CN107821922B

CN107821922B - Method for preparing whole shrimp pulp by debitterizing and enzymolysis of marine mysid shrimp and application thereof

Info

Publication number: CN107821922B
Application number: CN201711291095.7A
Authority: CN
Inventors: 许剑彬; 孙姜; 许福土; 王灵华
Original assignee: Yuhuan Wufeng Steamed And Degreased Fish Meal Factory
Current assignee: Yuhuan Wufeng Dry And Skim Fish Meal Factory
Priority date: 2017-12-08
Filing date: 2017-12-08
Publication date: 2020-10-27
Anticipated expiration: 2037-12-08
Also published as: CN107821922A

Abstract

The method for preparing the whole shrimp pulp rich in small peptide by the debitterizing and enzymolysis of the marine small mysidae comprises the following steps: 1) granulating, namely, classifying and crushing fresh marine small trash fish as raw material fish into protein granules with the maximum size of below 3 mm; 2) micronizing, and shearing at high speed to obtain protein microparticle slurry of 150 mesh or above; 3) micronizing, boiling at 90-100 deg.C for 10-20min, cooling to 55-75 deg.C, placing an ultrasonic probe into the tank, and performing micronization ultrasonic treatment to obtain micronized protein activated slurry; 4) carrying out enzymolysis, namely heating the shrimp to an enzymolysis reaction temperature of 40-65 ℃ in a microwave surrounding manner, adding protease which is 0.02-2% of the weight of the raw material shrimp into the particle protein slurry, and circularly and alternately applying the microwave, the ultrasonic wave and the enzymolysis reaction for 10-20 minutes/time, wherein the total enzymolysis reaction time is 20-60min, so as to obtain the enzymolysis protein slurry; inactivating enzyme, and vacuum concentrating to water content of 40-60wt% to obtain small peptide-rich enzymatic hydrolysate of whole shrimp pulp of marine Mysidacea; the preparation method can replace shrimp meal to be used as a feed protein source of an economic feed for aquaculture.

Description

Method for preparing whole shrimp pulp by debitterizing and enzymolysis of marine mysid shrimp and application thereof

Technical Field

The invention relates to the technical field of protein enzymolysis, in particular to a preparation method and application of whole shrimp paste rich in small peptides prepared by enzymolysis of marine furfur shrimps.

Background

An important and costly material in fish and shrimp feed is protein. The abundant marine fishes are the main objects of proteolysis, and one of the hydrolysis products of the marine proteolysis is small peptide. The small peptide is a general term of small molecular peptides, and comprises both molecular small peptides with 2-3 amino acid residues and oligopeptides with less than 10 amino acid residues, i.e. in practical application, small molecular small peptide products with about 10 amino acid residues can be classified as small peptide products. The small peptide has good functional characteristics, can be quickly absorbed by organisms in a complete form, directly participates in the synthesis of tissue protein, has physiological functions of resisting bacteria and improving immunity, and realizes the high-valued protein. Researches prove that the small peptide replaces a certain proportion of fish meal to be used as feed for feeding cultured fishes, can promote the growth of the cultured fishes, can also enhance the autoimmune function of the fed cultured fishes, and reduces the disease incidence rate of the fed cultured fishes.

Mysidacea, commonly known as a shrimp table, is a marine product of miniature shrimp, has a slightly flat side, gray body surface and 2-15 mm body length, and is distributed in semiisland of Liaodong, Zhejiang and coastal areas of Guangdong in China. The mysid has high nutritive value and rich protein, lipid and trace elements. Because the shape is tiny, the carapace is tiny, the meat quality is tender, the mysid can be dissolved by itself after being separated from the seawater, the freshness is difficult to maintain, and the edible part is less. Therefore, most of the mysid caught by fishermen is purchased by shrimp powder processing enterprises, and is processed into shrimp powder through the procedures of cooking, squeezing, drying, crushing and the like, and the shrimp powder is sold as a feed protein additive.

Therefore, how to well utilize low value shrimp to produce high value shrimp products is a great social value effort. The enzymolysis of the mysid to obtain shrimp paste rich in small peptides is one of the feasible ways.

However, the mechanism of proteolysis is very complex, and besides increasing the efficiency of proteolysis, the removal of bitter taste has been the goal sought. The problems are presented as follows:

1) the enzymolysis efficiency is low. The conventional water bath enzymolysis requires 55-65 ℃ for more than 7 hours, wastes time and labor and is easy to deteriorate; because of the defects of free enzyme, the enzymolysis reaction is not beneficial to the production and preparation of polypeptide, for example, the free enzyme is easy to be denatured in acid, alkali, heat, organic solvent and other media, and the activity is reduced or lost; the enzyme remains in the solution after the reaction, making it difficult to continue and automate the enzyme reaction.

2) The bitter taste of the protein hydrolysate is mainly caused by bitter peptides in the hydrolysate, and the enzyme hydrolyzed protein destroys the structure of the original protein, so that hydrophobic amino acids such as leucine, isoleucine, phenylalanine and the like originally hidden in the molecule are exposed on the surface of the molecule, and the enzyme hydrolysate has bitter taste of different degrees. These bitter peptides containing hydrophobic amino acid residues vary in chain length from 2, 3 to over ten amino acids. Bitter peptides become a main obstacle for applying the enzymatic hydrolysis mysid products to aquaculture. The shrimp paste is good and rich in small peptide, but is used for feeding and breeding prawns, and the prawns are not lovely to eat, which is also the most main obstacle for the popularization of the enzymolysis mysid products. Bitter peptides can be detected even in micromolar, and the bitter taste lasts longer than the sweet taste, the salty taste and the sour taste, so that the simple bitter taste is the root cause of refusing bitter substances of cultured fishes and shrimps.

How to debitterize, but it is also contemplated that the debitterizing operation does not affect the loss or destruction of the hydrolyzed peptide. The common method is as follows:

1) filtering and removing, and adsorbing by active carbon, or filtering and removing by an ultrafiltration membrane. However, when the fish/shrimp paste is produced by the enzymolysis method, firstly, color is not removed, activated carbon only can remove color, and ultrafiltration membranes are too fine and have high cost, so that the ultrafiltration membranes and the activated carbon are not suitable for use.

