Preparation method of eel viscera protease

Abstract

The invention relates to the technical field of fish processing, in particular to a preparation method of eel viscera protease, which comprises the following steps: degreasing, extracting and centrifuging viscera of fresh eels to obtain crude protease liquid: adding the degreased viscera into a buffer solution, and smashing by using a beater, the cutter set comprises an upper cutter body and a lower cutter body, gas circulation channels are arranged in the upper cutter body and the lower cutter body respectively, and the refrigeration assembly refrigerates gas to 4 DEG C and sends the gas into the circulation channels so that the temperature of the upper cutter body and the temperature of the lower cutter body can be kept constant. By modifying the knife group and additionally arranging the refrigeration assembly, the blades can be cooled, the temperature stability in the crushing process is ensured, the influence of the pH value change of a prepared buffer solution caused by the temperature change on the activity of protease is avoided, the deterioration of viscera is also avoided, and the influence of the pH value change is reduced; and less buffer solution and shorter extraction time can be needed, and the centrifugation time is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of fish processing, and in particular to a method for preparing eel visceral protease. Background Art

[0002] In the processing of eel meat products, eel viscera are usually discarded directly, and their nutritional value is not effectively utilized, resulting in a waste of resources. This not only causes a huge waste of aquatic resources but also causes serious environmental pollution. Therefore, the high-value utilization of eel viscera is an issue that needs to be solved urgently.

[0003] Proteases are a type of enzyme in organisms that can break down proteins and are widely found in animal viscera, plant stems and leaves, fruits, and microorganisms. Proteases are mainly extracted from plants, animals, and microorganisms, but proteases extracted from marine and aquatic resources have not been widely used. Nowadays, the demand for fish proteolytic enzymes in food processing is increasing. As one of the main by-products in the fishery industry, viscera have broad biotechnological potential in the extraction and application of digestive enzymes, especially proteases with high activity under a wide range of pH and temperature conditions and high catalytic activity at lower concentrations. These characteristics of fish proteases give them broad application prospects in food processing.

[0004] For example, the prior art application number is: CN201510195823.9, Isolation method of golden pomfret visceral protease, protease and its application, which discloses adding defatted viscera to 3-5 times the volume of a 4°C pH=7.5 Tris-HCl buffer solution, breaking it up with a pulper, and then leaching it at 4°C for 2-5 hours; however, in actual operation, due to the friction between the pulper blade and the viscera and the buffer solution, heat is generated during the breaking process, and then the actual pH value of the buffer may deviate from the target pH value, and the Tris-HCl buffer may be too high. The pH value of the flushing solution decreases with increasing temperature. Generally speaking, for every 1°C increase in temperature, the pH value of the Tris-HCl buffer solution decreases by about 0.03 units. The change in pH value affects the activity of the protease. At the same time, the increase in temperature will also cause changes in enzyme activity and stability, which is not conducive to preparation. Because the activity and stability of many enzymes depend on specific pH conditions and temperature, but the pH value and temperature will change during the crushing process, a preparation method for eel visceral protease is needed that can ensure that the temperature of the crushing process does not rise and achieve a stable pH value. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing eel visceral protease which can ensure that the temperature of the crushing process does not rise and achieve a stable pH value.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A method for preparing eel visceral protease, comprising:

[0008] Step 1: Defatting, leaching and centrifuging the viscera of fresh eels to obtain crude protease solution:

[0009] Cut the cleaned fresh eel viscera into sections, and defatting the viscera in 4°C acetone for 10-20 minutes according to a solid-liquid volume ratio of 1:2-3 to remove fat. After defatting, wash off the acetone on the viscera with 4°C distilled water; add the defatted viscera into 2-4 times the volume of 4°C Tris-HCl buffer solution with pH=7.5, and fully break it with a beater, and then extract it at 4°C for 1.5-3 hours; centrifuge at 4°C, 10000-20000 rpm for 10-20 minutes, and take the supernatant, which is the crude protease enzyme solution;

