Aquatic protein feed and preparation method thereof

Abstract

The invention discloses an aquatic protein feed and a preparation method thereof, and belongs to the technical field of aquaculture feeds, the feed takes enzymolysis hermetia illucens protein, wall-broken yeast protein and enzymolysis pea peptide as a compound protein source, and comprises a multilayer sustained-release microcapsule prepared by adopting a three-fluid electrostatic spraying technology, and probiotics and digestive enzymes are embedded in the multilayer sustained-release microcapsule, so that the aquatic protein feed is prepared. And the outer layer is a pH response type gel and a protective film. The preparation method comprises the following steps: carrying out enzymolysis and membrane separation pretreatment on a compound protein source, constructing the microcapsule by virtue of low-temperature electrostatic spraying, and carrying out low-temperature mixing extrusion and functional post-treatment under the protection of inert gas. According to the invention, time-sequence precise release and efficient protection of nutrients and active ingredients in digestive tracts are realized, the protein digestibility can be obviously improved, the feed coefficient and nitrogen emission are reduced, and the feed has the comprehensive functions of promoting growth, regulating intestinal tracts and enhancing immunity at the same time; the industrial problems that a traditional feed is high in dependence degree on fish meal and low in digestion and utilization rate, and functional additives are prone to inactivation are effectively solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquatic feed, in particular to an aquatic protein feed and a preparation method thereof. BACKGROUND

[0002] In the aquaculture industry, protein feed is the core of determining the efficiency, cost and environmental impact of aquaculture. Currently, the mainstream technology mainly relies on fish meal, soybean meal and other traditional protein sources, and is facing increasingly severe challenges. First, in terms of protein sources, overfishing leads to the depletion of fish meal resources and high prices, while plant proteins such as soybean meal contain various anti-nutritional factors, which seriously affect the digestion and absorption of fish and shrimp and other aquatic animals, resulting in low feed utilization and limited growth performance. Second, in terms of feed function and processing, the existing technology is mainly simple physical mixing, and heat-sensitive functional additives (such as probiotics and enzyme preparations) are easily inactivated in the high-temperature granulation process. At the same time, the nutrient release of conventional feed does not match the digestive rhythm of aquatic animals, resulting in nutrient waste and eutrophication pollution of water bodies. In addition, the problem of antibiotic abuse still exists for the prevention of diseases, and the ordinary feed added with immune enhancers often has single function, and the active ingredients are easily destroyed in the stomach acid environment, which cannot target the intestinal tract. Therefore, developing a new type of protein feed that can efficiently replace fish meal, significantly improve the utilization rate of nutrients, has the function of healthy aquaculture, and is environmentally friendly, and a preparation method thereof, has become a technical problem to be solved in the field. SUMMARY

[0003] The main purpose of the present application is to provide an aquatic protein feed and a preparation method thereof, which can effectively solve the problems mentioned in the background art.

[0004] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is: An aquatic protein feed, comprising the following components by weight percentage: 45-55% of a complex protein source, 25-35% of a slow-release carrier system, 4-8% of a functional fat source, 2-4% of a mineral premix, 1-3% of a vitamin premix, and 8-15% of a carrier and a binder; The complex protein source is composed of enzymatic black soldier fly larva protein, broken cell brewer's yeast protein and enzymatic pea peptide in a weight ratio of (4-6):(2-4):(1-3); The slow-release carrier system is a multi-layer microcapsule embedded with active substances, which comprises an inner core, an intermediate gel layer and an outer protective film from inside to outside; the inner core comprises probiotics and digestive enzymes, the intermediate gel layer is a pH-responsive sodium alginate gel, and the outer protective film is a composite film comprising chitosan and plant essential oil.

[0005] The proportion of composite protein sources is set to achieve the complementation and balance of essential amino acids, while the specific structure of the slow-release carrier system is the key to ensure the stability of heat-sensitive and acid-sensitive active ingredients such as probiotics and digestive enzymes during processing, storage and passing through the stomach, and to ensure their targeted release in the intestine.

