Preparation method and application of fermentation liquor of enzyme-producing fungi

Abstract

The invention discloses a preparation method and application of fermentation liquor of enzyme-producing fungi. The method comprises the following steps: fermenting enzyme-producing fungi, monitoring a pH value and a dissolved oxygen value in real time in the fermentation process, stopping fermentation when the pH value is observed to be reduced to the lowest point and then is increased by 0.03-0.05, and / or when the dissolved oxygen value is observed to be reduced by less than or equal to 5% within 2 hours, and preparing fermentation liquor. According to the method, the fermentation end point can be detected and judged on line in real time in the preparation process, judgment is accurate, the delay problem in a traditional method is avoided, the fermentation efficiency and yield can be improved, and a brand new thought is provided for large-scale enzyme production through fungus fermentation.

Description

Technical Field

[0001] The present application relates to the field of microbial fermentation, and specifically to a method for preparing a fermentation broth of an enzyme-producing fungus and its application. Background Art

[0002] Laccase, a member of the polyphenol oxidase family, has important applications in the food industry, removal of environmental pollutants, water purification, and biomass treatment due to its structural and functional properties. However, the industrial application of laccase still faces many challenges, including low production efficiency, low stability, short lifespan, and high price.

[0003] In recent years, two types of culture have been developed for Ganoderma lucidum: solid-state fermentation (SSF) and submerged fermentation (SmF). Numerous studies have been conducted on process optimization for each type of fermentation. Submerged fermentation is relatively simpler to regulate the physiological production of laccase than solid-state fermentation, and high laccase yields are generally easier to obtain using submerged fermentation. Currently, much research has been conducted on producing high-yield laccase using submerged fermentation in small laboratory fermenters (less than 50 L). Some literature has reported high enzyme activities, reaching 400-4000 U / mL, through submerged fermentation. However, most of this research focuses on medium formulation and fermentation conditions, with only a limited amount of research addressing the fermentation endpoint. Current studies on the fermentation endpoint typically rely on measuring a single or multiple substances to determine the endpoint. This presents two challenges: a lack of online detection methods and techniques; and the significant delays in sampling and measurement time required for obtaining results. This can lead to delays in tank loading, impacting fermentation efficiency and yield. Summary of the Invention

[0004] Based on this, it is necessary to provide a preparation method and application of the fermentation broth of enzyme-producing fungi.

[0005] The first aspect of the present application provides a method for preparing a fermentation broth of an enzyme-producing fungus, comprising the following steps:

[0006] The enzyme-producing fungus is fermented, and the pH value and dissolved oxygen value are monitored in real time during the fermentation process. When the pH value decreases to the lowest point and then increases by 0.03-0.05, and / or when the dissolved oxygen value decreases by less than or equal to 5% within 2 hours, the fermentation is terminated and a fermentation liquid is prepared.

[0007] In some embodiments, the fermenting step comprises:

[0008] Expanding and culturing the enzyme-producing fungus to obtain a seed solution;

[0009] The seed liquid is inoculated into a fermentation tank containing a fermentation medium for fermentation.

[0010] In some embodiments, the fermentation scale is 1000L~1200L.

[0011] In some embodiments, the fermentation medium is a liquid culture medium comprising one or more of 1 g / L to 5 g / L yeast extract, 20 g / L to 25 g / L corn steep liquor, 10 g / L to 15 g / L tobacco stem powder, 1.0 g / L to 1.5 g / L KH2PO4, 1.0 g / L to 1.5 g / LMgSO4·7H2O, 30 g / L to 35 g / L bran, and 0.1 g / L to 0.5 g / L vitamin B1.

[0012] In some embodiments, the fermentation temperature is 25°C to 30°C.

[0013] In some embodiments, the fermentation stirring speed is 70 rpm to 120 rpm.

[0014] In some embodiments, the fermentation has a ventilation ratio of 0.5 vvm to 0.8 vvm.