2) Masking, for example, the bitter taste of caffeine, is masked or otherwise inhibited when sucrose is added, i.e., the taste cells feel less bitter.

In a word, how to obtain the whole shrimp paste rich in small peptides by high-efficiency enzymolysis and low-cost debitterizing is a process problem which needs to be solved urgently.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to obtain the whole shrimp paste rich in small peptides with high-efficiency enzymolysis and clean debitterizing without damaging the peptides, and provides a preparation method and application for preparing the whole shrimp paste rich in the small peptides by the enzymolysis of the marine mysid.

The invention is realized in such a way that a method for preparing whole shrimp pulp rich in small peptide by debitterizing and enzymolysis of marine mysid shrimp comprises the following steps:

1) granulating, namely freeze-drying fresh marine small bran shrimps serving as raw materials until the water content is 50-70 wt%, and crushing to obtain protein granules with the maximum size of below 3 mm;

2) micronizing, adding 0-3 deg.C pure water into protein particles according to a mass ratio of 1:0.2, shearing at ultra high speed to obtain protein microparticle slurry with particle size of more than 150 meshes, and adding pure water until the mass ratio of shrimp to water is 1: 0.5-5;

3) micronizing, heating protein particle slurry by microwave surrounding, boiling at 90-100 deg.C for 10-20min, cooling to the micronization temperature of 55-75 deg.C, and allowing an ultrasonic probe to extend into the protein particle slurry to act on the micronization ultrasonic wave to reduce the diameter of the protein particles of the protein particle slurry to micron level or below to obtain protein activation slurry;

4) performing enzymolysis, namely performing microwave surrounding heating on the micro-particle protein activated slurry to the enzymolysis reaction temperature of 40-65 ℃, fixing protease which is 0.01-2 wt% of the weight of the raw material fish on the surfaces of dispersion carriers, then uniformly fixing the dispersion carriers on the surfaces of a plurality of preset bodies, immersing the micro-particle protein activated slurry into the surfaces of the preset bodies, and performing enzymolysis reaction for 10-20 min/time in an alternating manner, wherein the total enzymolysis reaction time is 20-60min, so as to obtain the enzymolysis protein slurry;

5) selective separation, namely performing multistage pressure filtration on the enzymolysis protein slurry, selectively separating oligopeptide with the molecular weight of 1000-4000Da from the enzymolysis protein slurry, wherein the rest enzymolysis protein slurry is debitterized enzymolysis protein slurry; vacuum concentrating the debitterized enzymatic protein slurry until the water content is 40-60wt%, and obtaining small peptide-rich marine mysid enzymatic whole shrimp slurry;

steps 1) to 5) are all carried out in a nitrogen protection environment with the residual oxygen content of less than 5 percent.

Furthermore, the preset body is a polyethylene plate, and the dispersion carriers are fixed on the surfaces of the plurality of preset bodies, namely the dispersion carriers are uniformly adhered to the upper surface and the lower surface of the thin polyethylene plate, and finally the thin polyethylene plates are assembled at intervals to form the grid plate consisting of a plurality of concentric circular rings.

Further, the preset body is a film, a plurality of isolating convex ribs are arranged on the upper surface of the film at intervals, the isolating convex ribs are sinusoidal curves with the same period, dispersing carriers are uniformly adhered to the upper surface and the lower surface of the film, and then the film is wound into a preset body winding body with a certain diameter around the peripheral surface of the shaft.

Furthermore, the microwave surrounding heating means that a plurality of microwave generators are arranged on the peripheral wall of the tubular container at intervals to perform microwave surrounding heating on the protein particle slurry.

Further, theApplying miniaturized ultrasonic wave, extending ultrasonic probes with diameter of 10-40mm into the depth of the tubular container from the center 1/3 of the tube, arranging multiple ultrasonic probes at intervals in the length direction of the tubular container, and controlling frequency at 20-40kHz and field intensity at 70-100w/cm²And performing ultrasonic action for 3-10 minutes.

Further, the protease is fixed on the surface of the dispersion carrier to prepare Fe₃O₄Magnetic nanoparticles silane-modified to Fe₃O₄And (3) obtaining a micro magnetic core on the surface of the magnetic nano particle, and covalently coupling protease to the surface of the micro magnetic core to obtain the dispersed silicon ribozyme particle.

An alternate action device used in the method for preparing the whole shrimp pulp rich in small peptides by debitterizing and enzymolysis of the marine small mysid is characterized in that the microwave, ultrasonic and micro-particle protein activated pulp is immersed into a preset body for enzymolysis reaction to be cyclically and alternately applied through the alternate action device, the alternate action device comprises an enzyme immobilization reaction tube, a microwave tube and an ultrasonic wave tube which are communicated end to end, a grating plate or a preset body winding body is fixed in the enzyme immobilization reaction tube, slurry is filled in the alternate action device at a certain pressure, and the slurry circulates in the alternate action device once after each certain interval time in the alternate action device; during the interval time, the slurry in the microwave tube is subjected to the action of microwaves, the slurry in the ultrasonic wave tube is subjected to the action of ultrasonic waves, and the enzymatic hydrolysis reaction occurs in the enzyme immobilization reaction tube.

The utility model provides a multistage pressure boost filter equipment that uses in marine product small chaff shrimp takes off bitter enzymolysis preparation full shrimp thick liquid that is rich in small peptide, including the annular section of thick bamboo of falling cone, the annular section of thick bamboo entry of falling cone, centre and export are fixed with first order filtration membrane, second grade filtration membrane and tertiary filtration membrane respectively, and interior conical wall is equipped with the recoil mouth, and the cone stopper slides on the cone wall axis in the annular section of thick bamboo of back taper is equipped with the awl stopper, and the awl stopper antedisplacement blocks up the recoil mouth, cone stopper rethread is released the recoil mouth.

The small peptide-rich marine small mysidacea enzymatic hydrolysis whole shrimp paste is obtained by the method for preparing the small peptide-rich whole shrimp paste by debitterizing and enzymatic hydrolysis of the marine small mysidacea, the marine small mysidacea enzymatic hydrolysis whole shrimp paste is liquid paste, and the content of small molecular small peptides with less than 10 amino acid residues is 15-20 wt%.

