Fermentation medium for improving enzyme activity of lignin degrading enzyme produced by white-rot fungi and application of fermentation medium

Abstract

The invention relates to the technical field of microorganisms, in particular to a fermentation medium for improving enzyme activity of lignin degrading enzyme produced by white-rot fungi and application thereof.The fermentation medium comprises a culture container and a controller, the culture container is provided with a driving part, and the driving part is used for driving the culture container to rotate; the controller is used for dividing a fermentation period based on the fermentation time, and the fermentation period comprises a hypha expansion period, an environment coordination period and a fermentation stable period; in the hypha expansion period, the controller controls the driving piece to enable the rotating speed of the culture container to be 400-800 r / min, so that the hypha is cut off by shearing force generated by rotation, and a new growing point is formed; in the environment coordination period, the controller controls the driving piece to enable the rotating speed of the culture container to be 300-400 r / min; and the fermentation stable phase controller controls the driving piece to enable the rotating speed of the culture container to be 100-200r / min. With the adoption of the technical scheme, the rotating speed of the fermentation culture medium is dynamically controlled, so that the effects of dispersing hyphae, cutting the hyphae and damaging the mycelia generated by shearing force of rotation can be regulated in a targeted manner in each hypha growth stage, and the improvement of enzyme activity is promoted.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi and its application. Background Art

[0002] White rot fungi are a type of filamentous fungi with important ecological and economic value. They can cause white rot of wood and have a selective degradation effect on lignocellulose. There are many types of white rot fungi, including Pleurotus, Phanerochaete, and Fomes. White rot fungi mainly belong to the Basidiomycetes class, and only a few belong to the Ascomycetes class. As filamentous fungi, white rot fungi grow following a branching pattern. The tubular hyphae growing from spores elongate at the tip, and at the same time, new branches are formed along the hyphae. The branches continue and form a porous three-dimensional hyphal network, thus forming the mycelium. The main components of lignocellulose are cellulose, hemicellulose, and lignin. The main bottleneck restricting the comprehensive utilization of straw with high added value is the effective degradation of lignocellulose. So far, the methods for lignin degradation mainly include physical methods, chemical methods, physicochemical methods, and biodegradation methods. Among them, biodegradation has become a research hotspot as a green and environmentally friendly method for lignin treatment. Biodegradation mainly includes microbial degradation method and enzyme degradation method. However, the microbial degradation method has problems such as long time (generally 21 - 60 days) and demanding growth conditions (sterile, excluding interference from external miscellaneous bacteria), thus limiting its commercial application in actual production. Therefore, researchers have shifted their focus to enzyme degradation. Therefore, improving the activities of microbial cellulose, hemicellulose, and lignin-degrading enzymes has become the research focus.

[0003] In the prior art, for example, Patent Publication No. CN109161535A discloses a fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi and its application. By adding manganese ions and / or copper ions to the initial fermentation medium, the activity of the degrading enzyme is enhanced. Patent Publication No. CN109055335B discloses a fermentation medium for improving the enzyme activity of enzymes produced by white rot fungi and its application. Using ramie, peptone, Tween, and urea as new components, combined with other inorganic salts, a medium suitable for the fermentation of white rot fungi to produce enzymes is formed, which can significantly improve the enzyme activities of cellulase, hemicellulase, and lignin-degrading enzymes produced by white rot fungi. However, there is still a need to study further ways to enhance the activity of the degrading enzyme.

[0004] Enzyme activity refers to the ability of an enzyme to catalyze a specific chemical reaction, usually expressed by the rate of substrate consumption or product formation per unit time. Therefore, an increase in the enzyme production amount usually leads to an increase in enzyme activity, and the improvement of enzyme activity can be further promoted by increasing the enzyme production amount. Summary of the Invention

[0005] To solve the above problems, the present invention provides a fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi and its application. By dynamically controlling the rotation speed of the fermentation medium, the shearing force generated by targeted rotation during each mycelial growth stage can disperse mycelia, cut mycelia, and cause injury to mycelia, thereby promoting the improvement of enzyme activity.

