Preparation method of protein-embedded sodium alginate microspheres and application of protein-embedded sodium alginate microspheres in aquaculture

Abstract

The invention discloses a preparation method of protein-embedded sodium alginate microspheres and application of the protein-embedded sodium alginate microspheres in aquaculture. According to the preparation method, after the prepared sodium alginate microspheres are used for feeding fish, the embedded protein can be prevented from being digested by gastric juice and is digested and absorbed in intestinal tracts, so that the problem of waste caused by feeding of protein or polypeptide active substances through digestive tracts is solved, efficient utilization of the protein or polypeptide active substances is realized, and the utilization rate of the protein or polypeptide active substances is increased. The method can be used for embedding various protein or polypeptide active substances. After tilapia mossambica is fed with the sodium alginate microspheres embedded with the functional neuropeptide UII, the sodium alginate microspheres can protect the functional neuropeptide, can be prevented from being digested by gastric juice, and can be digested and absorbed in intestinal tracts. After tilapia mossambica is fed with the UII embedded with sodium alginate, expression of tilapia mossambica reproduction related genes is improved, gonad development of male and female tilapia mossambica is remarkably promoted, and the UII has a good application prospect in fish culture and breeding.

Description

Technical Field

[0001] The present invention relates to the field of aquaculture, and more particularly to a method for preparing protein-embedded sodium alginate microspheres and application thereof in aquaculture. Background Art

[0002] Tilapia, commonly known as African crucian carp, belongs to the order Perciformes, suborder Perciformes, family Cichlidae, and genus Oreochromis. It has the characteristics of rapid growth, strong adaptability, tolerance to low oxygen, wide diet and short breeding cycle. It is suitable for large-scale intensive breeding and has very high economic value. Therefore, it is one of the most important freshwater farmed fish in my country and even the world.

[0003] Sodium alginate is a polysaccharide carbohydrate extracted from the brown algae Laminaria japonica or Sargassum. It undergoes rapid ion exchange and cross-linking reactions with various divalent metal ions to form alginate gel. Alginate gel is characterized by low production cost, biodegradability, and non-biotoxicity. It is also pH-sensitive (i.e., it is virtually insoluble in acidic environments but swells in weakly alkaline ones), making it widely used for the delivery of drugs and bioactive compounds. For example, patent CN118892458A discloses drug-loaded sodium alginate microspheres and their preparation method. Through the physical cross-linking of sodium alginate and carboxymethyl chitosan, the microspheres are encapsulated with drugs, allowing them to deliver drugs to patients for treatment.

[0004] Microencapsulation technology uses polymer film-forming materials as capsule walls or shells to encapsulate substances such as liquids, solids, or gases within microcapsules. Microcapsules typically range in size from micrometers to nanometers and can be categorized by size as nanocapsules (<200nm), microcapsules or microspheres (0.2-2000μm), and microbeads (>2nm).

[0005] Currently, the main methods for preparing sodium alginate microcapsules include ionic crosslinking, emulsion crosslinking, spray drying, and complex coacervation. Among them, the emulsion crosslinking, spray drying, and complex coacervation methods are relatively complex to operate, have high equipment requirements, and have high production costs, making them unsuitable for the preparation of small-scale sodium alginate microspheres. The ionic crosslinking method, which uses a sodium alginate solution and metal ions to react to form a gel, is more convenient. This method is divided into a sharp hole coagulation bath method and a drip method. The drip method is simple to operate, has mild reaction conditions, is easy to control the temperature, and has a rapid reaction, allowing for the rapid formation of gel microspheres. In recent years, the preparation method of dripping CaCl2 as a calcium source into sodium alginate has been widely used to prepare microspheres that encapsulate large molecular bioactive substances.

[0006] Functional neuropeptides are small polypeptides synthesized and released by neurons. They act as signaling molecules in the nervous system, participating in various physiological processes and possessing a wide range of physiological regulatory functions. Urotensin II (Urotensin II) has cardiovascular, osmotic, immunomodulatory, and neuromodulatory functions in fish. UII exerts its biological functions by binding to its receptor, the urotensin II receptor (UT). Urantide is a competitive antagonist of the UII receptor, UT, with high affinity for UT. Therefore, in studies of UII, the efficient binding of urantide to UT can be exploited to block the physiological functions of UII and thereby determine whether the changes are caused by UII. Previous research by our team has shown that Urotensin II can regulate the expression of reproductive-related genes in the brain-pituitary-gonadal axis of GIFT tilapia, affecting the synthesis and secretion of sex hormones, thereby promoting gonadal development in tilapia. Research on functional neuropeptides or proteins in fish is relatively in-depth and comprehensive, and researchers have a basic understanding of the physiological functions of different neuropeptides or proteins in fish. However, there are technical problems in the application of functional neuropeptides or proteins in specific research and actual production.

