Culture medium for protease production by Mucor fermentation and its application

Abstract

The invention discloses a culture medium for producing protease by fermentation of Mucor, comprising basic components and additives, wherein the basic components comprise a first citrus citrus mushroom, bran and water, wherein the mass ratio of the first citrus citrus mushroom to the bran is (4.5-5.5):(9.5-10.5), and the additives comprise an additional carbon source, an additional nitrogen source and an additional trace element compound, wherein the additional carbon source is selected from one or more of glucose, sucrose, maltose and starch, the additional nitrogen source is selected from one or more of a second citrus citrus mushroom, soybean meal, yeast extract and peptone, and the trace element is selected from one or more of calcium, zinc, magnesium and sodium. The invention also discloses a method for producing protease by fermentation of Mucor, wherein Mucor is fermented in the culture medium for producing protease by fermentation of Mucor of the above embodiment. The invention also discloses a protease obtained by the above method. The invention also discloses a method for catalyzing protein hydrolysis, comprising hydrolyzing the protein using the above protease.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, and in particular to a culture medium for producing protease by fermentation of Mucor and application thereof. Background Art

[0002] Proteases are widely found in animals, plants and microorganisms. They are a type of enzyme that catalyzes the hydrolysis of proteins to produce peptides and amino acids, and they perform different physiological functions and metabolic activities in organisms. Proteases have a wide range of applications in industry. According to statistics, proteolytic enzymes account for about 75% of all industrial enzyme preparations. Currently, commercially produced proteases mainly come from animals, plants and microorganisms. However, due to the lack of resources and the high cost of separation and purification technology, the preparation of animal and plant proteases faces the problem of difficulty in large-scale production. Microorganisms have attracted much attention due to their fast growth rate, strong flexibility, less space and time required, and biochemical diversity. Microbial proteases are the most important proteases in industrial production, accounting for about 40% of the total enzyme preparations in the world.

[0003] Some commercial neutral and alkaline proteases are derived from bacteria, while there are more types of proteases from fungi, including acidic, neutral and alkaline proteases. Many fungi, such as Penicillium, Aspergillus and yeast, can produce extracellular proteases. Fungal proteases are easier to obtain through solid-state fermentation. Compared with other microorganisms, fungi have the following advantages: most of them are generally recognized as safe substances (GRAS), and the proteases produced are mainly extracellular enzymes, which are easy to separate and purify from fermentation products. Aspergillus oryzae is the most important strain used in traditional plant fermentation. However, it often produces some bitterness in the process of preparing plant peptides. A very small amount of bitterness can have a serious negative impact on the taste of the peptide product, reducing consumer satisfaction with the product. It is an urgent problem to be solved when promoting the application of plant peptides.

[0004] Hair mould is also known as black mould and long hair mould. Common species include big hair mould and micro hair mould. They are widely distributed in wine koji, plant residues, corrupt organic matter and soil. Hair mould can synthesize and secrete a variety of extracellular proteases. Different types of extracellular proteases constitute a complex and orderly protease system, which forms a composite enzyme system with the cellulose and pectinase systems produced together, and catalyzes a variety of biochemical reactions at the same time. One of the reasons why the enzymatic hydrolysis products produce bitterness is the hydrophobic amino acids at the end of the peptide chain. When the protein is hydrolyzed, the structure is destroyed, the internal hydrophobic groups are exposed, and bitterness will be generated. Therefore, the higher the content of hydrophobic amino acids in the protein, the more they are exposed after hydrolysis, and the more bitter the hydrolyzate is. Since the hair mould protease is a composite protease system containing endonucleases and exonucleases, the exonucleases remove the bitterness when they cut off the hydrophobic amino acids from the end of the peptide chain.

[0005] Cibotium cibotium is a cultivable edible mushroom that is considered a healthy food because it contains high levels of protein and dietary fiber, is low in fat, and has pharmacological effects such as antioxidant, immunomodulatory, and anti-inflammatory responses. In addition, Cibotium cibotium has high levels of glutamic acid and aspartic acid, making it suitable for producing high-quality flavor bases. Using Cibotium cibotium protein to prepare bioactive peptides and deep-processing Cibotium cibotium to expand its application range has great economic and social benefits. However, commercial proteases have high costs and low yields, and the choice of food-grade commercial proteases is limited. Summary of the invention

[0006] Based on this, the present invention creatively combines the production of protease by Mucor fermentation with the fermentation of Pleurotus citrinopileatus to provide a culture medium for the production of protease by Mucor fermentation of Pleurotus citrinopileatus with low-cost enzymatic hydrolysis and application thereof.

