A feed additive for enhancing immunity of ruminants and a preparation method thereof

Abstract

The present application relates to the technical field of feed additive, in particular to a feed additive for enhancing immunity of ruminants and a preparation method thereof.The present application first reacts L-ascorbic acid with sodium trimetaphosphate to prepare an ascorbic acid phosphate intermediate, then esterifies and calcium-magnesium salts the ascorbic acid phosphate intermediate with lauric acid esterification and calcium-magnesium saltification under the catalysis of immobilized lipase to obtain a phosphorylated-lauric acylated calcium-magnesium ascorbate; subsequently, short-chain chitosan dispersion, first sodium phytate segment locking solution, long-chain chitosan dispersion and second sodium phytate segment locking solution are sequentially added to form modified ascorbic acid immune enhancement microparticles, which are then mixed with pregelatinized corn starch and malt dextrin and spray dried; the present application is conducive to improving the granulation stability, rumen retention and later-stage release and utilization of ascorbic acid active substances, and is suitable for ruminant immune stress period diet addition.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, specifically to a feed additive that enhances the immunity of ruminants and its preparation method. Background Technology

[0002] Ruminants experience increased oxidative stress levels during weaning and regrouping, the perinatal period, long-distance transport, and high-temperature heat stress. This leads to fluctuations in feed intake and rumen fermentation stability, as well as decreased peripheral immune cell activity, ultimately affecting growth, lactation, and disease resistance. Supplementing feed with ascorbic acid can help regulate antioxidant activity and maintain immune function. However, due to the unique digestive tract structure of ruminants, additives must undergo pelleting, storage, and rumen fermentation before reaching the abomasum and small intestine, making it difficult for ordinary ascorbic acid to maintain its effective state.

[0003] The existing method of directly adding L-ascorbic acid is simple, but its enediol structure is sensitive to oxygen, heat, moisture, and metal ions, and it is easily oxidized and degraded in steam conditioning, granulation, storage, and rumen fluid environments. Increasing the dosage to compensate for losses will increase feeding costs and reduce the economics of precision nutrition and environmentally friendly farming.

[0004] To improve stability, existing technologies often employ salt derivatives such as ascorbate phosphate and calcium ascorbate. While these substances can improve chemical stability to some extent, they are highly hydrophilic and easily dissolve and diffuse in the near-neutral aqueous environment of the rumen. This results in the premature release of the active ingredient in the rumen, leading to insufficient availability in the abomasum and small intestine.

[0005] Fat coatings can form an outer barrier through oils, waxes, or fatty acid salts, reducing the contact between active ingredients and water and rumen fluid. However, the coating layer is prone to softening, cracking, or wear during steam conditioning, pelleting, rumination, chewing, and rumen agitation. Furthermore, it lacks directional binding with ascorbic acid molecules, making it susceptible to premature release along defective channels.

[0006] Chitosan-based materials can form ionic networks with amino groups and multiple anions, which can be used for nutrient encapsulation and sustained release. However, conventional chitosan encapsulation is mostly random gel or monolithic coating, and there is a lack of molecular positioning relationship between the active substance and the shell material. Under continuous rumen fluid agitation and changes in ionic strength, the outer layer is prone to swelling, internal diffusion channels are formed, and the release rhythm is unstable. Summary of the Invention

[0007] In view of this, the purpose of this invention is to propose a feed additive that enhances the immunity of ruminants and its preparation method, so as to solve the problem that existing ascorbic acid additives for ruminants are easily inactivated or prematurely released in the pelleting, storage and rumen environment, making it difficult to meet the stable nutritional regulation needs during the immune stress period.

[0008] To achieve the above objectives, the present invention provides a method for preparing a feed additive that enhances the immunity of ruminants, comprising the following steps:

[0009] S1: L-ascorbic acid is reacted with sodium trimetaphosphate under alkaline, light-protected and nitrogen-protected conditions to obtain ascorbic acid phosphate intermediate;

[0010] S2: The ascorbate phosphate intermediate is esterified with lauric acid in the presence of immobilized lipase, and then salinated with calcium magnesium alkaline slurry to obtain phosphorylated-laurynoyl calcium magnesium ascorbate.