[0010] Among them, the beater includes a motor, a knife group and a refrigeration component, and the refrigeration component cools the gas to 4°C and keeps it constant; the knife group includes a knife shaft, an upper knife body, a lower knife body and a sealing slip ring, the knife shaft includes a connecting end and a mounting end, the connecting end is connected to the motor, and the upper knife body and the lower knife body are fixedly installed on the mounting end from top to bottom; an air inlet is arranged on one side surface of the mounting end, and an air outlet is arranged on the other side surface; a gas inlet and a gas outlet of different heights are arranged on the side wall in the middle of the knife shaft, and an air inlet channel and an air outlet channel are arranged inside the knife shaft, the air inlet channel is used to connect the gas inlet and the air inlet, and the air outlet channel is used to connect the gas outlet and the air outlet; the sealing slip ring sealing sleeve is arranged on the gas inlet and connected to the refrigeration component to deliver 4°C gas into the air inlet; the upper knife body and the lower knife body both have a middle part and two blades, and the middle part includes a first edge and a second edge that are mutually opposed , the distance from the first edge of the middle part of the upper blade body to the mounting end is greater than the distance from the second edge to the mounting end, and the two blades of the upper blade body are respectively connected to the first edge and the second edge at an angle upward; the distance from the first edge of the middle part of the lower blade body to the mounting end is less than the distance from the second edge to the mounting end, and the two blades of the lower blade body are respectively connected to the first edge and the second edge at an angle downward; the middle part of the upper blade body and the lower blade body overlapping each other is an upper overlapping part and a lower overlapping part, an upper air inlet groove and an upper air outlet groove are provided on the lower surface of the upper overlapping part, and a lower air inlet groove and a lower air outlet groove corresponding to the upper air inlet groove and the upper air outlet groove are provided on the upper surface of the lower overlapping part, the upper air inlet groove and the lower air inlet groove cooperate to form an air inlet pipe and are connected to the air inlet port, and the upper air outlet groove and the lower air outlet groove cooperate to form an air outlet pipe and are connected to the air outlet port; gas circulation channels are respectively provided in the four blades; the circulation channels are respectively connected to the air outlet pipe and the air inlet pipe;

[0011] Step 2: Use ammonium sulfate to roughly separate the crude protease solution to obtain a composite protease solution:

[0012] Step 3: Separate the composite protease solution obtained in step 2 to obtain a number of eel visceral proteases.

[0013] Preferably, the cleaned fresh eel viscera are cut into sections in the length direction of the viscera.

[0014] Preferably, step 2 further comprises:

[0015] Grind ammonium sulfate into fine powder, add it into the crude protease solution to the following mass fractions: 25%, 35%, 45%, 55%, 65%, respectively, and salt out to obtain the crude composite protease solution;

[0016] Taking casein as the standard substrate, the enzyme activity of the crude composite protease solution obtained by salting out at different ammonium sulfate mass fractions was determined by ultraviolet spectrophotometry, and the crude composite protease solution with the highest activity was selected;

[0017] The obtained crude composite protease solution with the highest activity is allowed to stand at 4°C for 2-5 hours, centrifuged at 4°C and 10000-15000 r / min for 10-20 min, the supernatant is discarded, the precipitate is dissolved with 1 / 5-1 / 4 volume of the crude protease solution of pH 7.5 Tris-Hcl buffer solution, dialyzed at 4°C with distilled water as the dialysis external fluid under magnetic stirring for 24-48 hours, during which the distilled water is changed several times, and the liquid in the dialysis bag is the composite protease solution.

[0018] Preferably, the step 3 further comprises: eluting the composite protease solution obtained in the separation step 2 with an eluent and freeze-drying the mixture to obtain a plurality of eel visceral proteases.

[0019] Preferably, the sealing slip ring is provided with a first interface and a second interface, the first interface and the second interface are arranged up and down, and the upper and lower intervals are the same as the intervals between the gas inlet and the gas outlet; the inner wall of the sealing slip ring is provided with a first annular groove corresponding to the first interface, and a second annular groove corresponding to the second interface;

[0020] The sealing sliding ring sealing sleeve is arranged on the outer periphery of the knife shaft and the first annular groove is arranged opposite to the gas inlet, the second annular groove is arranged opposite to the gas outlet, and is connected to the refrigeration component to send the gas at 4°C into the air inlet through the first interface, and at the same time, the gas discharged from the gas outlet is sent into the refrigeration component through the second interface, the gas is cooled to 4°C again, and then sent to the first interface to form a cycle.

[0021] Preferably, after the knife group is installed, magnetic fluid is passed through the first interface, gas inlet, air inlet channel, air inlet, air inlet pipe, blade circulation pipe, air outlet pipe, air outlet, air outlet channel, gas outlet, and second interface for polishing.

[0022] Preferably, the knife set is installed in an environment below 4°C.

[0023] Preferably, the blade has a cutting edge, and the gas circulation channel in the blade is arranged close to the cutting edge.

[0024] Preferably, the blade has an upper surface, a lower surface and a blade back, and the blade back is perpendicular to the upper surface and the lower surface respectively.