[0007] Commonly used methods for applying functional neuropeptides or proteins are feed spraying, intraperitoneal injection or muscle implantation. The feed spraying method causes a large loss of the polypeptides or proteins sprayed on the surface of the feed after entering the water, and even if part of them are ingested by the fish, they are digested or degraded in the stomach, resulting in the polypeptides or proteins being unable to exert their biological functions. Intraperitoneal injection and muscle implantation are more effective, but both methods have the problems of complex operation, time-consuming and labor-intensive, high cost, and causing acute stress to the fish. Therefore, their operability is significantly limited when used in the actual production of fish farming. Due to the above reasons, the application of functional neuropeptides or proteins in the actual production of aquaculture is greatly limited. Therefore, the development of technologies that can protect the activity of functional neuropeptides or proteins, allow them to enter the fish body through conventional feeding methods, and protect neuropeptides or proteins from digestion and degradation in the fish's gastric juice is of great significance for the application of functional neuropeptides or proteins in aquaculture. Summary of the Invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing protein-embedded sodium alginate microspheres and their application in aquaculture.

[0009] The first object of the present invention is to provide a method for preparing protein-embedded sodium alginate microspheres.

[0010] The second object of the present invention is to provide microspheres prepared by the preparation method.

[0011] The third object of the present invention is to provide the use of the microspheres in the preparation of oral aquatic preparations.

[0012] The fourth object of the present invention is to provide a method for preparing sodium alginate microspheres encapsulating urotensin II and using the microspheres in aquatic feed additives.

[0013] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0014] The present invention prepares sodium alginate microspheres under different production conditions, selects the optimal production conditions, and prepares sodium alginate microspheres without an embedding material and sodium alginate microspheres embedding bovine serum albumin (BSA), urotensin II (UⅡ), and an antagonist of the urotensin II receptor UT (Urantide). Tilapia are fed with these microspheres for one month to determine the embedding effect of the sodium alginate microspheres and the regulatory effect of feeding tilapia with sodium alginate embedding UⅡ on tilapia reproduction. The results show that the sodium alginate microspheres can effectively prevent the embedding material from being digested by acidic gastric juice, and release UⅡ in a weakly alkaline intestinal fluid environment. UⅡ can promote the gonadal development of tilapia and the expression of reproductive-related genes on the hypothalamus-pituitary-gonadal reproductive axis, thereby achieving the effect of improving reproductive speed and efficiency and improving economic benefits. Therefore, the present invention claims protection for the preparation method of sodium alginate microspheres embedding functional neuropeptides and their use in promoting tilapia gonadal development.

[0015] The present invention claims a method for preparing protein-embedded sodium alginate microspheres, comprising the following steps:

[0016] S1. Add the protein to be embedded into a solution of 1.5-2.5% sodium alginate by mass, remove bubbles, and obtain embedding solution;

[0017] S2. Add the embedding solution dropwise into a 0.5-1.5% calcium chloride solution;

[0018] S3. Cross-link for 0.5 to 1.5 hours.

[0019] Preferably, the mass fraction of the sodium alginate solution is 2% sodium alginate.

[0020] Preferably, the mass fraction of the calcium chloride solution is 1%.

[0021] Preferably, cross-linking is performed for 1 hour.

[0022] Preferably, the concentration of the protein to be embedded in the embedding solution is 0.4-0.6 g / 100 g.

[0023] More preferably, the concentration of the protein to be embedded in the embedding solution is 0.5 g / 100 g.

[0024] The present invention also claims protection for the microspheres prepared by the preparation method.

[0025] The present invention also claims protection for the use of the microspheres in preparing oral aquatic preparations.

[0026] On the other hand, the present invention claims to protect the use of sodium alginate microspheres embedded with urotensin II prepared by the preparation method in tilapia feed additives.

[0027] Preferably, the tilapia feed additive is an additive that promotes fish gonad development.