[0015] In some embodiments, the culture medium in the expanded culture comprises 20 g / L to 25 g / L potato dextrose water.

[0016] In some embodiments, the rotation speed of the expanded culture is 70 rpm to 120 rpm.

[0017] In some embodiments, the inoculation amount of the expanded culture is 5% to 10%.

[0018] In some embodiments, the temperature of the expanded culture is 25°C to 30°C.

[0019] In some embodiments, the expansion culture time is 72h~120h.

[0020] In some embodiments, the seed liquid is inoculated into a fermentation tank containing a fermentation medium at an inoculation rate of 5% to 15%.

[0021] In some embodiments, the enzyme-producing fungus includes a white-rot fungus; alternatively, the white-rot fungus includes Ganoderma lucidum.

[0022] The second aspect of the present application provides an enzyme production method, comprising the following steps:

[0023] Prepare fermentation broth using the method described in the first aspect of the present application; and

[0024] The enzyme is isolated from the fermentation broth.

[0025] In some embodiments, the enzyme comprises laccase.

[0026] The above-mentioned method for preparing the fermentation broth of enzyme-producing fungi can realize online real-time monitoring and judgment of the fermentation end point during the preparation process. The judgment result is accurate and avoids the delay problem in traditional methods. It helps to improve fermentation efficiency and yield, and provides a new idea for the large-scale production of enzymes by fungal fermentation. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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.

[0028] Figure 1 These are the curves showing changes in DO, pH, and laccase activity during fermentation of different batches in one embodiment of the present application, wherein A is the curve showing changes in DO, B is the curve showing changes in pH, and C is the curve showing changes in laccase activity.

[0029] Figure 2 These are the curves showing changes in pH, DO and laccase activity of the fermentation broth over fermentation time in Example 2 of the present application, wherein A is the curve showing changes in pH, B is the curve showing changes in DO, and C is the curve showing changes in laccase activity.

[0030] Figure 3 These are curves showing changes in pH, DO, and laccase activity of the fermentation broth over fermentation time in Example 3 of the present application, wherein A is the curve showing changes in pH, B is the curve showing changes in DO, and C is the curve showing changes in laccase activity. DETAILED DESCRIPTION

[0031] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application 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 disclosure of the present application.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0033] 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.

[0034] 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.

[0035] As used herein, the terms "having," "containing," "including," and "comprising" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0036] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0037] In this application, when referring to the unit of a data range, if the unit is only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0038] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0039] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0040] In this application, the terms "first," "second," and "third," etc., in "the first aspect," "the second aspect," "the third aspect," 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," and "third," etc., are only used for non-exhaustive enumeration and description purposes and should be understood not to constitute closed-ended limitations on quantity.

[0041] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values within the numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these 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 every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. "Numerical interval" allows for a broad range of numerical interval types including percentage intervals, ratio intervals, and ratio intervals.

[0042] In this application, "greater than or equal to", "greater than or equal to", and "≥" have the same meaning and can be used interchangeably; "less than or equal to", "less than or equal to", and "≤" have the same meaning and can be used interchangeably; "greater than" can be equivalently expressed as ">", and "less than" can be equivalently expressed as "<". In this application, unless otherwise specified, "greater than or equal to" and "≥" can be regarded as providing two solutions of "greater than" and "equal to". In this application, unless otherwise specified, "less than or equal to" and "≤" can be regarded as providing two solutions of "less than" and "equal to".

[0043] Currently, research on the fermentation endpoint is basically based on the determination of one or several substances. This has two pain points: one is the lack of online detection means and methods; the other is that the determination requires sampling time and measurement time. It takes a long time to get the measurement results, and the results will be delayed, which may lead to delays in tank loading and have a certain impact on fermentation efficiency and yield.

[0044] Based on this, the embodiments of the present application at least provide a method for preparing the fermentation broth of enzyme-producing fungi.

