Microcapsule coated emulsified feed for improving immunity of animals and its preparation method

Abstract

The application discloses a microcapsule-coated emulsified feed for improving immunity of animals and a preparation method thereof, and belongs to the technical field of animal feed processing and functional feed additive. Step one: mixing and dispersing yeast beta-glucan, yeast mannose oligosaccharide, yeast nucleotide, sodium butyrate, glycerol and deionized water, and adjusting pH by using food-grade acid or food-grade alkali if necessary, so as to obtain an inner water phase of an aqueous phase immune nutritional component with pH being 6.0-6.8; through a water-in-oil-in-water type composite emulsification structure, the water-dispersible immune nutritional components such as yeast beta-glucan, yeast mannose oligosaccharide, yeast nucleotide and sodium butyrate are arranged in the inner water phase, and the fat-soluble active components such as oregano oil and / or thymol, curcumin and vitamin E are arranged in the oil phase, so that the two types of immune-regulating components with relatively large property difference can be jointly loaded in the same microcapsule structure, and the problems of uneven dispersion of components, asynchronous release and unstable effective dose caused by simple dry mixing are improved.

Description

Technical Field

[0001] This invention belongs to the technical field of animal feed processing and functional feed additives, specifically relating to a microencapsulated emulsified feed for enhancing animal immunity and its preparation method. Background Technology

[0002] Weaned piglets, young poultry, and aquatic larvae often experience problems such as decreased feed intake, impaired intestinal barrier, increased diarrhea rate, decreased serum immunoglobulin levels, and increased inflammatory factors when subjected to conditions such as regrouping, weaning, feed changes, transportation, or high-density farming. To reduce antibiotic dependence, the feed industry has gradually adopted functional components such as plant essential oils, glyceryl monolaurate, butyrate, yeast β-glucan, mannan oligosaccharides, nucleotides, curcumin, and vitamin E to improve animal gut health and immune status.

[0003] However, in actual feed processing and application, the following specific problems exist when some fat-soluble active substances and water-dispersible immunonutrients are used in combination: First, plant essential oil components such as oregano oil and thymol have volatile and irritating odors, which are easily lost during conditioning, granulation, and storage, leading to a decrease in the effective dose entering the animal body; Second, fat-soluble substances such as curcumin and plant essential oils have different dispersion phases than water-dispersible substances such as yeast β-glucan, yeast nucleotides, and sodium butyrate. If simply dry-mixed, problems such as local enrichment, decreased mixing uniformity, and inconsistent release positions can easily occur; Third, existing common coated essential oil products mainly use oils, starch, or a single wall material to encapsulate fat-soluble components, making it difficult to simultaneously carry water-dispersible immunonutrient components, resulting in fat-soluble antibacterial regulatory components and water-dispersible immunostimulating components not being released synchronously in the intestine; Fourth, if the wall material disintegrates and releases prematurely in the gastric juice environment, the essential oil and butyrate are rapidly released in the stomach and proximal small intestine, which not only easily causes palatability and irritation problems, but also makes it difficult to maintain a sufficient effective concentration in the later intestinal tract.

[0004] While existing technologies such as spray-dried microcapsules, fat powder coating, cyclodextrin inclusion, and monolayer alginate gels exist, they still suffer from problems including simple wall material structures, insufficient loading capacity of oil-water biphasic active components, low retention rate of effective components after granulation, excessively rapid release in simulated gastric juice, and unstable immune-enhancing effects. Therefore, it is necessary to develop a microencapsulated emulsified feed suitable for industrial feed production, capable of simultaneously protecting both fat-soluble and water-dispersible immunomodulatory components, and exhibiting low release in gastric juice but relatively sufficient release in intestinal juice. Summary of the Invention

[0005] The purpose of this invention is to provide a microencapsulated emulsified feed for enhancing animal immunity and its preparation method. Through the synergistic construction of an inner aqueous phase, an oil phase, and an outer composite wall layer, water-dispersible immunonutrient components such as yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides, and sodium butyrate are encapsulated in the inner aqueous phase, while fat-soluble components such as oregano oil and / or thymol, curcumin, and vitamin E are dispersed in the oil phase. The composite wall layer is formed by sodium alginate, low-methoxyl pectin, calcium ions, and chitosan lactate, thereby improving the retention rate of active components during granulation and storage, reducing premature release in gastric juice, and enhancing intestinal release and animal immune improvement effects.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing microencapsulated emulsified feed for enhancing animal immunity includes the following steps: Step 1: Mix and disperse yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides, sodium butyrate, glycerol and deionized water. If necessary, adjust the pH with food-grade acid or food-grade alkali to obtain an internal aqueous phase containing the immunonutrient components with a pH of 6.0-6.8.

[0007] Step 2: Mix medium-chain triglycerides, glyceryl monolaurate, hydrogenated vegetable oil, oregano oil and / or thymol, curcumin, vitamin E, soybean lecithin and polyglycerol ricinoleate, heat to 45-60℃ to form a homogeneous oil phase, then add the aqueous phase obtained in Step 1 to the oil phase, and perform shear emulsification under the condition of keeping warm at 45-60℃ to obtain a water-in-oil type primary emulsion.

