CN107821728B

CN107821728B - Preparation method and application of whole fish paste rich in small peptides prepared by enzymolysis of small marine fishes

Info

Publication number: CN107821728B
Application number: CN201711291148.5A
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: 2020-10-20
Anticipated expiration: 2037-12-08
Also published as: CN107821728A

Abstract

A method for preparing whole fish paste rich in small peptides by enzymolysis of small marine fishes comprises the following steps: 1) granulating, namely taking fresh marine small trash fish as raw material fish, freeze-drying, and carrying out grading crushing to obtain protein granules; 2) micronizing, and shearing at high speed to obtain protein microparticle slurry of 150 mesh or above; 3) microminiaturizing, heating to the microminiaturization temperature of 55-75 ℃, and extending an ultrasonic probe into a holding tank to perform microminiaturization ultrasonic wave to prepare micro-particle protein activated slurry; 4) and (3) carrying out enzymolysis, namely heating the mixture to an enzymolysis reaction temperature of 40-65 ℃ by enclosing with microwaves, adding protease which is 0.01-2% of the weight of the raw material fish into the particle protein slurry, circularly and alternately applying the microwaves, ultrasonic waves and the enzymolysis reaction to obtain enzymolysis protein slurry, and then carrying out vacuum concentration until the water content is 40-60wt% to obtain the small peptide-rich marine trash fish enzymolysis whole fish slurry. The preparation method has high enzymolysis efficiency and low production cost, and can replace fish meal to be used as a feed protein source of an economic feed for aquaculture.

Description

Preparation method and application of whole fish paste rich in small peptides prepared by enzymolysis of small marine fishes

Technical Field

The invention relates to the technical field of protein enzymolysis, in particular to a preparation method and application of enzymatic hydrolysis whole fish paste of small marine trash fish.

Background

An important and costly raw material in fish 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.

The mechanism of proteolysis is very complex, since the substrate contains a large number of different kinds of insoluble proteins. The aim of obtaining more small peptides is to improve the enzymolysis efficiency by the same amount of protein.

1) Proteases are key to hydrolyzing proteins. The proteases studied by enzymolysis are various and are mainly classified into those from animals, plants and microorganisms according to their sources. Different proteases greatly influence the physicochemical functional properties of the hydrolysate, so the selection of protease is crucial in the marine proteolysis industry. The nutritional value and flavor of protein hydrolysates vary greatly depending on the enzyme used.

2) 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.

How to carry out enzymolysis on small marine trash fish with low cost and high efficiency to obtain whole fish pulp rich in small peptides is an urgent technical problem to be solved.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to obtain whole fish pulp rich in small peptides by carrying out enzymolysis on small marine fish with low cost and high efficiency, and provides a preparation method and application for preparing whole fish pulp rich in small peptides by carrying out enzymolysis on small marine fish.

The invention is realized in such a way that a preparation method for preparing whole fish paste rich in small peptides by enzymolysis of small marine fishes comprises the following steps:

1) granulating, namely freeze-drying the fish which is prepared from fresh marine small trash fish serving as a raw material until the water content is 50-70 wt%, classifying and crushing the fish into protein particles with the maximum size of below 5mm, and storing the protein particles in a dry and scattered manner;

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 fish to water is 1: 0.2-10;

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 protein particles of the protein particle slurry to below micrometer level to obtain protein activated 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; vacuum concentrating the enzymolysis protein slurry until the water content is 40-60wt%, and obtaining small peptide-rich small marine trash fish enzymolysis whole fish slurry;

steps 1) and 4) are all carried out in a nitrogen protection environment with the residual oxygen content of less than 5%.

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, the action is a miniaturized ultrasonic wave with a diameter of 10-40mmThe probe is inserted into the tubular container from the center 1/3 of the tube, and multiple ultrasonic probes are arranged at intervals in the length direction of the holding tank at frequency of 20-40kHz and field strength of 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.

Further, the immersion of the microwave, ultrasonic and micro-particle protein activated slurry into the preset body for the cyclic alternate application of the enzymolysis reaction is realized by an 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, the grating plate or the preset body winding body is fixed in the enzyme immobilization reaction tube, the alternate action device is filled with the slurry 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.

Further, the circulation once means that about all the slurry in the enzymolysis immobilization reactor flows into the microwave tube according to the tube flow, about all the slurry in the microwave tube flows into the ultrasonic wave tube, and the circulation time required for circulating once is determined as the pause time.