[0028] More preferably, the gonad is a testis or an ovary.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides a method for preparing protein-embedded sodium alginate microspheres. The method can ensure that after the prepared sodium alginate microspheres are fed to fish, the embedded protein can avoid being digested by gastric juice and instead be digested and absorbed in the intestine, thereby solving the waste of drugs fed through the digestive tract and achieving efficient utilization of drugs. The method can be used for embedding various drugs or active substances.

[0031] The sodium alginate microspheres encapsulating the functional neuropeptide UII, prepared using this method, were fed to tilapia. The results showed that the microspheres protected the functional neuropeptide, preventing it from being digested by gastric juice and allowing it to be absorbed in the intestine. The microspheres also increased the expression of reproductive-related genes in tilapia, thereby promoting gonadal development and promising application prospects in fish farming and breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Gastric emptying of tilapia after being fasted for 24 hours and then fed a diet that was 2% of their body weight. A to L represent gastric emptying at 30 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 7 hours, 9 hours, 11 hours, 13 hours, 16 hours, and 19 hours after feeding, respectively.

[0033] Figure 2 The digestion of tilapia stomach contents within 19 hours after fasting for 24 hours and then feeding them with 2% body weight feed and sodium alginate microspheres.

[0034] Figure 3Growth indicators (body length and weight) of male and female GIFT tilapia after feeding blank sodium alginate microspheres and sodium alginate microspheres embedded with BSA, UⅡ and UT antagonist Urantide for 15 and 30 days; A and B are the body length and weight of male and female tilapia after 15 days of feeding, respectively; C and D are the body length and weight of male and female tilapia after one month of feeding, respectively; data are expressed as mean ± SEM (n = 3-12), analyzed by independent sample T test, *p < 0.05.

[0035] Figure 4 Effects of feeding blank sodium alginate microspheres and sodium alginate microspheres embedded with BSA, UⅡ and UT antagonist Urantide on the mRNA levels of gnrh1, gnrh2 and gnrh3 genes in the hypothalamus of male tilapia; A shows the changes in gnrh1 gene mRNA level; B shows the changes in gnrh2 gene mRNA level; C shows the changes in gnrh3 gene mRNA level; data are expressed as mean ± standard error (n=3-6), one-way ANOVA, SNK multiple comparison, *p<0.05, **p<0.01, ***p<0.005.

[0036] Figure 5 Effects of feeding blank sodium alginate microspheres and sodium alginate microspheres embedded with BSA, UⅡ and UT antagonist Urantide on the mRNA levels of gnrh1, gnrh2 and gnrh3 genes in the hypothalamus of female tilapia; A shows the changes in gnrh1 gene mRNA level; B shows the changes in gnrh2 gene mRNA level; C shows the changes in gnrh3 gene mRNA level; data are expressed as mean ± standard error (n=6-12), one-way analysis of variance, SNK multiple comparison, *p<0.05, **p<0.01, ***p<0.005.

[0037] Figure 6 Effects of feeding blank sodium alginate microspheres and sodium alginate microspheres embedded with BSA, UⅡ and UT antagonist Urantide on the mRNA levels of lhβ, fshβ and gthα genes in the pituitary of male tilapia; A shows the changes in the mRNA level of lhβ gene; B shows the changes in the mRNA level of fshβ gene; C shows the changes in the mRNA level of gthα gene; data are expressed as mean ± standard error (n=3-6), one-way analysis of variance, SNK multiple comparison, *p<0.05, **p<0.01, ***p<0.005.

[0038] Figure 7Effects of feeding blank sodium alginate microspheres and sodium alginate microspheres embedded with BSA, UⅡ and UT antagonist Urantide on the mRNA levels of lhβ, fshβ and gthα genes in the pituitary of female tilapia; A shows the changes in the mRNA level of lhβ gene; B shows the changes in the mRNA level of fshβ gene; C shows the changes in the mRNA level of gthα gene; data are expressed as mean ± standard error (n=6-12), one-way analysis of variance, SNK multiple comparison, *p<0.05, **p<0.01, ***p<0.005.

[0039] Figure 8 Effects of feeding blank sodium alginate microspheres and sodium alginate microspheres embedded with BSA, UⅡ and UT antagonist Urantide on the mRNA levels of lhr and fshr genes in the testis of male tilapia; A shows the changes in the mRNA level of lhr gene; B shows the changes in the mRNA level of fshr gene; data are expressed as mean ± SD (n = 3-6), one-way ANOVA, SNK multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.005.