[0045] In a first aspect of the present application, a method for preparing a fermentation broth of an enzyme-producing fungus is provided, comprising the following steps:

[0046] The enzyme-producing fungi were fermented, and the pH value and dissolved oxygen value were monitored in real time during the fermentation process. When the pH value dropped to the lowest point and then increased by 0.03-0.05, the fermentation was terminated and a fermentation liquid was prepared.

[0047] Illustratively, in some embodiments, enzyme-producing fungi are fermented, and the pH value and dissolved oxygen value are monitored in real time during the fermentation process. When the pH value drops to the lowest point and then increases by 0.03, 0.04, 0.05 or a value or range between any two of the above values, the fermentation is terminated and the fermentation broth is prepared.

[0048] In some embodiments, a method for preparing a fermentation broth of an enzyme-producing fungus is provided, comprising the following steps:

[0049] The enzyme-producing fungi were fermented, and the pH value and dissolved oxygen value were monitored in real time during the fermentation process. When the dissolved oxygen value decreased by less than or equal to 5% within 2 hours, the fermentation was terminated and a fermentation liquid was prepared.

[0050] Exemplarily, in some embodiments, the enzyme-producing fungus is fermented, and the pH value and dissolved oxygen value are monitored in real time during the fermentation process. When the dissolved oxygen value decreases by 1%, 2%, 3%, 4%, 5% or a value or range between any two of the above values within 2 hours, the fermentation is terminated and the fermentation liquid is prepared.

[0051] In some embodiments, a method for preparing a fermentation broth of an enzyme-producing fungus is provided, comprising the following steps:

[0052] The enzyme-producing fungi were fermented, and the pH value and dissolved oxygen value were monitored in real time during the fermentation process. When the pH value dropped to the lowest point and then increased by 0.03-0.05, and when the dissolved oxygen value dropped by less than or equal to 5% within 2 hours, the fermentation was terminated and the fermentation liquid was prepared.

[0053] It should be noted that the method for judging the fermentation endpoint in the preparation method of the fermentation broth of the enzyme-producing fungus provided above does not rely on the pH value and DO value of the fermentation broth for judgment. Due to the amount of condensed water in the sterilization process and the error in the calibration pH, the initial pH of different batches of fermentation broth is different, and the DO of different batches is also different. If it is judged by the size of the data, it will cause errors. The correct method is to judge based on the trend. Observe that the pH value of the fermentation broth continues to decrease and then begins to increase. When the pH value increases by 0.03~0.05 over a period of time, it indicates that the pH value has reached the lowest point and is increasing. Fermentation is terminated. When the DO value of the fermentation broth drops rapidly and then decreases by less than or equal to 5% within 2 hours, it indicates that the DO value shows a stable trend after a rapid drop, and fermentation is terminated.

[0054] The above-mentioned method for preparing the fermentation broth of enzyme-producing fungi can realize online real-time monitoring and judgment of the fermentation endpoint during the preparation process, and the judgment result is accurate, and avoids the delay problem in traditional methods, which helps to improve fermentation efficiency and yield.

[0055] In some embodiments, the step of fermenting comprises:

[0056] S100: expanding the culture of the enzyme-producing fungus to obtain a seed solution;

[0057] S200: inoculating the seed liquid into a fermentation tank containing a fermentation medium for fermentation.

[0058] In some embodiments, in step S100, the culture medium used in the expanded culture comprises 20 g / L to 25 g / L of potato dextrose water. Without limitation, the concentration of potato dextrose water may be, but is not limited to, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, or a value or range between any two of the foregoing values.

[0059] In some embodiments, in step S100, the temperature of the expanded culture is 25° C. to 30° C. Without limitation, the temperature of the expanded culture can be, but is not limited to, 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., or a value or range between any two of the above values.

[0060] In some embodiments, in step S100, the ventilation ratio of the expanded culture is 0.5 vvm to 0.7 vvm. In a non-limiting manner, the ventilation ratio of the expanded culture can be, but is not limited to, 0.5 vvm, 0.6 vvm, 0.7 vvm, or a value or range between any two of the above values.