[0008] Step 3: Dissolve sodium alginate, low-methoxyl pectin, whey protein isolate, resistant dextrin, and maltodextrin in water. If necessary, adjust the pH to 5.8-6.5 using food-grade acid or food-grade alkali to obtain an external aqueous phase wall material solution. Add the water-in-oil primary emulsion obtained in Step 2 to the external aqueous phase wall material solution and homogenize and emulsify to obtain a water-in-oil-in-water composite emulsion.

[0009] Step 4: Under stirring conditions, calcium chloride solution is slowly added to the composite emulsion obtained in Step 3 to carry out ion gelation. Then, under stirring conditions, chitosan lactate solution is slowly added to carry out outer composite coating to obtain microcapsule-coated emulsion.

[0010] Step 5: Spray dry the microencapsulated emulsion obtained in Step 4 to obtain microencapsulated emulsified powder. Then mix the microencapsulated emulsified powder with the base feed at a ratio of 0.5-3.0% of the total feed mass. Pelletize or directly make into powdered feed under the condition of conditioning temperature not exceeding 75℃ to obtain microencapsulated emulsified feed for enhancing animal immunity.

[0011] Furthermore, in the internal aqueous phase, the mass ratio of yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides, sodium butyrate, glycerol, and deionized water is 8-18:12-24:3-8:5-12:2-6:60-100; the D90 particle size of the yeast β-glucan is not greater than 25 μm.

[0012] Furthermore, in the oil phase, the mass ratio of medium-chain triglycerides, glyceryl monolaurate, hydrogenated vegetable oil, oregano oil and / or thymol, curcumin, vitamin E, soybean lecithin, and polyglycerol ricinoleate is 45-65:8-18:6-15:1-4:0.2-1.0:0.2-0.8:3-7:1-4; the melting point of the hydrogenated vegetable oil is 48-58℃.

[0013] Furthermore, in the external aqueous phase wall material liquid, the mass ratio of sodium alginate, low-methoxyl pectin, whey protein isolate, resistant dextrin, maltodextrin, and water is 1.5-3.5:0.4-1.2:1-3:8-15:5-10:70-100; the food-grade acid is one or more of citric acid, lactic acid, and malic acid, and the food-grade alkali is one or more of sodium bicarbonate, sodium citrate, and sodium hydroxide.

[0014] Furthermore, in step two, the mass ratio of the internal aqueous phase to the oil phase is 1:2-5, the shear emulsification rate is 8000-12000 r / min, the shear emulsification time is 3-8 min, and the average droplet size of the resulting water-in-oil primary emulsion is 0.5-5 μm.

[0015] Furthermore, in step three, the mass ratio of the water-in-oil primary emulsion to the external aqueous wall material liquid is 1:3-8, the homogenization pressure is 10-25MPa, the homogenization times are 1-2 times, and the resulting water-in-oil composite emulsion has a D50 particle size of 20-80μm.

[0016] Further, in step four, the mass concentration of the calcium chloride solution is 0.3-1.2%, the mass concentration of the chitosan lactate solution is 0.05-0.30%; the ion gelation temperature is 25-40℃, the pH is 5.0-5.8, the stirring speed is 200-400 r / min, the ion gelation time is 15-40 min, the calcium chloride solution is added at a time of 10-30 min, and the chitosan lactate solution is added at a time of 10-25 min.

[0017] Furthermore, in step five, the inlet air temperature of the spray drying is 130-155℃, the outlet air temperature is 65-80℃, the water content of the microcapsule-coated emulsified powder obtained after spray drying is not higher than 8%, the water activity is not higher than 0.45, the D50 particle size is 80-250μm, and the encapsulation rate of oregano oil and / or thymol is not less than 80%.

[0018] Furthermore, the encapsulation efficiency is determined based on the total content of oregano oil and / or thymol in the microcapsule-coated emulsified powder and the surface free content, and is calculated according to the following formula: Encapsulation rate = (Total content - Surface free content) / Total content × 100%.

[0019] The content of oregano oil and / or thymol was determined by gas chromatography or high performance liquid chromatography.

[0020] The present invention also provides a microencapsulated emulsified feed for enhancing animal immunity, comprising a base feed and microencapsulated emulsified powder dispersed in the base feed, wherein the microencapsulated emulsified powder accounts for 0.5-3.0% of the total mass of the microencapsulated emulsified feed.

[0021] The microcapsule-coated emulsified powder comprises an inner aqueous phase containing yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides and sodium butyrate, an oil phase containing medium-chain triglycerides, glyceryl monolaurate, oregano oil and / or thymol, curcumin and vitamin E encapsulating the inner aqueous phase, and a composite wall material layer located outside the oil phase.

[0022] The composite wall material layer includes a calcium gel layer formed by calcium ion crosslinking of sodium alginate and low-methoxyl pectin, and a chitosan lactate polyelectrolyte composite coating layer located outside the calcium gel layer.