[0040] Figure 9 Effects of feeding blank sodium alginate microspheres and sodium alginate microspheres embedded with BSA, UⅡ and UT antagonist Urantide on the mRNA levels of lhr and fshr genes in the ovaries of female tilapia; A shows the changes in the mRNA level of the lhr gene; B shows the changes in the mRNA level of the fshr gene; data are expressed as mean ± standard error (n=6-12), one-way analysis of variance, SNK multiple comparison, *p<0.05, **p<0.01, ***p<0.005.

[0041] Figure 10 Changes in testicular tissue morphology in male tilapia after being fed with feed and sodium alginate microspheres encapsulating functional neuropeptides for 15 and 30 days; PS: primary spermatocyte; Sp: sperm; SS: secondary spermatocyte; St: spermatid; Scale bar: 100 μm.

[0042] Figure 11 Changes in ovarian tissue morphology in female tilapia after being fed with feed and sodium alginate microspheres encapsulating functional neuropeptides for 15 and 30 days; N: nucleus; Nu: nucleolus; YG: yolk granule; YV: yolk vesicle; ZR: zona radiata; Scale bar: 100 μm. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below with reference to the accompanying drawings and specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available unless otherwise specified.

[0044] The tilapia fry used in the experiment had a mass of 25-35 g / fish, a water temperature of 28-29°C, and were acclimated for more than 2 weeks under 12 h light and 12 h dark conditions.

[0045] Example 1 Preparation of protein-embedded sodium alginate microspheres

[0046] Bovine serum albumin (BSA) was used as the embedding protein, and the orthogonal method was used to determine the optimal parameters for preparing sodium alginate microspheres embedded with the protein. The mass fraction of calcium chloride, the mass fraction of sodium alginate, and the cross-linking time were selected as the main factors affecting the quality of the microspheres. 16 (3 4 ) orthogonal table (Table 1) for orthogonal experiment.

[0047] Table 1: L of BSA-embedded alginate microspheres prepared under different conditions 16 (3 4 ) of the orthogonal test table

[0048]

[0049] According to the parameters in Table 1 and the following preparation method, 16 groups of protein-embedded sodium alginate microspheres were prepared:

[0050] 1. Preparation of sodium alginate solution: Sodium alginate was mixed with deionized water (100 g in total), dissolved under magnetic stirring at 50°C, and allowed to stand at 4°C overnight to remove bubbles to obtain sodium alginate microcapsule solution;

[0051] 2. Protein embedding: Add 50 mg of BSA powder to the sodium alginate microcapsule solution, stir evenly, and let it stand at 4°C overnight to remove bubbles to obtain the embedding solution;

[0052] 3. Preparation of microspheres: Transfer the embedding solution to a syringe equipped with a medical No. 9 needle (avoid bubbles during the transfer process) and drip the embedding solution dropwise into a 27°C calcium chloride solution at a drop distance of about 8 cm to 10 cm. Maintain a drop rate of 1 mL / min while maintaining magnetic stirring (the speed of the magnetic stirrer is 300 rpm).

[0053] 4. Cross-linking: After the addition is complete, place the calcium chloride solution on ice and let it stand for 10 minutes;

[0054] 5. Cleaning: After cross-linking, remove the microspheres and rinse the gel microspheres repeatedly with deionized water to remove the surface liquid and transfer them to a culture dish;

[0055] 6. Freeze-drying: After freeze-drying for 12 hours, pack in sealed bags and store at -80℃.

[0056] Example 2 Gastric emptying of tilapia fed with 2% of their body weight after fasting for 24 hours

[0057] 1. Experimental Methods

[0058] Sixteen tilapia fry were fed a commercially available tilapia feed at 8:00 AM at a concentration of 2% g / body weight. Gastric emptying was observed at 30, 60, 2, 3, 4, 5, 7, 9, 11, 13, 16, and 19 hours after feeding.

[0059] 2. Experimental Results

[0060] Figure 1 This is the gastric emptying of tilapia fed after a 24-hour fast. The stomach of the tilapia swelled after feeding, but returned to normal size 19 hours later. Dissection revealed no visible stomach contents, indicating that the stomach empties approximately 19 hours after feeding.

[0061] Example 3 Results of protein content and drug loading rate of protein-embedded sodium alginate microspheres.

[0062] 1. Experimental Methods

[0063] The 16 groups of protein-embedded sodium alginate microspheres prepared in Example 1 were tested.