[0007] The first object of the present invention is to provide a culture medium for producing protease by Mucor fermentation, comprising basic components and additives, wherein the basic components include a first citrus pleurotus eryngii, bran and water, the mass ratio of the first citrus pleurotus eryngii to the bran is (4.5-5.5):(9.5-10.5), the additives include an external carbon source, an external nitrogen source and an external trace element compound, the external carbon source is selected from one or more of glucose, sucrose, maltose and starch, the external nitrogen source is selected from one or more of a second citrus pleurotus eryngii, soybean meal, yeast extract and peptone, and the trace element is selected from one or more of calcium, zinc, magnesium and sodium.

[0008] The second object of the present invention is to provide a method for producing protease by fermentation of Mucor, wherein Mucor is fermented in the culture medium for producing protease by fermentation of Mucor in the above-mentioned embodiment.

[0009] The third object of the present invention is to provide a protease obtained by the method described in any one of the above embodiments.

[0010] The fourth object of the present invention is to provide a method for catalyzing protein hydrolysis, comprising using the protease described in any of the above embodiments to hydrolyze the protein.

[0011] The present invention utilizes a microbial fermentation method to organically combine the fermentation production of protease and the enzymatic hydrolysis of citrus citrus polypeptide. By utilizing citrus citrus as a fermentation medium, Mucor is induced to secrete a protease suitable for enzymatic hydrolysis of citrus citrus polypeptide. While the citrus citrus polypeptide is released under the action of a mixed enzyme system generated by the metabolic activity of the microorganism, the microorganism improves the efficiency of growth and enzyme production with the help of a polypeptide hydrolyzate, thereby reducing the production cost of citrus citrus polypeptide. At the same time, the use of Mucor fermentation also overcomes the disadvantages of heavy bitterness and poor taste of polypeptide products prepared by an enzyme hydrolysis method, thereby having a good application prospect. The growth conditions of the Mucor fermentation are optimized, the effects of factors such as carbon source, nitrogen source, inorganic salt, time, temperature, water-to-material ratio and inoculation amount on the protease activity of the fermented citrus citrus polypeptide are explored, the culture medium components and culture conditions are further optimized, and the protease activity of Mucor is further improved, thereby providing a theoretical basis for the application of protease in citrus citrus. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a diagram showing the results of determining the types of proteases produced by Mucor according to an embodiment of the present invention;

[0013] Figure 2 This is a graph showing the effect of different carbon sources on the activity of Mucor protease according to an embodiment of the present invention;

[0014] Figure 3 This is a graph showing the effect of different nitrogen sources on the activity of Mucor protease according to an embodiment of the present invention;

[0015] Figure 4 This is a graph showing the effects of different trace elements on the activity of Mucor protease according to an embodiment of the present invention;

[0016] Figure 5 This is a graph showing the effect of the inoculation amount on the activity of Mucor protease according to an embodiment of the present invention;

[0017] Figure 6 This is a graph showing the effect of the material-water ratio on the activity of Mucor protease according to an embodiment of the present invention;

[0018] Figure 7 This is a graph showing the effect of fermentation temperature on Mucor protease activity according to an embodiment of the present invention;

[0019] Figure 8 This is a graph showing the effect of fermentation time on the activity of Mucor protease according to an embodiment of the present invention;

[0020] Fig. 9 This is an optimum temperature diagram of a protease according to an embodiment of the present invention;

[0021] Fig.10 This is a temperature stability diagram of a protease according to an embodiment of the present invention;

[0022] Fig.11 The optimum pH value and pH stability diagram of the protease according to one embodiment of the present invention;

[0023] Fig.12 This is a diagram showing the effect of metal ions on protease activity according to an embodiment of the present invention. DETAILED DESCRIPTION

[0024] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0026] In the first aspect, an embodiment of the present invention provides a culture medium for producing protease by Mucor fermentation, comprising basic components and additives, wherein the basic components include a first Pleurotus citrinum, bran and water, the mass ratio of the first Pleurotus citrinum to the bran is (4.5-5.5):(9.5-10.5), the additives include an external carbon source, an external nitrogen source and an external trace element compound, the external carbon source is selected from one or more of glucose, sucrose, maltose and starch, the external nitrogen source is selected from one or more of a second Pleurotus citrinum, soybean meal, yeast extract and peptone, and the trace elements are selected from one or more of calcium, zinc, magnesium and sodium.