The small-peptide-rich small-fish enzymatic hydrolysis whole fish paste is prepared by the preparation method for preparing small-peptide-rich whole fish paste through enzymatic hydrolysis of the small marine fishes, the small-peptide-rich marine fish enzymatic hydrolysis whole fish paste is in a liquid slurry state, and the content of small-molecule small peptides with less than 10 amino acid residues is 15-20 wt%.

Use of the small peptide-enriched enzymatic whole fish slurry of marine small trash fish of claim 9 in aquaculture, wherein fish meal is replaced by at least 20wt% of the total weight of fish meal, and the fish meal is continuously fed to the aquaculture fish.

The invention achieves the aim of obtaining the whole fish pulp rich in small peptides by the enzymolysis of the small marine trash fish with low cost and high efficiency through mainly improving the following points,

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 5mm, 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%.

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

The invention takes the low-value marine small trash fish as the raw material, and fully utilizes the abundant marine biological resources in China; the high-value pulp small 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.

Drawings

FIG. 1 is a schematic diagram of an alternate action device of the preparation method of whole fish paste rich in small peptides by enzymolysis of small marine fishes.

FIG. 2 is a cross-sectional view of a pre-arranged winding body of the preparation method of whole fish paste rich in small peptides by the enzymolysis of small marine fishes.

FIG. 3 is an expanded view of a preset winding body of the preparation method of whole fish paste rich in small peptides by the enzymolysis of small marine fishes.

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 spindle, 5.2 film, 5.3 spacer bead, 5.4 dispersion carrier.

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 fish paste rich in small peptides by enzymolysis of small marine fishes comprises the following steps:

1) granulating, namely cleaning fresh marine small trash fish which is taken as a raw material fish, freeze-drying until the water content is 50-70 wt%, grading and crushing to obtain protein granules with the particle size of less than 5mm, and storing in a freeze-dried mode;

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 fish to water is 1: 0.2-10.

Preferably, the mass ratio of the raw material fish feed water is 1: 0.5-5.

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.

4) performing enzymolysis, namely performing microwave surrounding heating on the small particle protein activated slurry to an enzymolysis reaction temperature of 40-65 ℃, fixing protease which is 0.01-2% of the weight of the raw material fish on the surface 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 microwave and ultrasonic for 10-20min, wherein the total enzymolysis reaction time is 20-60min, so as to obtain enzymolysis protein slurry; after the enzymolysis reaction is finished, vacuum concentrating until the water content is 40-60% by weight, and obtaining the small marine trash fish enzymolysis whole fish paste rich in small peptides.

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. The upper surface and the lower surface of the polyethylene sheet are coated with thermosetting resin, so that the silase particles have the same electrostatic charge, such as positive charge, the charged silase particles are flatly laid on the thermosetting resin, and the charged silase particles are mutually repelled by the same polarity, so that the silase particles are uniformly distributed on the polyethylene sheet, and the specific surface area of the polyethylene sheet is increased. 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 figure 1, in order to realize the alternate application of enzymolysis, microwave and ultrasonic wave, an alternate action device is designed, and 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 5 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.

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 conveyer belt, the output port of the first crusher is filtered by a 2-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 4-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 of 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 fish, and the addition amount of the flavourzyme is 0.05-0.1wt% of the weight of the raw material fish.

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 winding body 5 has more compact intervals, a sinusoidal curved passing path is provided between the isolation convex rib 5.3 and the upper and lower films, the curved passing path prolongs the opportunity that enzyme = protease contacts protein macromolecular chains, and good conditions are provided for dispersive enzymolysis contact.

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

The invention adopts an enzyme dispersion solidification technology, increases the contact specific surface area of the enzyme and the micro-particle protein activated slurry, omits a high-temperature enzyme deactivation step, and reduces the cost for enzymolysis production.

The other steps are the same as in example 1.

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

The application of small peptide-rich small trash fish enzymatic hydrolysis whole fish paste in aquaculture is characterized in that fish meal is replaced by at least 20wt% of small trash fish enzymatic hydrolysis whole fish paste in total weight of the fish meal to feed cultured fish.

Based on the consideration of the culture cost, 25-30wt% of fish meal is preferably replaced by the small trash fish enzymolysis whole fish paste, the small trash fish enzymolysis whole fish paste with 25-30wt% of marine products is added to continuously feed the small yellow croakers for three months, the average daily gain reaches 0.42-0.88g, the feed efficiency is improved by 10% compared with that of a control group added with the fish 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, and the disease incidence rate is reduced.