[0006] Preferably, in the enzymatic black soldier fly larva protein, the content of small peptides with a molecular weight of 500-2000 Da is not less than 40%; the broken wall rate of the broken wall saccharomyces cerevisiae protein is not less than 95%; and the content of trypsin inhibitor in the enzymatic pea peptide is less than 0.1 mg / g. High proportion of small peptides is easy to absorb and has physiological activity; high broken wall rate ensures the complete release of yeast intracellular nutrients; and strict control of the content of trypsin inhibitor eliminates the influence of the main anti-nutritional factors, which together constitute a high-efficiency, low-anti-nutritional, high-quality composite protein base.

[0007] Preferably, the probiotics are bacillus subtilis and / or bacillus licheniformis, and the effective viable count of the probiotics in the feed is not less than 1×10 6 CFU / g; and the digestive enzymes include protease and phytase. The selected bacillus strains have strong stress resistance, and the combination with the embedding technology can ensure that the product reaches the specified minimum viable count, thereby playing a probiotic function in intestinal colonization. The addition of protease and phytase aims to strengthen protein digestion and destroy phytic acid phosphorus in plant raw materials, respectively, from the perspective of endogenous supplementation, to improve the overall nutrient utilization rate.

[0008] Preferably, the functional fat source is fish oil treated with an antioxidant, and the antioxidant is rosemary extract; the mineral premix contains organic trace elements, including glycine iron and selenomethionine; and vitamins A, D and E in the vitamin premix are in the form of microcapsules. The use of natural antioxidants to treat fish oil improves the stability of polyunsaturated fatty acids. The use of organic trace elements and microencapsulated fat-soluble vitamins improves the bioavailability of minerals and the stability of vitamins during processing and storage, respectively, which together ensures the final effectiveness of trace active ingredients.

[0009] Preferably, the surface of the feed particles is also sprayed with a bacteriostatic coating containing a mixture of phages specific to aquatic pathogenic bacteria. This coating is an additional safety layer for the feed. Phages have high specificity and can target and inhibit the proliferation of common pathogenic bacteria (such as Vibrio) on the surface of the feed or in the aquaculture water, thereby introducing a biological protection mechanism at the feeding port, reducing the occurrence of diseases, and meeting the demand for reducing antibiotics in aquaculture.

[0010] A method for preparing the aforementioned aquatic protein feed, comprising the following steps: S1. Preparing a composite protein source: S1a. The black soldier fly larvae protein is subjected to stepwise enzymatic hydrolysis, first using alkaline protease hydrolysis, and then using flavor protease hydrolysis to obtain an enzymatically hydrolyzed black soldier fly larvae protein solution, which is dried for later use; S1b. The brewer's yeast is subjected to high-pressure homogenization combined with ultrasonic treatment to break the cell wall, obtaining a broken cell wall brewer's yeast protein slurry, which is dried for later use; S1c. After the pea protein is subjected to enzymatic hydrolysis, the anti-nutritional factors are removed using membrane separation technology to obtain an enzymatically hydrolyzed pea peptide solution, which is dried for later use; S2. Preparation of a slow-release carrier system: using a three-fluid coaxial electrostatic spraying device, the inner core liquid containing probiotics and digestive enzymes, the intermediate layer liquid of sodium alginate, and the outer layer liquid containing chitosan and plant essential oils are pumped out at the same time, and under the high-voltage electrostatic field, multi-layer microcapsules are formed, and then dried and solidified below 40°C; S3. Mixing and pelleting: the various complex protein source components prepared in step S1, the slow-release carrier system prepared in step S2, the functional fat source, the mineral premix, the vitamin premix, the carrier, and the binder are mixed, and under the protection of inert gas, low-temperature extrusion pelleting is carried out using a twin-screw extruder at 35-50°C to obtain primary feed pellets; S4. Post-treatment: the primary feed pellets obtained in step S3 are subjected to cold air drying until the water content is ≤10%, and then a bacteriostatic coating solution is sprayed on the surface, and finally dried to obtain the aquatic protein feed Preferably, in step S2, the process parameters of the three-fluid coaxial electrostatic spraying are: voltage 10-20 kV, flow rate ratio of the inner core liquid, the intermediate layer liquid, and the outer layer liquid is 1: (1.5-2.5): (0.8-1.2), and the total flow rate is 1.0-2.0 mL / min. Under these conditions, multi-layer microcapsules with complete "core-shell" structure, uniform particle size, and high embedding rate can be stably formed. The matching of voltage and flow rate is the key to ensure the stable breaking of the jet stream into droplets and the realization of layer-by-layer wrapping, which directly affects the performance of the slow-release system.