[0064] Method for detecting the amount of protein embedded in sodium alginate microspheres:

[0065] 1. Add 300±1.0 mg of freeze-dried sodium alginate microspheres to a 50 mL centrifuge tube and dissolve in 20 mL of phosphate buffer (pH = 6.8). Pour the microspheres and liquid in the centrifuge tube into a grinding dish and grind until no obvious microsphere particles are left. Transfer the suspension to a 50 mL centrifuge tube.

[0066] 2. Centrifuge the sample in the 50 mL centrifuge tube (4000 rpm) for 20 min, remove the supernatant and add it to a new 15 mL centrifuge tube and dilute to the smallest integer (e.g., if the suspension is 5.6 mL, dilute to 6 mL);

[0067] 3. Divide the liquid in the 15mL centrifuge tube into two EP tubes, 1mL per tube, centrifuge at 16000r / min for 5min, and transfer the supernatant to new EP tubes;

[0068] 4. Filter the liquid in the EP tube through a 0.2 μm filter membrane and adjust the filtrate to 1 mL with phosphate buffer (pH = 6.8);

[0069] 5. Detect protein concentration using BCA kit at 562 nm.

[0070] Embedded bovine serum albumin mass = protein concentration × 20mL

[0071] Drug loading rate = embedded bovine serum albumin mass / freeze-dried sodium alginate microsphere mass × 100%

[0072] 2. Experimental Results

[0073] The protein content and drug loading efficiency of the protein-embedded sodium alginate microspheres prepared with different parameters are shown in Table 2, where the drug loading efficiency was tested twice.

[0074] Table 2: Properties of BSA-encapsulated alginate microspheres prepared under different conditions

[0075]

[0076] Example 4 Digestion of protein-encapsulated sodium alginate microspheres in the stomach

[0077] 1. Experimental Methods

[0078] Sixteen tilapia fry were fed a 2% g / BW (body weight) diet at 8:00 AM along with 50 mg of BSA-encapsulated sodium alginate microspheres (Group 2 in Table 1). Fish were dissected 30, 60, 2, 3, 4, 5, 7, 9, 11, 13, 16, and 19 hours after feeding to observe digestion of the protein-encapsulated sodium alginate microspheres in the stomach.

[0079] 2. Experimental Results

[0080] The results are as follows Figure 2 As shown, after feeding tilapia with sodium alginate microspheres embedded with BSA, the stomach of the tilapia was dissected to observe the digestion of the feed and microspheres, which illustrates the digestion of the microspheres and feed by the tilapia.

[0081] As feeding time increases, the amount of feed in the stomach decreases and is gradually digested. Upon dissection, the sodium alginate microspheres in the stomach are propelled into the intestines through gastric peristalsis. The number of sodium alginate microspheres decreases over time, and no alginate microspheres were found in the tilapia stomachs 16 hours after feeding. Given that tilapia gastric emptying time is 19 hours, dissection of the intestines 19 hours after feeding revealed numerous alginate microspheres with normal morphology and structure. This suggests that the sodium alginate microspheres can resist digestion by tilapia gastric juice, protecting the encapsulated protein from digestion and absorption in the intestines.

[0082] Example 5 Release rate of protein-embedded sodium alginate microspheres in the gastrointestinal tract.

[0083] 1. Experimental Methods

[0084] 1. Prepare tilapia simulated intestinal fluid and simulated gastric fluid

[0085] Preparation of tilapia simulated gastric fluid: 16.4 mL of 24.4% dilute hydrochloric acid, 10.0 g of pepsin, add water and mix well, dilute to 1000 mL, pH 1.2;

[0086] Preparation of tilapia simulated intestinal fluid: Dissolve 6.8 g of potassium dihydrogen phosphate in 500 mL of water and adjust the pH to 6.8 with 0.1 mol / L sodium hydroxide solution; add 10 g of trypsin and dilute to 1000 mL with water, pH 6.8.

[0087] 2. Detection of the release rate of protein-encapsulated sodium alginate microspheres in simulated intestinal fluid

[0088] Weigh 1 microsphere (50 ± 1.0 mg) and place it in 20 mL of simulated intestinal fluid. Incubate at 28°C. Take 1 mL of sample at intervals and add the same volume of simulated intestinal fluid. The content of BSA released from the sodium alginate microspheres embedded with BSA in the intestinal fluid was detected by BCA Protein Assay Kit) and microplate reader.