[0006] To achieve the above object, the technical solution of the present invention is as follows: A fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi, comprising a culture container and a controller. The culture container is used to culture white rot fungal mycelia and provide a liquid fermentation environment. The culture container is provided with a driving member for driving the culture container to rotate self.

[0007] The controller is used to divide the fermentation cycle based on the fermentation time. The fermentation cycle includes a mycelial expansion period, an environmental coordination period, and a fermentation stability period.

[0008] During the mycelial expansion period, the controller controls the driving member to make the rotation speed of the culture container 400 - 800 r / min, so that the shearing force generated by the rotation cuts the mycelia to form new growth points.

[0009] During the environmental coordination period, the controller controls the driving member to make the rotation speed of the culture container 300 - 400 r / min, so that the shearing force generated by the rotation improves the distribution of oxygen and nutrients.

[0010] During the fermentation stability period, the controller controls the driving member to make the rotation speed of the culture container 100 - 200 r / min, reducing the injury to the mycelia caused by the shearing force generated by the rotation.

[0011] Adopting the above solution has the following beneficial effects:

[0012] 1. In this solution, the culture container can provide an environment for culturing white rot fungal mycelia and a fermentation environment. The liquid fermentation environment enables the shearing force during the rotation of the culture container to act on the internal fermentation environment.

[0013] 2. In this solution, the controller divides the fermentation cycle according to the fermentation time. During the fermentation process, the fermentation speed is affected by the change in the number of bacteria. In the early stage, the number of bacteria is much smaller than the amount of nutrients, and the bacteria reproduction conditions are limited by the small base number. In the middle stage, the number of bacteria is moderate and they reproduce normally. In the late stage, the amount of nutrients decreases while the number of bacteria is large. Therefore, the fermentation cycle is divided into a mycelial expansion period, an environmental coordination period, and a fermentation stability period.

[0014] By different rotation speeds, the shearing force on the mycelia in the culture container can be adjusted. In the early stage of fermentation, due to rich nutrients and a small amount of bacteria, a larger shearing force can be used to damage the mycelia to a certain extent at this time, thereby forming new growth points to promote the growth of bacteria and induce more mycelia.

[0015] After a certain base number of bacteria is present, the shear force is reduced, and an appropriate shear force is used to ensure uniform distribution of nutrients and hyphae, reducing the probability of excessive local bacterial colonies becoming viscous.

[0016] After the number of hyphae is large enough and stable in the later stage, the rotation speed is reduced, thereby reducing the injury to the mycelium caused by the shear force, reducing the artificial mycelium lethality rate and reducing the energy consumption of the driver.

[0017] Furthermore, the fermentation environment includes glucose 0.8 g / L - 1.2 g / L, peptone 0.8 g / L - 1.2 g / L, lignin-containing carrier 40 g / L, inducer 0.1 mmol / L - 1 mmol / L, MgSO4 1.2 g / L - 1.5 g / L, KH2PO4 1 g / L - 1.2 g / L, CaCl2 0.1 g / L - 0.12 g / L, and the balance is water.

[0018] Beneficial effects: A basic culture environment is constructed by glucose, peptone, MgSO4, KH2PO4, CaCl2, and water, and the lignin-containing carrier is the target for fermentation. White rot fungi secrete corresponding degrading enzymes to degrade the lignin in the carrier. The inducer can additionally enhance the enzyme activity, thereby promoting fermentation.

[0019] Furthermore, the lignin-containing carrier includes crop straw and wheat bran.

[0020] Beneficial effects: Crop straw includes the straw of common crops such as corn and rice, and wheat bran is the main by-product in the wheat processing process. This type of lignin carrier has a large application space and source, facilitating application and expansion.

[0021] Furthermore, the inducer includes one or more of ferulic acid, dimethylaniline, vanillic acid, cinnamic acid, guaiacol, veratryl alcohol, pyrogallol, syringic acid, and gallic acid.

