Compound microcapsule for preventing and treating necrotic enteritis of poultry and its preparation method

Abstract

This invention discloses a composite microcapsule for preventing necrotizing enteritis in poultry and its preparation method. The microcapsule has a W / O / W dual emulsion structure. The inner aqueous phase contains sodium butyrate, the oil phase contains cinnamaldehyde and free allicin, and the outer aqueous phase wall material layer contains whey protein isolate-allicin and thiol-chitosan-allicin conjugates. Allicin is anchored to the wall material in batches via disulfide bonds, achieving spatial isolation through the W / O / W emulsion. It is then cured by double cross-linking with TG enzyme and tannic acid, followed by low-temperature spray drying. The total encapsulation efficiency of the microcapsule is ≥88.6%, and the release rate in gastric juice after 2 hours is <6%. In vitro antibacterial experiments show that the MIC is only 62.5 μg / mL, which is 8 times lower than that of the physical mixture. After simulated gastrointestinal digestion, the antibacterial activity retention rate reaches 96.6%~97.7%, and the FIC value is 0.46~0.49, exhibiting a strong synergistic effect. Poultry challenge experiments have shown that microcapsules can reduce mortality to 5%, diarrhea rate to 11%, serum diamine oxidase activity to 15.2±3.0 U / L, and IL-10 concentration to 78.5±7.2 pg / mL, significantly protecting intestinal barrier function and anti-inflammatory ability, and effectively preventing avian Clostridium perfringens enteritis.

Description

Technical Field

[0001] This invention belongs to the field of feed additives, specifically relating to a composite microcapsule for preventing and treating necrotic enteritis in poultry and its preparation method. Background Technology

[0002] *Clostridium perfringens* is a major pathogen causing necrotic enteritis in poultry. Under the dual pressures of high-density intensive farming and the complete ban on antibiotics in feed, the incidence of necrotic enteritis continues to rise, causing significant economic losses to the poultry industry. Plant essential oils and organic acid salts have received widespread attention as antibiotic alternatives, among which cinnamaldehyde, allicin, and sodium butyrate are three active ingredients that have been extensively studied and applied. Existing research shows that cinnamaldehyde has a significant in vitro inhibitory effect on *Clostridium perfringens*, with a MIC of 125 μg / mL, while garlic oil has a MIC of 250 μg / mL; the combined effect of the two is additive (FIC=1). Sodium butyrate can provide energy to intestinal epithelial cells and repair the mucosal barrier. These three agents act on different targets of *Clostridium perfringens*—cinnamaldehyde disrupts cell membranes, allicin inhibits metabolic enzymes, and sodium butyrate regulates the intestinal microenvironment and enhances host immunity—showing potential for synergistic improvement of poultry gut health.

[0003] However, existing studies have clearly indicated that unprotected sodium butyrate alone does not improve the course of necrotizing enterocolitis. This is because sodium butyrate is absorbed in large quantities in the stomach and duodenum, making it difficult to effectively reach the posterior end of the intestine to exert its effects. Essential oil components also suffer from poor stability, high volatility, and rapid absorption in the upper digestive tract after oral administration. Therefore, they need to be protected during feed processing and in vivo transport to exert their effective function at the target site. Currently, the use of these three compounds in combination for the prevention of necrotic enteritis in poultry presents the following main problems: First, allicin is extremely sensitive to heat, and its thiosulfinate groups undergo significant degradation at temperatures exceeding 80°C. Existing microencapsulation processes mostly employ high-temperature spray drying (inlet air temperature 180~200°C), resulting in an allicin retention rate of only about 62%, severely impairing its biological activity. Second, sodium butyrate is water-soluble, while cinnamaldehyde and allicin are hydrophobic, exhibiting significant differences in their physicochemical properties. Traditional encapsulation techniques often target single core material designs, making it difficult to stably co-encapsulate water-soluble and hydrophobic components within the same system. Third, existing products mostly employ a single encapsulation structure, where the release behavior of different core materials is passively determined by their respective diffusion coefficients—water-soluble sodium butyrate diffuses quickly and releases early, while hydrophobic cinnamaldehyde releases later. The lack of active timing design means that the time windows for each component to reach the intestine cannot effectively match the infection process, making it difficult to achieve the synergistic therapeutic goal of first inhibiting bacteria and then repairing the infection. To address the aforementioned issues, existing technologies have explored methods such as amylose inclusion complexes (CN106580916B), double-layer film encapsulation (CN100367944C), and composite coagulation (CN122181707A). However, the relationship between the wall material and the core material mainly relies on non-covalent or physical cross-linking, lacking chemical bonding, thus limiting stability and targeted release performance. At the product level, most are single-component microcapsules or simple physical mixtures, such as a mixture of cinnamaldehyde and sodium butyrate (CN104187001A), a compound of cinnamaldehyde and allicin (CN117044846B), and a mixture of cinnamon extract, garlic extract, and sodium butyrate (CN106879826A). To stably co-encapsulate these three components with vastly different physicochemical properties in the same system and achieve time-sequential release, it is necessary not only to solve the interfacial compatibility problem of hydrophilic and hydrophobic core materials, but also to take into account the protection of heat-sensitive components during processing and the active regulation of the release rate of each component. The technology is quite challenging, and there are currently no publicly available technical solutions. Summary of the Invention

[0004] To address the shortcomings of existing technologies, such as the difficulty in stably co-encapsulating the three active ingredients (cinnamaldehyde, allicin, and sodium butyrate) in the same system, the easy degradation of heat-sensitive components during processing, and the lack of time-sequential release design in existing products, this invention provides a composite microcapsule for preventing necrotizing enteritis in poultry and its preparation method. This invention achieves spatial isolation of the hydrophilic and hydrophobic core material by chemically bonding allicin to the wall material in batches, combined with a W / O / W dual emulsion structure, followed by dual cross-linking curing with TG enzyme and tannic acid, and low-temperature spray drying, resulting in a ternary composite microcapsule with time-sequential release characteristics, thus achieving the protection and synergistic delivery of the antibacterial active ingredient.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a composite microcapsule for preventing and treating necrotic enteritis in poultry. The composite microcapsule has a W / O / W dual emulsion structure, comprising an inner aqueous phase, an oil phase, and an outer aqueous phase wall material layer. The inner aqueous phase contains sodium butyrate; the oil phase contains cinnamaldehyde and free allicin; the outer aqueous phase wall material layer contains whey protein isolate-allicin conjugate and thiol chitosan-allicin conjugate. The whey protein isolate-allicin conjugate is a product of whey protein isolate and allicin linked by disulfide bonds, and the thiol chitosan-allicin conjugate is a product of thiol chitosan and allicin linked by disulfide bonds.