[0023] Furthermore, after shaking the microencapsulated emulsified powder in simulated gastric juice containing pepsin at pH 2.0 for 2 hours at 37±1℃ and 80-120 r / min, the release rate of oregano oil and / or thymol is no higher than 25%; subsequently, after transferring it to simulated intestinal juice containing pancreatic enzymes and bile salts at pH 6.8 and shaking it for another 6 hours at 37±1℃ and 80-120 r / min, the cumulative release rate of oregano oil and / or thymol is no less than 65%.

[0024] Furthermore, after the microencapsulated emulsified feed is sealed and stored for 8 weeks at 25°C and 60% relative humidity, the retention rate of oregano oil and / or thymol is not less than 80%; the content of oregano oil and / or thymol in the release rate and retention rate is determined by gas chromatography or high performance liquid chromatography.

[0025] Compared with existing technologies, this invention provides a microencapsulated emulsified feed for enhancing animal immunity and its preparation method, which has the following beneficial effects: Through a water-in-oil-in-water composite emulsification structure, water-dispersible immunonutrient components such as yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides and sodium butyrate are placed in the inner aqueous phase, while fat-soluble active components such as oregano oil and / or thymol, curcumin, and vitamin E are placed in the oil phase. This allows two types of immunomodulatory components with significantly different properties to be co-loaded in the same microencapsulation structure, improving the problems of uneven component dispersion, asynchronous release and unstable effective dosage caused by simple dry mixing.

[0026] This invention introduces medium-chain triglycerides, glyceryl monolaurate, and hydrogenated vegetable oil into the oil phase. The medium-chain triglycerides are beneficial for dissolving and dispersing fat-soluble active components, while glyceryl monolaurate has interfacial stabilizing and auxiliary antibacterial regulating effects. The hydrogenated vegetable oil can improve the structural stability of the oil phase during spray drying and granulation, and reduce the volatilization loss of oregano oil and / or thymol during high-temperature conditioning and storage.

[0027] This invention constructs a first wall material layer through the composite calcium gelation of sodium alginate and low-methoxyl pectin, and then forms an outer polyelectrolyte composite coating layer using chitosan lactate. Compared to single alginate or single spray-dried wall material, this structure can significantly reduce premature release in simulated gastric juice and achieve more complete release under simulated intestinal juice conditions, thereby improving the utilization efficiency of the active ingredients after they reach the intestine.

[0028] This invention limits the pH of the external aqueous phase to 5.8-6.5 and the pH of ion gelation to 5.0-5.8, so that sodium alginate, low methoxyl pectin and chitosan lactate can form a uniform gel layer and a composite coating layer, avoiding local gel agglomeration, excessively large microcapsule particle size and decreased encapsulation rate.

[0029] This invention, by performing shear emulsification under a heat preservation condition of 45-60℃, can avoid premature coagulation of hydrogenated vegetable oil, which would cause discontinuity in the oil phase and increase the particle size of the primary emulsion. This makes the particle size of the composite emulsion more stable, thereby improving the encapsulation rate, granulation retention rate and storage retention rate after spray drying.

[0030] The microencapsulated emulsified feed obtained by this invention can improve the growth performance of weaned piglets, reduce the diarrhea rate, increase serum IgA, IgG and intestinal mucosal sIgA levels, and reduce inflammatory factor levels without the use of antibiotic growth promoters, indicating that the product can be used to improve animal immunity and intestinal health. Attached Figure Description

[0031] Figure 1 Scanning electron microscope images of the microcapsule-coated emulsified powders obtained in Example 3, Comparative Example 1, Comparative Example 2 and Comparative Example 5; Figure 2 Fourier transform infrared spectra of the microcapsule-coated emulsified powders obtained in Example 3, Comparative Example 1, and Comparative Example 2; Figure 3 Differential scanning calorimetry (DSC) spectra of the microcapsule-coated emulsified powders obtained in Example 3 and Comparative Example 3. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Please see Figures 1-3 This invention provides a technical solution for microencapsulated emulsified feed for enhancing animal immunity and its preparation method: In the following examples, yeast β-glucan is a feed-grade product with a β-glucan content of not less than 70% and a D90 particle size of not more than 25 μm after air jet milling; yeast mannan oligosaccharide is a feed-grade product with an effective substance content of not less than 80%; yeast nucleotides are feed-grade products with a total nucleotide content of not less than 40%; sodium butyrate is feed-grade sodium butyrate before coating; oregano oil uses carvacrol and thymol as the main detection markers; the degree of deacetylation of chitosan lactate is not less than 85%; the degree of esterification of low-methoxyl pectin is 25-40%; and the basal feed is a corn-soybean meal type weaned piglet basal feed without antibiotic growth promoters.

[0034] Example 1 A method for preparing microencapsulated emulsified feed for enhancing animal immunity includes the following steps: Step 1: Add yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides, sodium butyrate, glycerol and deionized water to the mixing tank in a mass ratio of 8:12:3:5:2:60. Stir for 20 minutes at 25℃ and 600r / min. Adjust the pH of the internal aqueous phase to 6.0 by lowering the pH with citric acid or raising the pH with sodium bicarbonate to obtain the internal aqueous phase.