[0022] Beneficial effects: The inducer includes aromatic inducers. Ferulic acid, dimethylaniline, vanillic acid, cinnamic acid, guaiacol, veratryl alcohol, pyrogallol, syringic acid, and gallic acid are all aromatic inducers, and aromatic inducers can increase the enzyme activity in fermentation.

[0023] Furthermore, the inducer also includes copper ion and manganese ion salt solutions.

[0024] Beneficial effects: Metal ions affect the activity of lignin degradation-related enzymes. When the concentration is appropriate, the effect of copper in promoting Lac secretion is very obvious. Beyond the critical concentration, an inhibitory effect will be shown.

[0025] Furthermore, the inducer also includes Tween 80 and TritonX-100.

[0026] Beneficial effects: Surfactants can promote the growth of white rot fungi and increase the activity of lignin-degrading enzymes. Both Tween 80 and Triton X-100 have good induction effects.

[0027] Furthermore, the pH value of the fermentation environment is 5 - 6.

[0028] Beneficial effects: Lignin-degrading enzymes secreted by white rot fungi, such as lignin peroxidase, manganese peroxidase, and laccase, etc., have the highest activity at pH 5 to 6, which is beneficial for efficient lignin degradation.

[0029] Furthermore, the temperature of the fermentation environment is 25 - 30 °C.

[0030] Beneficial effects: The optimal growth temperature of white rot fungi is usually between 25 - 30 °C. Within this temperature range, the metabolic activities of the fungi are vigorous, the mycelial growth is rapid, and biomass can be formed more quickly, providing a basis for lignin degradation.

[0031] Furthermore, the mycelia of white rot fungi are one or more types of mycelia among Pleurotus eryngii, Pleurotus ostreatus, Phanerochaete chrysosporium, and Irpex lacteus.

[0032] Beneficial effects: Different white rot fungi have different specificities when decomposing different lignin-containing carriers. Selecting different types of white rot fungi based on the lignin-containing carrier can promote the fermentation efficiency.

[0033] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is a logical schematic diagram of an embodiment of the fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi of the present invention;

[0035] Figure 2 It is an axonometric schematic diagram of an embodiment of the fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi of the present invention.

[0036] The reference numerals in the accompanying drawings of the specification include: 1, culture container; 2, driving member; 3, ventilation port. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The technical solutions of the present invention will be described clearly and completely below with reference to the accompanying drawings. Apparently, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0038] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by terms such as "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation to the present invention. In addition, the terms "first", "second", and "third" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0039] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the terms "installed", "connected", and "coupled" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0040] The following is a further detailed description through specific embodiments:

[0041] As shown in the attached Figure 1 - Figure 2 figure: A fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi, including a culture container 1 and a controller. The culture container 1 is a culture tank, and an air inlet 3 is provided on the culture tank. The air inlet 3 is used to provide an aeration ratio of 1.0 vvm. The culture container 1 is used to culture white rot fungal mycelia and provide a liquid fermentation environment. The culture container 1 is provided with a driving member 2, and the driving member 2 is a servo motor. The driving member 2 is used to drive the culture container 1 to rotate self.

[0042] The fermentation environment includes glucose at 0.8 g / L - 1.2 g / L, peptone at 0.8 g / L - 1.2 g / L, lignin-containing carrier at 40 g / L, inducer at 0.1 mmol / L - 1 mmol / L, MgSO4 at 1.2 g / L - 1.5 g / L, KH2PO4 at 1 g / L - 1.2 g / L, CaCl2 at 0.1 g / L - 0.12 g / L, and the balance is water. The lignin-containing carrier is in the form of corn straw powder, and the inducer uses a combined inducer composed of gallic acid, copper ion salt solution, and Tween 80.

[0043] Control the initial pH value of the fermentation environment to 5, and adjust the fermentation environment to 25 - 30 °C.

