Method for producing laccase through fermentation of ganoderma lucidum

Abstract

The invention belongs to the technical field of biological fermentation, and relates to a method for producing laccase through ganoderma lucidum fermentation. The method for producing laccase through fermentation of ganoderma lucidum comprises the following steps: inoculating a ganoderma lucidum seed solution into a culture medium, and carrying out first-stage fermentation, second-stage fermentation and third-stage fermentation, the second ventilation ratio in the second-stage fermentation process is smaller than the first ventilation ratio in the first-stage fermentation process, and the third ventilation ratio in the third-stage fermentation process is larger than the second ventilation ratio in the second-stage fermentation process; the second stirring rotating speed in the second-stage fermentation process is lower than the first stirring rotating speed in the first-stage fermentation process, and the third stirring rotating speed in the third-stage fermentation process is higher than the second stirring rotating speed in the second-stage fermentation process. The method provided by the invention can effectively reduce the generation of foam and improve the enzyme production capacity of ganoderma lucidum by adjusting the ventilation ratio and the rotating speed, and is simple to operate, quick in defoaming and high in repeatability.

Description

Technical Field

[0001] The present application belongs to the field of biological fermentation technology and relates to a method for producing laccase by fermentation of Ganoderma lucidum. Background Art

[0002] During the fermentation process of Ganoderma lucidum to produce laccase, foam will be produced. A moderate amount of foam is beneficial to fermentation. It can increase the contact between gas and liquid during liquid fermentation, promote gas exchange, and thus increase oxygen transfer. However, excessive foam produced during the fermentation process will have an adverse effect on the entire fermentation process, mainly in the following two aspects: 1. It prevents Ganoderma lucidum from absorbing oxygen and expelling carbon dioxide, affecting its production metabolism and thus its enzyme activity; 2. It leads to contamination by miscellaneous bacteria. After a large amount of foam bursts out, it will invade the shaft seal and make the shaft seal ineffective. Summary of the Invention

[0003] Based on this, it is necessary to provide a method for producing laccase by fermenting Ganoderma lucidum, so as to reduce the foam generated during the fermentation process of Ganoderma lucidum and improve the laccase activity produced by Ganoderma lucidum.

[0004] In some embodiments, a method for producing laccase by fermentation of Ganoderma lucidum is provided, comprising the following steps:

[0005] inoculating the Ganoderma lucidum seed liquid into the culture medium to carry out first stage fermentation, second stage fermentation and third stage fermentation;

[0006] wherein the second ventilation ratio during the second stage fermentation is less than the first ventilation ratio during the first stage fermentation, and the third ventilation ratio during the third stage fermentation is greater than the second ventilation ratio during the second stage fermentation;

[0007] The second stirring speed during the second stage fermentation is lower than the first stirring speed during the first stage fermentation, and the third stirring speed during the third stage fermentation is higher than the second stirring speed during the second stage fermentation.

[0008] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the first stage of fermentation, the first ventilation ratio is controlled to be 0.6-0.7.

[0009] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the first stage of fermentation, the first stirring speed is controlled to be 90 r / min to 120 r / min.

[0010] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the second stage of fermentation, the second ventilation ratio is controlled to be 0.3-0.4.

[0011] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the second stage of fermentation, the second stirring speed is controlled to be 60 r / min to 80 r / min.

[0012] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the third stage of fermentation, the third ventilation ratio is controlled to be 0.6-0.7.

[0013] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the third stage of fermentation, the third stirring speed is controlled to be 90 r / min to 120 r / min.