[0035] Step 2: Medium-chain triglycerides, glyceryl monolaurate, hydrogenated vegetable oil, oregano oil, curcumin, vitamin E, soybean lecithin, and polyglycerol ricinoleate were added to an emulsifying tank in a mass ratio of 45:8:6:1:0.2:0.2:3:1. The mixture was heated to 45°C and held at this temperature to form a homogeneous oil phase. Then, the aqueous phase obtained in Step 1 was added to the oil phase in a mass ratio of 1:2. Under the condition of holding at 45°C, the mixture was sheared and emulsified at 8000 r / min for 3 min to obtain a water-in-oil primary emulsion.

[0036] Step 3: Mix sodium alginate, low-methoxyl pectin, whey protein isolate, resistant dextrin, maltodextrin, and water in a mass ratio of 1.5:0.4:1:8:5:70. Adjust the pH of the external aqueous phase wall material solution to 5.8 using citric acid to lower the pH or sodium bicarbonate to raise the pH. Add the water-in-oil primary emulsion obtained in Step 2 to the external aqueous phase wall material solution at a mass ratio of 1:3. Homogenize once at 10 MPa to obtain a water-in-oil composite emulsion.

[0037] Step 4: Control the temperature of the composite emulsion at 25℃, adjust the pH to 5.0 with citric acid, and slowly add a 0.3% calcium chloride solution over 10 minutes while stirring at 200 rpm. Continue stirring for 15 minutes to carry out ion gelation. Then, slowly add a 0.05% chitosan lactate solution over 10 minutes while stirring for 10 minutes to carry out outer composite coating, thus obtaining a microcapsule-coated emulsion.

[0038] Step 5: Spray dry the microencapsulated emulsion at an inlet air temperature of 130°C and an outlet air temperature of 65°C to obtain microencapsulated emulsified powder. Mix the microencapsulated emulsified powder with the base feed at a ratio of 0.5% of the total feed mass, and granulate the mixture at a conditioning temperature of 75°C and a conditioning time of 30 seconds to obtain microencapsulated emulsified feed.

[0039] Examples 2-5 The preparation process of Examples 2-5 is the same as that of Example 1, the difference being the proportions of each raw material and the process parameters as shown in the table below.

[0040] Table 1

[0041] As shown in Table 1, Examples 1 and 5 respectively cover the low and high ends of multiple key numerical ranges in the claims, Example 3 is located in the middle region of the main parameters, and Examples 2 and 4 are located in the low-middle and high-middle regions, thus supporting the generalization of the numerical ranges in the claims.

[0042] Comparative Example 1 differs only in that, in step four, an equal volume of deionized water is added instead of chitosan lactate solution; all other raw material ratios and process parameters are the same as in Example 3. This comparative example was used to investigate the effects of the chitosan lactate outer polyelectrolyte composite coating layer on gastric juice controlled release, storage stability, and animal immune indicators.

[0043] Comparative Example 2 differed only in that low-methoxyl pectin was not added in step three, and maltodextrin was used instead of low-methoxyl pectin by mass. All other raw material ratios and process parameters were the same as in Example 3. This comparative example was used to investigate the effect of low-methoxyl pectin's participation in the construction of the calcium gel layer on the stability and controlled-release performance of the microcapsule structure.

[0044] Comparative Example 3 differs only in that hydrogenated vegetable oil is not added in step two, and an equal mass of medium-chain triglycerides is used to replace hydrogenated vegetable oil. All other raw material ratios and process parameters are the same as in Example 3. This comparative example is used to investigate the effect of hydrogenated vegetable oil on granulation thermal stability and storage retention.

[0045] Comparative Example 4 differs only in that yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides, and sodium butyrate are not included as the inner aqueous phase in step two for the preparation of water-in-oil colostrum. Instead, they are mixed in equal amounts with microcapsule powder and basal feed in dry powder form in step five. The remaining oil phase, outer aqueous phase, gelation, and spray drying process parameters are the same as in Example 3. This comparative example is used to examine the necessity of encapsulating water-dispersible immunonutrient components in the inner aqueous phase.

[0046] Comparative Example 5 differs only in that, in step two, after the oil phase is heated to 52°C, the holding temperature is stopped and the temperature is lowered to 35°C before the inner aqueous phase is added for shear emulsification. All other raw material ratios and process parameters are the same as in Example 3. This comparative example is used to investigate the effects of a 45-60°C holding temperature during shear emulsification on the continuity of the oil phase, droplet size, and encapsulation effect.

[0047] Performance testing methods 1. Determination of carvacrol and thymol content The contents of carvacrol and thymol in the samples were determined according to NY / T 3137-2017 "Determination of Carvacrol and Thymol in Feed by Gas Chromatography". Ethanol extraction was used, followed by purification and detection by gas chromatography-flame ionization detector (GC-FID), with external standard method for quantification. The oregano oil content was calculated based on the total amount of carvacrol and thymol markers in the sample.