[0006] The core material of the composite microcapsules consists of the following components by weight percentage: cinnamaldehyde 45%~55%, allicin 20%~35%, sodium butyrate 15%~30%, totaling 100%. The wall material of the composite microcapsules consists of the following components by weight percentage: whey protein isolate 70%~85%, thiol chitosan 10%~18%, tannic acid 0.5%~3%, and transglutaminase 0.2%~0.5%.

[0007] This invention also provides a method for preparing composite microcapsules for preventing and treating necrotic enteritis in poultry, comprising the following steps: (1) Preparation of protein-allicin conjugate: Whey protein isolate was dissolved in deionized water, and the first portion of allicin was added to carry out the conjugation reaction. The protein-allicin conjugate was obtained after purification. The specific preparation conditions of the protein-allicin conjugate were as follows: Whey protein isolate was dissolved in deionized water, and ultrasonically treated for 20 min at 40℃, 20~40 kHz, and 50 W / L. The pH was adjusted to 4.5, the first portion of allicin was added, and the mixture was stirred at 25℃ for 30 min. After the reaction was completed, the mixture was desalted and purified using an ultrafiltration system with a molecular weight cutoff of 3 kDa.

[0008] (2) Preparation of chitosan-allicin conjugate: Thiol-modified chitosan was dissolved in acetic acid solution, and a second part of allicin was added to carry out the conjugation reaction. The chitosan-allicin conjugate was obtained after purification. The specific preparation conditions of the chitosan-allicin conjugate were as follows: Thiol-modified chitosan was dissolved in 1% acetic acid solution, the pH was adjusted to 5.5, a second part of allicin was added, and the mixture was stirred at room temperature for 2 h. After the reaction was completed, the mixture was desalted and purified using an ultrafiltration system with a molecular weight cutoff of 3 kDa.

[0009] Allicin is divided into three parts: the first part accounts for 50% to 55% of the total allicin, the second part accounts for 30% to 40%, and the third part accounts for 10% to 20% and is retained in the free state.

[0010] (3) Preparation of oil phase: Cinnamaldehyde and the third part of allicin are mixed in a mass ratio of 5:1 to 25:1. Add the lipophilic emulsifier polyglycerol ricinoleate in an amount of 1% to 4% of the mass of the oil phase, stir evenly, and the oil phase is obtained.

[0011] (4) Preparation of W / O colostrum: Sodium butyrate is dissolved in deionized water to prepare an internal aqueous phase solution with a mass percentage concentration of 10%~25%; under high-speed shearing conditions of 10000~15000 rpm, the internal aqueous phase is slowly added to the oil phase in step (3) and sheared for 5~10 min. The volume ratio of the internal aqueous phase to the oil phase is 1:1~3:1 to obtain W / O colostrum.

[0012] (5) Preparation of external aqueous phase: The protein-allicin conjugate obtained in step (1) and the chitosan-allicin conjugate obtained in step (2) are mixed at a mass ratio of 4:1 to 8:1 and stirred evenly to obtain the external aqueous phase.

[0013] (6) Construction of W / O / W dual emulsion: Under shear conditions of 3000~8000 rpm, the W / O primary emulsion from step (4) is slowly added to the external aqueous phase from step (5), wherein the volume ratio of the W / O primary emulsion to the external aqueous phase is 1:2~1:6, and shearing is performed for 5~10 min; the pH is adjusted to 5.0, and the mixture is stirred at room temperature for 45~60 min; tannic acid is added until the system concentration is 0.05%~0.15%, and the mixture is stirred at room temperature for another 45~60 min.

[0014] (7) Double cross-linking curing: Add transglutaminase to the W / O / W system in step (6) at a dosage of 0.2% to 0.5% of the wall material mass, and cross-link and cure at 15 to 25°C for 4 to 8 hours; then add tannic acid to the system until the final concentration is 0.02% to 0.1%, and continue stirring for 30 minutes to complete the double cross-linking curing.

[0015] (8) Spray drying: The W / O / W double emulsion obtained in step (7) is spray dried with an inlet air temperature of 100~130℃, an outlet air temperature of 60~75℃, a feed rate of 10~15 mL / min, and an atomization pressure of 0.15~0.25 MPa to obtain the composite microcapsules.

[0016] In the above method, in step (1), the whey protein isolate is preferably prepared into a solution with a mass-to-volume ratio of 5% to 10%, more preferably 7.5%. The ultrasonic treatment conditions are preferably 40℃, 40 kHz, and 50 W / L for 20 min. The mass ratio of whey protein isolate to the first allicin can be adjusted according to the protein purity, preferably 4:1 to 8:1, and most preferably 4:1. During the ultrafiltration purification process, it is preferred to use an equal-volume washing method for desalting.

[0017] In the above method, the thiol content of the thiol-modified chitosan in step (2) is preferably 200~400 μmol / g, and it is preferably prepared into a solution with a mass-volume ratio of 1%~5% after being dissolved in 1% acetic acid solution. The mass ratio of thiol-modified chitosan to the second allicin is preferably 0.5:1~2:1.

[0018] In the above method, the mass ratio of cinnamaldehyde to the third allicin in step (3) can be further preferred to be 8:1 to 15:1, and most preferably 8.3:1. The amount of the lipophilic emulsifier polyglycerol ricinoleate can be further preferred to be 2% to 4% of the oil phase mass, and most preferably 3%.

[0019] In the above method, the concentration of the sodium butyrate aqueous solution in step (4) can be further preferably 15%~25%, and most preferably 20%. The high-speed shearing conditions can be further preferably 11000~13000 rpm, and the shearing time can be further preferably 6~10 min. The volume ratio of the aqueous phase to the oil phase can be further preferably 1.5:1~2.5:1, and most preferably 1.7:1.

[0020] In the above method, the mass ratio of protein-allicin conjugate to chitosan-allicin conjugate in step (5) can be further preferred to be 5:1 to 7:1, and most preferably 6:1.

[0021] In the above method, the shearing conditions in step (6) can be further preferred to be 4000~6000 rpm, and the shearing time can be further preferred to be 6~10 min. The volume ratio of W / O promulgated milk to external aqueous phase can be further preferred to be 1:3~1:5, and most preferably 1:4. After adjusting the pH to 5.0, the stirring time at room temperature is preferably 50~60 min; the amount of tannic acid added can be further preferred to be 0.08%~0.12% of the system concentration, and most preferably 0.1%; the stirring time after adding tannic acid is preferably 50~60 min.

[0022] In the above method, the amount of transglutaminase used in step (7) can be further preferably 0.25%~0.4% of the wall material mass, and most preferably 0.3%. The cross-linking curing temperature is preferably 18~22℃, and the cross-linking curing time is preferably 5~7 h, and most preferably 6 h. The tannic acid is added to bring the final concentration of the system to preferably 0.03%~0.08%.