The application of the small peptide-rich enzymatic hydrolysis whole shrimp paste of the marine small mysidae shrimps in aquaculture is characterized in that the shrimp powder is replaced by the enzymatic hydrolysis whole shrimp paste of the marine small mysidae shrimps accounting for at least 10wt% of the total weight of the fish meal to continuously feed the cultured shrimps.

The invention achieves the aim of how to obtain the whole shrimp paste rich in small peptide by high-efficiency enzymolysis and low-cost debitterizing by mainly improving the following points,

improvement in synergy:

1. granulation- > micronization- > microminiaturization, and creates the condition of small molecule for enzymolysis of protein source

(1) Stepwise cooperation for miniaturization

Proteolysis is divided into at least two stages, the first step, the attachment of enzyme molecules to small protein particles; in the second step, hydrolysis occurs, releasing a large number of soluble peptides and amino acids.

The steps 1) to 3) of the invention aim to obtain protein particles as small as possible, the step 1) is firstly classified and crushed to the maximum size below 3mm, the step 2) is sheared into particle pulp at high speed, the step 3) is carried out ultrasonic vibration and microminiaturization after cooking, ultrasonic waves are longitudinal waves with the vibration frequency of more than 20KHz, the ultrasonic action generates cavitation action in a liquid medium, the ultrasonic action can enable the liquid medium to form micro bubbles, and then the micro bubbles are ruptured, thereby being beneficial to the microminiaturization of the particle size of the protein.

(2) The cost is not easy to be reduced to more than 150 meshes.

The realization of the micronization belongs to the tiny innovation of the self, and the soil method is used. In the prior art, the grinding particle size of more than 150 meshes is realized, and an ultrafine grinder or a colloid mill or a fluidized bed airflow grinder must be purchased, and the devices are very expensive. The fish meal substitutes are small and micro enterprises, so that the investment and purchase of the high machines are unrealistic, and the purpose is achieved.

The high-speed shearing device comprises a high-speed shearing part, a small rotating blade, an inlet ultra-high-speed motor with the rotating speed exceeding 30000 rpm, and a taper sleeve which is additionally arranged around the blade for increasing the shearing effect and has a gathering effect on fluid, wherein the inner wall of the taper sleeve is provided with a convex edge for increasing the impact effect. After the taper sleeve is additionally arranged, the blades are stirred at a high speed for shearing under the gathering of the taper sleeve, the height of liquid in the taper sleeve is higher than that of the liquid around the taper sleeve to form a suction effect on the liquid around the taper sleeve, and protein particles around the taper sleeve are continuously sucked into the taper sleeve to be sheared and crushed. The particle size of the cut and crushed material is more than 150 meshes by laser detection. That is, the invention solves the technical problem of high-speed shearing to 150 mesh granularity with small investment of an ultra-high speed motor, and has low cost.

The key point of the realization is that the small blade is driven to shear in the taper sleeve at a high rotating speed, 5kg of particle pulp can be produced every 5-10 minutes, so that the production efficiency per minute is not high, and improvement is needed.

The activation of protein micro-particle slurry- > protein activation slurry is generated by the small molecule activation of heat, ultrasonic wave and microwave after freezing

Freezing in the step 1), heating and boiling by surrounding microwaves in the step 2), and alternately applying microwaves and ultrasound before enzymolysis reaction in the step 4), wherein on one hand, the tissue structure of protein particles is loosened by first freezing and then heating and cold and hot shock; on the other hand, by using microwaves, polar molecules of a liquid medium are repeatedly and rapidly oriented and rotated under the action of a microwave high-frequency electric field to generate heat by friction, and the heating is started from the inside of a substance and quickly reaches the required temperature; the ultrasonic vibration enables the polar molecules to repeatedly and rapidly orient and rotate, the protein structure is looser, the ultrasonic wave not only utilizes the cavitation effect to enable the protein particle size to be miniaturized, but also enables more hydrophobic groups to be exposed on the surface of the protein molecules by the ultrasonic wave with high field strength. Expose more action points capable of combining with enzyme, accelerate hydrolysis reaction and greatly improve hydrolysis degree

Therefore, the invention generates activation effect from protein particle slurry- > protein activation slurry through the combined action of the freezing and then the heating, the ultrasonic wave and the microwave.

The protease molecules are contacted with the liquid medium in a dispersing way, so that the enzymolysis time is greatly reduced

The two effects of the two aspects already enable the protein enzymolysis to have tiny and activated protein particles, which greatly accelerates the speed of protease enzymolysis and can realize rapid enzymolysis. The periodic stirring during the enzymolysis process is to make the protease disperse more evenly around more small molecule particles to be hydrolyzed.

Proteolysis is divided into at least two stages, the first step, the attachment of enzyme molecules to small protein particles; in the second step, hydrolysis occurs, releasing a large number of soluble peptides and amino acids. In order to more quickly contact more enzyme molecules with small protein particles in the first step, the invention adopts a method of dispersing carriers by protease, then uniformly dispersing and fixing the carriers on the surface of a preset body, such as a grating plate or a polyethylene rod, so that the dispersed contact is realized, the protease can be recovered at any time, the enzymolysis reaction speed is accelerated, and the enzymolysis time is reduced to 20-60min from more than 7 hours in the prior art.

Alternative action of microwave, ultrasonic and enzymolysis

For the enzymolysis in the step 4), an alternate action device is designed, and the micro-particle protein activated slurry is alternately applied by microwave, ultrasonic and enzymolysis, the alternate action device is formed by connecting the alternate action device end to end, and the alternate action device comprises an enzyme immobilization reaction tube, a microwave tube and an ultrasonic wave tube which are communicated end to end, the alternate action device has high efficiency, the enzymolysis degree of the slurry is greatly improved, and the content of small peptides reaches 15-20 wt%.

Improvement in debittering:

1. the inlet, the middle and the outlet of the inverted cone annular cylinder are provided with three-stage filter membrane plates

The annular section of thick bamboo of the back taper entry, centre, export dress tertiary filter diaphragm plate, liquid is used in the entry filtration membrane with certain pressure, and the fluid pressure through filtration membrane necessarily reduces, in order to compensate this kind of pressure and subtract progressively, has designed an annular section of thick bamboo of back taper, and an annular section of thick bamboo of back taper makes tertiary filtration cross section reduce step by step, and the osmotic pressure of first order preceding, first order to second level, second level to third level increases step by step.