[0025] Preferably, the upper blade body and the lower blade body are obtained by 3D printing.

[0026] The beneficial effects of the present invention are as follows: by modifying the existing blade group of the pulping machine and adding a refrigeration component, and by connecting the circulation channel in the blade with the air outlet pipe and the air inlet pipe formed in the middle of the upper blade body and the lower blade body, the gas at 4°C generated by the refrigeration component is circulated through the gas inlet, the air inlet channel, the air inlet, the air inlet pipe, the circulation pipe of the blade, the air outlet pipe, the air outlet, the air outlet channel, and the gas outlet, so that the blade can be cooled, and the temperature stability during the smashing process is ensured, and the change of the pH value of the configured Tris-HCl buffer solution caused by the temperature change is avoided to affect the activity of the protease, and the deterioration of the internal organs caused by the temperature change is also avoided. Since the influence of the change of the pH value is reduced, less Tris-HCl is required. The buffer solution and the prior art require a longer time to recover to 4°C due to the increase in temperature, resulting in the need for recovery time in the extraction step. However, the present application does not require recovery and can achieve a shorter extraction time. The reduction in the amount of solution also shortens the centrifugation time, thereby saving costs and improving efficiency. By making the upper and lower long ends of the middle parts of the upper and lower blade bodies inconsistent, the places where the blades pass can be different, thereby increasing the speed of crushing and improving efficiency. At the same time, the low-temperature and constant-temperature blades can ensure hardness. Since eels eat some fish or crustaceans, there may be some lumps or food residues in the eel's internal organs. The low-temperature and constant-temperature knives ensure hardness and can achieve cutting. Even if they are not cut, they can avoid excessive instantaneous friction and temperature rise that affects the pH value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A schematic diagram of a blade assembly of a beater used in a method for preparing eel visceral protease according to a specific embodiment of the present invention;

[0028] Figure 2 An exploded view of a blade assembly of a beater used in a method for preparing eel visceral protease according to a specific embodiment of the present invention;

[0029] Figure 3 An internal diagram of the blades of a beater knife group used in a method for preparing eel visceral protease according to a specific embodiment of the present invention (the thickness of a blade portion of the upper knife body is hidden to reflect the internal circulation channel);

[0030] Figure 4 for Figure 3 Another angle view of

[0031] Figure 5 for Figure 3 A bottom view at an oblique angle;

[0032] Explanation of reference numerals: 1. knife assembly; 11. knife shaft; 111. connection end; 112. mounting end; 113. air inlet; 114. air outlet; 115. gas inlet; 116. gas outlet; 12. upper knife body; 121. upper air inlet groove; 122. upper air outlet groove; 13. lower knife body; 131. lower air inlet groove; 132. lower air outlet groove; 14. sealing slip ring; 141. first interface; 142. second interface; 143. first annular groove; 144. second annular groove; 15. middle part; 16. blade; 161. circulation channel; 162. blade; 163. back of knife. DETAILED DESCRIPTION

[0033] In order to explain the technical content, achieved objectives and effects of the present invention in detail, the following is an explanation in conjunction with the implementation modes and the accompanying drawings.

[0034] Please refer to Figures 1 to 5 , a method for preparing eel visceral protease, comprising:

[0035] Step 1: Defatting, leaching and centrifuging the viscera of fresh eels to obtain crude protease solution:

[0036] Cut the cleaned fresh eel viscera into sections, and defatting the viscera in 4°C acetone for 10-20 minutes according to a solid-liquid volume ratio of 1:2-3 to remove fat. After defatting, wash off the acetone on the viscera with 4°C distilled water; add the defatted viscera into 2-4 times the volume of 4°C Tris-HCl buffer solution with pH=7.5, and fully break it with a beater, and then extract it at 4°C for 1.5-3 hours; centrifuge at 4°C, 10000-20000 rpm for 10-20 minutes, and take the supernatant, which is the crude protease enzyme solution;