[0023] In the above method, the inlet air temperature of spray drying in step (8) is preferably 110~125℃, and most preferably 120℃; the outlet air temperature is preferably 60~70℃, and most preferably 65℃; the feed rate is preferably 10~14 mL / min, and most preferably 12 mL / min; the atomization pressure is preferably 0.18~0.22 MPa, and most preferably 0.20 MPa.

[0024] In the above method, the purity of allicin is preferably ≥98%, the purity of cinnamaldehyde is preferably ≥98%, the purity of sodium butyrate is preferably ≥98%, and the moisture content of sodium butyrate is preferably ≤2%. The protein content of whey protein isolate is preferably ≥80%. The purity of tannic acid is preferably ≥93%. The enzyme activity of transglutaminase is preferably ≥100 U / g.

[0025] In the above method, the molecular weight cutoff of the ultrafiltration system in steps (1) and (2) is 3 kDa. It is preferred to use tangential flow ultrafiltration for desalination and purification to facilitate large-scale production.

[0026] In the above method, the temperature of the complex condensation reaction in step (6) is preferably room temperature (20~30℃), which does not require additional heating or cooling and is easy to operate.

[0027] In the above method, prior to the spray drying step, the W / O / W dual emulsion may be homogenized as needed to further optimize the particle size distribution and encapsulation uniformity of the microcapsules.

[0028] In the above method, the core material composition of the composite microcapsules can be further optimized as follows: 50% cinnamaldehyde, 30% allicin, and 20% sodium butyrate. Under this core material ratio, the antibacterial synergistic effect of the three active ingredients is most significant, and the FIC value is the lowest.

[0029] In the above method, the composite microcapsules are preferably used to prevent Clostridium perfringens enteritis in poultry, and the poultry are preferably broiler chickens.

[0030] The composite microcapsules prepared by the above method achieved an overall encapsulation efficiency of over 88.6%. In vitro simulated gastrointestinal digestion experiments showed that the cumulative release rate of the composite microcapsules in simulated gastric juice was less than 6% over 2 hours, effectively protecting the core material as it passed through the gastric environment. After transfer to intestinal juice, they exhibited a distinct sequential release pattern: allicin preferentially released, cinnamaldehyde intermediately released, and sodium butyrate released later. In vitro antibacterial experiments showed that the composite microcapsules achieved a minimum inhibitory concentration (MIC) of 62.5 μg / mL against Clostridium perfringens, with a retention rate of 96.6%–97.7% of antibacterial activity after simulated gastrointestinal digestion and an FIC value ≤0.49, indicating a synergistic effect. A poultry challenge protection experiment showed that the challenge group fed with this compound microcapsule experienced a reduction in mortality to 5%, diarrhea rate to 11%, Clostridium ileum count to 3.5 log10 CFU / g, serum diamine oxidase (DAO) activity to 15.2±3.0 U / L (no significant difference from the blank control group), and serum IL-10 concentration restored to 78.5±7.2 pg / mL (close to the blank control group level). The average daily weight gain reached 57 g / d, and the feed conversion ratio was 1.62. Testing revealed that the microcapsules effectively repaired intestinal barrier damage caused by the challenge and significantly restored the expression of anti-inflammatory factors; all indicators were significantly better than those of the challenge control group.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In this invention, allicin is chemically anchored to whey protein isolate and thiol-based chitosan via disulfide bonds. In vitro release experiments showed that the cumulative release rate of the composite microcapsules in simulated gastric juice was less than 6% after 2 hours, and the antibacterial activity retention rate after simulated gastrointestinal digestion reached 96.6%~97.7%, which was far superior to the comparative samples (48.8%~49.2%), effectively overcoming the problem of easy degradation of heat-sensitive components in the gastric environment during high-temperature processing in the prior art.

[0032] (2) The present invention uses a W / O / W dual emulsion structure to spatially position sodium butyrate, cinnamaldehyde and chemically anchored allicin in three phases. The encapsulation rates are 89.5%~91.2% for cinnamaldehyde, 88.2%~91.2% for allicin and 86.5%~88.0% for sodium butyrate, with a total encapsulation rate of ≥88.6%, which is significantly better than the comparative examples (total encapsulation rate of 78.6%~84.2%). This invention solves the problem that it is difficult to stably co-encapsulate hydrophilic and hydrophobic core materials in the same system in the prior art.

[0033] (3) The present invention adopts a dual cross-linking and curing process of TG enzyme and tannic acid. The stability of the gastric juice in the example is significantly better than that of the comparative example 3 which is cross-linked with only tannic acid (gastric juice release rate 14.2%~20.8%), which takes into account both the protection of the core material and the targeted release into the intestine during the processing.

[0034] (4) This invention utilizes the different spatial positioning of the three components in a W / O / W dual emulsion to achieve sequential release in intestinal fluid: the cumulative release rate of allicin in the wall material layer reaches 70.5%~78.2% from 0 to 4 hours; the cumulative release rate of cinnamaldehyde in the oil phase reaches 74.2%~82.3% from 4 to 8 hours; and the release of sodium butyrate in the inner aqueous phase is the most delayed, with a cumulative release rate of 76.2%~80.5% from 8 hours. The difference in release rate among the three components is most significant from 4 to 6 hours, forming a staggered release sequence. The FIC values ​​(0.46~0.49) demonstrate that the three components have a very strong synergistic effect, far superior to the physical mixture (FIC 0.96) and each comparative example (FIC 0.88~0.93). Further experiments on poultry challenge protection verified that the microcapsules of the present invention can significantly improve serum DAO activity, increase serum IL-10 concentration, effectively protect the integrity of the intestinal mucosal barrier and exert anti-inflammatory effects; all indicators of the example group, such as mortality rate, diarrhea rate, Clostridium ileum count, average daily weight gain and feed conversion ratio, were significantly better than those of the control group 2.

[0035] (5) All raw materials used in the method described in this invention are commercially available. The preparation process only involves conventional emulsification, crosslinking and spray drying equipment, without the need for special equipment or complex processes. Attached Figure Description

[0036] Figure 1 This is a flowchart of the preparation method described in this invention; Figure 2 Cinnamaldehyde, allicin, and sodium butyrate simulate gastrointestinal digestion release rates; Figure 3 The images show 96-well plate measurements of the in vitro antibacterial activity of the samples from Examples 1-3 and Comparative Examples 1-3 of this invention before (top) and after (bottom) simulated gastrointestinal digestion. The red circles in the images indicate the wells corresponding to the minimum inhibitory concentration (MIC). The text on the left side of the images represents blank control, physical mixture, comparative example 1, comparative example 2, comparative example 3, example 1, example 2, and example 3, respectively. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the reagents and materials used, unless otherwise specified, are commercially available. Experimental methods in the embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Those skilled in the art can make various modifications and changes to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and changes should be included within the protection scope of the present invention.