[0027] The present invention utilizes a microbial fermentation method to organically combine the fermentation production of protease and the enzymatic hydrolysis of citrus citrus polypeptide. By utilizing citrus citrus as a fermentation medium, Mucor is induced to secrete a protease suitable for enzymatic hydrolysis of citrus citrus polypeptide. While the citrus citrus polypeptide is released under the action of a mixed enzyme system generated by the metabolic activity of the microorganism, the microorganism improves the efficiency of growth and enzyme production with the help of a polypeptide hydrolyzate, thereby reducing the production cost of citrus citrus polypeptide. At the same time, the use of Mucor fermentation also overcomes the disadvantages of heavy bitterness and poor taste of polypeptide products prepared by an enzyme hydrolysis method, thereby having a good application prospect. The growth conditions of the Mucor fermentation are optimized, the effects of factors such as carbon source, nitrogen source, inorganic salt, time, temperature, water-to-material ratio and inoculation amount on the protease activity of the fermented citrus citrus polypeptide are explored, the culture medium components and culture conditions are further optimized, and the protease activity of Mucor is further improved, thereby providing a theoretical basis for the application of protease in citrus citrus. In some embodiments, the mass ratio of the added carbon source to the basic component is (5-7):100, for example, 5:100, 5.5:100, 6:100, 6.5:100, 7:100, etc.; the mass ratio of the added nitrogen source to the basic component is (3-6):100, for example, 3:100, 3.5:100, 4:100, 4.5:100, 5:100, 5.5:100, 5:100, etc.; the mass ratio of the added trace element compound to the basic component is (0.05-0.4):100, for example, 0.05:100, 0.1:100, 0.15:100, 0.2:100, 0.25:100, 0.3:100, 0.35:100, 0.4:100, etc.

[0028] In some embodiments, the added trace element compound can be selected from chlorides and sulfates of trace elements, such as one or more of calcium chloride, zinc chloride, magnesium chloride, sodium chloride, calcium sulfate, zinc sulfate, magnesium sulfate, and sodium sulfate.

[0029] In some specific embodiments, the added carbon source is selected from sucrose, and the mass ratio of the sucrose to the basic component is (6-6.5):100; the added nitrogen source is selected from the second citronella mushroom, and the mass ratio of the second citronella mushroom to the basic component is (4.5-5.5):100; the added trace element compound is selected from magnesium sulfate and calcium chloride, and the mass ratio of the magnesium sulfate to the basic component is (0.04-0.06):100, and the mass ratio of the calcium chloride to the basic component is (0.02-0.04):100.

[0030] In a second aspect, an embodiment of the present invention provides a method for producing protease by fermenting Mucor, wherein Mucor is fermented in a culture medium for producing protease by fermenting Mucor in the above embodiment.

[0031] The initial number of Mucor determines the reproduction of Mucor and the number of spores within a certain period of time, which in turn affects the ability of Mucor to produce protease. In some embodiments, the inoculation amount of the Mucor enzyme in the culture medium for the Mucor fermentation to produce protease is 0.5×10 6 / g~1.5×10 6 Pieces / g.

[0032] Changes in substrate moisture content have an important impact on the growth and metabolic capacity of microorganisms. Low moisture will reduce nutrient transfer, microbial growth, enzyme stability and matrix expansion; high moisture will cause particle agglomeration, poor ventilation and bacterial contamination. Therefore, during the microbial fermentation process, the initial moisture content must be controlled within an appropriate range. In some embodiments, the solid-to-liquid ratio of the culture medium for protease production by Mucor fermentation is 1:(1.2-1.5). "Solid-to-liquid ratio" refers to the mass ratio of the solid phase to the liquid phase in the culture medium.

[0033] Suitable temperature is necessary for bacterial growth. It directly affects the growth of microorganisms, protein synthesis, enzyme and cell activity, and metabolite synthesis. In addition, the culture temperature can extend or shorten the fermentation period accordingly, so the appropriate fermentation temperature must be selected during the fermentation process. The concentration of the product changes during the fermentation process, but the yield increase slows down or even decreases after a certain period of time. Therefore, whether it is to obtain bacterial cells or metabolites, microbial fermentation has an optimal time stage. In many cases, the fermentation time is related to the fermentation temperature. In some embodiments, the fermentation temperature is 27°C to 29°C, and the fermentation time is 72h to 120h.