[0037] The beater includes a motor, a knife group 1 and a refrigeration component, wherein the refrigeration component cools the gas to 4°C and keeps it constant; the knife group 1 includes a knife shaft 11, an upper knife body 12, a lower knife body 13 and a sealing slip ring 14, the knife shaft 11 includes a connecting end 111 and a mounting end 112, the connecting end 111 is connected to the motor, and the upper knife body 12 and the lower knife body 13 are fixedly mounted on the mounting end 112 from top to bottom; an air inlet 113 is provided on one side surface of the mounting end 112, and an air outlet 114 is provided on the other side surface; a side wall of the middle part 15 of the knife shaft 11 is provided A gas inlet 115 and a gas outlet 116 of different heights are arranged, and an air inlet channel and an air outlet channel are arranged inside the knife shaft 11, the air inlet channel is used to communicate with the gas inlet 115 and the air inlet 113, and the air outlet channel is used to communicate with the gas outlet 116 and the air outlet 114; the sealing sleeve of the sealing slip ring 14 is arranged on the gas inlet 115 and is connected to the refrigeration component to deliver 4°C gas into the air inlet 113; the upper knife body 12 and the lower knife body 13 both have a middle part 15 and two blades 16, and the middle part 15 includes a first edge and a second edge that are mutually opposite The distance from the first edge of the middle part 15 of the upper blade body 12 to the mounting end 112 is greater than the distance from the second edge to the mounting end 112, and the two blades 16 of the upper blade body 12 are respectively connected to the first edge and the second edge by tilting upward; the distance from the first edge of the middle part 15 of the lower blade body 13 to the mounting end 112 is less than the distance from the second edge to the mounting end 112, and the two blades 16 of the lower blade body 13 are respectively connected to the first edge and the second edge by tilting downward; the middle part 15 of the upper blade body 12 and the lower blade body 13 overlap each other to form an upper overlapping part and a lower overlapping part, and the lower part of the upper overlapping part An upper air inlet groove 121 and an upper air outlet groove 122 are provided on the surface of the lower overlapped portion, and a lower air inlet groove 131 and a lower air outlet groove 132 corresponding to the upper air inlet groove 121 and the upper air outlet groove 122 are provided on the upper surface of the lower overlapped portion, the upper air inlet groove 121 and the lower air inlet groove 131 cooperate to form an air inlet pipe and are connected to the air inlet port 113, and the upper air outlet groove 122 and the lower air outlet groove 132 cooperate to form an air outlet pipe and are connected to the air outlet port 114; gas circulation channels 161 are respectively provided in the four blades 16; the circulation channels 161 are respectively connected to the air outlet pipe and the air inlet pipe;

[0038] Step 2: Use ammonium sulfate to roughly separate the crude protease solution to obtain a composite protease solution:

[0039] Step 3: Separate the composite protease solution obtained in step 2 to obtain a number of eel visceral proteases.

[0040] From the above description, it can be known that by modifying the existing blade group 1 of the beater and adding a refrigeration component, and by connecting the circulation channel 161 in the blade 16 and the air outlet pipe and air inlet pipe formed in the middle part 15 of the upper blade body 12 and the lower blade body 13, the 4°C gas generated by the refrigeration component is circulated through the gas inlet 115, the air inlet channel, the air inlet port 113, the air inlet pipe, the circulation pipe of the blade 16, the air outlet pipe, the air outlet port 114, the air outlet channel, and the gas outlet 116, so that the blade 16 can be cooled, ensuring the temperature stability during the smashing process, avoiding the change of the pH value of the configured Tris-HCl buffer solution caused by the temperature change and affecting the activity of the protease, and also avoiding the deterioration of the internal organs caused by the temperature change. Since the influence of the change of the pH value is reduced, less is needed. The Tris-HCl buffer solution in the prior art takes a longer time to recover to 4°C due to the increase in temperature, resulting in the need for recovery time in the extraction step. However, the present application does not require recovery and can achieve a shorter extraction time. The reduction in the amount of solution also shortens the centrifugation time, thereby saving costs and improving efficiency. The upper and lower long ends of the middle portion 15 of the upper blade body 12 and the lower blade body 13 are inconsistent, so that the blade 16 passes through different places, thereby increasing the speed of crushing and improving efficiency. At the same time, the low-temperature and constant-temperature blade 16 can ensure hardness. Since eels eat some fish or crustaceans, there may be some lumps or food residues in the eel's internal organs. The low-temperature and constant-temperature knife ensures hardness and can achieve cutting. Even if it does not cut, it can avoid excessive instantaneous friction and temperature rise that affects the pH value.

[0041] Furthermore, the cleaned fresh eel viscera are cut into sections in the length direction of the viscera.

[0042] From the above description, we can see that since there is mucus in the eel's internal organs, even after cleaning, some will still remain. Cutting it in the length direction (that is, the eel is in the shape of long strips, the direction from head to tail of the eel is the length direction, and the length direction of the internal organs is also the direction from head to tail of the eel) can make the internal organs exposed without being covered by mucus, which is convenient for fat removal.