[0089] Specific steps: Prepare standards of different concentrations according to the instructions of the BCA kit. Use a microplate reader to detect the absorbance of the standards and samples at 562nm. Use the absorbance values ​​of the standards of different concentrations to establish a standard curve, obtain a regression equation, substitute the absorbance values ​​of the samples into the equation, and calculate the BSA content in the samples. The cumulative release rate of samples at different time periods is calculated according to the following formula:

[0090] Cumulative release rate = [20Ci + V∑C(i-1)] / m × 100%.

[0091] Where: Ci is the mass concentration of bovine serum albumin in the release medium (g / L), V is the volume of each sampling, which is 5 mL for this system, and m is the total drug loading (mg).

[0092] 3. Detection of the release rate of protein-encapsulated sodium alginate microspheres in simulated gastric fluid

[0093] Weigh 1 microsphere (50 ± 1.0 mg) and place it in 20 mL of simulated gastric fluid. Incubate at 28°C. Take 200 μL of sample at intervals and add the same volume of fresh simulated gastric fluid medium. The content of BSA released from sodium alginate microspheres embedded with BSA in gastric juice was detected by BCA Protein Assay Kit and microplate reader.

[0094] Specific steps: Prepare standards of different concentrations according to the instructions of the BCA kit. Use a microplate reader to detect the absorbance of the standards and samples at 562nm. Use the absorbance values ​​of the standards of different concentrations to establish a standard curve, obtain a regression equation, substitute the absorbance values ​​of the samples into the equation, and calculate the BSA content in the samples. The cumulative release rate of samples at different time periods is calculated according to the following formula:

[0095] Cumulative release rate = [20C i +V∑C (i-1) ] / m×100%.

[0096] In the formula: C i is the mass concentration of bovine serum albumin in the release medium (g / L), V is the volume of each sampling, which is 5 mL for this system, and m is the total drug loading (mg).

[0097] 2. Experimental Results

[0098] The results of Examples 2 and 4 show that the digestion time of sodium alginate microspheres in the tilapia stomach is 19 hours. Therefore, the 19-hour gastric juice release rate was tested. After that, the microspheres enter the intestine for further digestion. Existing technology and experience show that food is digested approximately 12 hours after entering the intestine, so the 12-hour intestinal juice release rate was tested. The drug loading rate, 12-hour intestinal juice release rate, and 19-hour gastric juice release rate were weighted averaged with weights of 0.4, 0.3, and 0.3, respectively. The results are shown in Table 3.

[0099] Table 3 Release rate of BSA-encapsulated sodium alginate microspheres in simulated intestinal fluid and simulated gastric fluid

[0100]

[0101]

[0102] The protein-encapsulated sodium alginate microspheres prepared under the second experimental condition of the orthogonal experiment achieved a drug loading rate of 2.51%, a release rate of 3% in simulated gastric fluid after 19 hours, and a release rate of 100% in simulated intestinal fluid after 12 hours. This indicates that the prepared sodium alginate microspheres effectively protect the encapsulated protein from gastric digestion, greatly reducing the loss of the encapsulated protein. Furthermore, the encapsulated protein is fully released in the intestinal fluid, ensuring that the encapsulated protein is not wasted and is fully absorbed and utilized by the intestine.

[0103] Taking the above factors into consideration, this set of experimental conditions (i.e., 1% by mass calcium chloride and 2% by mass sodium alginate solution, and a cross-linking time of 1 h for the preparation of protein-embedded microspheres) was selected as the subsequent experimental method for preparing protein-embedded sodium alginate microspheres.

[0104] Example 6 Preparation of Sodium Alginate Microspheres Encapsulating BSA, UII, and Urantide

[0105] 1. Preparation of sodium alginate solution: 2 g of sodium alginate was mixed with 98 g of deionized water, dissolved under magnetic stirring at 50°C, and allowed to stand at 4°C overnight to remove bubbles to obtain a sodium alginate solution;

[0106] 2. Embedment of three proteins (peptides):

[0107] (1) BSA: Add 1 mg of BSA to the sodium alginate solution, stir evenly, and let it stand at 4°C overnight to remove bubbles to obtain the embedding solution.

[0108] (2) Tilapia UII mature peptide: 1 mg of functional neuropeptide UⅡ (GGNSECFWKYCV) was added to the sodium alginate solution, stirred evenly, and allowed to stand at 4°C overnight to remove bubbles to obtain the embedding solution.