[0038] All raw materials used in the embodiments of this invention were commercially available: whey protein isolate was purchased from Zhengzhou Hongxiang Chemical Co., Ltd., with a protein content ≥80%; thiol-modified chitosan was purchased from Xi'an Qiyue Biotechnology Co., Ltd. (custom-synthesized), with a thiol content of 200~400 μmol / g; cinnamaldehyde was purchased from Wuhan Lingjia Yi Chemical Co., Ltd., with a purity ≥98%; allicin was purchased from Hebei Jiuxing Chemical Products Co., Ltd., with a purity ≥98%; sodium butyrate was purchased from Rizhao Dongrunde Agricultural and Animal Husbandry Development Co., Ltd., with a sodium butyrate content ≥98% and a moisture content ≤2%; polyglycerol ricinoleate (PGPR) was purchased from Wuhan Jiyesheng Chemical Co., Ltd.; tannic acid was purchased from Wuhan Jiyesheng Chemical Co., Ltd., with a purity ≥93%; transglutaminase (TG enzyme) was purchased from Jiangsu Yiming Biotechnology Co., Ltd., with an enzyme activity ≥100 U / g; in addition, pepsin, trypsin, potassium dihydrogen phosphate, soybean lecithin, bile salts, dilute hydrochloric acid and other reagents used to simulate digestive fluids were all commercially available analytical grade or biochemical reagents, used only for in vitro experimental characterization, and not used as feed additive components.

[0039] Example 1 Core material composition:

[0040] A composite microcapsule for preventing and treating necrotic enteritis in poultry and its preparation method, such as Figure 1 The process, as shown, includes the following steps: (1) Preparation of protein-allicin conjugate: Weigh 7.5 kg of whey protein isolate, dissolve it in deionized water, and add to a final volume of 100 kg (preparing a 7.5% w / v solution). Sonicate the solution for 20 min at 40℃, 20–40 kHz, and 50 W / L to promote the exposure of sulfhydryl groups within the protein. Adjust the pH to 4.5, add the first portion of allicin (1.875 kg, representing 50% of the total allicin content), and stir at 25℃ for 30 min. After the reaction is complete, desalt and purify the solution using an ultrafiltration system (molecular weight cutoff 3 kDa, equal volume washing), and concentrate to 50 kg.

[0041] (2) Preparation of chitosan-allicin conjugate: Weigh 1.25 kg of thiol-modified chitosan, dissolve it in 1% acetic acid solution, and add to a final volume of 60 kg. Adjust the pH to 5.5, add a second portion of allicin (1.125 kg, accounting for 30% of the total allicin), and stir the mixture at room temperature for 2 h. After the reaction is complete, desalt and purify the mixture using an ultrafiltration system (with the same parameters as in step (1)), and concentrate it to 30 kg.

[0042] (3) Preparation of oil phase: Weigh 6.25 kg of cinnamaldehyde and mix it with 0.75 kg of allicin (mass ratio 8.3:1), add 0.21 kg of PGPR (3% of the oil phase mass), stir evenly, and obtain 7.21 kg of oil phase.

[0043] (4) Preparation of W / O colostrum: Weigh 2.5 kg of sodium butyrate and dissolve it in deionized water to prepare a 20% (w / w) internal aqueous phase solution (total mass 12.5 kg). Under 12000 rpm, slowly add 12.5 kg of internal aqueous phase to 7.21 kg of oil phase and shear for 8 min to obtain 19.71 kg of W / O colostrum.

[0044] (5) Preparation of external aqueous phase: Mix the protein-allicin conjugate solution concentrated by ultrafiltration in step (1) with the chitosan-allicin conjugate solution concentrated by ultrafiltration in step (2). Adjust the pH to 5.0 with 1 mol / L acetic acid or NaOH (pre-adjust before adding the colostrum), stir at room temperature for 30 min, so that the two wall materials can be fully combined by electrostatic attraction.

[0045] (6) Construction of W / O / W dual emulsion: Under moderate shear conditions of 4000~6000 rpm, the W / O primary emulsion from step (4) was slowly added to the external aqueous phase (primary emulsion: external aqueous phase = 1:4) from step (5), and sheared for 8 min. The pH of the system was maintained at 5.0, and 0.1 kg of tannic acid (concentration 0.1% w / v) was added. The mixture was stirred at room temperature for 60 min to complete the coagulation reaction.

[0046] (7) Double cross-linking curing: Add 0.026 kg of TG enzyme (0.3% w / v of wall material mass), cross-link and cure at 20℃ for 6 h; add 0.05 kg of tannic acid, continue stirring for 30 min to complete the double cross-linking curing.

[0047] (8) Spray drying: inlet air temperature 120℃, outlet air temperature 65℃, feed rate 12mL / min, atomization pressure 0.20MPa, to obtain ternary composite microcapsule essential oil.

[0048] Example 2 Core material composition:

[0049] (1) Preparation of protein-allicin conjugate: Weigh 10 kg of whey protein isolate, dissolve it in deionized water, and add to a final volume of 120 kg. Sonicate the mixture for 20 min at 40℃, 20–40 kHz, and 50 W / L. Adjust the pH to 4.5, add 2.625 kg of the first portion of allicin (50% of the total allicin content), and stir at 25℃ for 30 min. After the reaction is complete, desalt and purify the mixture using an ultrafiltration system (molecular weight cutoff 3 kDa, equal volume washing), and concentrate to 40 kg.

[0050] (2) Preparation of chitosan-allicin conjugate: Weigh 2.0 kg of thiol-modified chitosan, dissolve it in 1% acetic acid solution and add to a final volume of 60 kg. Adjust the pH to 5.5, add 1.575 kg of allicin (30% of the total allicin), and stir at room temperature for 2 h. After the reaction is complete, desalt and purify using an ultrafiltration system (with the same parameters as in step (1)), and concentrate to 30 kg.

[0051] (3) Preparation of oil phase: Weigh 6.75 kg of cinnamaldehyde and mix it with 1.05 kg of allicin. Add 0.22 kg of PGPR (3% of the oil phase mass), stir well, and obtain 7.495 kg of oil phase.

[0052] (4) Preparation of W / O colostrum: Weigh 3.0 kg of sodium butyrate and dissolve it in deionized water to prepare a 25% (w / w) internal aqueous phase solution. Under the condition of 15000 rpm, slowly add 12 kg of internal aqueous phase to 8.02 kg of oil phase and shear for 6 min to obtain 19.495 kg of W / O colostrum.