The experimental data are as follows:

the same female parent of the small yellow croaker is selected as an experimental object, natural light is adopted for the experiment, the small yellow croaker is cultured in a culture barrel, the culture seawater is deep 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, stopping feeding for 24h, and selecting juvenile yellow croakers with uniform size, strong physique and no diseases on the body surface. 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 fish meal; group A: FH (commercial homologous hydrolysate) 30%, group B: the small marine trash fish obtained in example 1 was subjected to enzymolysis of the whole fish paste by 30%, the total culture experiment was conducted for 90 days, and the feed intake, feed rate (weight gain/feed), and death rate of the fish were recorded during the feeding, and the results are shown in table 1.

TABLE 1 comparison table of small yellow croaker bred by using enzymolysis of whole fish paste instead of fish meal

Daily ration treatment	Control	Group A	Group B
				Initial weight, g	80	80	80
Final weight, g	135	145	160
				Food intake, g	145	160	165
Feed efficiency, weight gain/feed	0.38	0.41	0.48
				The mortality rate is%	3.3	2.1	0.6

As shown in Table 1, when the small yellow croaker is continuously fed with 30wt% of the small trash fish enzymatic hydrolysis whole fish paste added with marine products for three months, the average daily gain reaches 0.88g, the feed efficiency is improved by 10% compared with that of a control group added with fish meal, the feed efficiency is improved by 7% compared with that of a group A of similar hydrolysate on the market, the immune function of the cultured fish is enhanced, the death rate of the cultured fish is reduced by 81% compared with that of a control group B added with fish meal, and the death rate of the cultured fish is reduced by 71% compared with that of a group A of similar hydrolysate on the market.

The invention takes the low-value marine small trash 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 fish paste rich in small peptides by enzymolysis of small marine fishes 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 fish 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; vacuum concentrating the enzymolysis protein slurry until the water content is 40-60wt%, and obtaining small peptide-rich small marine trash fish enzymolysis whole fish slurry;

2. The method for preparing whole fish slurry rich in small peptides by enzymolysis of marine small trash fish as claimed in claim 1, wherein the preset body is a polyethylene plate, and the dispersion carrier is fixed on the surface of the plurality of preset bodies by uniformly adhering the dispersion carrier to the upper and lower surfaces of the thin polyethylene plate, and finally assembling the plurality of thin polyethylene plate grids at intervals to form a plurality of concentric circular grid plates.

3. The method for preparing the whole fish paste rich in small peptides by the enzymolysis of the small marine fishes as claimed in claim 1, wherein 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, dispersion carriers are uniformly adhered to the upper and lower surfaces of the film (5.2), and then the film (5.2) is wound into a preset body winding body (5) with a certain diameter around the outer peripheral surface of the shaft (5.1).

4. The method for preparing whole fish slurry rich in small peptides by enzymolysis of marine small trash fish as claimed in claim 2 or 3, wherein the 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 slurry.

5. The method for preparing whole fish slurry rich in small peptides by enzymolysis of small marine fishes as claimed in claim 2 or 3, wherein the ultrasonic probe with a diameter of 10-40mm is extended into the tubular container from the center 1/3 of the tube by applying the miniaturized ultrasonic waves, and multiple ultrasonic probes are arranged at intervals in the length direction of the holding tank 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 whole fish slurry rich in small peptides by enzymatic hydrolysis of marine small trash fish as claimed in claim 1, wherein the protease is immobilized on the surface of the 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. The method for preparing whole fish slurry rich in small peptides by enzymolysis of marine small trash fish according to claim 2 or 3, wherein the alternate application of the microwave, the ultrasound and the enzymolysis cycle is realized by an 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 preset body winding body (5) is fixed in the enzyme immobilization reaction tube (1), the slurry fills 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 (1).

8. The method for preparing whole fish slurry rich in small peptides by enzymolysis of marine small trash fish according to claim 7, wherein the circulation is once, the whole slurry in the enzymolysis immobilization reactor (1) can be calculated to flow into the microwave tube (2) according to the tube flow, the whole slurry in the microwave tube (2) flows into the ultrasonic wave tube (3), and the circulation time (T) required for the circulation once is determined as the pause time.

9. The small peptide-rich enzymatic whole fish paste of marine small trash fish obtained by the method for preparing small peptide-rich whole fish paste by enzymatic hydrolysis of marine small trash fish according to any one of claims 1 to 8, wherein the small peptide-rich enzymatic whole fish paste of marine small trash fish is in a liquid paste state, and the content of small molecular small peptides with less than 10 amino acid residues is 15 to 20 wt%.

10. Use of the small peptide-enriched enzymatic whole fish pulp of marine small trash fish of claim 9 in the preparation of aquaculture feed, wherein at least 20wt% of the total weight of fish meal of the small trash fish pulp is used in place of fish meal for continuous feeding of farmed fish.