[0014] In some embodiments, the provided method for producing laccase by fermentation of Ganoderma lucidum satisfies one or more of the following conditions:

[0015] (1) The first stage of fermentation lasts 20h~24h;

[0016] (2) The second stage of fermentation lasts for 24 to 48 hours;

[0017] (3) The third stage of fermentation lasts 48 to 96 hours;

[0018] (4) In the step of inoculating the Ganoderma lucidum seed liquid into the culture medium, the inoculation amount is 5% to 15%;

[0019] (5) a pH of 4.2 to 4.7 during the first stage of fermentation, a pH of 4.2 to 4.9 during the second stage of fermentation, and a pH of 4.8 to 6.3 during the third stage of fermentation; and,

[0020] (6) The dissolved oxygen value during the first stage of fermentation was 50%~100%, the dissolved oxygen value during the second stage of fermentation was 14%~51%, and the dissolved oxygen value during the third stage of fermentation was 13%~17%.

[0021] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the culture medium includes: yeast extract powder 2.0 g / L~4.0 g / L, corn steep liquor 20.0 g / L~25.0 g / L, bran 31.0 g / L~35.0 g / L, tobacco stem powder 20.0 g / L~25.0 g / L, potassium dihydrogen phosphate 1.1 g / L~1.5 g / L, magnesium sulfate heptahydrate 1.0 g / L~2.0 g / L, vitamin B1 0.1 g / L~0.2 g / L and silicone oil defoamer 8 / 10000v / v~10 / 10000v / v.

[0022] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the fermentation pressure during the first stage fermentation, the second stage fermentation, and the third stage fermentation is independently 0.5 bar to 0.8 bar.

[0023] The above-mentioned method for producing laccase by fermentation of Ganoderma lucidum can effectively reduce the generation of foam and promote the absorption of oxygen by Ganoderma lucidum by adjusting the relationship between the ventilation ratio and the rotation speed in each fermentation stage. At the same time, the carbon dioxide produced by its metabolism can be smoothly eliminated, thereby effectively restoring the normal growth and metabolism of Ganoderma lucidum, improving the enzyme production capacity of Ganoderma lucidum, and achieving higher enzyme activity per unit time, greatly improving the efficiency of industrial production, saving manpower and material resources, and having the advantages of simple operation, fast defoaming, and high repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments and examples of this application and to provide a more complete understanding of the application and its beneficial effects, the following briefly introduces the drawings required for use in the description of the embodiments or examples. Obviously, the drawings described below are only some embodiments of this application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0025] Figure 1 This is a trend diagram of pH value changes during the fermentation process in different fermentation tanks in Example 1;

[0026] Figure 2 This is a graph showing the changing trend of DO (dissolved oxygen) during the fermentation process in different fermenters in Example 1. DETAILED DESCRIPTION

[0027] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the accompanying drawings. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the present disclosure.

[0028] It should be noted that when an element is referred to as being “fixed to” another element, it may be directly on the other element or there may be an intermediate element. When an element is referred to as being “connected to” another element, it may be directly connected to the other element or there may be an intermediate element.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0030] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0031] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of the related listed items, and the said any and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions: A, B and "a combination of A and B".

[0032] In this application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0033] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0034] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0035] In this application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this application.

[0036] In this application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of this application.

[0037] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0038] In the present invention, in the "first aspect," "second aspect," "third aspect," "fourth aspect," etc., the terms "first," "second," "third," "fourth," etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," "third," "fourth," etc. serve only as non-exhaustive enumeration and description and should be understood not to constitute a closed-ended limitation on quantity.

[0039] In this application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0040] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0041] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0042] In this application, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0043] The "room temperature" in this application generally refers to 5°C to 30°C, preferably 25±5°C.

[0044] In industrial production, the liquid-to-liquid ratio during microbial fermentation is a crucial indicator; a high liquid-to-liquid ratio indicates high production efficiency. The fermentation tank volume is crucial for laccase production; a 20% reduction in volume doubles laccase production. During microbial fermentation, foam forms in the fermentation broth. Excessive foam can reduce the liquid-to-liquid ratio, lower oxygen transfer efficiency, increase the risk of contamination, hinder bacterial respiration, and affect metabolism.