[0061] In some embodiments, in step S100, the inoculum size for the expanded culture is 5% to 10%. In a non-limiting manner, the inoculum size for the expanded culture can be, but is not limited to, 5%, 6%, 7%, 8%, 9%, 10%, or a value or range between any two of the above values.

[0062] In some embodiments, in step S100, the expansion culture time is 72 hours to 120 hours. In a non-limiting manner, the expansion culture time can be, but is not limited to, 72 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, or a value or range between any two of the above values.

[0063] In some embodiments, in step S200, the fermentation scale is 1000 L to 1500 L. Without limitation, the fermentation scale can be, but is not limited to, 1000 L, 1100 L, 1200 L, 1300 L, 1400 L, 1500 L, or a value or range between any two of the above values.

[0064] In some embodiments, in step S200, the fermentation medium is a liquid culture medium, and the liquid culture medium includes one or more of 1 g / L to 5 g / L yeast extract, 20 g / L to 25 g / L corn steep liquor, 10 g / L to 15 g / L tobacco stem powder, 1.0 g / L to 1.5 g / L KH2PO4, 1.0 g / L to 1.5 g / LMgSO4·7H2O, 30 g / L to 35 g / L bran, and 0.1 g / L to 0.5 g / L vitamin B1.

[0065] In some embodiments, the concentration of yeast extract can be, but is not limited to, 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, or a value or range between any two of the above values.

[0066] In some embodiments, the concentration of corn steep liquor can be, but is not limited to, 20 g / L, 21 g / L, 22 g / L, 23 g / L, 24 g / L, 25 g / L, or a value or range between any two of the above values.

[0067] In some embodiments, the concentration of tobacco stem powder can be, but is not limited to, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L, or a value or range between any two of the above values.

[0068] In some embodiments, the concentration of KH2PO4 can be, but is not limited to, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, or a value or range between any two of the above values.

[0069] In some embodiments, the concentration of MgSO4·7H2O may be, but is not limited to, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, or a value or range between any two of the foregoing values.

[0070] In some embodiments, the concentration of bran can be, but is not limited to, 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, or a value or range between any two of the above values.

[0071] In some embodiments, the concentration of vitamin B1 can be, but is not limited to, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, or a value or range between any two of the above values.

[0072] In some embodiments, in step S200, the fermentation temperature is 25° C. to 30° C. Without limitation, the fermentation temperature can be, but is not limited to, 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., or a value or range between any two of the above values.

[0073] In some embodiments, in step S200, the fermentation stirring speed is 70 rpm to 120 rpm. In a non-limiting manner, the fermentation stirring speed can be, but is not limited to, 70 rpm, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, or a value or range between any two of the above values.

[0074] In some embodiments, in step S200, the ventilation ratio of the fermentation is 0.5 vvm to 0.8 vvm. Without limitation, the ventilation ratio of the fermentation can be, but is not limited to, 0.5 vvm, 0.6 vvm, 0.7 vvm, 0.8 vvm, or a value or range between any two of the above values.

[0075] In some embodiments, in step S200, the seed solution is inoculated into the fermentation tank containing the fermentation medium at an inoculum level of 5% to 15%. In a non-limiting manner, the inoculum level may be, but is not limited to, 5%, 7%, 9%, 11%, 13%, 15%, or a value or range between any two of the foregoing values.

[0076] In some embodiments, the enzyme-producing fungi include white-rot fungi; further, the white-rot fungi include one or more of Ganoderma lucidum, Versicolor versicolor, and Trametes sp.

[0077] The second aspect of the present application provides an enzyme production method, comprising the following steps:

[0078] S100': preparing a fermentation broth using the above method; and

[0079] S200': Isolation of enzyme from fermentation broth.

[0080] In some embodiments, the enzyme comprises laccase.

