Krill-based enzymolysis delivery system as well as preparation method and application thereof

Abstract

The invention discloses a krill-based enzymolysis delivery system and a preparation method and application thereof. The preparation method comprises the following steps: mixing krill with water, homogenizing, adjusting the pH value of slurry, adding calcium chloride and dithiothreitol, and stirring to obtain a mixture; adding a compound enzyme, soybean lysophosphatide and chitosan into the mixture, and carrying out an enzymolysis reaction to obtain an enzymolysis mixture; the enzymolysis mixture is cooled and stirred for a period of time, then enzyme deactivation treatment and concentration are performed, and the krill-based enzymolysis delivery system is obtained. According to the preparation method disclosed by the invention, the nutrition of the krill is released to the maximum extent through a strategy of'activation of endogenous enzyme and exogenous enzyme synergism ', and more importantly, lipid and protein components released by the krill in the enzymolysis process are utilized to form a natural'nutrition delivery system' through in-situ self-assembly with an exogenously added specific auxiliary material; therefore, protection, targeted delivery and slow release of the active ingredients are realized, and the nutritional function of the active ingredients in aquatic animals is remarkably improved finally.

Description

Technical Field

[0001] This invention relates to the field of krill resource utilization technology, specifically to a krill-based enzymatic hydrolysis delivery system, its preparation method, and its application. Background Technology

[0002] Antarctic krill is the world's most abundant source of nutrients, with a biological resource of 600-1 billion tons. Even under strict management and sustainable development measures with ecological protection at its core, the exploitable amount is 620,000 tons, which is not currently being fully utilized.

[0003] Antarctic krill is rich in high-quality protein, with a protein content as high as 50%-60% in its dry matter. It contains all the essential amino acids required by humans and farmed animals, and has a high digestibility and absorption rate. In addition, Antarctic krill is rich in Omega-3 long-chain polyunsaturated fatty acids such as DHA and EPA. Moreover, compared with fish oil, the Omega-3 long-chain polyunsaturated fatty acids in krill oil are mainly in the phospholipid form, unlike the triglyceride form in fish oil, making them easier to digest and absorb. Regarding functional nutrients, Antarctic krill is rich in highly active astaxanthin, which can improve the antioxidant capacity and immune disease resistance of humans and farmed animals.

[0004] Currently, Antarctic krill has two main applications: human food and animal feed. Human food production focuses on functional health foods, therefore the current market size cannot fully absorb the catch and exploitable reserves of Antarctic krill. Meanwhile, global animal feed production is close to 1.4 billion tons, creating a strong demand for feed ingredients. High-end aquatic feed, in particular, has long relied on fishmeal supplies, and its sustainable development and improvement in feed quality are significantly limited by the fishmeal supply side.

[0005] When used in animal feed, especially aquatic feed, Antarctic krill not only significantly reduces fishmeal consumption, but its excellent nutritional composition also plays a vital role in attracting appetite, promoting growth, and maintaining health. However, current applications of Antarctic krill in aquatic feed fail to fully realize and release its superior functional nutritional benefits.

[0006] Current methods of applying Antarctic krill involve two main approaches: firstly, adding defatted or whole krill powder directly to formulated feed; and secondly, using traditional enzymatic hydrolysis techniques to enhance nutritional value and improve the utilization efficiency of krill. However, traditional enzymatic hydrolysis techniques, whether using single exogenous enzymes or simple formulations, often neglect the precise control of the krill's own highly efficient endogenous enzyme system, leading to incomplete hydrolysis or nutrient destruction. Furthermore, active substances in the hydrolysate, such as small peptides and unsaturated fatty acids, are easily deactivated or oxidized during feed processing (e.g., high-temperature pelleting) and storage. Krill's own autolytic enzyme system is an excellent protease hydrolysis method; with appropriate control, it can achieve twice the result with half the effort. Secondly, conventional methods of adding enzymatic hydrolysate often involve simply mixing it into the feed, exposing nutrients to the external environment. This results in rapid release and low absorption efficiency in the aquatic animal's digestive tract, and the nutrients are easily dissolved in water, causing waste and water pollution.

[0007] Existing technologies (such as CN111802454A) disclose methods for preparing feed additives using enzymatic hydrolysis of krill, but their core lies in the enzymatic hydrolysis process itself and does not solve the problems of subsequent nutrient stabilization and efficient delivery. Other technologies (such as CN113100378A) involve using pre-made liposomes or microcapsules to encapsulate nutrients, but such exogenous delivery carriers are costly, have complex processes, and poor compatibility with feed matrices, making them difficult to apply on a large scale in the feed industry.

[0008] Therefore, developing a technology that can simultaneously construct a low-cost, high-efficiency in-situ delivery system during enzymatic hydrolysis and perfectly integrate with feed processing technology is crucial for improving the nutritional value and utilization efficiency of krill in aquatic feed. Summary of the Invention

[0009] The purpose of this invention is to provide a krill-based enzymatic hydrolysis delivery system, its preparation method, and its application. The preparation method of this invention not only maximizes the release of krill nutrients through a strategy of "activating endogenous enzymes + synergistic exogenous enzymes," but more importantly, it utilizes the lipid and protein components released by the krill itself during the enzymatic hydrolysis process to form a natural "nutrient delivery system" through in-situ self-assembly with specific exogenously added excipients. This achieves the protection, targeted delivery, and sustained release of active ingredients, ultimately significantly enhancing their nutritional function in aquatic animals.