[0011] Preferably, in step S3, the temperature of the first zone of the twin-screw extruder is 45-50°C, and the temperature of the second zone is 30-35°C; the temperature of the cold air drying is 25-30°C. The low-temperature extrusion process with partitioning is set to only make the binder melt and plasticize at the temperature of the first zone, and the temperature of the second zone is further reduced to avoid irreversible damage to the added probiotics, enzyme preparations, and vitamins, etc. The subsequent cold air drying is also based on the consideration of protecting heat-sensitive components to ensure the activity of the final product.

[0012] Preferably, in step S4, before spraying the antibacterial coating solution, a nanometer silicon film is formed by spraying a nanometer silicon dioxide suspension on the surface of the primary feed particles. The pre-formed nanometer silicon film is an important enhancement step. The thin film can effectively fill the pores on the surface of the particles, significantly improve the stability of the feed particles in water, reduce the dissolution rate, thereby reducing feed waste and water pollution, and providing a denser and smoother substrate for subsequent spraying of the antibacterial coating.

[0013] Preferably, in step S1a, the specific conditions of the stepwise enzymatic hydrolysis are: first hydrolysis with alkaline protease at 50-55℃ and pH 8.5-9.5 for 2-3 hours, and then hydrolysis with flavor protease at 45-50℃ and pH 6.5-7.0 for 1-2 hours; in step S1b, the pressure of the high-pressure homogenization is 60-80 MPa, and the power of the ultrasonic treatment is 400-600 W for 5-10 minutes. The specific temperature, pH and time combination of the stepwise enzymatic hydrolysis can efficiently produce small peptides in the target molecular weight range; the specific parameters of high-pressure homogenization combined with ultrasonic treatment can achieve efficient disruption of yeast cell walls under low thermal load, balancing the effect and energy consumption.

[0014] Compared with the prior art, the present application has the following beneficial effects: The present application constructs a gradient slow-release aquatic feed based on a ternary compound protein source of "insect-yeast-plant peptide", the core of which is that through the unique multi-layer microcapsule embedding technology, under the low-temperature electrostatic spraying and extrusion process, the time sequence precise release and efficient protection of proteins and active ingredients in the digestive tract of farmed animals are realized. This not only synergistically improves the digestion and absorption rate of feed proteins, significantly reduces nitrogen emissions, but also synchronously realizes the multi-functional integration of promoting growth, regulating intestinal health and enhancing immunity by endogenously supplementing digestive enzymes, probiotics and immunologically active substances, thereby reducing the dependence on fish meal, the demand for antibiotics and the pollution of water environment at the source. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The present application is a schematic diagram of the preparation method of the aquatic protein feed. DETAILED DESCRIPTION

[0016] In order to make the technical means, creative features, purposes and effects of the present application easy to understand, the following will further describe the present application in combination with specific embodiments.

[0017] As shown in the preparation method flowchart, the following will be described in combination with a detailed embodiment. Figure 1

[0018] 1. Raw material preparation and pretreatment 1.1 Preparation of compound protein source raw material: ​Enzymatic hydrolysis of black soldier fly larvae protein: Dry black soldier fly larvae powder was mixed with deionized water at a ratio of 1:10. First, the pH was adjusted to 9.0 with 1M NaOH solution, and alkaline protease was added (enzyme-substrate ratio of 5000 U / g protein). The mixture was stirred in a 52°C water bath for 2.5 hours.

[0019] Principle: In this step, alkaline protease is used to extensively cut the peptide bonds of proteins in a slightly alkaline environment, generating a large number of medium and small molecular peptides.