[0034] Preferably, the fermentation temperature is 27.03°C, the fermentation time is 103.95h, and the water-to-material ratio is 1.43. For the convenience of configuration, preferably, the fermentation temperature is 27°C, the fermentation time is 104h, and the water-to-material ratio is 1.4.

[0035] Preferably, the solid-liquid ratio of the culture medium for producing protease by Mucor fermentation is 1:(1.4-1.5), the fermentation temperature is 27° C.-27.5° C., and the fermentation time is 100 h-110 h.

[0036] In a third aspect, the present invention provides a protease obtained by the method described in any of the above embodiments. The protease obtained by the fermentation method of the culture medium includes alkaline protease, acidic protease and neutral protease.

[0037] In a fourth aspect, an embodiment of the present invention provides a method for catalyzing protein hydrolysis, comprising hydrolyzing the protein using the protease described in any of the above embodiments.

[0038] Temperature is an important factor affecting enzymatic reactions. Temperature increases can promote the reaction rate of proteases, but when the temperature rises to a certain level, the enzyme protein begins to denature, resulting in a loss of enzyme activity. In some embodiments, the hydrolysis temperature is 45°C to 55°C. Specifically, it can be 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, or 55°C.

[0039] In some embodiments, the pH of the hydrolysis is 6.5 to 7.5, and specifically may be 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0040] In some embodiments, a metal ion phosphate buffer is added to the hydrolysis reaction system, and the metal ion is selected from Mg 2+ , K + and Ga 2+ Any one or more of. The metal ion may also be selected from Fe 2+ 、Zn 2+ Any one or both of .

[0041] The present invention is described in detail below with reference to specific embodiments.

[0042] The detection method used in the following examples is:

[0043] Determination of moisture content:

[0044] Direct drying method / GB5009.3—2016

[0045] National Food Safety Standard Determination of Water in Food.

[0046] Determination of crude protein:

[0047] Kjeldahl nitrogen determination method / GB5009.5—2016

[0048] National Food Safety Standard Determination of Protein in Foods.

[0049] Determination of protease activity:

[0050] Folin method / SB / T10317-1999, GB / T23527-2009

[0051] Industry Standard of the People's Republic of China SB / T10317-1999 Protease Activity Assay

[0052] National Standard of the People's Republic of China GB / T23527-2009 Protease preparations.

[0053] Example 1 Effect of Additives on Protease Production

[0054] The effects of carbon source, nitrogen source, trace elements and other factors on protease activity were explored.

[0055] Results of determination of protease types produced by Mucor:

[0056] Mucor can secrete a variety of proteases after fermentation. In order to determine the types of its high-yield proteases, it was cultured at 28°C for 96 hours to fully ferment and produce enzymes in the basic culture medium (5g of Pleurotus citrinopileatus, 10g of bran, and 15g of deionized water). The activity of three proteases in the crude enzyme solution was determined. The results are shown in Table 1. Figure 1 .

[0057] Depend on Figure 1 It can be seen that both Mucor fungi can secrete alkaline protease, acidic protease and neutral protease after fermentation and culture, among which the neutral protease has a higher activity. Therefore, the determination indicator of enzyme activity in subsequent experiments is the activity of Mucor neutral protease.

[0058] 1.1 Screening of external carbon sources

[0059] In the basic culture medium (5g of Pleurotus citrinopileatus, 10g of bran, 15g of deionized water), a certain amount of glucose, sucrose, maltose and starch were added as external carbon sources, and added to the basic culture medium at a rate of 6%, sterilized at 121℃ for 30min, shaken while hot, and cooled to about 30℃ at room temperature. 6 The culture medium was placed in a 28°C mold incubator and cultured for 96 hours before the protease activity was determined. The basal culture medium was used as a blank control and the protease activity was determined under the optimal fermentation conditions. The results are as follows Figure 2 shown.

[0060] The results showed that different carbon sources had different effects on the protease production of Mucor. Generally speaking, carbon sources that are easily utilized are beneficial to the growth of microorganisms, while carbon sources that decompose slowly are beneficial to the production of protease. Figure 2 It can be seen that Mucor uses a wide range of carbon sources, and the type of carbon source has a great influence on the synthesis of Mucor protease. Among them, sucrose is more conducive to Mucor protease production than other carbon sources, which may be due to the induction of sucrose on the synthesis of protease; glucose, maltose, and starch all inhibit the secretion of Mucor protease, which may be due to the inhibitory effect of their decomposition metabolites on protease. In summary, sucrose was selected as the carbon source for the fermentation medium.