[0010] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0011] The first aspect of this invention provides a method for preparing a krill-based enzymatic hydrolysis delivery system, the method comprising the following steps:

[0012] (a) Mix krill with water and homogenize to obtain a slurry. Adjust the pH of the slurry to alkaline, then add calcium chloride and dithiothreitol and stir to obtain a mixture.

[0013] (b) Add a complex enzyme, soybean lysophospholipids and chitosan to the mixture to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysate mixture, wherein the complex enzyme is composed of a neutral protease and phospholipase A2;

[0014] (c) Cool the enzymatic hydrolysate mixture and stir it for a period of time, then inactivate the enzyme, and then concentrate it to obtain the krill-based enzymatic hydrolysis delivery system.

[0015] Preferably, in step (a), the mass ratio of krill to water is 1:(1~3); the pH of the slurry is adjusted to 7.5~9.5; the stirring temperature is 45~55℃, and the time is 40~60min.

[0016] Preferably, in step (a), the final concentration of calcium chloride is 3-5 mM and the final concentration of dithiothreitol is 1-5 mM.

[0017] Preferably, in step (b), the amount of compound enzyme added is 0.1% to 0.8% of the mass of the gel solution; the mass ratio of neutral protease to phospholipase A2 is (1 to 3): 1.

[0018] Preferably, in step (b), the amount of soybean lysophospholipid added is 0.5% to 5% of the slurry mass; and the amount of chitosan added is 0.1% to 2% of the slurry mass.

[0019] Preferably, in step (b), the enzymatic hydrolysis reaction temperature is 45~55℃, the pH value is 6.5~7.5, and the time is 1~3h.

[0020] Preferably, in step (c), the temperature is lowered to 25~40℃, the stirring speed is 30~100rpm, and the stirring time is 30~90min; the concentration is carried out until the water content is 40%~60%.

[0021] A second aspect of the present invention provides a krill-based enzymatic hydrolysis delivery system prepared by the above-described preparation method.

[0022] A third aspect of the present invention provides the application of the krill-based enzymatic delivery system prepared by the above-described method in the preparation of aquatic feed.

[0023] A fourth aspect of the present invention provides an aquatic feed comprising a krill-based enzymatic hydrolysis delivery system prepared by the preparation method, wherein the krill-based enzymatic hydrolysis delivery system has a mass content of 3% to 6%.

[0024] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0025] This invention abandons the costly post-encapsulation technology and creatively constructs a delivery system simultaneously during enzymatic hydrolysis. It utilizes the krill's own components (lipids, peptides) and exogenously added soybean lysophospholipids and chitosan for in-situ self-assembly, forming a natural and biocompatible delivery carrier. This achieves "in-situ encapsulation" of core nutrients, effectively protecting the encapsulated astaxanthin and Omega-3 long-chain polyunsaturated fatty acids, significantly reducing oxidative losses during feed pelleting and storage. This delivery system mimics a biomembrane structure, facilitating intestinal recognition and absorption in aquatic animals, achieving slow-release and targeted delivery of nutrients, improving utilization, and reducing water pollution. Furthermore, the preparation method of this invention is characterized by low cost, high efficiency, and integrated process. The entire process does not require complex post-encapsulation equipment and can be completed in existing enzymatic hydrolysis tanks, making it highly suitable for upgrading existing feed additive production lines and possessing the potential for large-scale promotion.

[0026] In the preparation method of this invention, endogenous enzymes (targeted initial digestion) synergistically work with exogenous neutral proteases (efficiently generating small peptides) and phospholipase A2 (specifically hydrolyzing phospholipids to release functional fatty acids and lysophospholipids) to achieve comprehensive and efficient release of nutrients. In addition, soybean lysophospholipids, as efficient emulsifiers and biomembrane components, can significantly promote the formation of lipid vesicles. Chitosan, as a cationic polysaccharide, can not only bind to peptides through electrostatic interactions but also improve the stability of the delivery system. Due to its own immune-enhancing properties, it endows feed ingredients with additional health benefits. At the same time, the complete krill flavor is preserved and enhanced, resulting in a significant appetite-stimulating effect. Moreover, the combined action of abundant active peptides and chitosan, as well as the combined action of Omega-3 long-chain polyunsaturated fatty acids and astaxanthin, can enhance the immunity and disease resistance of farmed animals. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0028] Figure 1 The survival rates of different groups of shrimp in Experiment 1 of this invention are shown.