[0020] Subsequently, the pH of the system was adjusted to 6.8 with 1M HCl solution, and flavor protease was added (enzyme-substrate ratio of 3000 U / g protein). The mixture was continuously stirred and hydrolyzed at 48°C for 1.5 hours.

[0021] Principle: Flavor protease is an exopeptidase that can release amino acids and small peptides (especially dipeptides and tripeptides) from the ends of peptide chains, further reducing peptide molecular weight and improving flavor and absorption rate. After hydrolysis, the enzyme solution was inactivated at 95°C for 10 minutes. After cooling, the supernatant was centrifuged and spray dried (inlet temperature 180°C, outlet temperature 85°C) to obtain enzyme-hydrolyzed black soldier fly protein powder rich in small peptides. The content of small peptides with a molecular weight of 500-2000 Da in the obtained protein powder was about 45%.

[0022] Wall-broken Saccharomyces cerevisiae protein: Saccharomyces cerevisiae dry powder was suspended in phosphate buffer (concentration 10% w / v). First, high-pressure homogenization was performed at 75 MPa pressure for 3 cycles.

[0023] Principle: High-pressure homogenization physically breaks down the tough cell wall of Saccharomyces cerevisiae through strong shearing, cavitation, and impact.

[0024] Subsequently, the homogenized suspension was placed in an ultrasonic processor and treated with ultrasound at a power of 500 W for 8 minutes (working for 2 seconds, with 1 second interval).

[0025] Principle: The cavitation effect of ultrasound can further damage the cell structure and promote the release and dispersion of intracellular substances. After treatment, the slurry was centrifuged, and the supernatant was collected and spray dried to obtain wall-broken Saccharomyces cerevisiae protein powder with a wall-breaking rate of about 97%.

[0026] Enzymatic hydrolysis of pea peptides: Pea protein isolate was prepared into a 5% solution, and neutral protease was used for hydrolysis at 50°C for 4 hours. The hydrolysate was passed through an ultrafiltration membrane system with a molecular weight cutoff of 5000 Da.

[0027] Principle: Membrane separation technology can efficiently retain the insufficiently hydrolyzed macromolecular proteins, while allowing the target small peptides and amino acids to pass through, and can effectively remove anti-nutritional factors such as trypsin inhibitors combined with high molecular weight components. Collect the permeate, concentrate and spray dry to obtain the enzyme hydrolyzed pea peptide powder, which is detected to have a trypsin inhibitor content of less than 0.05 mg / g.

[0028] 1.2 Preparation of raw material solution for sustained-release carrier system: Inner core liquid: Mix Bacillus subtilis freeze-dried powder (1 x 10 11 CFU / g) and Bacillus licheniformis freeze-dried powder (same number of viable bacteria) at a ratio of 1:1, and disperse in phosphate buffer containing 10% trehalose and 5% glycerol (as freeze-drying protectant) together with protease and phytase, and stir uniformly.

[0029] Intermediate layer liquid: Prepare a 3% (w / v) aqueous sodium alginate solution.

[0030] Outer layer liquid: Dissolve chitosan in 1% (v / v) acetic acid solution, adjust the concentration to 2% (w / v), then add 1% (v / v) thyme essential oil, and emulsify uniformly.

[0031] 2. Construction of sustained-release carrier system (multilayer microcapsules) A three-fluid coaxial electrostatic spraying device is used. The inner core liquid, intermediate layer liquid and outer layer liquid prepared above are loaded into three syringe pumps, respectively.

[0032] Principle: The three-fluid coaxial needle forms concentric circular laminar flow of the three liquids at the outlet. When a high-voltage electrostatic field (set to 15 kV in this example) is applied, the droplets overcome the surface tension under the action of the electric field force, form a stable "Taylor cone" and spray out charged jets. The jets stretch, thin and break into small composite droplets in the air.

[0033] Example parameters: The flow rates of the inner core liquid, intermediate layer liquid and outer layer liquid are set to 0.4 mL / min, 0.8 mL / min and 0.4 mL / min (flow rate ratio 1:2:1), respectively. The droplets are sprayed onto the receiving device (connected to the negative electrode) and immediately enter a drying chamber maintained at 35°C.