[0048] 2. Encapsulation rate determination Weigh 2.000 g of microcapsule-coated emulsified powder, add 20 mL of n-hexane, vortex for 1 min, and then extract at 25 °C for 30 min to fully elute the free carvacrol and thymol on the particle surface. Then filter and determine the content of carvacrol and thymol in the filtrate as the surface free content.

[0049] Take another 1.000g of the same batch of microcapsule-coated emulsified powder, add 50mL of ethanol, sonicate for 30min, vortex for 5min, and then heat in a 60℃ water bath for 30min. During the water bath, vortex or oscillate once every 8-10min, for a total of 3-4 vortexing or oscillations, so as to fully destroy the microcapsule wall material and release the encapsulated carvacrol and thymol. After cooling, make up to volume, filter, and determine the content of carvacrol and thymol in the filtrate as the total content.

[0050] The embedding rate is calculated using the following formula: Encapsulation rate = (Total content - Surface free content) / Total content × 100%.

[0051] 3. Determination of water content and water activity The moisture content of the microencapsulated emulsified powder was determined in accordance with GB / T 6435-2014 "Determination of Moisture in Feed".

[0052] Water activity was determined using the diffusion method with a water activity meter, in accordance with GB 5009.238-2016, "National Food Safety Standard - Determination of Water Activity in Food". Before testing, samples were equilibrated in a sealed environment at 25℃ for 30 minutes. Each sample was measured in triplicate, and the average value was taken.

[0053] 4. Particle size determination The average droplet size of the water-in-oil primary emulsion and the D50 size of the water-in-oil composite emulsion were determined using a laser particle size analyzer. Before measurement, the sample was diluted with the corresponding continuous phase to avoid multiple scattering. Measurements were repeated three times, and the average value was taken.

[0054] 5. Granulation retention rate determination After mixing the microencapsulated emulsified powder with the basic feed, the mixture was granulated at a conditioning temperature of 75℃ and a conditioning time of 30s. The total content of carvacrol and thymol in the feed before and after granulation was determined.

[0055] Granulation retention rate is calculated using the following formula: Granulation retention rate = content after granulation / content before granulation × 100%.

[0056] 6. Determination of storage retention rate Microencapsulated emulsified feed was packaged into aluminum-plastic composite bags and sealed. It was stored at 25°C and 60% relative humidity for 8 weeks, and the total content of carvacrol and thymol before and after storage was determined.

[0057] Storage retention rate is calculated using the following formula: Storage retention rate = content after 8 weeks of storage / content before storage × 100%.

[0058] 7. Determination of release rates of simulated gastric and intestinal fluids Preparation of simulated gastric juice: Take 2.0g of sodium chloride, add approximately 900mL of water, adjust the pH to 2.0 with hydrochloric acid, add pepsin, and bring the volume to 1000mL to obtain simulated gastric juice. The pepsin used is derived from porcine gastric mucosa, with an enzyme activity of not less than 250U / mg; the final concentration of pepsin in the simulated gastric juice is 3.2g / L, which is approximately 800U / mL based on enzyme activity. If the enzyme activity of pepsin varies between different batches, the amount added should be calculated based on the enzyme activity to ensure that the pepsin activity in the simulated gastric juice remains consistent.

[0059] Preparation of simulated intestinal fluid: Adjust the pH to 6.8 with phosphate buffer, add pancreatic enzyme and bile salts, and mix thoroughly to obtain simulated intestinal fluid. The pancreatic enzyme is derived from the porcine pancreas, and its digestive capacity should meet the requirements of the Pancreatic Enzyme section in the Chinese Pharmacopoeia. The amount of pancreatic enzyme added to the simulated intestinal fluid is 10.0 g / L, and the pancreatic enzyme activity is converted to 100 ± 10 U / mL based on trypsin activity. The bile salts are bovine bile salts or porcine bile salts, with a bile salt content of not less than 50%, and the final concentration of bile salts in the simulated intestinal fluid is 10.0 g / L.

[0060] Weigh 1.000 g of microencapsulated emulsified powder and determine its total content of carvacrol and thymol, which is recorded as the total content of the sample. Add the sample to 100 mL of simulated gastric juice and shake at 37±1℃ and 100 r / min for 2 h. Take a sample and determine the content of carvacrol and thymol released in the simulated gastric juice, which is recorded as the amount released in the gastric juice.

[0061] The gastric juice release rate is calculated using the following formula: Gastric juice release rate = Amount released in gastric juice / Total content of sample × 100%.

[0062] After the gastric juice release test, the residual microcapsules were separated and transferred into 100 mL of simulated intestinal fluid. The mixture was shaken for 6 h at 37±1℃ and 100 r / min. The contents of carvacrol and thymol released in the simulated intestinal fluid were measured and recorded as the amount released in the intestinal fluid.

[0063] The cumulative release rate is calculated using the following formula: Cumulative release rate = (released amount in gastric juice + released amount in intestinal juice) / total sample content × 100%.

[0064] If multiple samples are taken and an equal volume of fresh release medium is added during the test, the release volume in gastric fluid and intestinal fluid will be corrected according to the sampling volume and the dilution factor of the replenishment.