[0053] (5) Preparation of external aqueous phase: Mix the protein-allicin conjugate solution concentrated by ultrafiltration in step (1) with the chitosan-allicin conjugate solution concentrated by ultrafiltration in step (2). Adjust the pH to 5.0 with 1 mol / L acetic acid or NaOH (pre-adjust before adding the colostrum), stir at room temperature for 30 min, so that the two wall materials can be fully combined by electrostatic attraction.

[0054] (6) Construction of W / O / W dual emulsion: Under moderate shear conditions of 4000~6000 rpm, the W / O primary emulsion (19.495 kg) from step (4) was slowly added to the external aqueous phase (primary emulsion: external aqueous phase = 1:3.5) from step (5), and sheared for 6 min. The pH of the system was maintained at 5.0, and 0.072 kg of tannic acid (concentration 0.08% w / v) was added. The mixture was stirred at room temperature for 60 min to complete the coagulation reaction.

[0055] (7) Double cross-linking curing: Add 0.048 kg of TG enzyme (0.4% of the wall material mass), cross-link and cure at 20℃ for 6 h; add 0.027 kg of tannic acid (0.03% w / v), continue stirring for 30 min to complete the double cross-linking curing.

[0056] (8) Spray drying: inlet air temperature 110℃, outlet air temperature 65℃, feed rate 10mL / min, atomization pressure 0.22MPa, to obtain ternary composite microcapsule essential oil.

[0057] Example 3 Core material composition:

[0058] (1) Preparation of protein-allicin conjugate: Weigh 5 kg of whey protein isolate, dissolve it in deionized water, and add to a final volume of 100 kg. Sonicate the mixture for 20 min at 40℃, 20–40 kHz, and 50 W / L. Adjust the pH to 4.5, add 1.0 kg of allicin (50% of the total allicin content), and stir at 25℃ for 30 min. After the reaction is complete, desalt and purify the mixture using an ultrafiltration system (molecular weight cutoff 3 kDa, equal volume washing), and concentrate to 50 kg.

[0059] (2) Preparation of chitosan-allicin conjugate: Weigh 0.75 kg of thiol-modified chitosan, dissolve it in 1% acetic acid solution, and bring the volume to 60 kg. Adjust the pH to 5.5, add 0.6 kg of the second portion of allicin (accounting for 30% of the total allicin), and stir the reaction at room temperature for 2 h. After the reaction is completed, desalt and purify the product using an ultrafiltration system (with the same parameters as in step (1)), and concentrate it to 30 kg.

[0060] (3) Preparation of oil phase: Weigh 5.5 kg of cinnamaldehyde and mix it with 0.4 kg of allicin (mass ratio 14:1), add 0.18 kg of PGPR (3% of the oil phase mass), stir evenly, and obtain 6.08 kg of oil phase.

[0061] (4) Preparation of W / O colostrum: Weigh 2.5 kg of sodium butyrate and dissolve it in deionized water to prepare a 20% (w / w) internal aqueous phase solution (total mass 12.5 kg). Under 10000 rpm conditions, slowly add 12.5 kg of internal aqueous phase to 6.08 kg of oil phase and shear for 10 min to obtain 18.58 kg of W / O colostrum.

[0062] (5) Preparation of external aqueous phase: Mix the protein-allicin conjugate solution concentrated by ultrafiltration in step (1) with the chitosan-allicin conjugate solution concentrated by ultrafiltration in step (2). Adjust the pH to 5.0 with 1 mol / L acetic acid or NaOH (pre-adjust before adding the colostrum), stir at room temperature for 30 min, so that the two wall materials can be fully combined by electrostatic attraction.

[0063] (6) Construction of W / O / W dual emulsion: Under moderate shear conditions of 4000 rpm, the W / O primary emulsion from step (4) was slowly added to the external aqueous phase from step (5) (primary emulsion: external aqueous phase = 1:4.3), and sheared for 10 min. The pH of the system was maintained at 5.0, and 0.12 kg of tannic acid (concentration 0.12% w / v) was added. The mixture was stirred at room temperature for 45 min to complete the coagulation reaction.

[0064] (7) Double cross-linking curing: Add 0.012 kg of TG enzyme (0.2% of the wall material mass), cross-link and cure at 20℃ for 5 h; add 0.05 kg of tannic acid (0.05% w / v concentration), continue stirring for 30 min to complete the double cross-linking curing.

[0065] (8) Spray drying: inlet air temperature 125℃, outlet air temperature 70℃, feed rate 14 mL / min, atomization pressure 0.18MPa, to obtain ternary composite microcapsule essential oil.

[0066] Comparative Example 1: Allicin was not pre-bonded with the wall material.

[0067] (1) Preparation of external aqueous phase: Weigh 7.5 kg of whey protein isolate and dissolve it in deionized water, weigh 1.25 kg of mercapto-modified chitosan and dissolve it in 1% acetic acid solution, mix the two, add water to 80 kg, adjust the pH to 5.0, and stir at room temperature for 30 min.

[0068] (2) Preparation of oil phase: Weigh 6.25 kg of cinnamaldehyde and 3.75 kg of allicin (all added, regardless of batch), mix them, add 0.21 kg of PGPR, stir evenly, and obtain 10.21 kg of oil phase.

[0069] (3) Preparation of W / O colostrum: The preparation of the internal aqueous phase and the preparation of W / O colostrum are the same as in Example 1.

[0070] (4) W / O / W construction, double crosslinking curing and spray drying are the same as in Example 1.

[0071] Comparative Example 2: Traditional gelatin-gum arabic composite wall material is used.

[0072] Since this wall material combination cannot chemically bond with allicin, the batch addition of allicin is retained in this comparative example as a physical mixing process and does not involve a chemical anchoring mechanism. Due to the different types of wall materials, the pH value was also adjusted according to their optimal re-setting pH to compare the advantages of the new chemically cross-linked wall materials.

[0073] (1) Allicin was added in batches as in Example 1 (50%:30%:20%).

[0074] (2) The preparation of the internal aqueous phase, oil phase, and W / O primary emulsion are the same as in Example 1.

[0075] (3) Preparation of external aqueous phase: Weigh 7.5 kg of gelatin and 1.25 kg of gum arabic, dissolve them in deionized water and add to 80 kg, mix and adjust the pH to 4.0 (isoelectric point of gelatin-gum arabic coagulation), stir at room temperature for 30 min to obtain external aqueous phase.

[0076] (4) Construction of W / O / W dual emulsion: The W / O colostrum was slowly added to the external aqueous phase and sheared for 8 min; the pH was kept at 4.0 and stirred at room temperature for 50 min.

[0077] (5) No TG enzyme or tannic acid is added; spray drying is carried out directly.

[0078] (6) Spray drying: inlet air temperature 120℃, outlet air temperature 65℃, feed rate 12 mL / min, atomization pressure 0.20 MPa.