[0034] Principle: The sodium alginate rapidly ionically crosslinks and gels when it contacts the calcium chloride solution on the receiving device (or during drying), forming a solidified intermediate gel layer. The outer layer of chitosan-essential oil composite film is formed under mild drying. Finally, multilayer microcapsules with uniform particle size distribution (about 100-300 μm) and obvious "core-shell" structure are obtained. The whole process is at low temperature, effectively protecting the activity of probiotics and enzymes.

[0035] 3. Feed mixing, low-temperature pelleting and forming The components are weighed in the following percentages by weight: complex protein source (black soldier fly: yeast: pea peptide = 5:3:2) 50%, the above prepared multi-layer microcapsules 30%, fish oil treated with 0.05% rosemary extract 6%, organic mineral premix (containing ferrous glycinate, selenomethionine, etc.) 3%, microencapsulated vitamin premix 2%, microcrystalline cellulose 9%, algin 1%.

[0036] All solid powder components are put into a three-dimensional motion mixer and mixed for 25 minutes in a nitrogen-filled environment until uniform. Then add fish oil and continue mixing for 10 minutes. The mixed material is sent to a twin-screw extruder.

[0037] Process control: The temperature of the extruder is strictly controlled, with zone 1 (melting zone) set to 48°C to allow the adhesive such as algin to be moderately plasticized; zone 2 (extrusion zone) is set to 33°C.

[0038] Principle introduction: This low-temperature extrusion process aims to avoid the heat damage to probiotics, digestive enzymes and added microencapsulated vitamins in the microcapsules caused by traditional high-temperature pelleting (usually > 80°C). After extrusion through the die, the material is cut into cylindrical wet soft particles with a particle size of about 1.5 mm by the cutter.

[0039] 4. Post-treatment and functional modification Primary drying and stability treatment: The wet particles are immediately sent to a fluidized bed dryer, and dehumidified cold air at 28°C is used for drying until the moisture content of the particles is reduced to below 9.5%. Subsequently, a 10% nanosilica water dispersion (particle size 30-50 nm) is sprayed onto the surface of the particles in the fluidized bed.

[0040] Principle introduction: Nanosilica can form a dense hydrophobic film on the surface of the particles, significantly filling the surface pores, so that water does not easily penetrate into the feed after it is put into water, thereby improving its stability in water and reducing nutrient loss.

[0041] Bacteriostatic functional coating application: After the nanosilica film is dried, a layer of gelatin-glycerol solution containing a cocktail of phages against Vibrio parahaemolyticus and Vibrio harveyi (gelatin concentration 5%, total phage titer ≥1×10 9 PFU / mL) is sprayed.

[0042] Principle introduction: The gelatin solution forms a film at low temperature, fixing the phages on the surface of the feed. When the feed is put into water or eaten, the phages can be released locally, inhibiting the proliferation of specific pathogenic bacteria in the water or intestinal tract, playing a targeted biological protection role.

[0043] After the spraying is completed, the product is dried at 30℃ under ventilation until the final moisture content of the product is less than 10%, and the aquatic protein feed product is obtained.

[0044] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. Those skilled in the art should understand that the present application is not limited to the above-mentioned embodiments, and the above-mentioned embodiments and descriptions in the specification are only to illustrate the principles of the present application. Without departing from the spirit and scope of the present application, various changes and improvements can be made to the present application, and these changes and improvements all fall within the scope of the claimed present application. The scope of protection of the present application is defined by the appended claims and their equivalents.

Claims

1. An aquaculture protein feed, characterized in that: By weight percentage, it comprises the following components: Compound protein source 45-55%, slow-release carrier system 25-35%, functional fat source 4-8%, mineral premix 2-4%, vitamin premix 1-3%, carrier and binder 8-15%; The compound protein source is composed of enzymatic Hermetia illucens larvae protein, broken cell Saccharomyces cerevisiae protein and enzymatic pea peptide in a weight ratio of (4-6):(2-4):(1-3); The slow-release carrier system is a multi-layer microcapsule embedded with active substances, which comprises an inner core, an intermediate gel layer and an outer protective film from inside to outside; the inner core contains probiotics and digestive enzymes, the intermediate gel layer is a pH-responsive sodium alginate gel, and the outer protective film is a composite film containing chitosan and plant essential oil.