[0043] Furthermore, step 2 further includes:

[0044] Grind ammonium sulfate into fine powder, add it into the crude protease solution to the following mass fractions: 25%, 35%, 45%, 55%, 65%, respectively, and salt out to obtain the crude composite protease solution;

[0045] Taking casein as the standard substrate, the enzyme activity of the crude composite protease solution obtained by salting out at different ammonium sulfate mass fractions was determined by ultraviolet spectrophotometry, and the crude composite protease solution with the highest activity was selected;

[0046] The obtained crude composite protease solution with the highest activity is allowed to stand at 4°C for 2-5 hours, centrifuged at 4°C and 10000-15000 r / min for 10-20 min, the supernatant is discarded, the precipitate is dissolved with 1 / 5-1 / 4 volume of the crude protease solution of pH 7.5 Tris-Hcl buffer solution, dialyzed at 4°C with distilled water as the dialysis external fluid under magnetic stirring for 24-48 hours, during which the distilled water is changed several times, and the liquid in the dialysis bag is the composite protease solution.

[0047] Furthermore, the step 3 further comprises: eluting the composite protease solution obtained in the separation step 2 with an eluent and freeze-drying the mixture to obtain a plurality of eel visceral proteases.

[0048] Furthermore, the sealing slip ring 14 is provided with a first interface 141 and a second interface 142, and the first interface 141 and the second interface 142 are arranged up and down, and the upper and lower intervals are the same as the intervals between the gas inlet 115 and the gas outlet 116; the inner wall of the sealing slip ring 14 is provided with a first annular groove 143 corresponding to the first interface 141, and a second annular groove 144 corresponding to the second interface 142;

[0049] The sealing sleeve of the sealing slip ring 14 is arranged on the outer periphery of the knife shaft 11 and the first annular groove 143 is arranged opposite to the gas inlet 115, the second annular groove 144 and the gas outlet 116 are arranged opposite to each other, and are connected to the refrigeration component to deliver the gas at 4°C into the air inlet 113 through the first interface 141, and at the same time, the gas discharged from the gas outlet 116 is delivered into the refrigeration component through the second interface 142, the gas is cooled to 4°C again, and then delivered to the first interface 141 to form a cycle.

[0050] It can be seen from the above description that energy consumption can be reduced by recycling gas.

[0051] Furthermore, after the installation of the knife group 1 is completed, magnetic fluid is passed through the first interface 141, the gas inlet 115, the air inlet channel, the air inlet 113, the air inlet pipe, the circulation pipe of the blade 16, the air outlet pipe, the air outlet 114, the air outlet channel, the gas outlet 116, and the second interface 142 for polishing.

[0052] From the above description, it can be seen that by using magnetic fluid for polishing, the resistance of gas operation can be reduced and the constant temperature effect can be ensured.

[0053] Furthermore, the knife set 1 is installed in an environment below 4°C.

[0054] From the above description, it can be seen that since the application scenario of the present application is 4°C, by installing in an environment below 4°C, the sealing can be guaranteed, and even if the temperature rises and expands, better sealing can be achieved.

[0055] Furthermore, the blade 16 has a blade edge 162 , and a gas circulation channel 161 in the blade 16 is disposed close to the blade edge 162 .

[0056] It can be seen from the above description that since the blade 162 generates the most heat during cutting, the blade 162 can be quickly cooled by arranging the gas circulation channel 161 close to the blade 162.

[0057] Furthermore, the blade 16 has an upper surface, a lower surface and a blade back 163, and the blade back 163 is perpendicular to the upper surface and the lower surface respectively.

[0058] From the above description, it can be seen that by making the blade back 163 perpendicular to the upper surface and the lower surface respectively, a vortex can be formed at the blade back 163 during the rotation process, which facilitates the collision and mixing of the eel viscera at the blade back 163 and improves the crushing effect.

[0059] Furthermore, the upper blade body 12 and the lower blade body 13 are obtained by 3D printing.

[0060] From the above description, it can be seen that by using 3D printed upper blade body 12 and lower blade body 13, due to the presence of various grooves and channels, traditional processing is difficult, and directly using 3D printing can solve the above problem.