The back flushing opening of the inner conical wall is matched with the conical plug

Through the back flush mouth that sets up interior conical wall, the cooperation of awl stopper for but filter equipment steam sterilization, also can be with holding back the liquid back flush, from third level, second grade, first order filtration lamina membranacea during the back flush, the cross section of passing through increases step by step, the required pressure of back flush reduces step by step, has made things convenient for the back flush greatly and then does benefit to recycle, greatly reduced separation cost, than purchase ultrafiltration equipment, only the expense of debitterizing process, per ton than purchase and use ultramicro filtration equipment and reduce 500 and give an once more charge 700 yuan.

The preparation method for preparing the whole shrimp paste rich in small peptides by enzymolysis of the marine small mysidae has high enzymolysis efficiency and low production cost, can replace shrimp meal to be used for aquaculture, and can be used as a feed protein source of economic feed for aquaculture.

The invention takes the low-value marine small mysidacea as the raw material, fully utilizes the abundant marine organism resources in China, prepares the high-value pulpy small peptide product by utilizing the enzymolysis technology, has low energy consumption and short process flow, and reserves the nutrient components of the whole fish; opens up a new way for the transformation of products in the fish meal processing industry in China.

Drawings

FIG. 1 is a schematic diagram of an alternate action device of the method for preparing whole shrimp paste rich in small peptide by enzymolysis of the marine small mysidacea.

FIG. 2 is a cross-sectional view of a pre-arranged winding body of the method for preparing whole shrimp paste rich in small peptide by enzymolysis of the marine mysid.

FIG. 3 is an expanded view of the pre-arranged winding body of the method for preparing the whole shrimp paste rich in small peptide by the enzymolysis of the marine small mysidacea.

FIG. 4 is a front sectional view of a multistage pressure filtration device of the method for preparing whole shrimp slurry rich in small peptides by enzymatic hydrolysis of marine pityriasis shrimp in a pressure filtration state.

FIG. 5 is a front sectional view of a multistage pressure filtration device in a pressure-reducing backwashing state of a method for preparing a small peptide-rich whole shrimp paste by enzymatic hydrolysis of marine pityriasis shrimp according to the present invention.

FIG. 6 is a schematic view of the multistage pressure filtration device of the method for preparing shrimp slurry rich in small peptides by enzymatic hydrolysis of marine pityriasis shrimp.

1 enzyme immobilization reaction tube, 2 microwave tube, 3 ultrasonic tube, 4 microwave generator, 5 pre-set winding body, 6 ultrasonic probe, 7 inlet pump, 8 circulating pump, 9 emptying pump, 10 intermediate valve, 11 inlet valve, 12 outlet valve, 13 air inlet valve, 14 exhaust valve,

5.1 axes, 5.2 films, 5.3 spacer ribs, 5.4 dispersion carriers,

20 multistage supercharging filtering device, 21 inverted cone annular cylinder, 22 first stage filtering membrane, 23 second stage filtering membrane, 24 third stage filtering membrane, 25 conical plug, 26 guiding piece, 27 liquid inlet pipe, 28 liquid outlet pipe, 29 back flushing pipe, 30 first back flushing port, 31 second back flushing port,

32 the water plug is closed.

Detailed Description

The following detailed description of the embodiments of the present invention is provided in connection with the accompanying drawings, but is not intended to limit the scope of the invention.

Example 1

A method for preparing whole shrimp pulp rich in small peptide by enzymolysis of marine small mysidacea comprises the following steps:

1) granulating, namely freezing and drying fresh marine small chaff shrimps serving as raw material shrimps until the water content is 50-70 wt%, and grading and crushing the shrimps into protein granules below 3 mm;

2) micronizing, namely adding 0-3 ℃ pure water into protein particles according to the mass ratio of the protein particles to water of 1:0.2, shearing the protein particles to protein particle slurry with the particle size of more than 150 meshes at a high speed, adding the pure water until the mass ratio of the raw material shrimps to the water is 1: 0.5-5, and putting the protein particle slurry into a holding tank; preferably, the mass ratio of the material to the water is 1: 0.5-2.

Specifically, the rotating diameter of the shearing blade is 20-40mm, the rotating speed of the motor is over 30000 r, a conical gathering cover is arranged around the blade, collision convex edges are arranged on the inner wall of the conical cover at intervals, and the convex edges are 3-5mm higher than the inner conical surface. The maximum length of the particles in the particle size slurry is below 0.1mm, namely above 150 meshes by laser particle size detection. From the particle distribution, the fish bones and fish scales are hard and have the smallest crushing particle size and are concentrated on more than 200 meshes, while the fish proteins are softened in water and have larger particle sizes and are concentrated on more than 150-170 meshes.

3) Micronizing, heating protein particle slurry by microwave surrounding, boiling at 90-100 deg.C for 10-20min, cooling to microminiaturization temperature of 55-75 deg.C, placing ultrasonic probe into the containing tank, and performing microminiaturization ultrasonic wave to reduce the diameter of protein particle of the protein particle slurry to micrometer level or below to obtain micro-granule protein activated slurry;

4) performing enzymolysis, namely performing microwave surrounding heating on the small particle protein activated slurry to the enzymolysis reaction temperature of 40-65 ℃, fixing protease which weighs 0.01-2% of the raw material shrimps on the surfaces of a dispersion carrier, then fixing the dispersion carrier on the surfaces of a plurality of preset bodies, immersing the small particle protein activated slurry into the preset bodies for enzymolysis reaction, and circularly and alternately applying the microwave and the ultrasonic for 10-20min, wherein the total enzymolysis reaction time is 20-60min, so as to obtain the enzymolysis protein slurry;

5) selective separation, namely performing multistage pressure filtration on the enzymolysis protein slurry, selectively separating oligopeptide with the molecular weight of 1000-4000Da from the enzymolysis protein slurry, wherein the rest enzymolysis protein slurry is debitterized enzymolysis protein slurry; vacuum concentrating to water content of 40-60wt% to obtain small peptide-rich enzymatic hydrolysate of whole shrimp pulp of marine Mysidacea;

For step 4), the protease dispersion vector is carried out to prepare Fe with the particle size of 30-500nm₃O₄Magnetic nanoparticles, p.Fe₃O₄Performing surface treatment on the magnetic nanoparticles, and modifying the magnetic nanoparticles to Fe by adopting silane₃O₄The magnetic nano particle surface obtains a micro magnetic core, which specifically comprises the following steps: 3-aminopropyl-triethoxysilane. And (3) covalently coupling protease to the surface of the micro-magnetic core by using dithiocyano-benzyl or glutaraldehyde as a coupling agent to obtain the dispersed silicon ribozyme particles.