[0029] Figure 2 The final average weight of shrimp in different groups in Experiment Example 1 of this invention;

[0030] Figure 3 The weight gain rate of different groups of shrimp in Experiment Example 1 of this invention;

[0031] Figure 4The feed conversion ratios of different groups of shrimp in Experiment 1 of this invention;

[0032] Figure 5 The feeding rates of different groups of shrimp in Experiment Example 1 of this invention;

[0033] Figure 6 The astaxanthin content of shrimp muscle in different groups in Experiment Example 1 of this invention;

[0034] Figure 7 The final average weight of shrimp in different groups in Experiment Example 2 of this invention;

[0035] Figure 8 The weight gain rate of different groups of shrimp in Experiment Example 2 of this invention;

[0036] Figure 9 The feed conversion ratios of different groups of shrimp in Experimental Example 2 of this invention;

[0037] Figure 10 This represents the feeding rate of different groups of shrimp in Experiment Example 2 of this invention. Detailed Implementation

[0038] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0039] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0040] This invention provides a method for preparing a krill-based enzymatic hydrolysis delivery system, the method comprising the following steps:

[0041] (a) Mix krill with water and homogenize to obtain a slurry. Adjust the pH of the slurry to alkaline, then add calcium chloride and dithiothreitol and stir (to precisely activate the autolytic enzyme system in the krill, initiate controlled autolysis, and initially release nutrients such as proteins and peptides) to obtain a mixture.

[0042] (b) Add a complex enzyme, soybean lysophospholipids and chitosan to the mixture to carry out an enzymatic hydrolysis reaction (during this process, neutral protease and phospholipase A2 further refine the macromolecules, while soybean lysophospholipids and the lipids released by the autolysis of krill, chitosan and the enzymatically hydrolyzed peptide chains form a mixed micelle / vesicle structure with encapsulation ability through in situ self-assembly via intermolecular forces), to obtain an enzymatic hydrolysis mixture, wherein the complex enzyme is composed of neutral protease and phospholipase A2;

[0043] (c) Cool the enzymatic hydrolysis mixture and stir it for a period of time (this cooling and maturation process is conducive to the full encapsulation and embedding of newly released hydrophobic nutrients (phospholipid Omega-3, astaxanthin) and hydrophilic nutrients (small molecule peptides) by the mixed micelle / vesicle structure, thus completing the construction and stabilization of the in-situ nutrient delivery system), then perform enzyme inactivation treatment, and then concentrate it to obtain the krill-based enzymatic hydrolysis delivery system.

[0044] In one embodiment, in step (a), the mass ratio of krill to water is 1:(1~3); the pH of the slurry is adjusted to 7.5~9.5; the stirring temperature is 45~55℃, and the time is 40~60min.

[0045] In one embodiment, in step (a), the final concentration of calcium chloride is 3-5 mM and the final concentration of dithiothreitol is 1-5 mM.

[0046] In one embodiment, in step (b), the amount of compound enzyme added is 0.1% to 0.8% of the mass of the gel; the mass ratio of neutral protease to phospholipase A2 is (1 to 3): 1.

[0047] In one embodiment, in step (b), the amount of soybean lysophospholipid added is 0.5% to 5% of the slurry mass; the amount of chitosan added is 0.1% to 2% of the slurry mass.

[0048] In one embodiment, in step (b), the enzymatic hydrolysis reaction temperature is 45~55℃, the pH value is 6.5~7.5, and the time is 1~3h.

[0049] In one embodiment, in step (c), the temperature is lowered to 25~40°C, the stirring speed is 30~100 rpm, and the stirring time is 30~90 min; the concentration is carried out until the water content is 40%~60%.

[0050] Another embodiment of the present invention provides a krill-based enzymatic hydrolysis delivery system prepared by the above preparation method.

[0051] Another embodiment of the present invention provides the application of the krill-based enzymatic delivery system prepared by the above method in the preparation of aquatic feed.

[0052] Another embodiment of the present invention provides an aquatic feed, the aquatic feed comprising a krill-based enzymatic hydrolysis delivery system prepared by the preparation method, wherein the krill-based enzymatic hydrolysis delivery system has a mass content of 3% to 6%.

[0053] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0054] Example 1

[0055] This embodiment describes a method for preparing a krill-based enzymatic hydrolysis delivery system, the preparation method comprising the following steps:

[0056] (a) Take 100 kg of frozen krill, thaw it, add 150 kg of water to homogenize it to obtain a slurry, adjust the pH to 7.5 with NaOH, add 100 g of CaCl2 and 100 g of dithiothreitol, adjust the temperature to 50 °C, stir for 50 minutes to obtain a mixture;

[0057] (b) Add 300g of neutral protease (200,000 U / g) and 150g of phospholipase A2 (100,000 U / g) to the mixture, along with 3kg of soybean lysophospholipid and 0.5kg of chitosan (dissolved in dilute acetic acid); continue enzymatic hydrolysis at 50°C and pH 7.0 for 2 hours to obtain the enzymatically hydrolyzed mixture;

[0058] (c) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis mixture is cooled to 30°C by circulating water and then gently stirred (50 rpm) at this temperature for 60 minutes to mature. Finally, the product is sterilized at 90°C for 15 minutes to inactivate the enzyme and then concentrated at 60°C to a water content of 50% to obtain the krill-based enzymatic hydrolysis delivery system.