[0061] 1.2 Screening of external nitrogen sources

[0062] In the basic culture medium (5g of Pleurotus citrinopileatus, 10g of bran, and 15g of deionized water), a certain amount of Pleurotus citrinopileatus, soybean meal, yeast extract, and peptone were added as external nitrogen sources at a rate of 3% to the basic culture medium, sterilized at 121℃ for 30min, shaken while hot, and cooled to about 30℃ at room temperature. 6 The culture medium was placed in a 28°C mold incubator and cultured for 96 hours before the protease activity was determined. The basal culture medium was used as a blank control and the protease activity was determined under the optimal fermentation conditions. The results are as follows Figure 3 shown.

[0063] The results showed that compared with the blank control, soybean meal, yeast extract and peptone as external nitrogen sources were not conducive to the production of proteases. Pleurotus citrinopileatus as an external nitrogen source was conducive to the production of proteases.

[0064] 1.3 Screening of added trace elements

[0065] In the basic culture medium (5g of Pleurotus citrinopileatus, 10g of bran, and 15g of deionized water), a certain amount of calcium chloride, zinc sulfate, magnesium sulfate, and sodium chloride were added as trace elements of the culture medium at a rate of 0.3%, sterilized at 121℃ for 30min, shaken while hot, and cooled to about 30℃ at room temperature. 6 The culture medium was placed in a 28°C mold incubator and cultured for 96 hours before the protease activity was determined. The basal culture medium was used as a blank control and the protease activity was determined under the optimal fermentation conditions. The results are as follows Figure 4 shown.

[0066] Depend on Figure 4 It can be seen that the type of inorganic salt has a greater impact on the activity of proteases. Calcium chloride and magnesium sulfate have a more significant effect on improving the activity of proteases than other inorganic salts. Among them, the protease activity of Mucor is the highest in the medium with magnesium sulfate added. Since magnesium ions are activators of various enzymes in biological metabolic pathways, magnesium sulfate can increase the activity of proteases. The enzymatic activity of Mucor protease is closely related to calcium ions, which is consistent with the characteristics of fungal proteases. Calcium ions are an effective inducer for the synthesis of this protease. In summary, the inorganic salts selected for Mucor fermentation medium are magnesium sulfate and calcium chloride.

[0067] 1.4 Optimization of external additives

[0068] In order to further optimize the composition of the fermentation medium, based on the preliminary exploration of the effects of each single factor on the activity of the protease, the uniform design table U10 (108) was selected according to the factor level table shown in Table 1, and a uniform design experiment was carried out to optimize the added additives to determine the optimal fermentation medium composition for Mucor protease production.

[0069] Table 1 Uniform design factor level table

[0070]

[0071] According to the results of the single factor experiment, carbon source, nitrogen source and inorganic salt were selected as factors, and the protease activity in the culture medium was used as an indicator to optimize the optimal ratio of the culture medium for protease production by Mucor fermentation. The experimental results are shown in Table 2.

[0072] Table 2U 10 (10 8 ) Optimization of Mucor protease culture medium results

[0073]

[0074]

[0075] 1.5 Validation test of protease production by Mucor fermentation

[0076] The optimal ratio of the culture medium was obtained according to the uniform design optimization, that is, the addition amount of Pleurotus citrinum was 5.0%, the addition amount of sucrose was 6.1437%, the addition amount of magnesium sulfate was 0.0513%, and the addition amount of calcium chloride was 0.03%. The verification test was carried out under the conditions of the optimized culture medium ratio, and the results are shown in Table 3. The results show that the protease activity obtained under the conditions of the optimized culture medium ratio is 99.7229U / g.

[0077] Table 3 Model prediction values ​​and test verification results

[0078]

[0079] Example 2 Effect of fermentation conditions on protease production

[0080] 2.1 Screening of inoculum size

[0081] Take 5 250mL Erlenmeyer flasks, add the fermentation medium with the optimal ratio of 1.5 in Example 1, sterilize at 121°C for 30 min, shake while hot, and cool to about 30°C at room temperature. The inoculation amount of Mucor is 1×10 4 pcs / g, 1×10 5 pcs / g, 1×10 6 pcs / g, 1×10 7 pcs / g, 1×10 8 The cells were placed in a 28°C mold incubator for 96 hours and the protease activity was determined. Figure 5 As shown in the figure, 4:1.0×10 4 / g; 5: 1.0×10 5 / g; 6: 1.0×10 6 / g; 7: 1.0×107 / g; 8: 1.0×10 8 Pieces / g.