[0065] 8. Determination of β-glucan gastric juice leakage rate Weigh 1.000 g of microencapsulated emulsified powder and determine its total β-glucan content, which is recorded as the total β-glucan content. Add the sample to 100 mL of simulated gastric fluid, shake at 37±1℃ and 100 r / min for 2 h, and centrifuge to collect the supernatant.

[0066] The content of β-glucan-related polysaccharides in the supernatant was determined using the phenol-sulfuric acid method. Specifically, a standard solution was prepared with glucose and a standard curve was plotted. An appropriate amount of supernatant was taken and phenol solution and concentrated sulfuric acid were added for color development. After the reaction was allowed to proceed at room temperature, the absorbance was measured at 490 nm. The polysaccharide content in the supernatant was calculated based on the glucose standard curve and converted into the β-glucan leakage amount.

[0067] The gastric juice leakage rate of β-glucan is calculated using the following formula: β-glucan gastric juice leakage rate = β-glucan leakage amount / total β-glucan amount × 100%.

[0068] 9. Measurement of mixing uniformity After the microencapsulated emulsified powder is mixed with the basic feed, the mixing uniformity is tested in accordance with GB / T 5918-2008 "Determination of Mixing Uniformity of Feed Products".

[0069] Because the microencapsulated emulsified feed of this invention contains sodium butyrate, calcium chloride residues, and other components that may affect the chloride ion background, the methyl violet method is preferentially used to determine the mixing uniformity to avoid interference from background ions in the chloride ion selective electrode method. Sampling, extraction, and determination are performed according to GB / T 5918-2008, and the coefficient of variation (CV) is calculated based on the change in the content of the methyl violet tracer.

[0070] The mixed coefficient of variation (CV) is calculated using the following formula: CV = standard deviation / mean × 100%.

[0071] 10. Animal feeding trials and statistical analysis Weaned piglets of 28 days age, similar weight and health status were selected and randomly divided into a basal control group, Example 1-5 groups and Comparative Example 1-5 groups, with 6 replicates in each group and 6 piglets in each replicate. The experimental period was 28 days.

[0072] The basal control group was fed a basal diet without antibiotic growth promoters; the example groups and comparative groups were fed the corresponding microencapsulated emulsified diets, respectively. Feed intake, body weight, and diarrhea were recorded during the experiment. At the end of the experiment, blood and ileal mucosal samples were collected, and serum IgA, IgG, TNF-α, IL-6, and intestinal mucosal sIgA were measured using ELISA.

[0073] Data are expressed as mean ± standard deviation. One-way ANOVA was used for statistical analysis, and Duncan's method was employed for multiple comparisons. P < 0.05 indicated statistical significance, and different lowercase letters in the same column indicated statistical significance. Test Results Table 2

[0074] Table 2 shows that the microcapsule-coated emulsified powders obtained in Examples 1-5 all meet the requirements of water content not exceeding 8%, water activity not exceeding 0.45, encapsulation rate not less than 80%, high retention rate after granulation, and retention rate not less than 80% after 8 weeks of storage. Among them, Example 3 has the best overall effect. In Comparative Example 1, the encapsulation rate and storage retention rate decreased after removing the chitosan lactate outer coating; in Comparative Example 2, the encapsulation rate decreased to 79.8% after removing the low-methoxyl pectin, indicating that the low-methoxyl pectin plays an important role in the construction of the calcium gel layer and the encapsulation stability; in Comparative Example 3, the granulation retention rate decreased significantly to 68.4% after removing the hydrogenated vegetable oil, indicating that the hydrogenated vegetable oil makes a significant contribution to thermal stability; in Comparative Example 5, shear emulsification was not performed under the heat preservation condition of 45-60℃, which led to an increase in the initial emulsion particle size, a coarser composite emulsion particle size, an increase in water content and water activity, and a decrease in encapsulation rate and storage retention rate.

[0075] Table 3

[0076] As shown in Table 3, the release rates of carvacrol / thymol in simulated gastric fluid for 2 hours in Examples 1-5 were all no higher than 25%, and the cumulative release rates after 6 hours of continued shaking in simulated intestinal fluid were all no less than 65%, indicating that the microcapsule structure of the present invention can reduce premature release in the stomach and achieve effective release in the intestinal fluid environment. Figure 3 As shown, the release curve of Example 3 was relatively flat in the simulated gastric juice stage, and the release rate increased significantly after entering the simulated intestinal juice; while Comparative Examples 1, 2 and 5 showed faster release in the simulated gastric juice stage, indicating that the outer coating of chitosan lactate, the participation of low methoxy pectin in the construction of the gel layer and the incubation and shear emulsification at 45-60℃ all play a key role in the controlled release structure.

[0077] Comparative Example 1, without the chitosan lactate outer layer, showed a simulated gastric juice release rate of 38.2%; Comparative Example 2, without the addition of low-methoxyl pectin, showed a simulated gastric juice release rate of 43.5%; although the release performance of the lipid-soluble component in Comparative Example 4 was close to that of Example 3, the gastric juice leakage rate of β-glucan reached 82.6% because the water-dispersible immunonutrient component was not encapsulated in the inner aqueous phase, indicating that the water-in-oil-in-water structure is necessary for the protection of the water-dispersible immunonutrient component. In Comparative Example 5, the oil phase partially coagulated during shear emulsification, forming large-particle-size, non-uniform microcapsules, resulting in a gastric juice release rate of 55.3%, significantly reducing the controlled-release performance.