[0081] Some examples are provided below.

[0082] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0083] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0084] In the following examples, the definition of laccase activity (U / L) is: the amount of enzyme required to convert 1 µmol of ABTS (2,2'-azino-bis-(3-ethylbenzothiazole-6-sulfonic acid)) in 1 min is one activity unit.

[0085] In the following examples, the fermentation medium contains 3 g / L yeast extract, 20 g / L corn steep liquor, 10 g / L tobacco stem powder, 1.2 g / L KH2PO4, 1.04 g / L MgSO4·7H2O, 31.6 g / L bran, and 0.1 g / L vitamin B1.

[0086] Example 1

[0087] The most suitable tanking time for the fermentation of Ganoderma lucidum (BNCC No. BNCC382030 Ganoderma lucidum, purchased from Beina Biotechnology) to produce laccase was determined by checking the online detection curves of DO meter and pH meter.

[0088] The activated Ganoderma lucidum was inoculated at a 5% inoculum into a shake flask containing culture medium (potato dextrose water, 24.5 g / L). The culture was expanded at 25°C-30°C, 100 rpm, and a ventilation ratio of 0.7 vvm for 80 h to obtain a seed solution. The seed solution was inoculated at a 10% inoculum into a 1200 L stirred tank containing fermentation medium. Fermentation was carried out at a temperature of 25°C-30°C, a stirring speed of 100 rpm, and a ventilation ratio of 0.7 vvm. The DO (dissolved oxygen), pH, and laccase activity curves of different batches were collected during the fermentation process. The laccase activity was detected as follows: 2 mL of pH 3 sodium hydrogen phosphate-citrate buffer was added to a 5 mL EP tube, 0.5 mL of appropriately diluted fermentation supernatant (the absorbance of the diluted fermentation supernatant was in the range of 0.2-0.8), and 0.5 mL of 1 mmol / L ABTS solution was quickly placed in a 45°C constant temperature water bath to react for 5 minutes. After the reaction was complete, the OD 420 .

[0089] Enzyme activity calculation formula: Laccase (U / L) =

[0090] Where, ε is 3.6×10 4 , L / (mol⋅cm);

[0091] V 总 —Total volume of reaction system, mL;

[0092] V 酶 —Volume of fermentation broth added, mL;

[0093] Δt—reaction time, min.

[0094] The change curves of DO, pH and laccase activity during the fermentation process of different batches are shown as follows: Figure 1 As shown in A, B and C in the figure, it can be seen that the pH of the fermentation broth with different enzyme production levels has a trend of first being stable, then decreasing and then increasing, and the higher the enzyme activity, the longer the fermentation time corresponding to the lowest pH point. After the pH begins to rise, the enzyme activity is basically stable or even has a downward trend. This shows that after the pH begins to rise, the Ganoderma lucidum no longer produces laccase, and the corresponding decrease in enzyme activity is caused by the instability of laccase to storage temperature and time. Therefore, the corresponding laccase also has the highest enzyme activity from the lowest pH point to the beginning of the increase.

[0095] The DO of fermentation broths with different enzyme production levels all showed a trend of first decreasing slowly, then decreasing rapidly, and then stabilizing. The three groups with high laccase activity levels had corresponding DO at a higher level. The dissolved oxygen curves of batches with low enzyme activity levels all shifted to the right regularly, that is, the time of rapid decrease in dissolved oxygen was reached earlier, indicating that premature decrease in dissolved oxygen means lower enzyme activity. By comparing the pH, DO and enzyme activity curves, it can be found that the three time points from the lowest point of pH to the beginning of its rise, the beginning of the decrease and stabilization of dissolved oxygen, and the moment the enzyme activity reached its highest point were consistent.