[0082] from Figure 5 It can be seen that the inoculation size has a great influence on the synthesis of protease. The general rule of protease activity changing with inoculation size is that when the inoculation size is less than 1×10 6 When the inoculation amount was 1×10 / g, the enzyme activity increased with the increase of inoculation amount, and then the enzyme activity gradually decreased with the increase of inoculation amount. This may be because a high inoculation amount will cause the strain to reproduce too fast, resulting in the nutrients in the culture medium being unable to meet the normal growth metabolism of the bacteria, resulting in competitive inhibition, which is not conducive to the growth of the strain, and thus the ability of Mucor to produce protease is reduced. If the inoculation amount is too low, it exceeds the minimum growth limit of Mucor, the strain grows slowly, the mycelium yield is small, and the protease activity produced is low. Therefore, the inoculation amount is selected to be 1×10 6 Pieces / g.

[0083] 2.2 Screening of material-water ratio

[0084] Take five 250mL triangular flasks, add 5g of Pleurotus citrinopileatus and 10g of bran to each, and then add 7.5g, 10.5g, 15g, 19.5g, and 22.5g of deionized water to make a basic culture medium, sterilize at 121℃ for 30min, shake while hot, and cool to about 30℃ at room temperature. The inoculum size of Mucor is 1×10 6 The cells were placed in a 28°C mold incubator for 96 hours and the protease activity was determined. Figure 6 shown.

[0085] Mucor needs a certain amount of water to grow, but the water content should not be too high. Figure 6 It can be seen that when the material-water ratio is 1:1.3, the activity of Mucor protease is the greatest. When the material-water ratio is too large, it will reduce the volume of gas in the culture medium, affect gas exchange, and easily stick together, which is not conducive to the ventilation and heat dissipation of the solid culture medium, and there is no mycelium growth, and the enzyme activity is naturally not high; when the material-water ratio is too small, the water content of the culture medium is low and cannot provide enough water for Mucor growth. Therefore, the water content of the culture medium must be controlled within a certain range to be suitable for Mucor protease production. The material-water ratio of 1:1.3 is selected as the optimal ratio.

[0086] 2.3 Fermentation temperature screening

[0087] Take 5 250mL Erlenmeyer flasks, add the fermentation medium with the optimal ratio of 1.5 in Example 1, sterilize at 121°C for 30 min, shake while hot, and cool to about 30°C at room temperature. Mucor inoculum amount 1×10 6 The culture medium was cultured at 20℃, 24℃, 28℃, 32℃, and 36℃ for 96h, and the protease activity was determined. Figure 7 shown.

[0088] It can be concluded that the culture temperature has a great influence on the protease production of Mucor. When the temperature is low, Mucor grows slowly and the protease synthesis is relatively delayed. When the temperature is high, Mucor still grows well, but the strain decays quickly and the protease synthesis slows down accordingly. Therefore, the culture temperature of Mucor is selected to be 28℃.

[0089] 2.4 Screening of fermentation time

[0090] Take 6 250mL Erlenmeyer flasks, add the fermentation medium with the optimal ratio of 1.5 in Example 1, sterilize at 121°C for 30 min, shake while hot, and cool to about 30°C at room temperature. Mucor inoculum amount 1×10 6 The culture medium was placed in a 28°C mold incubator, and samples were taken at 24h, 48h, 72h, 96h, 120h, and 144h to determine the protease activity. Figure 8 shown.

[0091] from Figure 8 It can be seen that the activity of Mucor protease is different at different culture times. The relative enzyme activity of Mucor can reach a high level after 72 hours of culture, and the enzyme activity reaches the highest peak after 96 hours of culture. After 120 hours of culture, the enzyme activity decreases. There is an optimal time stage for bacterial fermentation. If the time is too short, the mycelium will not grow completely and the protease activity produced will be low; if the time is too long, the growth environment is not conducive to the growth of Mucor, the strain will decline, and the protease production capacity will decrease. Therefore, the culture time of Mucor is selected as 96 hours.