The preset body is a grating plate, the dispersing carriers are fixed on the surfaces of a plurality of grating plates, and the method is characterized in that the silase particles are uniformly adhered to the upper surface and the lower surface of a thin polyethylene plate, and finally, a plurality of thin polyethylene plates are assembled at intervals to form the grating plate. The spacing distance of the grating plates is 5-8 mm. Coating thermosetting resin on the upper and lower surfaces of polyethylene sheet to make the silase particles have the same electrostatic charge, such as positive charge, and spreading the charged silase particles on the thermosetting resin, so that the charged silase particles are repelled by the same polarity, which is helpful for uniformly distributing the silase particles on the polyethylene sheet to increase the density of the silase particlesSpecific surface area. Heating the thermosetting resin, and fixing the silicon nucleuse particles after curing. The concentration of the immobilized enzyme is measured by a biquinoline formic acid method to reach 2-3 mu g/cm²Practice proves that the immobilized enzyme has good activity.

As shown in fig. 1-3, in order to realize the alternate application of enzymolysis, microwave and ultrasonic wave, an alternate action device is designed, which comprises an enzyme immobilization reaction tube 1, a microwave tube 2 and an ultrasonic wave tube 3 which are communicated end to end, a slurry inlet tube is arranged between the microwave tube 2 and the ultrasonic wave tube 3, the slurry inlet tube is provided with an inlet valve 11 and an inlet pump 7, a slurry outlet tube is arranged between the enzyme immobilization reaction tube 1 and the microwave tube 2, the slurry outlet tube is provided with an outlet valve 12 and an evacuation pump 9, and a circulating pump 8 is arranged between the ultrasonic wave tube 3 and the enzyme immobilization reaction tube 1. The peripheral wall of the ultrasonic wave tube 3 is provided with ultrasonic probes 6 at intervals, and the peripheral wall of the microwave tube 2 is provided with a plurality of microwave generators 4 at intervals. An intermediate valve 10 is arranged at the inlet of the microwave tube 2, a nitrogen inlet pipe and an exhaust pipe are respectively arranged on two sides of the intermediate valve 10, and the nitrogen inlet pipe is provided with an air inlet valve 13 and an exhaust valve 14. The grid plates are assembled into a cylinder shape and are loaded into the enzyme immobilization reaction tube 1.

When in operation, firstly, nitrogen is filled, and the residual oxygen amount in the alternating action device is less than 5 percent. The method comprises the following specific operations: the intermediate valve 10 is closed, the inlet valve 13 is opened to fill with nitrogen, the outlet valve 14 is opened until it is detected that the residual oxygen content in the exhaust pipe is less than 5%, and the inlet valve 13 and the outlet valve 14 are closed.

And (2) filling slurry, opening an intermediate valve 10 and an inlet valve 11, opening an exhaust valve 14, starting an inlet pump 7, filling the micro-particle protein activated slurry obtained in the step 4) into the alternate action device, starting a circulating pump 8 at the same time, closing the exhaust valve 14 until slurry is filled into an exhaust pipe, and closing the inlet pump 7 and the circulating pump 8 when the pressure in the alternate action device reaches 1-5 MPa.

Thirdly, alternately acting, starting a microwave generator 4 to perform microwave surrounding heating on the microwave tube 2, starting a generator of an ultrasonic probe 6 to perform ultrasonic activation treatment on the slurry in the ultrasonic tube, and simultaneously performing enzymolysis reaction on the slurry in the enzymolysis immobilization reactor 1 by dispersing and contacting protease; for the total enzymolysis time 60min process, after the microwave, the ultrasonic wave and the enzymolysis respectively act for 20min, a primary circulating pump 8 is started, the slurry in the enzymolysis immobilization reactor 1 can be calculated according to the pipe flow, and all the slurry flows into the microwave pipe 2, the slurry in the microwave pipe 2 flows into the ultrasonic wave pipe 3, the slurry in the ultrasonic wave pipe 3 flows into the enzymolysis immobilization reactor 1, the circulating time T required by one circulation is achieved, and the circulating pump 8 is closed after the circulating time T. And after the third circulating pump 8 is started, namely, the slurry initially filled in the enzymolysis and immobilization reactor 1 flows into the enzymolysis and immobilization reactor 1 again for the second time to be subjected to enzymolysis, after the third circulating pump 8 is started again, the slurry initially filled in the enzymolysis and immobilization reactor 1 flows into the enzymolysis and immobilization reactor 1 for the third time to be subjected to enzymolysis, and the total enzymolysis time reaches 60min, so that the enzymolysis protein slurry is obtained. For the process with the total enzymolysis time of 20min, after the primary pump circulation, the microwave, the ultrasonic wave and the enzymolysis respectively act for 10min, the slurry initially filled in the enzymolysis immobilization reactor 1 after the tertiary pump circulation flows into the enzymolysis immobilization reactor 1 again for the second time to be subjected to enzymolysis, and the total enzymolysis time reaches 20 min. In order to realize secondary enzymolysis of the slurry, the microwave, the ultrasonic wave and the enzymolysis cycle are alternately applied for at least 3 pump cycles.

And fourthly, emptying, closing the intermediate valve 10, opening the outlet valve 12, starting the emptying pump 9, opening the air valve 13 after half a minute until the emptying pump 9 can not discharge the slurry, completing the task of emptying the enzymatic protein slurry in the alternate action device, closing the outlet valve 12 and stopping the emptying pump 9.

Thus, the next cycle (i- > c) is performed.