[0045] The present application provides a method for producing laccase by fermenting Ganoderma lucidum. The provided method for producing laccase by fermenting Ganoderma lucidum comprises the following steps: inoculating Ganoderma lucidum seed liquid into a culture medium, and performing first-stage fermentation, second-stage fermentation, and third-stage fermentation; the second ventilation ratio during the second-stage fermentation process is less than the first ventilation ratio during the first-stage fermentation process, and the third ventilation ratio during the third-stage fermentation process is greater than the second ventilation ratio during the second-stage fermentation process; the second stirring speed during the second-stage fermentation process is less than the first stirring speed during the first-stage fermentation process, and the third stirring speed during the third-stage fermentation process is greater than the second stirring speed during the second-stage fermentation process. The provided method can effectively reduce the generation of foam and improve the enzyme production capacity of Ganoderma lucidum by adjusting the ventilation ratio and the speed. The operation is simple, the defoaming is fast, and the repeatability is high.

[0046] In some embodiments, a method for producing laccase by fermentation of Ganoderma lucidum is provided, comprising the following steps:

[0047] inoculating the Ganoderma lucidum seed liquid into the culture medium to carry out first stage fermentation, second stage fermentation and third stage fermentation;

[0048] The second ventilation ratio during the second stage fermentation is smaller than the first ventilation ratio during the first stage fermentation, and the third ventilation ratio during the third stage fermentation is larger than the second ventilation ratio during the second stage fermentation.

[0049] The second stirring speed during the second stage fermentation is lower than the first stirring speed during the first stage fermentation, and the third stirring speed during the third stage fermentation is higher than the second stirring speed during the second stage fermentation.

[0050] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the first stage of fermentation, the first ventilation ratio is controlled to be 0.6-0.7.

[0051] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the first stage of fermentation, the first stirring speed is controlled to be 90 r / min~120 r / min, for example, 90 r / min, 100 r / min, 110 r / min, 120 r / min, etc., or a range consisting of any two of the aforementioned values.

[0052] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the second stage of fermentation, the second ventilation ratio is controlled to be 0.3-0.4.

[0053] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the second stage of fermentation, the second stirring speed is controlled to be 60r / min~80r / min, for example, 60r / min, 70r / min, 80r / min, etc., or a range consisting of any two of the aforementioned values.

[0054] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the third stage of fermentation, the third ventilation ratio is controlled to be 0.6-0.7.

[0055] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, during the third stage of fermentation, the third stirring speed is controlled to be 90 r / min~120 r / min, for example, 90 r / min, 100 r / min, 110 r / min, 120 r / min, etc., or a range consisting of any two of the aforementioned values.

[0056] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the time of the first stage fermentation is 20h~24h. For example, the time of the first stage fermentation can be 20h, 21h, 22h, 23h, 24h, etc., or a range consisting of any two of the aforementioned values.

[0057] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the time for the second stage fermentation is 24h~48h. For example, the time for the second stage fermentation can be 24h, 28h, 30h, 36h, 40h, 44h, 48h, etc., or a range consisting of any two of the foregoing values.

[0058] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the time for the third stage fermentation is 48h~96h. For example, the time for the third stage fermentation can be 48h, 50h, 56h, 60h, 64h, 72h, 96h, etc., or a range consisting of any two of the foregoing values.

[0059] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, in the step of inoculating the Ganoderma lucidum seed liquid into the culture medium, the inoculation amount is 5% to 15%.

[0060] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the pH value during the first fermentation stage is 4.2-4.7, the pH value during the second fermentation stage is 4.2-4.9, and the pH value during the third fermentation stage is 4.8-6.3.

[0061] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the dissolved oxygen value during the first stage of fermentation is 50%-100%, the dissolved oxygen value during the second stage of fermentation is 14%-51%, and the dissolved oxygen value during the third stage of fermentation is 13%-17%.