[0109] (3) Urantide, an antagonist of UII receptor UT: 1 mg of Urantide (H-Glu-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), an antagonist of UIII receptor UT, was added to the sodium alginate solution, stirred evenly, and allowed to stand at 4°C overnight to remove bubbles to obtain the embedding solution.

[0110] 3. Preparation of microspheres: Transfer the embedding solution to a syringe equipped with a medical No. 9 needle (avoid bubbles during the transfer process) and drip the embedding solution dropwise into a 1% mass fraction calcium chloride solution at 27°C at a drop distance of about 8 cm to 10 cm. Maintain a drip rate of 1 mL / min while maintaining magnetic stirring (magnetic stirrer speed of 300 rpm);

[0111] 4. Cross-linking: After the addition is complete, place the calcium chloride solution on ice and let it stand for 10 minutes;

[0112] 5. Cleaning: After cross-linking, remove the microspheres and rinse the gel microspheres repeatedly with deionized water to remove the surface liquid and transfer them to a culture dish;

[0113] 6. Freeze-drying: After freeze-drying for 12 hours, pack in sealed bags and store at -80℃.

[0114] Example 7 Feeding Experiment of Sodium Alginate Microspheres Encapsulated with BSA, UII and Urantide

[0115] 1. Experimental Methods

[0116] Four treatment groups were set up for tilapia:

[0117] Control group: fed with 2% g / BW feed;

[0118] BSA-fed group: fed with 2% g / BW feed and 350 mg of BSA-encapsulated sodium alginate microspheres of Example 6;

[0119] UⅡ feeding group: fed with 2% g / BW (feed and 350 mg of sodium alginate microspheres embedded with UⅡ of Example 6;

[0120] Urantide-fed group: fed with 2% g / BW feed and 350 mg of sodium alginate microspheres containing Urantide, an antagonist of UⅡ receptor UT, as described in Example 6.

[0121] Each treatment consisted of 8 male and female groups, with 15 tilapia per group. Tilapia were fed twice daily, morning and evening, at a 2% g / BW (body weight) diet and 350 mg of sodium alginate microspheres encapsulated with proteins or peptides. On the 15th day of feeding, 7 tilapia were randomly selected from each group, and on the 30th day, the remaining 8 tilapia were sampled for body length and weight measurement, blood was drawn, and gonads were collected, fixed, and paraffin-sectioned for HE staining.

[0122] 2. Experimental Results

[0123] like Figure 3 As shown in Figure 2, there was no significant change in the body length of male and female tilapia after 15 and 30 days of feeding ( Figure 3 A and Figure 3 C in the figure). After 15 days of feeding, the weight of male tilapia in the UII feeding group was lower than that in the other three groups, while the weight of tilapia in the Urantide feeding group was higher than that in the control group and the UII feeding group, but the difference did not reach significance ( Figure 3 B in the figure). After 30 days of feeding, the weight of male tilapia in the UII-fed group was lower than that in the other three groups, while the weight of tilapia in the Urantide-fed group was higher than that in the other three groups, but the difference did not reach significance ( Figure 3 D) in.

[0124] like Figure 4 As shown in the figure, after 15 days of feeding, the expression level of gnrh1 in the hypothalamus of tilapia in the UII-fed group increased compared with the control group, but the difference did not reach significance. However, the expression of gnrh1 gene in the hypothalamus of tilapia in the Urantide-fed group decreased significantly compared with the control group ( Figure 4A in the figure); After 30 days of feeding, the expression of gnrh2 gene in the hypothalamus of tilapia in the Urantide-fed group was significantly decreased compared with that in the control group ( Figure 4 B), indicating that Urantide can effectively bind to the UII receptor UT, thereby weakening the regulatory effect of endogenous UII on the expression of reproductive-related genes in the hypothalamus; the expression of gnrh3 gene in the hypothalamus of tilapia in the Urantide-fed group did not change significantly compared with the control group ( Figure 4 C).

[0125] like Figure 5 As shown in Figure 2, after 15 days of feeding, the expression of gnrh1 gene in the hypothalamus of female tilapia in the Urantide-fed group was significantly decreased compared with that in the control group ( Figure 5 A in the figure); After 30 days of feeding, the expression of gnrh1, gnrh2, and gnrh3 genes in the hypothalamus of tilapia in the Urantide-fed group was significantly decreased compared with that in the control group ( Figure 5 A, B and C in the above example).