For step 5), as shown in fig. 3, the multistage pressure filtration is: arranging an inverted cone annular cylinder, arranging a first-stage filter membrane capable of intercepting molecular weight larger than 4000Da at an inlet of the inverted cone annular cylinder, arranging a second-stage filter membrane capable of intercepting molecular weight larger than 2000Da in the middle of the inverted cone annular cylinder, and arranging a third-stage filter membrane capable of intercepting molecular weight larger than 1000Da at an outlet of the inverted cone annular cylinder, wherein the enzymolysis protein slurry obtained in the step 4) passes through the inverted cone annular cylinder, oligopeptides with molecular weight M being more than or equal to 2000Da and less than or equal to 4000Da are intercepted between the first-stage filter membrane and the second-stage filter membrane, and oligopeptides of a third-stage filter membrane with molecular weight M being more than or equal to 1000Da and less than or equal to 2000Da are intercepted between the second-stage; mixing the enzymolysis protein slurry flowing out of the outlet of the inverted cone annular cylinder into the enzymolysis protein slurry which does not pass through the inverted cone annular cylinder; because the cross-sectional areas of the inlet, the middle and the outlet of the inverted cone-shaped cylinder are gradually reduced, the pressure of the enzymolysis protein slurry inlet is 0.2Mpa, the pressure of the middle and the outlet is gradually increased, and the larger passing pressure of the filtering membrane is ensured.

A multistage supercharging filter device 20 used in a preparation method for preparing small peptide-rich whole shrimp paste by enzymolysis of marine small mysidae shrimps comprises an inverted cone-shaped cylinder 21, wherein the inverted cone-shaped cylinder 21 comprises a first section cone 21.1 and a second section cone 21.2 which are separated, a first-stage filter membrane 22 is fixed at an inlet of the first section cone 21.1, a third-stage filter membrane 24 is fixed at an outlet of the second section cone 21.2, a second-stage filter membrane 23 is fixed between the first section cone 21.1 and the second section cone 21.2, a cone plug 25 is matched in the inverted cone-shaped cylinder 21, and the head and the tail of the cone plug 25 are in sliding fit in a guide piece 26. The inlet of the first cone cylinder 21.1 is connected with a liquid inlet pipe 27, the outlet of the second cone cylinder 21.2 is connected with a liquid outlet pipe 28, and a branch of the liquid outlet pipe 28 is connected with a back flushing pipe 29.

The inner wall of the first section cone cylinder 21.1 is provided with a first backwashing port 30, and the inner wall of the second section cone cylinder 21.2 is provided with a second backwashing port 31. The first stage filtration membrane 22, the second stage filtration membrane 23 and the third stage filtration membrane 24 are installed in a filter screen plate frame.

The multistage supercharging filtering device has two working states, namely a supercharging filtering state and a decompression backwashing state, wherein the supercharging filtering state is that a conical plug moves forward to block a first backwashing port 30 and a second backwashing port 31, a backwashing pipe 29 is closed, enzymolysis protein slurry flows into an inverted conical annular cylinder 21 from a liquid inlet pipe 27, oligopeptide with the molecular weight M being more than or equal to 2000Da and less than or equal to 4000Da is intercepted between a first-stage filtering membrane 22 and a second-stage filtering membrane 23, and oligopeptide with the molecular weight M being more than or equal to 1000Da and less than or equal to 2000Da is intercepted between the second-stage filtering membrane 23 and a third-stage filtering membrane 24; and after the filtration is finished, mixing the enzymatic protein slurry flowing out of the outlet of the inverted cone annular cylinder into the enzymatic protein slurry which does not pass through the inverted cone annular cylinder to obtain the debitterized enzymatic protein slurry.

And in the decompression backwashing state, the front part of the first stage filter membrane 22 is blocked by a water blocking plug 32, the liquid outlet pipe 28 is blocked by the water blocking plug 32, the conical plug moves backwards, the backwashing pipe 29 is opened, and the intercepted protein slurry is flushed out from the first backwashing port 30 and the second backwashing port 31 by flushing water. From the outlet to the inlet, the cross section where the three-stage filtering membrane is located is gradually increased, and the pressure is gradually reduced, so that decompression washing of the inverted cone-shaped cylinder is formed. Until the state shown in fig. 3, that is, the three-stage filtration membrane is washed clean.

The working process is as follows: backwashing- > draining- > steam sterilizing- > cooling- > filtering- > backwashing and discharging pulp;

the details of the other steps are explained as follows:

the grading crushing is two-stage crushing, the first-stage crushing is that the marine small trash fish is conveyed to a first crusher by a conveying belt, the output port of the first crusher is filtered by a 4-mesh screen, the marine small trash fish is not circulated to the feed inlet of the first crusher by materials and is output to a second crusher by the materials, the output port of the second crusher is filtered by a 6-mesh screen, the marine small trash fish is not circulated to the feed inlet of the second crusher by the materials, and the marine small trash fish is output as qualified protein particles by the materials;

boiling at 90-100 deg.C for 10-20min before micronization to ensure that fish protein is fully denatured and space structure is opened, and facilitate enzymolysis.

The microwave surrounding heating means that a plurality of microwave generators are arranged on the peripheral wall of the microwave tube at intervals to perform microwave surrounding heating on the particle protein slurry. The microwave surrounding heating adopts a microwave generator with microwave frequency of 2450MHz and microwave power of 15-30W, and the heating time of each time depends on the size of the holding tank.

The microwave is made of ceramic or polyethylene resin; the diameter of the microwave is about 2 times of the half attenuation depth of the microwave, so that the microwave penetrates through the wall of the microwave tube to act on the micro-particle protein slurry, the uniform surrounding heating from the peripheral wall is ensured, and the consistency of the temperature distribution in the micro-particle protein slurry from inside to outside and from top to bottom is ensured. Preferably, the microwave tube is made of ceramic materials, the diameter of the microwave tube is 1400mm, and the length of the microwave tube is 3000 mm.

The ultrasonic probe with the diameter of 10-40mm extends into the ultrasonic tube from the center 1/3 of the tube by 20KHz-40KHz and the field intensity of 70-100w/cm²And performing ultrasonic action for 3-10 minutes. The ultrasonic probes are arranged at intervals in the length direction of the ultrasonic tube.