[0062] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the culture medium includes: yeast extract powder 2.0 g / L~4.0 g / L, corn steep liquor 20.0 g / L~25.0 g / L, bran 31.0 g / L~35.0 g / L, tobacco stem powder 20.0 g / L~25.0 g / L, potassium dihydrogen phosphate 1.1 g / L~1.5 g / L, magnesium sulfate heptahydrate 1.0 g / L~2.0 g / L, vitamin B1 0.1 g / L~0.2 g / L and silicone oil defoamer 8 / 10000v / v~10 / 10000v / v.

[0063] In some embodiments, in the provided method for producing laccase by fermentation of Ganoderma lucidum, the fermentation pressure during the first stage fermentation, the second stage fermentation, and the third stage fermentation is independently 0.5 bar to 0.8 bar.

[0064] The present invention adjusts the ventilation volume and rotation speed during the process, so that the foam generated by the culture medium can be eliminated, and the normal growth and metabolism of Ganoderma lucidum can be promoted. After the adjustment, the laccase activity produced by Ganoderma lucidum is increased by an average of 10.3 times, with obvious effects.

[0065] In order to make it easier to understand and implement the present invention, the following easier-to-implement, more specific and detailed embodiments and comparative examples are provided below as reference.

[0066] The following will further illustrate the concept, specific examples and technical effects of the present invention in conjunction with the accompanying drawings to fully understand the present invention. The purpose of providing these illustrations is only to help explain the present invention and should not be used to limit the scope of the claims of the present invention.

[0067] The following are specific examples, which describe the present disclosure in more detail. These examples are intended for illustrative purposes only, as various modifications and variations within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available. Unless otherwise stated, the raw materials used in the following experiments are all commercially available.

[0068] The Ganoderma lucidum used in the examples was sourced from Beina Biotechnology Co., Ltd. BNCC382030; the silicone oil defoamer was sourced from Deqing County Kangle Fine Chemical Factory, with the product model being silicone oil defoamer ZDS-2; and the membrane filter press was X12AWG30.

[0069] Example 1

[0070] (1) Air sterilization: Three 1200 L fermentation tanks numbered 301, 302, and 303 were sterilized under the following conditions: sterilization temperature 121 °C, sterilization time 30 min, primary temperature 95 °C, cooling temperature 30 °C, sterilization pressure 1.180 bar, sterilization air intake 800 L / min, and exhaust ratio 8.0. 600 L fermentation enzyme production medium was prepared for each tank; the formula of the fermentation enzyme production medium was 3.0 g / L yeast extract powder, 20.0 g / L corn steep liquor, 31.0 g / L bran, 20.0 g / L tobacco stem powder, 1.1 g / L potassium dihydrogen phosphate, 1.0 g / L magnesium sulfate heptahydrate, 0.1 g / L vitamin B1, and 8 / 10000 silicone oil defoamer (v / v).

[0071] (2) The sterilization of fermentation tanks 301, 302, and 303 was carried out under the following conditions: sterilization temperature of 121°C, sterilization time of 30 min, primary temperature of 95°C, cooling temperature of 30°C, sterilization pressure of 1.180 bar, sterilization air intake of 800 L / min, and exhaust ratio of 8.0. Before installing the dissolved oxygen probe, the dissolved oxygen value was calibrated to 0.5 bar. After the sterilization was completed, the air intake valve and each exhaust valve were closed, and sterile air was introduced into the tank to maintain a pressure of 0.5 bar. After the temperature in the tank dropped to 95°C, the stirring speed was turned on at 100 r / min, and the jacket circulating water was turned on for cooling. After the tank temperature dropped to 30-32°C, the tank was incubated in an air-conditioning system for 48 hours, and the incubation process was monitored in real time.