[0061] Embodiment 1

[0062] A method for preparing eel visceral protease, comprising:

[0063] Step 1: Defatting, leaching and centrifuging the viscera of fresh eels to obtain crude protease solution:

[0064] Cut the cleaned fresh eel viscera into sections along the length of the viscera, defat the viscera in acetone at 4°C for 20 minutes at a solid-liquid volume ratio of 1:2 to remove fat, and wash off the acetone on the viscera with 4°C distilled water after defatting; add the defatted viscera to 2 volumes of 4°C Tris-HCl buffer solution with a pH of 7.5, fully break it up with a beater, and then soak it at 4°C for 1.5 hours; centrifuge at 4°C, 10,000 rpm for 10 minutes, and take the supernatant, which is the crude protease solution;

[0065] Step 2: Use ammonium sulfate to roughly separate the crude protease solution to obtain a composite protease solution:

[0066] Grind ammonium sulfate into fine powder, add it into the crude protease solution to the following mass fractions: 25%, 35%, 45%, 55%, 65%, respectively, and salt out to obtain the crude composite protease solution;

[0067] Taking casein as the standard substrate, the enzyme activity of the crude composite protease solution obtained by salting out at different ammonium sulfate mass fractions was determined by ultraviolet spectrophotometry, and the crude composite protease solution with the highest activity was selected;

[0068] The obtained crude composite protease solution with the highest activity is allowed to stand at 4°C for 2-5 hours, centrifuged at 4°C and 10000-15000 r / min for 10-20 min, the supernatant is discarded, the precipitate is dissolved with 1 / 5-1 / 4 volume of the crude protease solution of pH 7.5 Tris-Hcl buffer solution, dialyzed at 4°C with distilled water as the dialysis external fluid under magnetic stirring for 24-48 hours, during which the distilled water is changed several times, and the liquid in the dialysis bag is the composite protease solution.

[0069] Step 3: Separate the composite protease solution obtained in step 2, elute it with an eluent, and freeze-dry it to obtain a number of eel visceral proteases.

[0070] The beater includes a motor, a knife group 1 and a refrigeration component, wherein the refrigeration component cools the gas to 4°C and keeps it constant; the knife group 1 includes a knife shaft 11, an upper knife body 12, a lower knife body 13 and a sealing slip ring 14, the knife shaft 11 includes a connecting end 111 and a mounting end 112, the connecting end 111 is connected to the motor, and the upper knife body 12 and the lower knife body 13 are fixedly mounted on the mounting end 112 from top to bottom; an air inlet 113 is provided on one side surface of the mounting end 112, and an air outlet 114 is provided on the other side surface; a side wall of the middle part 15 of the knife shaft 11 is provided A gas inlet 115 and a gas outlet 116 of different heights are arranged, and an air inlet channel and an air outlet channel are arranged inside the knife shaft 11, the air inlet channel is used to communicate with the gas inlet 115 and the air inlet 113, and the air outlet channel is used to communicate with the gas outlet 116 and the air outlet 114; the sealing sleeve of the sealing slip ring 14 is arranged on the gas inlet 115 and is connected to the refrigeration component to deliver 4°C gas into the air inlet 113; the upper knife body 12 and the lower knife body 13 both have a middle part 15 and two blades 16, and the middle part 15 includes a first edge and a second edge that are mutually opposite The distance from the first edge of the middle part 15 of the upper blade body 12 to the mounting end 112 is greater than the distance from the second edge to the mounting end 112, and the two blades 16 of the upper blade body 12 are respectively connected to the first edge and the second edge by tilting upward; the distance from the first edge of the middle part 15 of the lower blade body 13 to the mounting end 112 is less than the distance from the second edge to the mounting end 112, and the two blades 16 of the lower blade body 13 are respectively connected to the first edge and the second edge by tilting downward; the middle part 15 of the upper blade body 12 and the lower blade body 13 overlap each other to form an upper overlapping part and a lower overlapping part, and the lower part of the upper overlapping part An upper air inlet groove 121 and an upper air outlet groove 122 are provided on the surface of the lower overlapped portion, and a lower air inlet groove 131 and a lower air outlet groove 132 corresponding to the upper air inlet groove 121 and the upper air outlet groove 122 are provided on the upper surface of the lower overlapped portion, the upper air inlet groove 121 and the lower air inlet groove 131 cooperate to form an air inlet pipe and are connected to the air inlet port 113, and the upper air outlet groove 122 and the lower air outlet groove 132 cooperate to form an air outlet pipe and are connected to the air outlet port 114; gas circulation channels 161 are respectively provided in the four blades 16; the circulation channels 161 are respectively connected to the air outlet pipe and the air inlet pipe;

[0071] The sealing slip ring 14 is provided with a first interface 141 and a second interface 142, and the first interface 141 and the second interface 142 are arranged up and down, and the upper and lower intervals are the same as the intervals between the gas inlet 115 and the gas outlet 116; the inner wall of the sealing slip ring 14 is provided with a first annular groove 143 corresponding to the first interface 141, and a second annular groove 144 corresponding to the second interface 142;