Preferably, the enzymolysis process is kept at constant temperature and high pressure, and the constant temperature is realized by acting microwave for a time t at intervals of time t₁Keeping the enzymolysis reaction temperature of the particle protein slurry at 40-65 ℃; the high pressure is such that the particulate protein slurry is subjected to a pressure of 1-5 MPa.

The protease is alkaline protease generated by microbial fermentation or the protease is a compound enzyme selected from the alkaline protease and flavourzyme, wherein the addition amount of the alkaline protease is 0.5-1.0wt% of the weight of the raw material shrimp, and the addition amount of the flavourzyme is 0.05-0.1wt% of the weight of the raw material shrimp.

Example 2

As shown in fig. 2 and 3, in step 4), the preset body is a film 5.2, a plurality of isolating ribs 5.3 are arranged on the upper surface of the film 5.2 at intervals, and the isolating ribs 5.3 are at intervals of a sinusoidal curve with the same period. The upper and lower surfaces of the film 5.2 are uniformly adhered with dispersion carriers 5.4, such as silicon nucleus enzyme particles, and the concentration of the immobilized enzyme is measured by a biquinoline formic acid method to reach 2-3 mu g/cm²Practice proves that the immobilized enzyme has good activity. The film 5.2 is wound around the outer circumference of the shaft 5.1 to form a preset body winding body 5 with a certain diameter, and the preset body winding body 5 is fixed in the cylindrical immobilized enzyme reactor 1. Because the preset body winding body 5 has more compact intervals, a sinusoidal channel is provided between the isolation convex edge 5.3 and the upper and lower films, the sinusoidal channel is opened from one end of the preset body winding body 5, and is discharged from the other end, the sinusoidal channel prolongs the opportunity that protease contacts with a protein macromolecular chain, and good conditions are provided for dispersive enzymolysis contact.

And (3) vacuum concentrating the debitterized enzymatic hydrolysis protein slurry until the water content is 50-60% by weight, thus obtaining the small peptide-rich marine mysid enzymatic hydrolysis whole shrimp slurry.

During the enzymolysis reaction period in the step 4), the enzymolysis can be accelerated by extending a new preset body winding body 5, and the small peptide yield is increased.

In the embodiment 2, an enzyme dispersion solidification technology is adopted, so that the contact specific surface area of the enzyme and the micro-particle protein activated slurry is increased, the step of high-temperature enzyme deactivation is omitted, and the cost is reduced for enzymolysis production.

Through detection, the enzymatic hydrolysis whole shrimp pulp of the marine small mysidacea prepared by the invention has the advantages of 20-35 wt% of crude protein, 15-20wt% of small peptide, 3-6 wt% of crude fat, 3-8 wt% of ash, 2-6 wt% of lysine, 1-3 wt% of methionine and 40-60wt% of water. Wherein the protein hydrolysate with molecular weight below 1000Da accounts for 40-90%.

An application of small peptide-rich enzymolysis whole shrimp pulp of marine small bran shrimps in aquaculture, wherein the fish meal is replaced by at least 10wt% of the enzymolysis whole shrimp pulp of the marine small bran shrimps, which accounts for the total weight of the fish meal, to feed cultured fishes.

Based on the consideration of the culture cost, the shrimp powder is preferably replaced by 25-40wt% of the total weight of the shrimp powder, the shrimp powder is continuously fed with the whole enzymatic hydrolyzed shrimp paste of the marine small chaff shrimps, 25-30 wt% of the total enzymatic hydrolyzed shrimp paste of the marine small chaff shrimps is added, the shrimp is continuously fed with the Penaeus vannamei for three months, the average daily gain reaches 0.42-0.66g, the feed efficiency is improved by 15% compared with that of a control group added with the shrimp powder, the feed efficiency is improved by 7% compared with that of similar hydrolysate in the market, meanwhile, the immune function of the cultured shrimps is enhanced, and the disease incidence rate is reduced.

The experimental data are as follows:

the same female parent penaeus vannamei boone is selected as an experimental object in the experiment, natural light is adopted in the experiment, the penaeus vannamei boone is cultured in a culture barrel, the culture seawater is deep sea seawater, a running water culture mode is adopted, the flow rate is 5L/min, the water temperature is 15 +/-0.5 ℃, the dissolved oxygen is higher than 7mg/L, the salinity is 32 per thousand, and the pH is 7.5-8.0. Before the experiment is started, the food is stopped for 24 hours, and the juvenile Penaeus vannamei Boone with uniform size, strong physique and no diseases on the body surface is selected. The fish were randomly divided into 15 breeding barrels, 3 treatment groups, 5 treatments each repeated, 25 fish in each barrel, fed twice a day with artificial satiation 6:30 and 16:30, the amount of residual bait in each barrel was counted after 30 minutes of feeding, and the weight of residual bait not ingested was calculated from the average weight of 100 residual baits. The feed is added into common fish meal according to the following proportion for feeding:

control group: 30% of shrimp meal; group A: FH (commercial homologous hydrolysate) 30%, group B: the results of 30% of the enzymatic debitterized whole shrimp paste of marine mysidacea obtained in example 1, 90 days in total in the culture experiment, and the feed intake, feed rate (weight gain/feed), and death rate of the fish were recorded when the fish were raised, respectively, are shown in table 1.

TABLE 1 comparison table of Penaeus vannamei Boone cultured by enzymolysis of Debittered whole shrimp pulp instead of fish meal

Daily ration treatment	Control	Group A	Group B
				Initial weight, g	80	80	80
Final weight, g	110	125	140
				Food intake, g	125	140	155
Feed efficiency, weight gain/feed	0.24	0.32	0.39
				The mortality rate is%	3.3	2.1	0.6

As shown in Table 1, when 30wt% of marine small mysidacea enzymatic hydrolysis debitterized whole shrimp pulp is added to continuously feed the Penaeus vannamei Boone for three months, the average daily gain reaches 0.66g, the feed efficiency is improved by 15% compared with that of a control group B added with shrimp meal, the feed efficiency is improved by 7% compared with that of a group A of similar hydrolysate in the market, the immune function of the cultured fish is enhanced, the death rate is reduced by 81% compared with that of the control group B added with shrimp meal, and the death rate is reduced by 71% compared with that of the group A of similar hydrolysate in the market.