[0092] 2.5 Optimization of the optimal fermentation conditions for protease production

[0093] Firstly, single factor analysis was performed on the fermentation conditions, and then the Design-Expert 8.06 response surface analysis method was used to optimize the fermentation conditions to determine the optimal fermentation conditions.

[0094] According to the results of the single factor experiment, fermentation temperature, fermentation time and water-to-material ratio were selected as the central combination, and protease activity was used as the test index for response surface analysis, in order to explore the optimal fermentation condition combination for protease production by Mucor fermentation. The experimental results are shown in Table 4.

[0095] Table 4 Experimental design and results of optimizing Mucor growth conditions using response surface methodology

[0096]

[0097] The results of variance analysis of the response surface model of protease activity are shown in Table 5. As can be seen from Table 5, the model F value is 69.62, "Prob>F" <0.0001, indicating that the overall model is highly significant. The "Prob>F" of the lack of fit term of the model is 0.3972, which is greater than 0.05, and the lack of fit term is not significant, indicating that the model is suitable and can be used to analyze the changes in protease activity.

[0098] Table 5 Results of variance analysis of the response surface quadratic model for protease activity

[0099]

[0100]

[0101] Note: * indicates significant at the 0.05 level; ** indicates significant at the 0.01 level; *** indicates significant at the 0.001 level

[0102] The response surface model of protease activity was mathematically analyzed, and the optimal conditions for koji making were obtained by maximizing protease activity: fermentation temperature 27.03℃, fermentation time 103.95h, water-to-material ratio 1.43. The predicted value of protease activity under these conditions was 129.192U / g. Considering the feasibility of actual operation and simplifying the process, the culture conditions were revised to: fermentation temperature 27℃, fermentation time 104h, water-to-material ratio 1.4. The actual fermentation test was carried out under these culture conditions, and the average protease activity was 132.4536U / g.

[0103] Example 3 Study on the enzymatic properties of protease

[0104] 3.1 Extraction of protease

[0105] 3.1.1 Preparation of crude enzyme solution

[0106] Add 5 times the volume of sterile water to the fermentation product, disperse it evenly, extract it at 4°C for 24 hours, filter it with 8 layers of sterile gauze, centrifuge the filtrate (10000r / min, 20min), and the supernatant obtained is the crude enzyme solution.

[0107] 3.1.2 Ammonium sulfate fractional precipitation

[0108] Take 7 samples of 20ml crude enzyme solution, then slowly add solid ammonium sulfate in turn, so that the mass fraction of ammonium sulfate is 20%, 40%, 60%, 80%, and 100%, respectively. After dissolution, extract at 4℃ for 24h, and then centrifuge at 4℃ (10000r / min, 30min) to remove precipitation. Take the supernatant, dialyze with distilled water for 24h, and then centrifuge at 4℃ (10000r / min, 30min). Take the supernatant and adjust it to the same volume, and then measure the protein content and protease activity respectively.

[0109] 3.2 Optimal temperature of protease and the effect of temperature on protease activity

[0110] The enzyme solution was placed in a water bath at 30°C, 40°C, 45°C, 50°C, 55°C, 60°C and 70°C for reaction, and the protease activity was determined. The relative enzyme activity under different temperature conditions was calculated, and the temperature with the highest enzyme activity was the optimum temperature.

[0111] The protease samples were precisely kept in a water bath at 30°C, 40°C, 50°C and 60°C for 20 min, 40 min, 60 min, 80 min and 100 min, respectively, and their protease activities were measured. The enzyme activity before keeping warm was defined as 100%, and the relative enzyme activity under other conditions was calculated.

[0112] like Fig. 9 As shown in the figure, the activity of the protease increases at 30-50°C, and the activity is the highest at 50°C. When the temperature rises above 60°C, the activity of the protease drops sharply, indicating that the protease denatures and inactivates rapidly at too high a temperature. Therefore, the optimal temperature range of the protease is 45-55°C.

[0113] like Fig.10 As shown in the figure, the protease activity remained relatively stable at 30℃ and 40℃ due to the low temperature, which had little effect on the protease. After 100min at 30℃ and 40℃, the enzyme activity was retained at 88.14% and 92.64%, respectively. At 50℃ and 60℃, the protease activity decreased rapidly with the extension of the insulation time. After 100min, the enzyme activity was retained at 34.62% and 4.8%, respectively, indicating that the protease was not a high temperature resistant protease.