[0059] Example 2

[0060] This embodiment describes a method for preparing a krill-based enzymatic hydrolysis delivery system, the preparation method comprising the following steps:

[0061] (a) Take 100kg of frozen krill, thaw it, add 150kg of water to homogenize it to obtain a slurry, adjust the pH to 7.5 with NaOH, add 200g of CaCl2 and 200g of dithiothreitol, adjust the temperature to 50℃, stir for 50 minutes to obtain a mixture;

[0062] (b) Add 300g of neutral protease (200,000 U / g) and 150g of phospholipase A2 (100,000 U / g) to the mixture, along with 5kg of soybean lysophospholipid and 1.5kg of chitosan (dissolved in dilute acetic acid); continue enzymatic hydrolysis at 50°C and pH 7.0 for 2 hours to obtain the enzymatically hydrolyzed mixture;

[0063] (c) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis mixture is cooled to 30°C by circulating water and then gently stirred (50 rpm) at this temperature for 60 minutes to mature. Finally, the product is sterilized at 90°C for 15 minutes to inactivate the enzyme and then concentrated at 60°C to a water content of 50% to obtain the krill-based enzymatic hydrolysis delivery system.

[0064] Comparative Example 1

[0065] This comparative example illustrates a method for preparing a krill-based enzymatic hydrolysis delivery system, the method comprising the following steps:

[0066] (a) Take 100 kg of frozen krill, thaw it, add 150 kg of water to homogenize it to obtain a slurry, adjust the pH to 7.5 with NaOH, adjust the temperature to 50℃, stir for 50 minutes to obtain a mixture;

[0067] (b) Add 300g of neutral protease (200,000 U / g) and 150g of phospholipase A2 (100,000 U / g) to the mixture, and continue enzymatic hydrolysis at 50°C and pH 7.0 for 2 hours to obtain the enzymatic hydrolysate;

[0068] (c) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis mixture is cooled to 30°C by circulating water and then gently stirred (50 rpm) at this temperature for 60 minutes to mature. Finally, the product is sterilized at 90°C for 15 minutes to inactivate the enzyme and then concentrated at 60°C to a water content of 50% to obtain the krill-based enzymatic hydrolysis delivery system.

[0069] Comparative Example 2

[0070] This comparative example illustrates a method for preparing a krill-based enzymatic hydrolysis delivery system, the method comprising the following steps:

[0071] (a) Take 100 kg of frozen krill, thaw it, add 150 kg of water to homogenize it to obtain a slurry, adjust the pH to 7.5 with NaOH, add 200 g of CaCl2 and 200 g of dithiothreitol, adjust the temperature to 50 °C, stir for 50 minutes to obtain a mixture;

[0072] (b) Add 300g of neutral protease (200,000 U / g) and 150g of phospholipase A2 (100,000 U / g) to the mixture, and continue enzymatic hydrolysis at 50°C and pH 7.0 for 2 hours to obtain the enzymatic hydrolysate;

[0073] (c) After the enzymatic hydrolysis is completed, the enzymatic hydrolysis mixture is cooled to 30°C by circulating water and then gently stirred (50 rpm) at this temperature for 60 minutes to mature. Finally, the product is sterilized at 90°C for 15 minutes to inactivate the enzyme and then concentrated at 60°C to a water content of 50% to obtain the krill-based enzymatic hydrolysis delivery system.

[0074] Experimental Example 1

[0075] According to the test results, the nutritional indicators of the enzymatically hydrolyzed krill prepared in Example 1 of the present invention are as follows: moisture 55.17%, crude protein 30.14%, acid-soluble protein 73.21%, crude fat 2.25%, and ash 11.11%.

[0076] Experimental grouping and feed preparation:

[0077] The experimental feed was prepared according to the dry matter formula shown in Table 1.

[0078] The fish meal control group was used as the positive control (PC group), the krill-based enzymatic delivery system prepared in Comparative Example 1 was used to replace 5% fish meal as the negative control group (NC group), and the krill-based enzymatic delivery system prepared in Example 1 of this invention was used to replace 5% fish meal as the experimental group (HK group).

[0079] In the feed preparation process, all ingredients are first ground and passed through an 80-mesh sieve. Then, the feed components are thoroughly mixed. Next, oil is added and thoroughly mixed into the feed mixture. Finally, water is added, and a single-screw pellet mill is used to prepare feed pellets with a diameter of 1.0 mm. The pellets are then placed in trays and dried in a 55°C oven. After the pellets are dried and cooled, they are packaged and stored in a -20°C cold storage until use. The feed is designed to have a crude protein content of approximately 41% and a crude fat content of approximately 7%.

[0080] Table 1 Experimental Feed Formulation (% Dry Matter)

[0081] raw material PC NC HK soybean meal 22 22 22 Peanut Meal 10 10 10 Imported fishmeal 20 15 15 Shrimp powder 10 10 10 Conventional methods for enzymatic hydrolysis of krill 5 This method enzymatically hydrolyzes krill. 5 rapeseed meal 10 10 10 High-gluten flour 19.13 19.13 19.13 fish oil 2.5 2.9 2.9 Soy lecithin 2.5 2.5 2.5 calcium dihydrogen phosphate 1.4 1.4 1.4 Mineral premix 0.5 0.5 0.5 Vitamin premix 1 1 1 Vitamin C 0.2 0.2 0.2 choline chloride 0.5 0.5 0.5 Ethoxyquinoline 0.02 0.02 0.02 Calcium propionate 0.1 0.1 0.1 Yttrium oxide 0.15 0.15 0.15 total 100 100 100

[0082] Experimental shrimp and their farming management:

[0083] This experiment used Litopenaeus vannamei (initial weight 2.4 g) as the experimental subject. After being temporarily reared with commercial feed for 2 weeks to acclimatize to the environment, the shrimp were cultured at the Langya Base of the Yellow Sea Fisheries Research Institute (Qingdao, Shandong). Before the feeding experiment began, the shrimp were fasted for 24 hours and then randomly assigned to 9 polyethylene culture tanks (300L). Each group had 3 parallel tanks, with 30 shrimp in each tank. During the experiment, the shrimp were fed at 7:00, 12:00, 17:00, and 22:00 daily at apparent saturation. During the 8-week culture period, the circulating seawater system was turned on for 2 hours daily to remove suspended solids, and the rest of the time the water was kept still. Uneaten feed and feces were removed daily using a siphon, and the culture tanks were regularly cleaned, with half of the water changed daily. The water temperature during the culture period was 26-31℃, the salinity was 28-31, the dissolved oxygen was >8.1mg / L, and the pH range was 7.3-7.9.

[0084] Sample collection:

[0085] At the end of the culture experiment, shrimp were anesthetized with eugenol, and all shrimp in each culture tank were weighed and counted. Additionally, eight shrimp were randomly selected from each tank for sampling. Hemolymph was collected from the pericardial cavity of the shrimp using a 1ml syringe and mixed with an anticoagulant at a 1:2 (v / v) ratio into a 1.5ml centrifuge tube. After standing at 4°C for 4 hours, the tube was centrifuged (4000×g, 10 minutes, 4°C) to obtain the supernatant. After blood collection, three shrimp were randomly selected from each tank for subsequent nutritional analysis. These shrimp were then dissected, and the hepatopancreas and muscle were collected for further parameter determination. All tissue samples were immediately flash-frozen in liquid nitrogen and stored at -80°C in the laboratory for later use.

[0086] Body composition analysis and fatty acid determination:

[0087] The fatty acid composition of muscle was analyzed by gas chromatography (GC-2010 Pro, Shimadzu, Japan). First, the sample was freeze-dried for 48 h. After extracting the fat using the chloroform-methanol method, 30 μl of the sample was placed in a 10 ml glass tube, and 2 ml of 0.5 mol / L potassium hydroxide-methanol solution was added. The tube was then placed in a 75°C water bath for 30 min. After cooling, 1 ml of boron trifluoride-methanol solution was added, and the tube was placed in a 75°C water bath for 30 min. After cooling, 1 ml of pure water and 1 ml of n-hexane were added, vortexed, and allowed to stand on ice for 1 h. The supernatant was then used for analysis. Gas chromatography was performed using a silica capillary column (SH-RT-2560, 100 m × 0.25 mm × 0.20 μm, Shimadzu, Japan; dicyanopropyl polysiloxane as the stationary phase) and a flame ionization detector. The column temperature program was as follows: from 150°C to 200°C at a rate of 15°C min⁻¹; then from 200°C to 250°C at a rate of 2°C min⁻¹. The injector and detector temperatures were both 250°C. Results are expressed as a percentage of each fatty acid relative to total fatty acids (%TFA).

[0088] Astaxanthin content analysis in muscle:

[0089] The astaxanthin content in the muscle was determined by Qingdao Yuanxin Testing Technology Co., Ltd. The specific method was as follows: Approximately 1g of sample was accurately weighed and placed in a 50mL round-bottom centrifuge tube. 4mL of extraction solution was added, and the tube was homogenized thoroughly for 2 minutes. The blade was rinsed with 4mL of extraction solution, and the washings were added to the sample centrifuge tube. The mixture was vortexed and homogenized. The mixture was ultrasonically extracted at 15℃ or below for 10 minutes, centrifuged at 8000 rpm for 5 minutes, and all supernatant was collected in a 50mL centrifuge tube. The residue was added to 4mL of extraction solution, and the above process was repeated. The extracts were combined and mixed. The extract was dehydrated by filtration through anhydrous sodium sulfate and filtered into a 100mL brown rotary evaporator flask. The anhydrous sodium sulfate was rinsed twice with approximately 15mL of extraction solution, and the rinses were added to the evaporator flask. The mixture was concentrated under reduced pressure at 40℃±25℃ on a rotary evaporator until nearly dry. The mixture was dried under nitrogen, and 1.0mL of 0.1% BHT ethanol solution was accurately added. The flask was capped, and the extract was thoroughly dissolved. After filtration through a 0.22 μm organic membrane, the sample was analyzed by high performance liquid chromatography (LC-2030 C 3 D, Shimadzu, Japan).

[0090] Data Analysis:

[0091] Formula for calculating growth indicators:

[0092] Weight gain (g) = Final average weight - Initial average weight;

[0093] Weight gain rate (%) = 100 × (final average weight - initial average weight) / initial average weight;

[0094] Feed intake rate (%) = 100 × total feed consumption / ((initial average weight + final average weight) / 2) / number of days of rearing;

[0095] Feed conversion ratio (%) = 100 × (final average weight - initial average weight) / total feed consumption;

[0096] Survival rate (%) = 100 × final number / initial number;

[0097] All experimental data were analyzed using SPSS 19.0 with one-way ANOVA and Tukey multiple comparisons. A p-value < 0.05 was considered statistically significant. Results are expressed as mean ± standard error.