[0072] (3) After 48 hours, observe the results of the sample coating of 301, 302, and 303. There is no abnormality in the flat plate coating. The seed liquid in the three 60L seed tanks numbered 201, 202, and 203 is inoculated into the fermentation enzyme production medium of 301, 302, and 303 through pressure difference; after the inoculation, close the inoculation valves of the three fermentation tanks and the bottom valve of the seed tank, adjust the pressure in each fermentation tank to 0.5 bar, turn on the stirring speed to 300r / min, stir for 10 minutes to disperse the bacteria, and then adjust the stirring speed to 100r / min. Click on the 100% calibration of DO, adjust the ventilation ratio to 0.6, and sample coating is performed on 301, 302, and 303.

[0073] (4) After 24 h of incubation, before foam generation, the ventilation ratio and rotation speed of 301 in (3) were kept unchanged, and the ventilation ratio of 302 and 303 was reduced to 0.3 and the stirring speed was reduced to 60 r / min to reduce the foam generation of 302 and 303.

[0074] (5) Continue culturing for 24 hours, i.e., after a total culturing time of 48 hours, maintain the ventilation ratio and speed of 301 and 302 unchanged as in (4), adjust the ventilation ratio and speed of 303 to the initial values in (3), adjust the stirring speed to 100 r / min, and the ventilation ratio to 0.6. During the culturing period, monitor the parameters of the fermentation enzyme production process in real time. Continue culturing for 48 hours, i.e., after a total culturing time of 96 hours, filter the obtained bacterial solution using a membrane filter press.

[0075] Three replicate experiments were performed, and the pH and dissolved oxygen values during the fermentation process were recorded.

[0076] Figure 1 is the pH value recorded during the fermentation process of Example 1, Figure 2 It is the dissolved oxygen value recorded during the fermentation process of Example 1. Figure 1 In tank No. 301, the ventilation ratio and rotation speed were not adjusted during the fermentation process, resulting in a large amount of foam generated during the fermentation process, which seriously affected the metabolism of the Ganoderma lucidum and caused little change in the pH value during the fermentation process. In tanks No. 302 and 303, the ventilation ratio and rotation speed were adjusted during the fermentation process, and normal growth and metabolism were carried out. Therefore, the pH value change trend was to first decrease and then slowly increase until it reached a peak enzyme production point. Figure 2 In tank No. 301, a large amount of foam was generated during the fermentation process because the ventilation ratio and rotation speed were not adjusted, which seriously affected the growth of Ganoderma lucidum. Ganoderma lucidum is originally an aerobic bacteria, but the process inhibited its growth, resulting in a decrease in oxygen absorption, so the dissolved oxygen has been tending to a stable trend. In tanks No. 302 and 303, the ventilation ratio and rotation speed were adjusted during the fermentation process, and normal growth was carried out. The process was always absorbing oxygen, so the DO trend was decreasing until it tended to an equilibrium value.

[0077] Example 2

[0078] (1) Air sterilize a 1200 L fermentation tank under the following conditions: sterilization temperature 121 °C, sterilization time 30 min, primary temperature 95 °C, cooling temperature 30 °C, sterilization pressure 1.180 bar, sterilization air intake 800 L / min, exhaust ratio 8.0. Prepare 600 L fermentation enzyme production medium; the formula of the fermentation enzyme production medium is: yeast extract powder 3.0 g / L, corn steep liquor 20.0 g / L, bran 31.0 g / L, tobacco stem powder 20.0 g / L, potassium dihydrogen phosphate 1.1 g / L, magnesium sulfate heptahydrate 1.0 g / L, vitamin B1 0.1 g / L, silicone oil defoamer 8 / 10000 (v / v).

[0079] (2) The fermentation tank was sterilized under the following conditions: sterilization temperature of 121°C, sterilization time of 30 min, primary temperature of 95°C, cooling temperature of 30°C, sterilization pressure of 1.180 bar, sterilization air intake of 800 L / min, and exhaust ratio of 8.0. Before installing the dissolved oxygen probe, the dissolved oxygen value was calibrated to 0.0. After the sterilization was completed, the air intake valve and each exhaust valve were closed, and sterile air was introduced into the tank to maintain a pressure of 0.5 bar. After the temperature in the tank dropped to 95°C, the stirring speed was turned on at 100 r / min, and the jacket circulating water was turned on for cooling. After the tank temperature dropped to 30-32°C, the tank was incubated for 48 hours, and the incubation process was monitored in real time.