[0011] S3: Prepare short-chain chitosan dispersion, long-chain chitosan dispersion and sodium phytate segmented locking liquid respectively, and divide the sodium phytate segmented locking liquid into a first sodium phytate segmented locking liquid and a second sodium phytate segmented locking liquid;

[0012] S4: The phosphorylated-laurylated calcium magnesium ascorbate salt is dispersed in a mixture of purified water and anhydrous ethanol, and then the short-chain chitosan dispersion, the first sodium phytate segmented locking solution, the long-chain chitosan dispersion and the second sodium phytate segmented locking solution are added in sequence to obtain modified ascorbic acid immune-enhancing microparticles.

[0013] S5: The modified ascorbic acid immune-enhancing microparticles are mixed with pregelatinized corn starch and maltodextrin and then spray-dried to obtain a feed additive that enhances the immunity of ruminants.

[0014] The ratio of L-ascorbic acid to sodium trimetaphosphate in step S1 is 160-190:170-200 by weight.

[0015] The ratio of ascorbate phosphate intermediate, lauric acid, immobilized lipase and calcium magnesium alkaline slurry in step S2 is 90-110:105-135:15-22:100 by weight.

[0016] The ratio of phosphorylated-laurylated calcium magnesium ascorbate, short-chain chitosan dispersion, first sodium phytate segmented locking solution, long-chain chitosan dispersion and second sodium phytate segmented locking solution in step S4 is 225-275:500:90-110:500:190-210 by weight.

[0017] By weight, the ratio of modified ascorbic acid immune-enhancing microparticles, pregelatinized corn starch, and maltodextrin in step S5 is 720-880:500-540:180-220.

[0018] Preferably, the alkaline conditions described in step S1 are adjusted by using sodium bicarbonate and a 20% sodium hydroxide aqueous solution, with the pH maintained at 8.8-9.2.

[0019] Preferably, the preparation of the ascorbic acid phosphate intermediate in step S1 includes: adding L-ascorbic acid to purified water at 0-5°C, followed by adding sodium bicarbonate and sodium hydroxide to adjust the pH of the system to 8.8-9.2; then adding an aqueous solution of sodium trimetaphosphate dropwise to the system, maintaining the temperature at 0-5°C and the pH at 8.8-9.2 during the dropwise addition; after the sodium trimetaphosphate aqueous solution is added, continuing the reaction at 0-5°C for 2-3 hours, then raising the temperature to 23-27°C and reacting for 5-7 hours; adjusting the pH to 6.2-6.5, desalting through a nanofiltration membrane with a molecular weight cutoff of 200 Da, concentrating to a solids mass fraction of 35%-45%, and then adding ethanol for precipitation to obtain the ascorbic acid phosphate intermediate.

[0020] Preferably, the esterification reaction in step S2 is carried out under a nitrogen atmosphere at a temperature of 42-46°C for a reaction time of 8-12 hours.

[0021] Preferably, during the immobilized lipase reaction in step S2, the moisture content of the system is controlled to be no higher than 1% by mass using a 3Å molecular sieve.

[0022] Preferably, the calcium-magnesium alkaline slurry in step S2 is prepared by 4-6 parts calcium hydroxide, 1-3 parts magnesium hydroxide and the remainder purified water, based on a weight ratio of 100 parts.

[0023] Preferably, the calcium-magnesium alkaline slurry salinization in step S2 includes: adding the calcium-magnesium alkaline slurry to a dispersion system formed by the concentrated residue obtained from the esterification reaction, anhydrous ethanol, and purified water, stabilizing the pH of the system at 6.5-6.8, then adding anhydrous ethanol for precipitation, and obtaining phosphorylated-laurylated calcium magnesium ascorbate after filtration, washing, and drying.

[0024] Preferably, the short-chain chitosan dispersion in step S3 is prepared in 500 parts by weight, consisting of 1-2 parts glacial acetic acid, 12-18 parts chitosan oligosaccharide lactate, and the remainder purified water, and the pH is adjusted to 5.5-5.8 with sodium hydroxide aqueous solution.