[0098] Experimental results:

[0099] Regarding growth outcomes, there was no significant difference in shrimp survival rates, all exceeding 95%. Figure 1 This demonstrates the accuracy of the experimental results. The final average weight of the HK group ( Figure 2 ), weight gain rate ( Figure 3 The feed conversion ratio (FCR) of the HK group was significantly higher than that of the PC and NC groups (P < 0.05). The final average weight of the HK group was 19.3% and 22.3% higher than that of the PC and NC groups, respectively, demonstrating a good growth-promoting effect. Figure 4The feed intake rate of the HK group was significantly lower than that of the PC group (P < 0.05), 8.1% lower than that of the PC group, indicating that enzymatic hydrolysis of krill can improve the feed utilization efficiency of shrimp. Figure 5 The levels of the α group were significantly higher than those of the PC group (P < 0.05), indicating that the α group had a better appetite-inducing effect.

[0100] Regarding the astaxanthin content in shrimp muscle ( Figure 6 The astaxanthin content in the muscle of the HK group was 136% higher than that of the PC group (P<0.05), demonstrating a significantly optimized application delivery effect. Figures 1-6 In this context, different letters represent significant differences, while the same letter represents no significant differences.

[0101] Regarding the fatty acid composition of shrimp muscle (Table 2), the content of Omega-3 long-chain polyunsaturated fatty acids such as DHA and EPA was significantly higher in the HK group than in the PC group (P < 0.05). This demonstrates their in-situ delivery as a precise way to deliver high-quality nutrients.

[0102] Table 2

[0103] fatty acid PC NC HK 14:0 0.31 ± 0.03 0.20 ± 0.01 0.31 ± 0.04 16:0 18.54 ± 0.29 19.35 ± 0.14 20.17 ± 1.00 18:0 9.20 ± 0.09 9.91 ± 0.24 9.22 ± 0.16 20:0 0.19 ± 0.01 0.17 ± 0.00 0.18 ± 0.01 SFA 29.25 ± 0.28 29.83 ± 0.06 29.98 ± 0.76 16:1n-7 1.11 ± 0.07 b ]] 0.78 ± 0.04 a ]] 1.17 ± 0.04 b ]] 18:1n-9 13.34 ± 0.13 ab ]] 12.23 ± 0.15 a ]] 13.60 ± 0.21 b ]] 22:1n-9 0.14 ± 0.02 0.15 ± 0.01 0.18 ± 0.02 MUFA 14.93 ± 0.11 b ]] 13.91 ± 0.19 a ]] 15.36 ± 0.15 b ]] 18:2n-6 18.48 ± 0.32 a ]] <![CDATA[18.55 ± 0.41 ab ]]> <![CDATA[19.77 ± 0.04 b ]]> 20:2n-6 1.54 ± 0.02 1.62 ± 0.08 1.52 ± 0.05 n-6 PUFA <![CDATA[21.72 ± 0.31 a ]]> <![CDATA[22.22 ± 0.41 ab ]]> <![CDATA[23.23 ± 0.13 b ]]> 18:3n-3 1.15 ± 0.03 0.89 ± 0.06 1.11 ± 0.02 20:5n-3 <![CDATA[9.39 ± 0.10 a ]]> <![CDATA[9.73 ± 0.11 a ]]> <![CDATA[10.55 ± 0.12 b ]]> 22:5n-3 <![CDATA[0.84 ± 0.05 ab ]]> <![CDATA[0.77 ± 0.04 a ]]> <![CDATA[0.93 ± 0.03 b ]]> 22:6n-3 <![CDATA[10.41 ± 0.19 a ]]> <![CDATA[10.14 ± 0.24 a ]]> <![CDATA[13.58 ± 0.23 b ]]> n-3 PUFA <![CDATA[22.38 ± 0.07 a ]]> <![CDATA[21.48 ± 0.16 a ]]> <![CDATA[25.73 ± 0.64 b ]]>

[0104] Note: Different superscript letters in the same row of data indicate significant differences (P<0.05). SFA: saturated fatty acids; MUFA: monounsaturated fatty acids; PUFA: polyunsaturated fatty acids.

[0105] Experiment Example 2

[0106] Experimental grouping and feed preparation:

[0107] The fish meal control group was used as the positive control (PC group), the krill-based enzymatic delivery system prepared in Comparative Example 2 was used to replace 5% fish meal as the negative control group (NC group), and the krill-based enzymatic delivery system prepared in Example 2 of this invention was used to replace 5% fish meal as the experimental group (HK group).

[0108] The basic preparation method of the experimental feed is the same as that of Experiment 1. However, since turbot and Litopenaeus vannamei have different nutritional requirements, the amounts of fish meal, soybean meal, rapeseed meal, corn gluten meal, brewer's yeast and wheat flour in the basic formula are different in order to match the different nutritional needs of the animals (Table 3).