[0096] Example 2

[0097] Using the same fermentation conditions as in Example 1, a batch of fermentation broth was taken and the pH, DO and enzyme activity of the fermentation broth were tested. Figure 2 It can be seen from A, B and C that the pH first decreases and then increases. Since the enzyme activity of this batch is not high, the time to reach the lowest point is relatively short, that is, it reaches the lowest point at 47 hours. The corresponding dissolved oxygen also reaches stability at 47 hours, and the enzyme activity is also the highest at this time.

[0098] Example 3

[0099] Using the same fermentation conditions as in Example 1, a batch of fermentation broth was taken and the pH, DO and enzyme activity of the fermentation broth were tested. Figure 3 It can be seen from A, B and C that the pH first decreases and then increases. Since the enzyme activity of this batch is relatively high, it takes a longer time to reach the lowest point, that is, it reaches the lowest point at 84 hours. The corresponding dissolved oxygen also reaches stability at around 84 hours, and the enzyme activity is also the highest at this time.

[0100] 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.

[0101] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A method for preparing a fermentation broth of an enzyme-producing fungus, characterized in that: The following steps are involved: The enzyme-producing fungus is fermented, and the pH value and dissolved oxygen value are monitored in real time during the fermentation process. When the pH value decreases to the lowest point and then increases by 0.03-0.05, and / or when the dissolved oxygen value decreases by less than or equal to 5% within 2 hours, the fermentation is terminated and a fermentation liquid is prepared.

2. The method for preparing the fermentation liquid of the enzyme-producing fungus according to claim 1, wherein The fermentation step comprises: Expanding and culturing the enzyme-producing fungus to obtain a seed solution; The seed liquid is inoculated into a fermentation tank containing a fermentation medium for fermentation.

3. The method for preparing the fermentation liquid of the enzyme-producing fungus according to claim 2, wherein: The scale of the fermentation is 1000L~1500L.

4. The method for preparing the fermentation liquid of the enzyme-producing fungus according to claim 3, wherein: The fermentation medium is a liquid medium, which includes one or more of 1g / L-5g / L yeast extract powder, 20g / L-25g / L corn steep liquor, 10g / L-15g / L tobacco stem powder, 1.0g / L-1.5g / L KH2PO4, 1.0g / L-1.5g / L MgSO4·7H2O, 30g / L-35g / L bran, and 0.1g / L-0.5g / L vitamin B1.

5. The method for preparing the fermentation liquid of the enzyme-producing fungus according to claim 4, wherein: The fermentation satisfies one or more of the following conditions: The fermentation temperature is 25°C to 30°C; The stirring speed of the fermentation is 70 rpm to 120 rpm; The ventilation ratio of the fermentation is 0.5vvm~0.8vvm.

6. The method for preparing the fermentation liquid of the enzyme-producing fungus according to claim 2, wherein: The expanded culture satisfies one or more of the following conditions: The culture medium in the expanded culture comprises 20 g / L to 25 g / L potato glucose water; The temperature of the expanded culture is 25°C to 30°C; The rotation speed of the expanded culture is 70 rpm to 120 rpm; The ventilation ratio of the expanded culture is 0.5 vvm to 0.7 vvm; The inoculation amount of the expanded culture is 5% to 10%; The time of the expanded culture is 72h~120h.

7. The method for preparing the fermentation liquid of the enzyme-producing fungus according to claim 2, wherein: The seed liquid is inoculated into a fermentation tank containing a fermentation medium in an inoculation amount of 5% to 15%.

8. The method for preparing the fermentation broth of the enzyme-producing fungus according to any one of claims 1 to 7, characterized in that: The enzyme-producing fungus includes a white-rot fungus; optionally, the white-rot fungus includes a Ganoderma lucidum.

9. A method for producing an enzyme, characterized in that: The following steps are involved: The fermentation liquid is prepared by the method for preparing the fermentation liquid of the enzyme-producing fungus according to any one of claims 1 to 8; and The enzyme is isolated from the fermentation broth.

10. The enzyme production method according to claim 9, characterized in that The enzymes include laccases.

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