[0080] (3) After 48 hours, observe the results of the sample coating. There are no abnormalities in the plate coating. The seed liquid in the 60L seed tank is inoculated into the fermentation enzyme production medium through pressure difference. After the inoculation is completed, close the fermentation tank inoculation valve and the seed tank bottom valve, adjust the pressure in each fermentation tank to 0.5 bar, turn on the stirring speed to 300r / min, stir for 10 minutes to disperse the bacteria, and then adjust the stirring speed to 120r / min. Click on the 100% calibration of DO, adjust the ventilation ratio to 0.7, and sample and coat.

[0081] (4) After 24 h of incubation, reduce the ventilation ratio to 0.4 and the stirring speed to 80 r / min to reduce foaming.

[0082] (5) Continue culturing for 24 hours, i.e., after a total culturing time of 48 hours, adjust the stirring speed to 120 r / min and the ventilation ratio to 0.7. During the culturing period, monitor the fermentation enzyme production process parameters in real time. Continue culturing for 48 hours, i.e., after a total culturing time of 96 hours, filter the obtained bacterial solution using a membrane filter press.

[0083] Example 3

[0084] Similar to Example 2, the changes are as follows: in step (3), the stirring speed is 90 r / min and the ventilation ratio is 0.6; in step (4), the ventilation ratio is increased to 0.3 and the stirring speed is increased to 60 r / min; in step (5), the stirring speed is 90 r / min and the ventilation ratio is 0.6.

[0085] Enzyme activity assay

[0086] The filtrate obtained in Example 1 was tested for laccase activity using the ABTS method. The specific steps of the ABTS method are as follows: 2.5 mL of pH 3.0 sodium hydrogen phosphate-citrate buffer was added to three 5 mL EP tubes (one blank and one control). 2.5 μL of appropriately diluted enzyme solution was then added to each tube; the blank tube was left without enzyme solution. 500 μL of a 2 mmol / L 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution was then added. The tubes were then quickly placed in a 45°C water bath to react for 5 minutes. The absorbance at 420 nm was then measured using a Pulse 1810 UV spectrophotometer.

[0087] ,

[0088] Among them, ε is 3.6×10 4 , L / (mol·cm); Vtotal is the total volume of the reaction system / mL; Venzyme is the volume of the added enzyme solution / mL, is the reaction time / min.

[0089] The measured laccase activities obtained from different fermentation tanks in Example 1 are shown in Table 1 below.

[0090] Table 1

[0091]

[0092] From the results of laccase activity test in Table 1, it can be seen that the stability of the three repeated experiments was good. In the 301 jar, the ventilation ratio and rotation speed were not adjusted during the fermentation process, resulting in a large amount of foam. The foam occupied the entire fermentation liquid surface, preventing the Ganoderma lucidum from absorbing oxygen and releasing carbon dioxide, seriously affecting its production metabolism, resulting in a very low enzyme activity of only 9633U / L~12265 U / L; after 24 hours of cultivation in jar 302, before foam was produced, the ventilation ratio and rotation speed were reduced to effectively reduce the production of foam. However, during the entire cultivation process, Ganoderma lucidum is an aerobic bacterium and requires sufficient oxygen to promote its growth and metabolism. Therefore, in the late fermentation period, the Ganoderma lucidum in 302 did not reach its maximum enzyme production capacity, and the enzyme activity was 61993U / L~70014U / L; after 24 hours of cultivation in jar 303, before foam was produced, the ventilation ratio and rotation speed were reduced to effectively reduce the production of foam. After 48 hours of cultivation, the ventilation ratio and rotation speed were adjusted to the initial values. The growth and metabolic capacity of Ganoderma lucidum in the late fermentation period was restored, reaching its maximum enzyme production capacity of 106886U / L~113941U / L.