[0079] Comparative Example 3 Only tannic acid cross-links (no TG enzyme).

[0080] (1) The preparation of protein-allicin conjugate and chitosan-allicin conjugate l is the same as in Example 1.

[0081] (2) The preparation of internal aqueous phase, oil phase, and W / O colostrum is the same as in Example 1.

[0082] (3) The preparation of the external water phase and the construction of W / O / W are the same as in Example 1.

[0083] (4) Add tannic acid crosslinking at once: Add 0.15 kg of tannic acid and stir at room temperature for 30 min (without adding TG enzyme).

[0084] (5) Spray drying: inlet air temperature 120℃, outlet air temperature 65℃, feed rate 12mL / min, atomization pressure 0.20MPa, to obtain microcapsule essential oil products.

[0085] Experimental Example 1: Encapsulation rate determination.

[0086] (1) Measurement method Surface core material content determination: Accurately weigh approximately 0.5 g of sample and place it in a 50 mL centrifuge tube. Add 10 mL of n-hexane, vortex for 2 min, and centrifuge (5000 rpm, 10 min). Collect the supernatant. Repeat the extraction three times, combine the n-hexane phases, evaporate the solvent, weigh, and calculate the surface cinnamaldehyde and allicin content. For sodium butyrate content, replace n-hexane with deionized water using the same method.

[0087] Total core material content determination: Take another sample of about 0.5g, weigh accurately, place it in a 50mL centrifuge tube, add 10mL of n-hexane, sonicate to break the cell wall (40kHz, 30min), centrifuge and take the supernatant, repeat the extraction until no oil phase is detected, combine the n-hexane phases, evaporate to dryness and weigh, calculate the content of surface cinnamaldehyde and allicin. For sodium butyrate content, n-hexane needs to be replaced with deionized water, and the method is the same as above.

[0088] Content determination of each component: Cinnamaldehyde and allicin were determined by gas chromatography-FID (GC-FID) using an HP-5 column (30m × 0.25mm × 0.25μm), with the column temperature programmed from 80℃ to 230℃, and quantification using the internal standard method. Sodium butyrate was determined by high performance liquid chromatography (HPLC-UV).

[0089] Embedding rate calculation: Embedding rate (%) = (Total core material content - Surface core material content) / Total core material content × 100% Total encapsulation efficiency = (cinnamaldehyde encapsulation efficiency + allicin encapsulation efficiency + sodium butyrate encapsulation efficiency) / 3 (2) Experimental results Table 1 Summary of Embedding Rate Results

[0090] Table 1 shows that the overall encapsulation efficiency of Examples 1-3 was ≥88.6%, significantly better than that of the comparative examples. The allicin encapsulation efficiency of Comparative Example 1 (without allicin pre-binding) was only 72.8%, indicating that the pre-binding of allicin with the wall material significantly improved the encapsulation efficiency. The sodium butyrate encapsulation efficiency of Comparative Example 2 (gelatin-gum arabic wall material) was only 68.2%, while the sodium butyrate encapsulation efficiency of Examples 1-3 was 86.5%~88.0%, demonstrating the necessity of the W / O / W dual emulsion structure for encapsulating the water-soluble core material. The overall encapsulation efficiency of Comparative Example 3 (tannic acid cross-linking only) (84.2%) was lower than that of Example 1 (89.7%), verifying the contribution of TG enzyme + tannic acid dual cross-linking curing to improving the encapsulation efficiency. Sodium butyrate (water-soluble core material) achieved an encapsulation rate of up to 87.3% in the examples, far exceeding the conventional expectations of those skilled in the art for encapsulating hydrophilic core materials in complex cohesive systems (usually considered to be <70%). This unexpected technical effect directly proves the superiority of the W / O / W dual emulsion structure of the present invention.

[0091] Experimental Example 2: In vitro gastrointestinal simulation of digestion.

[0092] (1) Experimental scheme In vitro simulated digestion and release experiments were conducted on each sample using the dissolution determination method (paddle method) as specified in the Chinese Pharmacopoeia (2020 edition) 0931. Simulated gastric fluid preparation: 32.8 mL of dilute hydrochloric acid was added to 1600 mL of water, along with 20 g of pepsin, and the volume was adjusted to 2000 mL, pH 1.2. Simulated intestinal fluid preparation: 13.6 g of potassium dihydrogen phosphate was dissolved in 1000 mL of water, and the pH was adjusted to 6.8 with 0.1 mol / L NaOH; separately, 20 g of pancreatin, 40 mg of soybean lecithin, and 32 g of bile salts were dissolved in 400 mL of water. The two solutions were mixed and the volume was adjusted to 2000 mL.

[0093] Accurately weigh 0.5000g of each sample and place it in a dissolution vessel containing 400mL of simulated gastric fluid. Run at 39℃ and 100r / min for 2 hours. Take 5mL samples at 1h and 2h to determine the contents of cinnamaldehyde, allicin, and sodium butyrate, and calculate the cumulative release rate in the gastric fluid at each time point. Then, transfer the remaining sample from the dissolution vessel to a dissolution vessel containing 400mL of simulated intestinal fluid and run at the same temperature and speed for 8 hours. Take 5mL samples at 2h, 4h, 6h, and 8h to determine the contents of each component and calculate the cumulative release rate in the intestinal fluid at each time point. Cinnamaldehyde and allicin were detected by gas chromatography-FID (GC-FID), and sodium butyrate was detected by high-performance liquid chromatography-UV (HPLC-UV).

[0094] (2) Experimental Results Depend on Figure 2 The results showed that the cumulative release rate of the microcapsules in simulated gastric fluid was less than 6% over 2 hours, significantly better than the control group (14.2%-35.2%). The chemical anchoring and double cross-linking network of the wall material effectively inhibited the gastric acid-triggered burst release behavior, ensuring the core material entered the intestine intact. After being transferred into the intestinal fluid, all three examples showed obvious time-sequential release characteristics: allicin in the wall material layer was rapidly released from 0 to 4 hours (cumulative rate of 70.5%-78.2%), playing a leading role in antibacterial activity; cinnamaldehyde in the oil phase was continuously and slowly released from 4 to 8 hours (cumulative rate of 74.2%-82.3%), further enhancing the antibacterial effect; sodium butyrate in the aqueous phase was the slowest throughout, with a cumulative rate of only 76.2%-80.5% over 8 hours, and was released last to repair the intestinal mucosa. The difference in release rate among the three was most significant from 4 to 6 hours, forming a staggered release gradient of pharmacological effects. Among them, Example 2 had the slowest release in intestinal fluid over 2 hours (42.5%), with no obvious time gradient, and its synergistic efficiency was lower than that of Examples 1 and 3.

[0095] Experimental Example 3: In vitro antibacterial experiment.