[0109] Table 3 Experimental feed formulation (% dry matter basis)

[0110] Element PC NC HK Fish meal 40 35 35 Corn protein powder 6 6 6 soybean meal 8 8 8 Conventional methods for enzymatic hydrolysis of krill 5 This method enzymatically hydrolyzes krill. 5 rapeseed meal 10 10 10 brewer's yeast 5 5 5 wheat flour 22.88 22.28 21.68 Mineral premix 0.5 0.5 0.5 Vitamin premix 1 1 1 calcium dihydrogen phosphate 1 1 1 Vitamin C 0.2 0.2 0.2 choline chloride 0.2 0.2 0.2 Ethoxyquinoline 0.02 0.02 0.02 Yttrium oxide 0.1 0.1 0.1 Calcium propionate 0.1 0.1 0.1 fish oil 2 2 2 Soybean oil 2 2 2 Soy lecithin 1 1 1 total 100 100 100

[0111] Experimental fish and aquaculture management:

[0112] Before the experiment, the turbot fry used in the experiment were temporarily held in 2000L cylindrical polyethylene tanks (100 cm high; 230 cm in diameter; 210 fish per tank) for 14 days. During the temporary holding period, they were fed commercial feed twice a day (07:30 and 17:30). For the experimental grouping, 360 uniformly sized, healthy turbot fry with an initial body weight of (7.88±0.04) g were selected as the research subjects and randomly assigned to 12 polyethylene tanks (220L, 42 × 72 × 72 cm). Each group was fed 3 tanks of experimental feed, with 30 fish per tank. The culture experiment was conducted in an indoor flowing seawater system. The seawater used was deep well water. Feeding was done twice daily (07:30 and 17:30), both times with full feeding, for a 56-day feeding and growth experiment. During the aquaculture experiment, natural light was used, with water temperature ranging from 15.1 to 21.3℃, salinity from 24 to 29, pH from 7.5 to 8.5, and dissolved oxygen levels between 6 and 8.5 mg / L. After each feeding, feces and uneaten feed were promptly removed, and the experimental tanks were regularly scrubbed. Dead fish were removed immediately, and their number and weight were recorded.

[0113] Experimental sample collection:

[0114] After the rearing experiment, the experimental fish were starved for 24 hours and anesthetized with eugenol (1:10,000). The fish were then weighed (total weight per tank), and the number of surviving fish per tank was counted. Next, three turbot were randomly selected from each tank (nine fish per group), and their body weight, length, visceral mass weight, and liver weight were measured to calculate the fish's morphological indicators. Then, six fish were randomly selected from each tank, and blood was collected from the tail vein using a disposable syringe (1 ml). The blood was placed in a centrifuge tube (1.5 ml) and allowed to coagulate naturally at room temperature for 2 hours, then at 4°C for 6 hours, followed by centrifugation (836 × g; 4°C; 10 min). The supernatant was then collected as serum. After blood collection, the fish were dissected on ice, and samples of muscle, liver, and subcutaneous adipose tissue near the fins were collected. All samples were immediately flash-frozen in liquid nitrogen after sampling, and then transferred to a -76°C freezer for storage and future use. After sampling, four fish were randomly selected and placed in a -20°C freezer for later use.

[0115] Fatty acid composition analysis of the sample:

[0116] Gas chromatography was used to determine the fatty acid composition of various fish tissues and feed. First, the samples were vacuum dried using a freeze dryer. Then, they were subjected to methylation treatment in a 72℃ water bath (using KOH-methanol and HCl-methanol sequentially), followed by extraction with n-hexane. After overnight extraction, the supernatant was collected for analysis. The gas chromatograph used was a Shimadzu GC-2010 Pro, equipped with a flame ionization detector. The column temperature program was as follows: increasing from 150℃ to 200℃ at a rate of 15℃ / min, then increasing from 200℃ to 250℃ at a rate of 2℃ / min. The injection port and detector temperatures were both set to 250℃.

[0117] Data Analysis:

[0118] Formula for calculating growth indicators:

[0119] Weight gain (g) = Final average weight - Initial average weight;

[0120] Weight gain rate (%) = 100 × (final average weight - initial average weight) / initial average weight;

[0121] Feed intake rate (%) = 100 × total feed consumption / ((initial average weight + final average weight) / 2) / number of days of rearing;

[0122] Feed conversion ratio (%) = 100 × (final average weight - initial average weight) / total feed consumption;

[0123] Survival rate (%) = 100 × final number / initial number;

[0124] All experimental data were analyzed using SPSS 19.0 with one-way ANOVA and Tukey multiple comparisons. A p-value < 0.05 was considered statistically significant. Results are expressed as mean ± standard error.

[0125] Experimental results:

[0126] Regarding growth results, the survival rate was 100% in all cases, and no fish died during the rearing process, demonstrating the accuracy of the data. The final average weight of the HK group ( Figure 7 The weight gain rate in the HK group was significantly higher than that in the PC and NC groups (P < 0.05). Figure 8 The HK group showed significantly higher growth rate than the PC group (P < 0.05). The final average weight of the HK group was 10.7% and 7.9% higher than that of the PC and NC groups, respectively, demonstrating a good growth-promoting effect. The feed conversion ratio of the HK group (…) was significantly higher than that of the PC group (P < 0.05). Figure 9 The feed intake rate of the HK group was significantly lower than that of the PC group (P < 0.05), 15.6% lower than that of the PC group, indicating that enzymatic hydrolysis of krill can improve the feed utilization efficiency of shrimp. Figure 10 The levels were significantly higher than those in the PC and NC groups (P < 0.05), with increases of 10% and 8.5% compared to the PC and NC groups, respectively, indicating that it has a good appetite-inducing effect. Figures 7-10 In this context, different letters represent significant differences, while the same letter represents no significant differences.

