production equipment, low energy consumption, less generated waste residue and waste water, small environmental pollution and the like, and is beneficial to production enterprises to realize energy conservation, emission reduction and clean production;
(3) the laccase product produced by the method is solid laccase, is convenient to store and transport, has small enzyme activity loss in the storage process, and is convenient to store for a long time.
Detailed Description
Example 1 this example uses radix Notoginseng residue as raw material, Ganoderma (Ganoderma lucidum)Ganoderma lucidum5.896) is used as a strain to produce laccase by solid state fermentation. The method comprises the following specific steps:
1. pretreatment of pseudo-ginseng residue
Firstly, drying the pseudo-ginseng dregs in an oven at the temperature of 60 ℃ under normal pressure for the limited time that the pseudo-ginseng dregs are easy to crush. Pulverizing dried Notoginseng radix residue, sieving, and selecting Notoginseng radix residue with particle size of 0.1-0.3mm for subsequent process;
2. compounding and compounding
Firstly, 1.0 g of yeast powder, 1.0 g of urea, 0.04 g of copper sulfate, 0.2g of monopotassium phosphate, 0.2g of magnesium sulfate, 0.02 g of tween-80 and 0.03g of veratryl alcohol are added into 200 g of water and mixed uniformly, and then the solution and 100 g of pretreated pseudo-ginseng residue powder are mixed uniformly;
3. sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
4. fermentation of
Inoculating Ganoderma into the sterilized mixtureGanoderma lucidum5.896), the inoculation amount is 20%, the fermentation conditions are as follows: the fermentation period is 11 days, the pressure is normal pressure, and the temperature is 27 ℃;
5. drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
Example 2 this example uses radix Notoginseng residue as raw material, Ganoderma Applanatum (Ganoderma Applanatum)Ganoderma applanatum 3.15579) is used as a strain to produce laccase by solid state fermentation. The method comprises the following specific steps:
1. pretreatment of pseudo-ginseng residue
Firstly, drying the pseudo-ginseng dregs in an oven at the temperature of 60 ℃ under normal pressure for the limited time that the pseudo-ginseng dregs are easy to crush. Pulverizing dried Notoginseng radix residue, sieving, and selecting Notoginseng radix residue with particle size of 0.1-0.3mm for subsequent process;
2. compounding and compounding
Firstly, adding 4.0 g of peptone, 1.0 g of yeast powder, 0.02 g of copper sulfate, 0.1 g of monopotassium phosphate, 0.1 g of magnesium sulfate, 0.04 g of tween-80 and 0.06 g of veratryl alcohol into 100 g of water, uniformly mixing, and then uniformly mixing the solution and 100 g of pretreated pseudo-ginseng residue powder;
3. sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
4. fermentation of
Inoculating Ganoderma Applanatum (Ganoderma applanatum) into the sterilized mixtureGanoderma applanatum 3.15579), the inoculation amount is 20%, the fermentation conditions are as follows: the fermentation period is 12 days, and the fermentation period is 12 days,the pressure is normal pressure and the temperature is 29 ℃;
5. drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
Example 3 this example uses radix Notoginseng residue as raw material, and Ganoderma lucidum (C. tropicalis)Ganoderma tropicum3.7533) is used as a strain to produce laccase by solid state fermentation. The method comprises the following specific steps:
1. pretreatment of pseudo-ginseng residue
Firstly, drying the pseudo-ginseng dregs in an oven at the temperature of 60 ℃ under normal pressure for the limited time that the pseudo-ginseng dregs are easy to crush. Pulverizing dried Notoginseng radix residue, sieving, and selecting Notoginseng radix residue with particle size of 0.1-0.3mm for subsequent process;
2. compounding and compounding
Firstly, adding 4.0 g of yeast powder, 0.03g of copper sulfate, 0.10 g of monopotassium phosphate, 0.15 g of magnesium sulfate, 0.03g of tween-80 and 0.05 g of veratryl alcohol into 180 g of water, uniformly mixing, and then uniformly mixing the solution and 100 g of pretreated pseudo-ginseng residue powder;
3. sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
4. fermentation of
Inoculating Ganoderma tropicalis (Ganoderma lucidum (Fr.) karst) into the sterilized mixtureGanoderma tropicum3.7533) was fermented, the inoculum size was 25%, the fermentation conditions: the fermentation period is 13 days, the pressure is normal pressure, and the temperature is 28 ℃;
5. drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
Example 4 this example uses radix Notoginseng residue as raw material, and Ganoderma weberianum (A. weberianum, B. weberianum, C. weberianuGanoderma weberianum 5.1572) is used as a strain to produce laccase by solid state fermentation. The method comprises the following specific steps:
1. pretreatment of pseudo-ginseng residue