[0096] (1) Experimental protocol The minimal inhibitory concentration (MIC) of each sample against *Clostridium perfringens* (ATCC 13124) was determined by microbroth dilution method. *Clostridium perfringens* was inoculated into fluid thioglycollate medium, cultured anaerobically at 37°C to the logarithmic growth phase, and diluted with sterile physiological saline to 1×10 8 CFU / mL. The samples of Examples 1 to 3 and Comparative Examples 1 to 3 were dissolved in dimethyl sulfoxide and serially diluted two-fold, so that the final concentration range of cinnamaldehyde was 0.244 to 500 μg / mL, and mixed with the bacterial suspension in a 96-well plate (final concentration 5×10 5 CFU / mL), cultured anaerobically at 37°C for 24 h. The lowest concentration at no visible bacterial growth was recorded as MIC.

[0097] Meanwhile, to evaluate the protective effect of microencapsulation on active ingredients, each sample was first digested with simulated gastric fluid (pH 1.2, containing pepsin, 39°C, 2 h) and simulated intestinal fluid (pH 6.8, containing pancreatin and bile salts, 39°C, 4 h), then the supernatant was taken to determine MIC by the same method, and the retention rate of bacteriostatic activity was calculated (undigested MIC / digested MIC × 100%). After digestion, the supernatant of the sample was taken, and the actual cinnamaldehyde concentration was determined by GC. The measured concentration was used as the mother solution for two-fold serial dilution, and the MIC result was subject to the actual calculated value. The experimental group without adding sample was used as blank control.

[0098] By checkerboard microdilution method, cinnamaldehyde (A), allicin (B) and sodium butyrate (C) were serially diluted two-fold respectively to form a concentration combination matrix in a 96-well plate, and the Fractional Inhibitory Concentration (FIC) index was determined: FIC = MIC (A in combination) / MIC (A alone) + MIC (B in combination) / MIC (B alone) + MIC (C in combination) / MIC (C alone). Evaluation criteria: FIC ≤ 0.5 indicates synergism, 0.5 < FIC ≤ 1.0 indicates addition, 1.0 < FIC ≤ 2.0 indicates no interaction, and FIC > 2.0 indicates antagonism.

[0099] (2) Experimental results The intuitive results of the in vitro bacteriostatic experiment are shown in Figure 3 . The figure shows the culture status of each sample in 96-well plate before digestion (upper panel) and after digestion (lower panel). Red circles mark the well position of the lowest concentration (MIC) with no bacterial growth in each sample group. Combined with the data in Table 2, it can be seen that the MIC of Examples 1 to 3 is significantly lower than that of the comparative example groups, and they still maintain excellent bacteriostatic activity after simulated gastrointestinal digestion, which intuitively demonstrates the protective effect of the composite microcapsule of the present invention on heat-sensitive active ingredients.

[0100] Table 2 Summary of in vitro bacteriostatic experiment results

[0101] Note* Since the MIC of the physical mixture before and after digestion both reach the upper limit of detection in this experiment, it is impossible to calculate the meaningful activity retention rate based on its own MIC before digestion. Therefore, it is marked with "—" to indicate that it is not applicable.

[0102] As shown in Table 2, the MIC of the physical mixture before digestion was 500 μg / mL, while the MIC of the present invention was only 62.5 μg / mL, which is 8 times lower than that of the physical mixture. This demonstrates that the W / O / W dual emulsion microencapsulation structure can produce a strong synergistic effect of the three active ingredients, significantly improving the antibacterial efficiency. Meanwhile, the antibacterial activity retention rates of Examples 1-3 after simulated gastrointestinal digestion were all above 96.6% (96.6%~97.7%), while the retention rates of the comparative examples were only 48.8%~49.2%. This proves that the present invention, through the chemical bonding anchoring of allicin and the TG enzyme + tannic acid double cross-linking solidification process, has a significantly better protective effect on heat-sensitive components than existing encapsulation technologies. The FIC index measurement showed that the FIC values ​​of Example 1 (0.46), Example 2 (0.49), and Example 3 (0.47) were all ≤0.5, indicating a synergistic effect; while the FIC of the physical mixture was 0.96, and the FIC values ​​of each comparative example were 0.88~0.93, all showing only an additive effect.

[0103] Experiment Example 4: Poultry challenge protection experiment.

[0104] (1) Experimental scheme A two-strain infection model of Clostridium perfringens (ATCC 13124+ clinical isolate) was used for challenge protection experiments. Two hundred healthy 1-day-old AA broiler chickens were randomly divided into 5 groups (blank control group, challenge control group, Example 1 group, Example 3 group, and comparative example 2 group), with 4 replicates per group and 10 chickens per replicate.

[0105] The experiment was divided into three phases: the adaptation period was from day 1 to day 14, during which all groups were fed a basal diet; the challenge period was from day 15 to day 18, during which all groups except the blank control group were administered the virus twice daily by gavage. CFU / animal Clostridium perfringens mixed bacterial suspension (5 mL / animal), and each challenged group was fed a basal diet supplemented with 1.5 kg / t of the corresponding microcapsule sample; the recovery observation period was from day 19 to day 21.

[0106] After the experiment, mortality and diarrhea rates were recorded. Eight chickens were randomly selected from each group, and approximately 2-3 mL of blood was collected from the subwing vein. Serum was separated, and serum diamine oxidase (DAO) activity (U / L) and serum IL-10 concentration (pg / mL) were measured using a commercially available kit. Ileal contents were diluted and spread onto blood agar plates for Clostridium perfringens counting (log...). 10 (CFU / g); and calculate the average daily weight gain and feed conversion ratio.

[0107] (2) Experimental Results Table 3 Summary of the results of the Clostridium perfringens challenge protection test

[0108] Note * Different letters in the same row indicate significant differences (P<0.05), while the same letter indicates no significant differences (P>0.05).

[0109] In summary, the serum DAO activity in the challenge control group (38.7±5.3 U / L) was significantly higher than that in the blank control group, while the serum IL-10 concentration (42.5±6.3 pg / mL) was significantly lower, indicating that the challenge caused severe damage to the intestinal barrier and inhibited the expression of anti-inflammatory factors. In contrast, the serum DAO activity in the group described in Example 1 of this invention decreased to 15.2±3.0 U / L (no significant difference from the blank group), and the serum IL-10 concentration recovered to 78.5±7.2 pg / mL (close to the level of the blank control group), demonstrating that this product can effectively protect the integrity of the intestinal mucosal barrier and exert a significant anti-inflammatory effect. In Example 1, the Clostridium ileum count (3.5 log10 CFU / g) was 56.8% lower than that of the challenged control group (8.1 log10 CFU / g), the mortality rate (5%) was 84.8% lower than that of the challenged control group (33%), the diarrhea rate (11%) was 86.1% lower than that of the challenged control group (79%), the average daily weight gain recovered to 57 g / d, and the feed conversion ratio decreased to 1.62. All indicators were superior to those of Example 3 and Comparative Example 2, demonstrating that the product of this invention has a highly significant intestinal protective and anti-inflammatory effect against Clostridium perfringens challenged broilers. Based on all experiments, Example 1 is the optimal implementation scheme of this invention.