[0114] 3.3 Optimal pH of proteases and the effect of pH on protease activity

[0115] The enzyme solution was adjusted to the corresponding pH value with buffer solutions of pH 5.0, pH 6.0, pH 7.0, pH 8.0, pH 9.0 and pH 10.0, respectively, and the protease activity was measured, and the relative enzyme activity at different pH values ​​was calculated.

[0116] The pH value of the enzyme solution was adjusted to 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0 with different buffers, and the solution was kept in a constant temperature water bath at 40°C for 120 min. The protease activity was determined, and the enzyme activity before the incubation was defined as 100%. The relative enzyme activity under other conditions was calculated.

[0117] like Fig.11As shown in the figure, the enzyme activity showed an increasing trend from pH 5 to 7, and the highest enzyme activity was at pH 7. When the pH value continued to increase, the enzyme activity showed a downward trend, but at pH 9.0, the enzyme activity had an obvious peak, indicating that Mucor contained alkaline protease. In summary, the optimal pH value of the protease was 7.0. Each enzyme has an optimal pH value because of the Hofmeister effect. In systems with different pH values, the charge distribution of the amino group and the carboxyl group on the enzyme surface is affected, which in turn affects the binding of the enzyme to the substrate and the catalytic efficiency.

[0118] 3.4 Effects of metal ions on the activity of Mucor protease

[0119] The crude enzyme solution was mixed with an equal amount of 4.0 mmol / L different metal ions (K + 、Zn 2+ , Ca 2+ , Fe 2+ Mg 2+ , Cu 2+ ) phosphate buffer solution was mixed to obtain a metal ion solution with a concentration of 2.0 mmol / L, and the protease activity was determined. The enzyme activity without adding the metal ion solution was defined as 100%, and the relative enzyme activity under different metal ion conditions was calculated.

[0120] Fungal proteases are generally metalloproteases, whose activity mainly depends on certain metal cations, so their activity will be affected by metal chelates. Fig.12 The figure shows the effects of six metal ions on protease activity. 2+ , K + , Ga 2+ It significantly promoted the activity of protease (P<0.05). 2+ 、Zn 2+ The activity of protease was slightly promoted (P>0.05), while Cu 2+ It has a significant inhibitory effect on proteases and an inhibitory effect on proteins of about 30%. This may be due to the interaction between metal ions and related groups in the active center of the enzyme, which changes the conformation of the enzyme.

[0121] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0122] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solution of the present invention in detail, but it cannot be understood as limiting the scope of the invention patent protection. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A method for producing protease by fermentation of Mucor, characterized in that: fermenting Mucor in a culture medium for Mucor fermentation to produce protease; The invention is composed of a basic component and additives, wherein the basic component is composed of a first citrus citrus mushroom, bran and water, the mass ratio of the first citrus citrus mushroom to the bran is (4.5-5.5):(9.5-10.5), and the additives include an external carbon source, an external nitrogen source and an external trace element compound; The added carbon source is selected from sucrose, and the mass ratio of the sucrose to the basic component is (6-6.5):100; the added nitrogen source is selected from the second citronella mushroom, and the mass ratio of the second citronella mushroom to the basic component is (4.5-5.5):100; the added trace element compound is selected from magnesium sulfate and calcium chloride, and the mass ratio of the magnesium sulfate to the basic component is (0.04-0.06):100, and the mass ratio of the calcium chloride to the basic component is (0.02-0.04):100; The solid-liquid ratio of the culture medium for producing protease by Mucor fermentation is 1:(1.2-1.5); The fermentation temperature is 27°C to 29°C; The fermentation time is 72h to 120h.

2. The method for producing protease by fermentation of Mucor according to claim 1, characterized in that: The inoculation amount of the Mucor in the culture medium for the Mucor fermentation to produce protease is 0.5×10 6 / g~1.5×10 6 Pieces / g.

3. The method for producing protease by fermentation of Mucor according to claim 2, characterized in that: The solid-liquid ratio of the culture medium for producing protease by Mucor fermentation is 1:(1.4-1.5), the fermentation temperature is 27°C-27.5°C, and the fermentation time is 100h-110h.

4. The method for producing protease by fermentation of Mucor according to claim 3, characterized in that: The solid-liquid ratio of the culture medium for producing protease by Mucor fermentation is 1.4, the fermentation temperature is 27° C., and the fermentation time is 104 h.

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