[0025] Preferably, the long-chain chitosan dispersion in step S3 is prepared in 500 parts by weight, consisting of 2-4 parts glacial acetic acid, 8-12 parts long-chain chitosan, and the remainder purified water, and the pH is adjusted to 5.5-5.8 with an aqueous sodium hydroxide solution.

[0026] Preferably, the chitosan oligosaccharide lactate in step S3 has a number-average molecular weight of 4000 Da-6000 Da and a degree of deacetylation greater than 90%.

[0027] Preferably, the long-chain chitosan in step S3 has an average weight-average molecular weight of 50 kDa.

[0028] Preferably, the sodium phytate segmented locking solution in step S3 is prepared in 300 parts by weight, consisting of 1 part glacial acetic acid, 5-7 parts sodium phytate saline, and the remainder purified water. The sodium phytate segmented locking solution includes a first sodium phytate segmented locking solution and a second sodium phytate segmented locking solution; the weight ratio of the first sodium phytate segmented locking solution to the second sodium phytate segmented locking solution is 90-110:190-210.

[0029] Preferably, the addition of the short-chain chitosan dispersion in step S4 takes 15-25 minutes, during which the stirring speed is 650-750 rpm and the pH is 5.5-5.8. After the addition is complete, stirring continues for 25-35 minutes.

[0030] Preferably, in step S4, the first sodium phytate segmented locking solution is added after the short-chain chitosan dispersion and before the long-chain chitosan dispersion, and the addition time is 8-12 minutes. After the addition is completed, stirring is continued for 10-20 minutes.

[0031] Preferably, the long-chain chitosan dispersion in step S4 is added after the first sodium phytate segmented locking solution is added, with an addition time of 25-35 minutes, and stirring is continued for 35-45 minutes after the addition is completed; the second sodium phytate segmented locking solution is added after the long-chain chitosan dispersion is added, with an addition time of 25-35 minutes, and stirring is continued for 40-50 minutes after the addition is completed.

[0032] Preferably, the moisture content of the modified ascorbic acid immune-enhancing microparticles in step S4 is controlled to be 55%-70%.

[0033] Preferably, the inlet air temperature of the spray drying in step S5 is 105-110℃, the outlet air temperature is 58-62℃, and after spray drying, it is dried at 40℃ and vacuum degree not higher than -0.08MPa for 3-5 hours, and then granulated through a 40-mesh sieve.

[0034] A feed additive to enhance the immunity of ruminants, with a moisture content not exceeding 6%, an effective ingredient content of 12%-16% based on ascorbic acid equivalent, and a median particle size of 25-55 μm.

[0035] The beneficial effects of this invention are:

[0036] This invention prepares an ascorbic acid phosphate intermediate from L-ascorbic acid, and reacts it with lauric acid under the action of immobilized lipase to form phosphorylated-laurylated calcium magnesium ascorbate, so that the ascorbic acid molecule has both a phosphate terminus and a lauroyl terminus. The phosphate terminus helps to reduce the risk of oxidative inactivation in the processing and rumen environment, while the lauroyl terminus helps to form hydrophobic microdomains, thus providing both stability protection and aqueous release inhibition.

[0037] This invention allows the short-chain chitosan dispersion to first interact with the phosphate end, and then uses a first sodium phytate segmented locking solution for inner layer fixation, so that the ascorbic acid derivative forms a positioning structure inside the microparticles, rather than relying solely on the outer layer coating; this structure helps to reduce the premature diffusion of active ingredients in rumen fluid and improves the problem of excessively rapid hydrophilic release of ordinary ascorbic acid phosphate.

[0038] The present invention further incorporates a long-chain chitosan dispersion and uses a second sodium phytate segmented locking solution for outer layer locking, so that a flexible film-forming layer and segmented ion network are formed on the outer side of the particles. This structure helps to reduce structural damage during granulation, mixing and rumen agitation, and allows the effective structure of ascorbic acid to be gradually released in the abomasum and small intestine environment.