[0110] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composite microcapsule for preventing and treating necrotic enteritis in poultry, characterized in that, The composite microcapsules have a W / O / W dual emulsion structure, including an inner aqueous phase, an oil phase, and an outer aqueous phase wall material layer; The internal aqueous phase contains sodium butyrate; The oil phase contains cinnamaldehyde and free allicin; The outer aqueous phase wall material layer contains whey protein isolate-allicin conjugate and thiol chitosan-allicin conjugate. The whey protein isolate-allicin conjugate is a product of whey protein isolate and allicin linked by disulfide bonds, and the thiol chitosan-allicin conjugate is a product of thiol chitosan and allicin linked by disulfide bonds. The core material of the composite microcapsule is composed of the following components by mass percentage: cinnamaldehyde 45%~55%, allicin 20%~35%, sodium butyrate 15%~30%, totaling 100%; The wall material of the composite microcapsules is composed of the following components by mass percentage: 70%~85% whey protein isolate, 10%~18% thiol chitosan, 0.5%~3% tannic acid, and 0.2%~0.5% transglutaminase.

2. A method for preparing the composite microcapsules for preventing and treating necrotic enteritis in poultry as described in claim 1, characterized in that, Includes the following steps: (1) Preparation of protein-allicin conjugate: Dissolve whey protein isolate in deionized water, add the first portion of allicin to carry out the conjugation reaction, so that allicin is anchored to whey protein isolate molecules through disulfide bonds, and obtain protein-allicin conjugate after purification; (2) Preparation of chitosan-allicin conjugate: Thiol-modified chitosan was dissolved in acetic acid solution, and a second part of allicin was added to carry out the conjugation reaction, so that allicin was anchored to chitosan molecules through disulfide bonds, and chitosan-allicin conjugate was obtained after purification; Allicin is divided into three parts: the first part accounts for 50% to 55% of the total allicin, the second part accounts for 30% to 40%, and the third part accounts for 10% to 20% and is retained in the free state. (3) Preparation of oil phase: Cinnamaldehyde and the third part of allicin are mixed at a mass ratio of 5:1 to 25:1, and a lipophilic emulsifier is added. The mixture is stirred evenly to obtain the oil phase. (4) Preparation of W / O primary emulsion: Sodium butyrate was dissolved in deionized water to prepare an internal aqueous phase solution. Under high-speed shearing conditions, the internal aqueous phase was slowly added to the oil phase in step (3) to obtain W / O primary emulsion. (5) Preparation of external aqueous phase: The protein-allicin conjugate obtained in step (1) and the chitosan-allicin conjugate obtained in step (2) are mixed at a mass ratio of 4:1 to 8:1 and stirred evenly to obtain the external aqueous phase; (6) Construction of W / O / W dual emulsion: Under shear conditions, the W / O primary emulsion from step (4) is slowly added to the external aqueous phase from step (5), the pH is adjusted to 5.0, and the wall material is stirred to form a complex aggregate through electrostatic attraction and thiol-disulfide bond exchange reaction. Tannic acid is added and stirring is continued. (7) Double cross-linking curing: Add transglutaminase to the W / O / W system in step (6) for cross-linking curing, then add tannic acid and continue stirring to complete the double cross-linking curing; (8) Spray drying: The W / O / W dual emulsion obtained in step (7) is spray dried to obtain the composite microcapsules.

3. The preparation method according to claim 2, characterized in that, The specific preparation conditions for the protein-allicin conjugate in step (1) are as follows: Dissolve whey protein isolate in deionized water, sonicate at 40℃, 20~40kHz, and 50W / L for 20min, adjust the pH to 4.5, add the first portion of allicin, stir and react at 25℃ for 30min, and after the reaction is completed, use an ultrafiltration system with a molecular weight cutoff of 3kDa for desalting and purification.

4. The preparation method according to claim 2, characterized in that, The specific preparation conditions for the chitosan-allicin conjugate in step (2) are as follows: dissolve thiol-modified chitosan in 1% acetic acid solution, adjust the pH to 5.5, add the second part of allicin, stir the reaction at room temperature for 2 hours, and after the reaction is completed, use an ultrafiltration system with a molecular weight cutoff of 3kDa for desalting and purification.

5. The preparation method according to claim 2, characterized in that, The lipophilic emulsifier mentioned in step (3) is polyglycerol ricinoleate, and the amount used is 1% to 4% of the oil phase mass.

6. The preparation method according to claim 2, characterized in that, In step (4), the mass percentage concentration of sodium butyrate in the aqueous phase solution is 10%~25%; the high-speed shearing conditions are 10000~15000 rpm, shearing for 5~10 min; and the volume ratio of the aqueous phase to the oil phase is 1:1~3:

1.

7. The preparation method according to claim 2, characterized in that, The mass ratio of the protein-allicin conjugate to the chitosan-allicin conjugate in step (5) is 4:1 to 8:

1.

8. The preparation method according to claim 2, characterized in that, The shearing conditions in step (6) are 3000~8000 rpm, shearing for 5~10 min; the volume ratio of the W / O primary emulsion to the external aqueous phase is 1:2~1:6; the pH is adjusted to 5.0 and then stirred at room temperature for 45~60 min; the amount of tannic acid added is 0.05%~0.15% of the system concentration, and after addition, stirring is continued at room temperature for 45~60 min.

9. The preparation method according to claim 2, characterized in that, In step (7), the amount of transglutaminase used is 0.2% to 0.5% of the wall material mass, the cross-linking curing temperature is 15 to 25°C, and the cross-linking curing time is 4 to 8 hours; the addition of tannic acid to the final concentration of the system is 0.02% to 0.1%, and stirring is continued for 30 minutes to complete the double cross-linking curing. The inlet air temperature of the spray drying in step (8) is 100~130℃, the outlet air temperature is 60~75℃, the feed rate is 10~15mL / min, and the atomization pressure is 0.15~0.25MPa.

10. The composite microcapsules according to claim 1 or the composite microcapsules prepared by the preparation method according to any one of claims 2 to 9, characterized in that, The core material of the composite microcapsule is composed of the following components by mass percentage: 50% cinnamaldehyde, 30% allicin, and 20% sodium butyrate; the wall material of the composite microcapsule has a whey protein isolate to thiol chitosan mass ratio of 6:1.

Images