[0072] The sealing sleeve of the sealing slip ring 14 is arranged on the outer periphery of the knife shaft 11 and the first annular groove 143 is arranged opposite to the gas inlet 115, the second annular groove 144 and the gas outlet 116 are arranged opposite to each other, and are connected to the refrigeration component to deliver the gas at 4°C into the air inlet 113 through the first interface 141, and at the same time, the gas discharged from the gas outlet 116 is delivered into the refrigeration component through the second interface 142, the gas is cooled to 4°C again, and then delivered to the first interface 141 to form a cycle.

[0073] After the knife group 1 is installed, magnetic fluid is passed through the first interface 141, the gas inlet 115, the air inlet channel, the air inlet 113, the air inlet pipe, the circulation pipe of the blade 16, the air outlet pipe, the air outlet 114, the air outlet channel, the gas outlet 116, and the second interface 142 for polishing.

[0074] The knife set 1 is installed in an environment below 4°C.

[0075] The blade 16 has a blade edge 162 , and a gas circulation channel 161 in the blade 16 is disposed close to the blade edge 162 .

[0076] The blade 16 has an upper surface, a lower surface and a blade back 163 , and the blade back 163 is perpendicular to the upper surface and the lower surface respectively.

[0077] The upper blade body 12 and the lower blade body 13 are obtained by 3D printing.

[0078] Embodiment 2

[0079] A method for preparing eel visceral protease, which is the same as that of Example 1 and will not be repeated herein, wherein:

[0080] The viscera were defatted in acetone at 4°C for 15 min at a solid-to-liquid volume ratio of 1:2.5;

[0081] The defatted viscera were added into 2.5 volumes of 4°C pH 7.5 Tris-HCl buffer solution, and the viscera were fully broken up with a pulper and then soaked at 4°C for 2 hours; the viscera were centrifuged at 4°C, 15000 rpm for 15 minutes, and the supernatant was taken, which was the crude protease solution.

[0082] Embodiment 3

[0083] The viscera were defatted in acetone at 4°C for 20 min at a solid-to-liquid volume ratio of 1:3;

[0084] A method for preparing eel visceral protease, which is the same as that of Example 1 and will not be repeated herein, wherein:

[0085] The defatted viscera were added into 3 volumes of 4°C pH 7.5 Tris-HCl buffer solution, and the viscera were fully broken up with a pulper and then extracted at 4°C for 3 hours; the viscera were centrifuged at 4°C, 20,000 rpm for 20 minutes, and the supernatant was taken, which was the crude protease solution.

[0086] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention's specification and drawings, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for preparing eel visceral protease, characterized in that: include: Step 1: Defatting, leaching and centrifuging the viscera of fresh eels to obtain crude protease solution: Cut the cleaned fresh eel viscera into sections, and defatting the viscera in 4°C acetone for 10-20 minutes according to a solid-liquid volume ratio of 1:2-3 to remove fat. After defatting, wash off the acetone on the viscera with 4°C distilled water; add the defatted viscera into 2-4 times the volume of 4°C Tris-HCl buffer solution with pH=7.5, and fully break it with a beater, and then extract it at 4°C for 1.5-3 hours; centrifuge at 4°C, 10000-20000 rpm for 10-20 minutes, and take the supernatant, which is the crude protease enzyme solution; Among them, the beater includes a motor, a knife group and a refrigeration component, and the refrigeration component cools the gas to 4°C and keeps it constant; the knife group includes a knife shaft, an upper knife body, a lower knife body and a sealing slip ring, the knife shaft includes a connecting end and a mounting end, the connecting end is connected to the motor, and the upper knife body and the lower knife body are fixedly installed on the mounting end from top to bottom; an air inlet is arranged on one side surface of the mounting end, and an air outlet is arranged on the other side surface; a gas inlet and a gas outlet of different heights are arranged on the side wall in the middle of the knife shaft, and an air inlet channel and an air outlet channel are arranged inside the knife shaft, the air inlet channel is used to connect the gas inlet and the air inlet, and the air outlet channel is used to connect the gas outlet and the air outlet; the sealing slip ring sealing sleeve is arranged on the gas inlet and connected to the refrigeration component to deliver 4°C gas into the air inlet; the upper knife body and the lower knife body both have a middle part and two blades, and the middle part includes a first edge and a second edge that are mutually opposed , the distance from the first edge of the middle part of the upper blade body to the mounting end is greater than the distance from the second edge to the mounting end, and the two blades of the upper blade body are respectively connected to the first edge and the second edge at an angle upward; the distance from the first edge of the middle part of the lower blade body to the mounting end is less than the distance from the second edge to the mounting end, and the two blades of the lower blade body are respectively connected to the first edge and the second edge at an angle downward; the middle part of the upper blade body and the lower blade body overlapping each other is an upper overlapping part and a lower overlapping part, an upper air inlet groove and an upper air outlet groove are provided on the lower surface of the upper overlapping part, and a lower air inlet groove and a lower air outlet groove corresponding to the upper air inlet groove and the upper air outlet groove are provided on the upper surface of the lower overlapping part, the upper air inlet groove and the lower air inlet groove cooperate to form an air inlet pipe and are connected to the air inlet port, and the upper air outlet groove and the lower air outlet groove cooperate to form an air outlet pipe and are connected to the air outlet port; gas circulation channels are respectively provided in the four blades; the circulation channels are respectively connected to the air outlet pipe and the air inlet pipe; Step 2: Use ammonium sulfate to roughly separate the crude protease solution to obtain a composite protease solution: Step 3: Separate the composite protease solution obtained in step 2 to obtain a number of eel visceral proteases.