[0093] In summary, after 24 hours of cultivation and before foam is generated, the ventilation ratio and rotation speed are reduced, and after 48 hours of cultivation, the ventilation ratio and rotation speed are adjusted to the initial values. This can reduce the foam generated during the fermentation process of Ganoderma lucidum and increase the laccase activity of Ganoderma lucidum to achieve its maximum enzyme production capacity.

[0094] The above embodiments are merely illustrative of the effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical concepts disclosed herein are intended to be covered by the claims of the present invention.

[0095] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0096] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent. It should be noted that a person of ordinary skill in the art may make various modifications and improvements without departing from the spirit of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be based on the appended claims, and the description and drawings shall be used to interpret the content of the claims.

Claims

1. A method for producing laccase by fermentation of Ganoderma lucidum, characterized in that: The steps include: inoculating the Ganoderma lucidum seed liquid into the culture medium to carry out first stage fermentation, second stage fermentation and third stage fermentation; wherein the second ventilation ratio during the second stage fermentation is less than the first ventilation ratio during the first stage fermentation, and the third ventilation ratio during the third stage fermentation is greater than the second ventilation ratio during the second stage fermentation; The second stirring speed during the second stage fermentation is lower than the first stirring speed during the first stage fermentation, and the third stirring speed during the third stage fermentation is higher than the second stirring speed during the second stage fermentation.

2. The method according to claim 1, characterized in that During the first stage of fermentation, the first ventilation ratio is controlled to be 0.6-0.

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3. The method according to claim 1, characterized in that During the first stage of fermentation, the first stirring speed is controlled to be 90 r / min~120 r / min.

4. The method according to claim 1, wherein During the second stage of fermentation, the second ventilation ratio is controlled to be 0.3-0.

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5. The method according to claim 1, characterized in that During the second stage of fermentation, the second stirring speed is controlled to be 60 r / min~80 r / min.

6. The method according to claim 1, wherein During the third stage of fermentation, the third ventilation ratio is controlled to be 0.6-0.

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7. The method according to claim 1, characterized in that During the third stage of fermentation, the third stirring speed is controlled to be 90 r / min~120 r / min.

8. The method according to claim 1, characterized in that One or more of the following conditions are met: (1) The first stage of fermentation lasts 20h~24h; (2) The second stage of fermentation lasts for 24 to 48 hours; (3) The third stage of fermentation lasts 48 to 96 hours; (4) In the step of inoculating the Ganoderma lucidum seed liquid into the culture medium, the inoculation amount is 5% to 15%; (5) a pH of 4.2 to 4.7 during the first stage of fermentation, a pH of 4.2 to 4.9 during the second stage of fermentation, and a pH of 4.8 to 6.3 during the third stage of fermentation; and, (6) The dissolved oxygen value during the first stage of fermentation was 50%~100%, the dissolved oxygen value during the second stage of fermentation was 14%~51%, and the dissolved oxygen value during the third stage of fermentation was 13%~17%.

9. The method according to claim 1, characterized in that The culture medium comprises: 2.0 g / L to 4.0 g / L yeast extract powder, 20.0 g / L to 25.0 g / L corn steep liquor, 31.0 g / L to 35.0 g / L bran, 20.0 g / L to 25.0 g / L tobacco stem powder, 1.1 g / L to 1.5 g / L potassium dihydrogen phosphate, 1.0 g / L to 2.0 g / L magnesium sulfate heptahydrate, 0.1 g / L to 0.2 g / L vitamin B1, and 8 / 10000 v / v to 10 / 10000 v / v silicone oil defoaming agent.

10. The method according to any one of claims 1 to 9, characterized in that During the first stage fermentation, the second stage fermentation and the third stage fermentation, the fermentation pressure is independently 0.5 bar to 0.8 bar.

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