The invention takes the low-value marine mysorethorn whole fish as the raw material, and fully utilizes the abundant marine biological resources in China; the high-value pulpy short peptide product is prepared by utilizing an enzymolysis technology, the energy consumption is low, the process flow is short, and the nutritional ingredients of the whole fish are reserved; opens up a new way for the transformation of products in the fish meal processing industry in China.

Claims

1. A method for preparing whole shrimp pulp rich in small peptide by debitterizing and enzymolysis of marine small mysidae is characterized by comprising the following steps:

4) the method comprises the following steps of (1) circularly and alternately applying microwaves, ultrasound and enzymolysis, wherein the microwave application comprises the following steps: carrying out microwave surrounding heating on the micro-particle protein activated slurry to the enzymolysis reaction temperature of 40-65 ℃; the enzymolysis is carried out as follows: fixing protease which is 0.01-2 wt% of the weight of raw material shrimps on the surface of a dispersion carrier, then uniformly fixing the dispersion carrier on the surfaces of a plurality of preset bodies, and immersing the micro-particle protein activated slurry on the surfaces of the preset bodies; the ultrasound was applied as: applying ultrasonic waves to the activated slurry of the microglobulin; circularly and alternately applying microwave, ultrasound and enzymolysis to the same quantitative micro-particle protein activated slurry for 10-20 min/time, wherein the total enzymolysis reaction time is 20-60min, so as to obtain enzymolysis protein slurry;

2. The method for preparing shrimp pulp rich in small peptides by debitterizing and hydrolyzing marine mysid shrimps as claimed in claim 1, wherein said preset body is a polyethylene plate, and the dispersion carrier is fixed on the surface of a plurality of preset bodies, that is, the dispersion carrier is uniformly adhered to the upper and lower surfaces of the thin polyethylene plate, and finally a plurality of thin polyethylene plate grids are assembled at intervals to form a grid plate consisting of a plurality of concentric circular rings.

3. The method for preparing whole shrimp paste rich in small peptide by debitterizing and enzymolysis of marine small mysid as claimed in claim 1, characterized in that the preset body is a film (5.2), a plurality of isolating convex ridges (5.3) are arranged on the upper surface of the film (5.2) at intervals, the isolating convex ridges (5.3) are sinusoidal curves with the same period, the upper and lower surfaces of the film (5.2) are uniformly adhered with dispersing carriers (5.4), and then the film (5.2) is wound into a preset body winding body (5) with a certain diameter around the outer circumferential surface of the shaft (5.1).

4. The method for preparing shrimp paste rich in small peptides by debitterizing and hydrolyzing marine pityriasis miniata as claimed in claim 2 or 3, wherein said microwave surrounding heating means that a plurality of microwave generators are installed at intervals on the peripheral wall of the tubular container to perform microwave surrounding heating on the protein particle paste.

5. The method for preparing whole shrimp paste rich in small peptide by debitterizing and hydrolyzing marine Mysidacea as claimed in claim 2 or 3, wherein said application of the ultrasonic waves for miniaturization is carried out by inserting an ultrasonic probe having a diameter of 10-40mm into the tubular container at a depth of 1/3 from the center of the tube, and a plurality of ultrasonic probes are arranged at intervals in the length direction of the tubular container at a frequency of 20-40kHz and a field strength of 70-100w/cm²And performing ultrasonic action for 3-10 minutes.

6. The method for preparing a whole shrimp paste rich in small peptides by debittering marine mysid as claimed in claim 1, wherein said protease is immobilized on the surface of a dispersion carrier by preparing Fe₃O₄Magnetic nanoparticles silane-modified to Fe₃O₄And (3) obtaining a micro magnetic core on the surface of the magnetic nano particle, and covalently coupling protease to the surface of the micro magnetic core to obtain the dispersed silicon ribozyme particle.

7. An alternate action device used in the method for preparing the whole shrimp pulp rich in small peptide by the debittering and enzymolysis of the marine small mysid as per any one of the claims 2 to 5, characterized in that the alternate application of the microwave, the ultrasound and the enzymolysis is realized by a cyclic alternate action device, the alternate action device comprises an enzyme immobilization reaction tube (1), a microwave tube (2) and an ultrasonic wave tube (3) which are communicated end to end, the grating plate or the pre-arranged body winding body (5) is fixed in the enzyme immobilization reaction tube (1), the slurry is filled in the alternate action device under certain pressure, and the slurry circulates in the alternate action device once after each certain interval time in the alternate action device; during the interval time, the slurry in the microwave tube is subjected to the action of microwaves, the slurry in the ultrasonic wave tube is subjected to the action of ultrasonic waves, and the enzymatic hydrolysis reaction occurs in the enzyme immobilization reaction tube (1).

8. A multistage pressure-boosting filtering device used in a method for preparing whole shrimp paste rich in small peptides by debittering and enzymolysis of marine small mysid as claimed in any one of claims 1 to 6, which comprises an inverted cone annular cylinder (21), wherein a first-stage filtering membrane (22), a second-stage filtering membrane (23) and a third-stage filtering membrane (24) are respectively fixed at an inlet, a middle and an outlet of the inverted cone annular cylinder (21), a backwashing port (30, 31) is formed in an inner conical wall of the inverted cone annular cylinder (21), a cone plug (25) is arranged on an axial line of the inner conical wall in the inverted cone annular cylinder (21) in a sliding manner, the cone plug (25) moves forwards to block the backwashing port, and the cone plug (25) moves backwards to release the backwashing port.

9. A small peptide-rich marine pityriasis shrimp enzymatic hydrolysis whole shrimp paste obtained by the method for preparing small peptide-rich whole shrimp paste by debitterizing and enzymatic hydrolysis of marine pityriasis shrimp according to any one of claims 1 to 6, wherein the marine pityriasis shrimp enzymatic hydrolysis whole shrimp paste is in a liquid paste state, and the content of small molecular small peptides with less than 10 amino acid residues is 15-20 wt%.

10. Use of a small peptide enriched enzymatic whole shrimp paste of marine small bran shrimps as claimed in claim 9 in the preparation of an aquaculture feed by continuously feeding cultured shrimps with said enzymatic whole shrimp paste of marine small bran shrimps in an amount of at least 10% by weight based on the total weight of the shrimp meal instead of shrimp meal.