[0044] The preparation of white rot fungus mycelium includes strain activation. The slant seeds of white rot fungus are inoculated on PDA medium and cultured in a constant temperature shaker at 25-28 °C at 120-160 r / min for 3-5 days. After visible mycelium grows, a seed solution is prepared. Under the protection of a flame, the inoculation port of the culture tank is opened, and the prepared seed solution is put into the culture tank according to a preset ratio.

[0045] The controller is used to divide the fermentation cycle based on the fermentation time. The fermentation cycle includes a mycelium expansion period, an environment coordination period, and a fermentation stabilization period. The mycelium expansion period, environment coordination period, and fermentation stabilization period are set according to different white rot fungus strains. Taking Phanerochaete chrysosporium as an example, when the inoculation amount is 15%, the mycelium expansion period is 1-2 days after the start of fermentation, the environment coordination period is 3-8 days, and the fermentation stabilization period is from the 9th day to the end of fermentation.

[0046] During the mycelium expansion period, the controller controls the driving member 2 to make the rotation speed of the culture container 1 be 400-800 r / min, so that the shear force generated by the rotation cuts the mycelium to form new growth points.

[0047] During the environment coordination period, the controller controls the driving member 2 to make the rotation speed of the culture container 1 be 300-400 r / min, so that the shear force generated by the rotation improves the distribution of oxygen and nutrients.

[0048] During the fermentation stabilization period, the controller controls the driving member 2 to make the rotation speed of the culture container 1 be 100-200 r / min, reducing the injury to the mycelium caused by the shear force generated by the rotation.

[0049] The culture container 1 can provide an environment for cultivating white rot fungus mycelium and a fermentation environment. The liquid fermentation environment enables the shear force during the rotation of the culture container 1 to act on the internal fermentation environment. Different shear force magnitudes can be obtained according to different rotations, and different shear force magnitudes will have different effects on the mycelium.

[0050] The growth of mycelium mainly depends on the extension of the tip. When the mycelium breaks, each broken end can reactivate the growth mechanism to form new growth points. However, it does not mean that mycelium breakage will necessarily promote mycelium growth. Other factors such as the distribution and viscosity of the mycelium are also involved, which will directly or indirectly affect the nutritional competition of the mycelium. During the mycelium expansion period, the number of the bacterial population is much smaller than the amount of nutrients. The conditions for the reproduction of the bacterial population are suitable but the base number is small. Therefore, high rotation speed is used to cause mycelium breakage, thereby constructing more growth points, rapidly increasing the number of the bacterial population, and at the same time promoting the dispersion of the mycelium so as to evenly contact the lignin-containing carrier.

[0051] Hyphae are prone to excessive local mycelial growth, resulting in local broth viscosity, which causes a decrease in the enzyme activity of the microbial community. Therefore, after the mycelial expansion stage is completed, the shear force that can change the position of the hyphae needs to be maintained to ensure the interaction between the hyphae and nutrients in the culture container 1, reduce local broth viscosity, and prevent the decrease in the enzyme activity of the microbial community, and continuously promote the growth of the microbial community.

[0052] In the later stage, when the number of hyphae is large enough and stable, it is difficult to improve the local broth viscosity even when maintaining a rotation speed of 300 - 400 r / min. The enzyme activity of the microbial community will inevitably decline. At this time, the rotation speed can be reduced to reduce the injury to the mycelium caused by the shear force generated by rotation, thereby reducing the withering amount of the microbial community, stabilizing the number of the microbial community, and reducing energy consumption.

[0053] A basic culture environment is constructed by glucose, peptone, MgSO4, KH2PO4, CaCl2, and water. The carrier containing lignin is the target for fermentation. White rot fungi secrete corresponding degrading enzymes to degrade the lignin in the carrier. Inducers can additionally enhance enzyme activity, thereby promoting fermentation. Among them, aromatic / phenolic compounds represented by gallic acid are effective ways to increase the production of lignin-degrading enzymes by white rot fungi and can significantly increase the production of laccase. Copper ions and manganese ions can promote the enzyme activity of fungi at low concentrations. Copper ions and manganese ions are essential elements for the transcription of lignin-degrading enzymes and promote the production of MnP. Surfactants represented by Tween 80 improve their stability by preventing enzyme denaturation. The reverse micelle system formed by surfactants can simulate the suitable environment of the enzyme and maintain the enzyme activity. At the same time, surfactants are a reaction medium, can become an insoluble substrate and an excellent solvent for lignin. Surfactants can also reduce the crystallinity of cellulose, making the biomass produce a porous disordered structure and increasing the specific surface area of cellulose.