[0127] Regarding the fatty acid composition of shrimp muscle (Table 4), the HK group had significantly lower levels of 16:0 and 18:3n-3 than the PC group, while its DHA (22:6n-3) and EPA (20:5n-3) content was significantly higher than the control group (P<0.05), demonstrating efficient delivery of fatty acids.

[0128] Table 4. Effects of experimental diets on fatty acid composition of turbot muscle (% total fatty acids)

[0129] fatty acid PC NC HK 14:0 1.51±0.05 1.55±0.10 1.63±0.04 16:0 <![CDATA[18.44±0.15 a ]]> <![CDATA[16.22±0.41 b ]]> <![CDATA[16.29±0.14 b ]]> 18:0 6.21±0.20 6.44±0.22 5.85±0.10 20:0 0.24±0.03 0.22±0.03 0.27±0.02 SFA 26.99±0.34 27.88±0.54 27.25±0.88 18:1n-9 16.55±0.64 16.53±0.13 17.83±0.33 22:1n-9 0.21±0.02 0.17±0.02 0.21±0.03 24:1n-9 0.14±0.02 0.13±0.01 0.17±0.01 MUFA 18.88±0.21 16.92±0.20 18.59±0.33 18:2n-6 23.54±0.23 23.62±0.18 24.52±0.41 18:3n-6 0.05±0.01 0.06±0.01 0.12±0.03 20:3n-6 0.03±0.01 0.05±0.01 0.06±0.01 20:4n-6 1.26±0.03 1.19±0.02 1.28±0.04 n-6PUFA 24.87±0.15 24.46±0.21 25.82±0.26 18:3n-3 <![CDATA[1.72±0.00 a ]]> <![CDATA[1.54±0.00 b ]]> <![CDATA[1.43±0.04 b ]]> 20:3n-3 0.21±0.02 0.18±0.02 0.25±0.02 20:5n-3 <![CDATA[4.40±0.02 a ]]> <![CDATA[4.31±0.00 a ]]> <![CDATA[4.97±0.07 b ]]> 22:6n-3 <![CDATA[16.38±0.91 a ]]> <![CDATA[17.02±0.41 a ]]> <![CDATA[18.17±0.34 b ]]> n-3PUFA 23.34±0.96 24.24±0.64 25.44±0.61

[0130] Note: If there are no identical letters between data from different treatment groups in the same row, it is considered that there is a significant difference (P<0.05). SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; n-6PUFA: n-6 series polyunsaturated fatty acids; n-3PUFA: n-3 series polyunsaturated fatty acids.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing a krill-based enzymatic hydrolysis delivery system, characterized in that, The preparation method includes the following steps: (a) Mix krill with water and homogenize to obtain a slurry. Adjust the pH of the slurry to alkaline, then add calcium chloride and dithiothreitol and stir to obtain a mixture. (b) Add a complex enzyme, soybean lysophospholipids and chitosan to the mixture to carry out an enzymatic hydrolysis reaction to obtain an enzymatic hydrolysate mixture, wherein the complex enzyme is composed of a neutral protease and phospholipase A2; (c) Cool the enzymatic hydrolysate mixture and stir it for a period of time, then inactivate the enzyme, and then concentrate it to obtain the krill-based enzymatic hydrolysis delivery system.

2. The preparation method according to claim 1, characterized in that, In step (a), the mass ratio of krill to water is 1:(1~3); the pH of the slurry is adjusted to 7.5~9.5; the stirring temperature is 45~55℃ and the time is 40~60min.

3. The preparation method according to claim 1, characterized in that, In step (a), the final concentration of calcium chloride is 3-5 mM, and the final concentration of dithiothreitol is 1-5 mM.

4. The preparation method according to claim 1, characterized in that, In step (b), the amount of compound enzyme added is 0.1% to 0.8% of the mass of the gel solution; the mass ratio of neutral protease to phospholipase A2 is (1 to 3):

1.

5. The preparation method according to claim 1, characterized in that, In step (b), the amount of soybean lysophospholipid added is 0.5% to 5% of the slurry mass; the amount of chitosan added is 0.1% to 2% of the slurry mass.

6. The preparation method according to claim 1, characterized in that, In step (b), the enzymatic hydrolysis reaction temperature is 45~55℃, the pH value is 6.5~7.5, and the time is 1~3h.

7. The preparation method according to claim 1, characterized in that, In step (c), the temperature is lowered to 25~40℃, the stirring speed is 30~100rpm, and the stirring time is 30~90min; the concentration is carried out until the water content is 40%~60%.

8. The krill-based enzymatic delivery system prepared by the method of any one of claims 1 to 7.

9. The application of the krill-based enzymatic delivery system prepared by any one of claims 1 to 7 in the preparation of aquatic feed.

10. An aquatic feed, characterized in that, The aquatic feed includes the krill-based enzymatic hydrolysis delivery system prepared by the preparation method of any one of claims 1 to 7, wherein the krill-based enzymatic hydrolysis delivery system has a mass content of 3% to 6%.

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