[0078] Table 4

[0079] As shown in Table 4, the microencapsulated emulsified powders in Examples 1-5 can be uniformly dispersed in the basal feed, with a mixing coefficient of variation of less than 10%, indicating that the microencapsulated powders obtained by this invention are suitable for conventional feed mixing and pelleting processes. Combined with... Figure 2 As shown in the preparation process, the microcapsule powder obtained by this invention is spray-dried and then mixed with the basic feed. This avoids the local enrichment and volatilization loss caused by directly adding liquid essential oil to the feed, and also helps conventional feed production equipment to achieve uniform mixing.

[0080] Table 5

[0081] Note: Data in the table are mean ± SD, n=6; different lowercase letters in the same column indicate significant differences, P<0.05.

[0082] As shown in Table 5, compared with the baseline control group, Examples 1-5 significantly improved the average daily weight gain of weaned piglets, reduced the feed conversion ratio and diarrhea rate, increased serum IgA, IgG, and ileal mucosal sIgA levels, and decreased TNF-α and IL-6 levels. Example 3 showed the best overall effect, indicating that the emulsion coating structure formed under the intermediate region parameters of this invention is stable, releases appropriately, and can better improve the animal's immune status.

[0083] Compared with Example 3, in Comparative Example 1, the removal of the chitosan lactate outer coating increased the premature release of simulated gastric juice and decreased animal immune indicators; in Comparative Example 2, the removal of low-methoxyl pectin decreased the encapsulation rate and gastric juice protection ability, and increased the diarrhea rate; in Comparative Example 3, the removal of hydrogenated vegetable oil significantly reduced the granulation retention rate and storage retention rate, resulting in a decrease in the actual effective dose ingested; in Comparative Example 4, the water-dispersible immunonutrient components were not encapsulated in the inner aqueous phase, resulting in a significant increase in the β-glucan gastric juice leakage rate and a significant decrease in the improvement of serum IgA and ileal mucosal sIgA; in Comparative Example 5, the lack of shearing emulsification under incubation conditions of 45-60℃ resulted in uneven microcapsule structure, excessively rapid gastric juice release, and the animal immune improvement effect was close to that of the baseline control group.

[0084] Therefore, this invention is not simply a mixture of plant essential oils, immunopolysaccharides, and conventional wall materials. Instead, it achieves simultaneous improvement in granulation and storage stability, gastrointestinal controlled-release capability, and animal immune enhancement through the synergistic effect of an inner aqueous phase encapsulation, a water-in-oil-in-water composite emulsion, a sodium alginate-low methoxy pectin calcium gel layer, a chitosan lactate outer composite coating, and a hydrogenated vegetable oil thermally stable oil phase. This invention has outstanding substantive features and significant progress.

Claims

1. A method for preparing microencapsulated emulsified feed for enhancing animal immunity, characterized in that, Includes the following steps: Step 1: Mix and disperse yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides, sodium butyrate, glycerol and deionized water. If necessary, adjust the pH with food-grade acid or food-grade alkali to obtain an internal aqueous phase containing the immunonutrient components with a pH of 6.0-6.

8. Step 2: Mix medium-chain triglycerides, glyceryl monolaurate, hydrogenated vegetable oil, oregano oil and / or thymol, curcumin, vitamin E, soybean lecithin and polyglycerol ricinoleate, heat to 45-60℃ to form a homogeneous oil phase, then add the aqueous phase obtained in Step 1 to the oil phase, and perform shear emulsification under the condition of keeping warm at 45-60℃ to obtain a water-in-oil type primary emulsion; Step 3: Dissolve sodium alginate, low-methoxyl pectin, whey protein isolate, resistant dextrin and maltodextrin in water. If necessary, adjust the pH to 5.8-6.5 with food-grade acid or food-grade alkali to obtain an external aqueous phase wall material solution. Add the water-in-oil type primary emulsion obtained in Step 2 to the external aqueous phase wall material solution and homogenize and emulsify to obtain a water-in-oil-in-water composite emulsion. Step 4: Under stirring conditions, calcium chloride solution is slowly added to the composite emulsion obtained in Step 3 to carry out ion gelation. Then, under stirring conditions, chitosan lactate solution is slowly added to carry out outer layer composite coating to obtain microcapsule-coated emulsion. Step 5: Spray dry the microencapsulated emulsion obtained in Step 4 to obtain microencapsulated emulsified powder. Then mix the microencapsulated emulsified powder with the base feed at a ratio of 0.5-3.0% of the total feed mass. Pelletize or directly make into powdered feed at a conditioning temperature not exceeding 75℃ to obtain microencapsulated emulsified feed for enhancing animal immunity.