[0054] The lignin-degrading enzymes secreted by white rot fungi, such as lignin peroxidase, manganese peroxidase, and laccase, etc., have the highest activity at pH 5 to 6, which is conducive to the efficient degradation of lignin.

[0055] The optimal growth temperature of white rot fungi is usually between 25 - 30 °C. Within this temperature range, the metabolic activities of the fungi are vigorous, the hyphae grow rapidly, and biomass can be formed faster, providing a basis for lignin degradation.

[0056] An application of using the above fermentation medium to improve the enzyme activity of lignin-degrading enzymes produced by white rot fungi.

[0057] By directly using the above fermentation medium for fermentation operations, the enzyme activity of lignin-degrading enzymes produced by white rot fungi can be enhanced.

[0058] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

1. A fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi, characterized in that, It includes a culture container (1) and a controller. The culture container (1) is used for culturing white-rot fungus hyphae and providing a liquid fermentation environment. The culture container (1) is provided with a driving member (2), and the driving member (2) is used to drive the culture container (1) to rotate self. The controller is used to divide the fermentation cycle based on the fermentation time. The fermentation cycle includes a hypha expansion period, an environment coordination period, and a fermentation stable period. During the hypha expansion period, the controller controls the driving member (2) to make the rotation speed of the culture container (1) be 400 - 800 r / min, so that the shear force generated by the rotation cuts the hyphae to form new growth points. During the environment coordination period, the controller controls the driving member (2) to make the rotation speed of the culture container (1) be 300 - 400 r / min, so that the shear force generated by the rotation improves the distribution of oxygen and nutrients. During the fermentation stable period, the controller controls the driving member (2) to make the rotation speed of the culture container (1) be 100 - 200 r / min, reducing the injury to the mycelium caused by the shear force generated by the rotation.

2. The fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi according to claim 1, characterized in that, The fermentation environment includes glucose 0.8 g / L - 1.2 g / L, peptone 0.8 g / L - 1.2 g / L, lignin-containing carrier 40 g / L, inducer 0.1 mmol / L - 1 mmol / L, MgSO4 1.2 g / L - 1.5 g / L, KH2PO4 1 g / L - 1.2 g / L, CaCl2 0.1 g / L - 0.12 g / L, and the balance is water.

3. The fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi according to claim 2, wherein The lignin-containing carrier includes crop straws and wheat bran.

4. The fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi according to claim 3, characterized in that, The inducer includes one or more of ferulic acid, dimethylaniline, vanillic acid, cinnamic acid, guaiacol, veratryl alcohol, pyrogallol, syringic acid, gallic acid.

5. The fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi according to claim 4, characterized in that, The inducer also includes copper ion and manganese ion salt solutions.

6. The fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi according to claim 5, characterized in that, The inducer also includes Tween 80 and Triton X-100.

7. The fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi according to claim 6, characterized in that The pH value of the fermentation environment is 5 - 6.

8. The fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi according to claim 7, characterized in that, The temperature of the fermentation environment is 25 - 30 °C.

9. The fermentation medium for improving the enzyme activity of lignin-degrading enzymes produced by white rot fungi according to claim 8, characterized in that, The white-rot fungus hyphae are one or more kinds of hyphae of Pleurotus eryngii, Pleurotus ostreatus, Phanerochaete chrysosporium, and Irpex lacteus.

10. Application of a fermentation medium according to claim 9 in improving the enzyme activity of lignin-degrading enzymes produced by white-rot fungi.

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