2. The aquatic protein feed of claim 1, wherein: In the enzymatic Hermetia illucens larvae protein, the content of small peptides with a molecular weight of 500-2000 Da is not less than 40%; the broken cell rate of the broken cell Saccharomyces cerevisiae protein is not less than 95%; and the content of trypsin inhibitor in the enzymatic pea peptide is less than 0.1 mg / g.

3. The aquatic protein feed of claim 1, wherein: The probiotics are Bacillus subtilis and / or Bacillus licheniformis, and the effective viable count thereof in the feed is not less than 1x10 6 CFU / g; and the digestive enzyme comprises protease and phytase.

4. The aquatic protein feed of claim 1, wherein: The functional fat source is fish oil treated with an antioxidant, and the antioxidant is rosemary extract; the mineral premix contains organic trace elements, and the organic trace elements include ferric glycine and selenomethionine; and vitamins A, D and E in the vitamin premix are in the form of microcapsules.

5. The aquatic protein feed of claim 1, wherein: The surface of the feed granules is also sprayed with a bacteriostatic coating containing a bacteriophage mixture specific to aquatic pathogenic bacteria.

6. A method of preparing an aqua-protein feed according to any one of claims 1-5, characterized in that: The method comprises the following steps: S1. Preparation of compound protein source: S1a. Stepwise enzymolysis of Hermetia illucens larvae protein, first hydrolysis with alkaline protease, then hydrolysis with flavor protease to obtain enzymatic Hermetia illucens larvae protein solution, which is dried for later use; S1b. Broken cell Saccharomyces cerevisiae protein slurry is obtained by high-pressure homogenization combined with ultrasonic treatment for broken cell, which is dried for later use; S1c. After enzymolysis of pea protein, membrane separation technology is used to remove anti-nutritional factors to obtain enzymatic pea peptide solution, which is dried for later use; S2. Preparation of slow-release carrier system: using a three-fluid coaxial electrostatic spraying device, the inner core liquid containing probiotics and digestive enzymes, the intermediate layer liquid of sodium alginate and the outer layer liquid containing chitosan and plant essential oil are pumped out at the same time, and multi-layer microcapsules are formed in a high-voltage electrostatic field, and then dried and solidified below 40℃; S3. Mixing and granulation: mixing the compound protein source components prepared in step S1, the slow-release carrier system prepared in step S2, the functional fat source, the mineral premix, the vitamin premix, the carrier and the binder, and then low-temperature extruding and granulating under the protection of inert gas by using a double-screw extruder at 35-50℃ to obtain primary feed granules; S4. Post-treatment: the primary feed granules obtained in step S3 are dried by cold air to a water content of ≤10%, and then a bacteriostatic coating solution is sprayed on the surface, and finally dried to obtain the aquatic protein feed.

7. The method of claim 6, wherein: In step S2, the process parameters of the three-fluid coaxial electrostatic spraying are as follows: voltage 10-20 kV, flow rate ratio of inner core liquid, middle layer liquid and outer layer liquid 1:(1.5-2.5):(0.8-1.2), and total flow rate 1.0-2.0 mL / min.

8. The method of claim 6, wherein: In step S3, the temperature of the first zone of the double-screw extruder is 45-50℃, the temperature of the second zone is 30-35℃, and the temperature of the cold air drying is 25-30℃.

9. The method of claim 6, wherein: In step S4, before spraying the antibacterial coating solution, a nano-silicon film is formed by spraying a nano-silicon suspension on the surface of the primary feed particles.

10. The method of claim 6, wherein: In step S1a, the specific conditions of the stepwise enzymatic hydrolysis are as follows: first, alkaline protease hydrolysis at 50-55℃ and pH 8.5-9.5 for 2-3 hours, and then flavor protease hydrolysis at 45-50℃ and pH 6.5-7.0 for 1-2 hours; in step S1b, the pressure of the high-pressure homogenization is 60-80 MPa, and the power of the ultrasonic treatment is 400-600 W for 5-10 minutes.

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