2. The method for preparing eel visceral protease according to claim 1, characterized in that: When cutting the cleaned fresh eel viscera into sections, cut along the length of the viscera.

3. The method for preparing eel visceral protease according to claim 1, characterized in that: Step 2 further includes: Grind ammonium sulfate into fine powder, add it into the crude protease solution to the following mass fractions: 25%, 35%, 45%, 55%, 65%, respectively, and salt out to obtain the crude composite protease solution; Taking casein as the standard substrate, the enzyme activity of the crude composite protease solution obtained by salting out at different ammonium sulfate mass fractions was determined by ultraviolet spectrophotometry, and the crude composite protease solution with the highest activity was selected; The obtained crude composite protease solution with the highest activity is allowed to stand at 4°C for 2-5 hours, centrifuged at 4°C and 10000-15000 r / min for 10-20 min, the supernatant is discarded, the precipitate is dissolved with 1 / 5-1 / 4 volume of the crude protease solution of pH 7.5 Tris-Hcl buffer solution, dialyzed at 4°C with distilled water as the dialysis external fluid under magnetic stirring for 24-48 hours, during which the distilled water is changed several times, and the liquid in the dialysis bag is the composite protease solution.

4. The method for preparing eel visceral protease according to claim 1, characterized in that: The step 3 further comprises: eluting the composite protease solution obtained in the separation step 2 with an eluent and freeze-drying the mixture to obtain a plurality of eel visceral proteases.

5. The method for preparing eel visceral protease according to claim 1, characterized in that: The sealing slip ring is provided with a first interface and a second interface, the first interface and the second interface are arranged up and down, and the upper and lower intervals are the same as the intervals between the gas inlet and the gas outlet; the inner wall of the sealing slip ring is provided with a first annular groove corresponding to the first interface, and a second annular groove corresponding to the second interface; The sealing sliding ring sealing sleeve is arranged on the outer periphery of the knife shaft and the first annular groove is arranged opposite to the gas inlet, the second annular groove is arranged opposite to the gas outlet, and is connected to the refrigeration component to send the gas at 4°C into the air inlet through the first interface, and at the same time, the gas discharged from the gas outlet is sent into the refrigeration component through the second interface, the gas is cooled to 4°C again, and then sent to the first interface to form a cycle.

6. The method for preparing eel visceral protease according to claim 5, characterized in that: After the knife set is installed, magnetic fluid is passed through the first interface, the gas inlet, the air inlet channel, the air inlet, the air inlet pipe, the circulation pipe of the blade, the air outlet pipe, the air outlet, the air outlet channel, the gas outlet, and the second interface for polishing.

7. The method for preparing eel visceral protease according to claim 6, characterized in that: The knife set is installed in an environment below 4°C.

8. The method for preparing eel visceral protease according to claim 1, characterized in that: The blade has a blade edge, and the gas circulation channel in the blade is arranged close to the blade edge.

9. The method for preparing eel visceral protease according to claim 1, characterized in that: The blade comprises an upper surface, a lower surface and a blade back, and the blade back is perpendicular to the upper surface and the lower surface respectively.

10. The method for preparing eel visceral protease according to claim 1, characterized in that: The upper blade body and the lower blade body are obtained by 3D printing.

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