[0126] like Figure 6 As shown, after 30 days of feeding, the expression of lhβ and gthα genes in the pituitary of male tilapia in the UII feeding group was significantly increased compared with the control group ( Figure 6 A and C in the figure indicate that UII can significantly promote the expression of lhβ and gthα genes in the pituitary of tilapia; the expression of fshβ and gthα genes in the pituitary of tilapia in the Urantide-fed group was significantly decreased compared with that in the control group ( Figure 6 B and C in Figure 3 indicate that Urantide can effectively bind to the UII receptor UT, thereby weakening the promoting effect of endogenous UII on the expression of pituitary reproductive-related genes.

[0127] like Figure 7 As shown in Figure 2, after 15 days of feeding, the expression of lhβ gene in the pituitary of female tilapia in the UII feeding group was significantly increased compared with that in the control group ( Figure 7 A in the figure); After 30 days of feeding, the pituitary lhβ and gthα of tilapia in the UII feeding group increased significantly compared with the control group ( Figure 7 A and C in the figure), the expression of fshβ gene in the pituitary of tilapia in the Urantide-fed group was significantly decreased compared with that in the control group ( Figure 7 B) in.

[0128] like Figure 8 As shown, after 15 days of feeding, the expression of fshr gene in the testis of male tilapia in the UII feeding group was significantly increased compared with the control group ( Figure 8 B); the expression of lhr gene in tilapia testis showed no significant change compared with the control group ( Figure 8 A).

[0129] like Figure 9As shown in Figure 2, after 15 days of feeding, the expression of fshr gene in the ovaries of female tilapia in the Urantide-fed group was significantly decreased compared with the control group ( Figure 9 B), indicating that Urantide can effectively bind to the UII receptor UT, thereby weakening the regulatory effect of endogenous UII on the expression of ovarian reproductive-related genes; after 30 days of feeding, the expression of lhr gene in the ovaries of tilapia in the UII-fed group was significantly increased compared with the control group ( Figure 9 A).

[0130] like Figure 10 As shown, feeding male tilapia with feed and sodium alginate microspheres encapsulating functional neuropeptides significantly affected testicular morphology. Stained sections of male tilapia testes after 15 days of feeding revealed numerous primary and secondary spermatocytes. Spermatids were present in the control and UII groups, with the UII group exhibiting a higher number of spermatids. After 30 days of feeding, spermatids appeared in the testes of male tilapia in the BSA and Urantide groups, while mature sperm were present in sections from the control and UII groups, with the UII group exhibiting a higher number of spermatids.

[0131] like Figure 11 As shown, feeding female tilapia with alginate microspheres containing functional neuropeptides in their diet significantly impacted ovarian morphology. HE staining of gonadal sections after 15 days of feeding revealed that the histological stages of the control, BSA, Urantide, and UII groups were mid-stage III, early-stage IV, late-stage III, and mid-stage IV, respectively. HE staining of gonadal sections after 30 days of feeding revealed that the histological stages of the control, BSA, Urantide, and UII groups were late-stage III, mid-stage IV, early-stage IV, and stage V, respectively. Tilapia fed the diet containing UII-containing alginate microspheres exhibited faster ovarian development, with the majority of ovaries containing phase V oocytes, characterized by dissolved nuclear membranes, absent nucleoli, and cytoplasmic suffocation of large yolk granules.

Claims

1. A method for preparing protein-embedded sodium alginate microspheres, characterized in that: The following steps are involved: S1. Add the protein to be embedded into a solution of 1.5-2.5% sodium alginate by mass, remove bubbles, and obtain embedding solution; S2. Add the embedding solution dropwise into a 0.5-1.5% calcium chloride solution; S3. Cross-link for 0.5 to 1.5 hours.

2. The preparation method according to claim 1, characterized in that The mass fraction of the sodium alginate solution is 2% sodium alginate.

3. The preparation method according to claim 1, characterized in that The mass fraction of calcium chloride solution is 1%.

4. The preparation method according to claim 1, characterized in that Cross-link for 1 hour.

5. The microspheres prepared by the preparation method according to claim 1.

6. Use of the microspheres according to claim 5 in the preparation of oral aquatic preparations.

7. Use of sodium alginate microspheres encapsulating urotensin II prepared by the preparation method according to claim 1 in aquatic feed additives.

8. The use according to claim 7, characterized in that The aquatic feed additive is an additive for promoting the gonadal development of tilapia.

9. The use according to claim 8, characterized in that The gonad is a testis.

10. The use according to claim 8, characterized in that The gonad is an ovary.

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