Firstly, drying the pseudo-ginseng dregs in an oven at the temperature of 60 ℃ under normal pressure for the limited time that the pseudo-ginseng dregs are easy to crush. Pulverizing dried Notoginseng radix residue, sieving, and selecting Notoginseng radix residue with particle size of 0.1-0.3mm for subsequent process;
2. compounding and compounding
Firstly, 3.5 g of corn protein powder, 0.04 g of copper sulfate, 0.10 g of monopotassium phosphate, 0.15 g of magnesium sulfate, 0.02 g of tween-80 and 0.03g of veratryl alcohol are added into 160 g of water and uniformly mixed, and then the solution and 100 g of pretreated pseudo-ginseng residue powder are uniformly mixed;
3. sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
4. fermentation of
Inoculating Ganoderma weberianum (Wei Bo) into the sterilized mixtureGanoderma weberianum 5.1572), the inoculation amount is 20%, the fermentation conditions are as follows: the fermentation period is 12 days, the pressure is normal pressure, and the temperature is 28 ℃;
5. drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
Example 5 this example uses radix Notoginseng residue as raw material, Ganoderma (Ganoderma lucidum and/or Ganoderma sinense)Ganoderma lucidum5.896/tropical Ganoderma (Ganoderma lucidum and/or Ganoderma sinense)Ganoderma tropicum3.7533) is used as a strain to produce laccase by mixed solid fermentation. The method comprises the following specific steps:
1. pretreatment of pseudo-ginseng residue
Firstly, drying the pseudo-ginseng dregs in an oven at the temperature of 60 ℃ under normal pressure for the limited time that the pseudo-ginseng dregs are easy to crush. Pulverizing dried Notoginseng radix residue, sieving, and selecting Notoginseng radix residue with particle size of 0.1-0.3mm for subsequent process;
2. compounding and compounding
Firstly, adding 4.0 g of yeast powder, 0.04 g of copper sulfate, 0.10 g of monopotassium phosphate, 0.10 g of magnesium sulfate, 0.04 g of tween-80 and 0.06 g of veratryl alcohol into 190 g of water, uniformly mixing, and then uniformly mixing the solution and 100 g of pretreated pseudo-ginseng residue powder;
3. sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
4. fermentation of
Fermenting in asynchronous inoculation mode, inoculating Ganoderma (Ganoderma lucidum) into sterilized mixtureGanoderma lucidum5.896), fermenting for 24 hr, inoculating Ganoderma lucidum (Zygorhiza japonica (Zygorhiza) to the fermentation tankGanoderma tropicum3.7533 fermenting, and mixing with Ganoderma (Ganoderma lucidum and/or Ganoderma lucidum)Ganoderma lucidum5.896 and tropical Ganoderma lucidumGanoderma tropicum3.7533) inoculation ratio of 1:1, total inoculation amount of 20%, fermentation conditions: the fermentation period is 12 days, the pressure is normal pressure, and the fermentation temperature is 30 ℃;
5. drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
Example 6 this example uses radix Notoginseng residue as raw material, Ganoderma Applanatum (Ganoderma Applanatum)Ganoderma applanatum 3.15579/Ganoderma weberianum (Fr.) Sing.))Ganoderma weberianum 5.1572) is used as a strain to produce laccase by mixed solid fermentation. The method comprises the following specific steps:
1. pretreatment of pseudo-ginseng residue
Firstly, drying the pseudo-ginseng dregs in an oven at the temperature of 60 ℃ under normal pressure for the limited time that the pseudo-ginseng dregs are easy to crush. Pulverizing dried Notoginseng radix residue, sieving, and selecting Notoginseng radix residue with particle size of 0.1-0.3mm for subsequent process;
2. compounding and compounding
Firstly, adding 5.0 g of peptone, 0.04 g of copper sulfate, 0.15 g of monopotassium phosphate, 0.15 g of magnesium sulfate, 0.04 g of tween-80 and 0.05 g of veratryl alcohol into 185 g of water, uniformly mixing, and then uniformly mixing the solution and 100 g of pretreated pseudo-ginseng residue powder;
3. sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
4. fermentation of
Fermenting in asynchronous inoculation mode, inoculating Ganoderma Applanatum into sterilized mixtureGanoderma applanatum 3.15579), then inoculating weber after fermenting for 24 hoursGanoderma lucidum (Ganoderma lucidum)Ganoderma weberianum 5.1572 fermenting, and mixing with Ganoderma Applanatum (Ganoderma applanatum)Ganoderma applanatum 3.15579 and Ganoderma weberianum (Ganoderma lucidum and/or Ganoderma sinense)Ganoderma weberianum 5.1572) at a ratio of 2:1, a total inoculum size of 21%, fermentation conditions: the fermentation period is 13 days, the pressure is normal pressure, and the fermentation temperature is 29 ℃;
5. drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
Example 7 this example uses radix Notoginseng residue as raw material, Ganoderma (Ganoderma lucidum and/or Ganoderma sinense)Ganoderma lucidum5.896/tropical Ganoderma (Ganoderma lucidum and/or Ganoderma sinense)Ganoderma tropicum3.7533/Ganoderma weberianum (Fr.) Sing.))Ganoderma weberianum 5.1572) is used as a strain to produce laccase by mixed solid fermentation. The method comprises the following specific steps:
1. pretreatment of pseudo-ginseng residue
Firstly, drying the pseudo-ginseng dregs in an oven at the temperature of 60 ℃ under normal pressure for the limited time that the pseudo-ginseng dregs are easy to crush. Pulverizing dried Notoginseng radix residue, sieving, and selecting Notoginseng radix residue with particle size of 0.2-0.3mm for subsequent process;
2. compounding and compounding
Firstly, 4.5 g of corn protein powder, 0.03g of copper sulfate, 0.15 g of monopotassium phosphate, 0.20 g of magnesium sulfate, 0.04 g of tween-80 and 0.04 g of veratryl alcohol are added into 200 g of water and uniformly mixed, and then the solution and 100 g of pretreated pseudo-ginseng residue powder are uniformly mixed;
3. sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
4. fermentation of
Fermenting by synchronous inoculation, and inoculating Ganoderma (Ganoderma lucidum) into the sterilized mixtureGanoderma lucidum5.896), tropical Ganoderma (Ganoderma lucidum (Fr.) karst: (Ganoderma lucidum karst)Ganoderma tropicum3.7533), Ganoderma weberianum (Fr.) karst.)Ganoderma weberianum 5.1572 fermenting, and mixing with Ganoderma (Ganoderma lucidum and/or Ganoderma lucidum)Ganoderma lucidum5.896), tropical Ganoderma (Ganoderma lucidum (Fr.) karst: (Ganoderma lucidum karst)Ganoderma tropicum3.7533), Ganoderma weberianum (Fr.) karst.)Ganoderma weberianum 5.1572) at a ratio of 1:1:1, with a total inoculum size of 24%, fermentation conditions: the fermentation period is 12 days, the pressure is normal pressure, and the fermentation temperature is 28 ℃;
5. drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
Claims (4)
1. A method for producing laccase by taking pseudo-ginseng residue as a raw material and performing solid state fermentation on lucid ganoderma is characterized by comprising the following specific process steps:
(1) pretreatment of pseudo-ginseng residue
Drying wet pseudo-ginseng residue, pulverizing, sieving, and selecting pseudo-ginseng residue with particle size of 0.1-0.3mm for subsequent process;
(2) compounding and compounding
Uniformly mixing the pretreated pseudo-ginseng residue, a proper amount of nitrogen source, water, copper sulfate, magnesium sulfate, monopotassium phosphate, tween-80 and veratryl alcohol, wherein the specific batching mode is as follows:
100 parts of pretreated pseudo-ginseng residue, 2-5 parts of nitrogen source, 0.02-0.04 part of copper sulfate, 0.1-0.2 part of monopotassium phosphate, 0.1-0.2 part of magnesium sulfate, 0.02-0.04 part of tween-80, 0.03-0.06 part of veratryl alcohol, wherein the mass ratio of solid material to water is 1:1.0-1: 2.0; when in material preparation, adding the nitrogen source, the copper sulfate, the monopotassium phosphate, the magnesium sulfate, the Tween-80 and the veratryl alcohol into water, uniformly mixing, and then uniformly mixing the prepared solution and the pseudo-ginseng residue;
(3) sterilization
Sterilizing the mixture by adopting a high-pressure steam sterilization method: steam pressure is 1atm, temperature is 121 ℃, and time is at least 30 minutes;
(4) fermentation of
Performing solid state fermentation on the sterilized mixture, inoculating strains according to 20-25% of inoculation amount, and performing fermentation conditions: the pressure is normal pressure, the temperature is 27-29 ℃, and the fermentation period is 11-13 days;
(5) drying
And drying the fermentation culture for 72 hours in an oven at 60 ℃ under normal pressure to obtain the laccase product.
2. The method of claim 1, wherein the nitrogen source used in the compounding is one or more selected from yeast powder, corn gluten meal, and peptone.
3. The method according to claim 1, wherein the fermentation is carried out using a strain selected from the group consisting of Ganoderma lucidum (Fr.)), (Ganoderma lucidum (Fr.))Ganoderma lucidum5.896), Ganoderma Applanatum (Ganoderma applanatum)Ganoderma applanatum 3.15579), tropical Ganoderma (Ganoderma lucidum (Fr.) karst: (Ganoderma lucidum karst)Ganodermatropicum3.7533), Ganoderma weberianum (Fr.) karst.)Ganoderma weberianum 5.1572) is used.
4. A laccase enzyme obtainable by the process of any one of claims 1 to 3.
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| CN113046334A (en) * | 2021-04-14 | 2021-06-29 | 成都信息工程大学 | Method for producing laccase by mixed-strain solid-state fermentation of pseudo-ginseng residue |
| CN114015663A (en) * | 2021-11-25 | 2022-02-08 | 成都信息工程大学 | Composition for producing laccase, fermentation substrate and method for producing laccase by using fermentation substrate |
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| CN114015663A (en) * | 2021-11-25 | 2022-02-08 | 成都信息工程大学 | Composition for producing laccase, fermentation substrate and method for producing laccase by using fermentation substrate |
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