2. The method for preparing a microencapsulated emulsified feed for enhancing animal immunity according to claim 1, characterized in that, In the internal aqueous phase, the mass ratio of yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides, sodium butyrate, glycerol and deionized water is 8-18:12-24:3-8:5-12:2-6:60-100; the D90 particle size of the yeast β-glucan is not greater than 25 μm.

3. The method for preparing a microencapsulated emulsified feed for enhancing animal immunity according to claim 1, characterized in that, In the oil phase, the mass ratio of medium-chain triglycerides, glyceryl monolaurate, hydrogenated vegetable oil, oregano oil and / or thymol, curcumin, vitamin E, soybean lecithin, and polyglycerol ricinoleate is 45-65:8-18:6-15:1-4:0.2-1.0:0.2-0.8:3-7:1-4; the melting point of the hydrogenated vegetable oil is 48-58℃.

4. The method for preparing a microencapsulated emulsified feed for enhancing animal immunity according to claim 1, characterized in that, In the external aqueous phase wall material solution, the mass ratio of sodium alginate, low-methoxyl pectin, whey protein isolate, resistant dextrin, maltodextrin, and water is 1.5-3.5:0.4-1.2:1-3:8-15:5-10:70-100; the food-grade acid is one or more of citric acid, lactic acid, and malic acid; and the food-grade alkali is one or more of sodium bicarbonate, sodium citrate, and sodium hydroxide.

5. The method for preparing a microencapsulated emulsified feed for enhancing animal immunity according to claim 1, characterized in that, In step two, the mass ratio of the internal aqueous phase to the oil phase is 1:2-5, the shear emulsification rate is 8000-12000 r / min, the shear emulsification time is 3-8 min, and the average droplet size of the resulting water-in-oil primary emulsion is 0.5-5 μm.

6. The method for preparing a microencapsulated emulsified feed for enhancing animal immunity according to claim 1, characterized in that, In step three, the mass ratio of the water-in-oil primary emulsion to the external aqueous wall material liquid is 1:3-8, the homogenization pressure is 10-25MPa, the homogenization times are 1-2 times, and the resulting water-in-oil composite emulsion has a D50 particle size of 20-80μm.

7. The method for preparing a microencapsulated emulsified feed for enhancing animal immunity according to claim 1, characterized in that, In step four, the mass concentration of the calcium chloride solution is 0.3-1.2%, and the mass concentration of the chitosan lactate solution is 0.05-0.30%; the ion gelation temperature is 25-40℃, the pH is 5.0-5.8, the stirring speed is 200-400 r / min, the ion gelation time is 15-40 min, the calcium chloride solution is added at a time of 10-30 min, and the chitosan lactate solution is added at a time of 10-25 min.

8. The method for preparing a microencapsulated emulsified feed for enhancing animal immunity according to claim 1, characterized in that, In step five, the inlet air temperature of the spray drying is 130-155℃, the outlet air temperature is 65-80℃, the water content of the microcapsule-coated emulsified powder obtained after spray drying is not higher than 8%, the water activity is not higher than 0.45, the D50 particle size is 80-250μm, and the encapsulation rate of oregano oil and / or thymol is not less than 80%. The encapsulation efficiency was determined based on the total content of oregano oil and / or thymol in the microcapsule-coated emulsified powder and the surface free content, and was calculated according to the following formula: Encapsulation rate = (Total content - Surface free content) / Total content × 100%; The content of oregano oil and / or thymol was determined by gas chromatography or high performance liquid chromatography.

9. A microencapsulated emulsified feed for enhancing animal immunity, characterized in that, The product comprises a base feed and microencapsulated emulsified powder dispersed in the base feed, wherein the microencapsulated emulsified powder accounts for 0.5-3.0% of the total mass of the microencapsulated emulsified feed; The microcapsule-coated emulsified powder comprises an inner aqueous phase containing yeast β-glucan, yeast mannan oligosaccharide, yeast nucleotides and sodium butyrate, an oil phase containing medium-chain triglycerides, monolaurate glycerides, oregano oil and / or thymol, curcumin and vitamin E coating the inner aqueous phase, and a composite wall material layer located on the outside of the oil phase. The composite wall material layer includes a calcium gel layer formed by calcium ion crosslinking of sodium alginate and low-methoxyl pectin, and a chitosan lactate polyelectrolyte composite coating layer located outside the calcium gel layer.

10. A microencapsulated emulsified feed for enhancing animal immunity according to claim 9, characterized in that, The microencapsulated emulsified powder, after being shaken for 2 hours at 37±1℃ and 80-120 r / min in simulated gastric juice containing pepsin at pH 2.0, showed a release rate of no more than 25% for oregano oil and / or thymol. Subsequently, the mixture was transferred to simulated intestinal fluid containing pancreatic enzymes and bile salts at pH 6.8, and shaken for 6 hours at 37±1℃ and 80-120 r / min. The cumulative release rate of oregano oil and / or thymol was not less than 65%. After the microencapsulated emulsified feed is sealed and stored for 8 weeks at 25°C and 60% relative humidity, the retention rate of oregano oil and / or thymol is not less than 80%; the content of oregano oil and / or thymol in the release rate and retention rate is determined by gas chromatography or high performance liquid chromatography.

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