[0039] In summary, compared with conventional direct addition of ascorbic acid, ascorbate salt formulation, and simple fat coating, this invention achieves synergistic effects of granulation protection, rumen release inhibition, and post-release through phosphorylation-laurylylation of calcium and magnesium ascorbate, inner layer positioning of short-chain chitosan, outer layer film formation of long-chain chitosan, and segmented locking with sodium phytate. It is suitable for dietary supplementation of weaned calves, periparturient dairy cows, transport-stressed beef cattle, and sheep flocks under high-temperature heat stress. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments.

[0041] The sources and specifications of the raw materials used in this embodiment are as follows:

[0042] Hydrochloric acid: 37% by mass, ACS grade;

[0043] 3Å molecular sieve: Merck, type 208582;

[0044] Immobilized lipase: Merck, L4777 type lipase acrylic resin, enzyme activity not less than 5000U / g;

[0045] Chitosan oligosaccharide lactate: Merck, type 523682, number average molecular weight 5000 Da, degree of deacetylation greater than 90%;

[0046] Long-chain chitosan: Merck, type 900341, non-animal source, high purity, average weight-average molecular weight 50 kDa;

[0047] Sodium phytate salt hydrate: Merck, P8810;

[0048] Pregelatinized corn starch: Roquette LYCATAB C-LM type, corn-derived, partially pregelatinized, low moisture grade;

[0049] Maltodextrin: Merck, type 419672.

[0050] Example 1: A method for preparing a feed additive to enhance the immunity of ruminants, the specific steps of which are as follows:

[0051] (a) Preparation of ascorbate phosphate intermediate:

[0052] Add 900g of purified water deoxygenated by bubbling under nitrogen for 30 min to a reaction vessel equipped with a mechanical stirrer, thermometer, and dropping device. Add 176g of L-ascorbic acid at 0-5℃ and stir in the dark until completely dissolved. Then add 84g of sodium bicarbonate in three portions, waiting for the bubbling to weaken significantly after each addition. After the addition is complete, continue stirring for 20 min to partially convert L-ascorbic acid to ascorbate. Add a 20% sodium hydroxide aqueous solution over 30 min to maintain the pH of the system at 8.8-9.2. Separately, take 300g of purified water deoxygenated by bubbling under nitrogen for 30 min, add 184g of sodium trimetaphosphate, and stir until completely dissolved to obtain a sodium trimetaphosphate aqueous solution. Under continuous nitrogen purging and in the dark, incubate the sodium trimetaphosphate aqueous solution for 90 min. The solution was added dropwise to the ascorbate solution, maintaining a temperature of 0-5℃ and a pH of 8.8-9.2 during the addition. After the addition was completed, the reaction was continued at 0-5℃ for 2 hours, and then the temperature was raised to 25℃ for 6 hours. After the reaction was completed, 1 mol / L hydrochloric acid aqueous solution was added to adjust the pH of the system to 6.2-6.5. The inorganic small molecule salts were removed by conventional nanofiltration membrane with a molecular weight cutoff of 200 Da, and the solution was concentrated to a solid mass fraction of 40%. Then, 2600 g of 95% ethanol was added for precipitation. After filtration, the solution was dried at 45℃ and a vacuum degree not exceeding -0.08 MPa for 8 hours to obtain the ascorbate phosphate intermediate.

[0053] (II) Preparation of phosphorylated-laurylated calcium magnesium ascorbate:

[0054] 100g of ascorbic acid phosphate intermediate, 120g of lauric acid, 45g of 3Å molecular sieve activated at 250℃ for 4h and cooled to room temperature, 660g of tert-amyl alcohol, and 60g of dimethyl sulfoxide were added to a dry reaction vessel. The mixture was stirred for 30min under nitrogen protection to form a uniform dispersion of the ascorbic acid phosphate intermediate. Subsequently, 18g of immobilized lipase was added, and the reaction was carried out at 44℃ for 10h. During the reaction, nitrogen protection was maintained, and the moisture content of the system was controlled to be no higher than 1% by mass using the 3Å molecular sieve. After the reaction, the immobilized lipase and 3Å molecular sieve were removed by filtration. The filter cake was washed with tert-amyl alcohol, and the filtrates were combined and concentrated under reduced pressure below 45℃ until no obvious solvent distillation occurred. Add 300g anhydrous ethanol and 200g purified water to the concentrated residue, stir to disperse, and then add 100g of calcium-magnesium alkaline slurry prepared in advance from 5g calcium hydroxide, 2g magnesium hydroxide and purified water. Control the addition time to 30min, and continue stirring for 40min after addition to stabilize the pH of the system at 6.5-6.8. Then add 1000g anhydrous ethanol, let stand at 7℃ for 2h for precipitation, filter to collect the precipitate, and wash it successively with 500g of 80% ethanol aqueous solution and 500g of anhydrous ethanol. After washing, dry at 45℃ and vacuum degree not higher than -0.08MPa for 10h to obtain phosphorylated-laurylated calcium magnesium ascorbate.

[0055] (III) Preparation of short-chain chitosan dispersion, long-chain chitosan dispersion and sodium phytate segmented locking solution:

[0056] Add purified water, 1g glacial acetic acid, and 15g chitosan oligosaccharide lactate to a container and stir at 25°C for 2 hours. Then add a 10% sodium hydroxide aqueous solution to stabilize the pH at 5.5-5.8, and add purified water to make the total mass 500g to obtain a short-chain chitosan dispersion. Separately, add purified water, 3g glacial acetic acid, and 10g long-chain chitosan to a container and stir at 25°C for 6 hours. Then add a 10% sodium hydroxide aqueous solution to stabilize the pH at 5.5-5.8, and add purified water to make the total mass 500g to obtain a long-chain chitosan dispersion. Next, add purified water, 1g glacial acetic acid, and 6g sodium phytate hydrate to a container and stir for 40 minutes until completely dissolved to obtain 300g of sodium phytate segmented locking solution, which is then divided into a first sodium phytate segmented locking solution and a second sodium phytate segmented locking solution.

[0057] (iv) Preparation of modified ascorbic acid immune-enhancing microparticles:

[0058] Add 975g purified water and 525g anhydrous ethanol to a dispersion container, stir until homogeneous, then add 250g phosphorylated-laurylated calcium magnesium ascorbate. Stir for 20 min at 24℃ and 600 rpm to form a uniform dispersion. Then, add 500g short-chain chitosan dispersion over 20 min, maintaining a stirring speed of 700 rpm and a pH of 5.5-5.8 during the addition. Continue stirring for 30 min after the addition is complete. Next, add 100g of the first sodium phytate fractional locking solution over 10 min, and continue stirring for 15 min after the addition is complete. Then, add 600g of long-chain chitosan dispersion over 30 min, and continue stirring for 40 min after the addition is complete. Finally, add 200g of the second sodium phytate fractional locking solution over 30 min, and continue stirring for 45 min after the addition is complete. Desulfurize under reduced pressure at 38℃ and a vacuum degree not exceeding -0.06 MPa. Remove ethanol until no obvious ethanol is distilled off and the ethanol mass fraction in the initial dispersion is no higher than 0.5%; after removing ethanol under reduced pressure, cool the system to 25℃, adjust the pH to 5.5-5.8 with a 1% acetic acid aqueous solution, add purified water, and then stir at 1000 rpm for 10 min to redisperse the particles evenly. Filter through an 80-mesh sieve to remove a small amount of large particle agglomerates and collect the sieve-underflow dispersion; centrifuge the sieve-underflow dispersion at 4000 rpm for 10 min, discard the supernatant, collect the wet particles, wash the wet particles once with 500 g of purified water, centrifuge again at 4000 rpm for 10 min, discard the supernatant; dehydrate the obtained wet particles at low speed for 20 min at 25℃ and a vacuum degree no higher than -0.04 MPa to control the water content of the wet particles to 65%, obtaining modified ascorbic acid immune-enhancing particles;

[0059] (V) Drying and finished product preparation:

[0060] 800g of modified ascorbic acid immune-enhancing microparticles were added to 1800g of purified water and stirred at 25℃ and 1000rpm for 20min to fully disperse the wet microparticles, resulting in a redispersible solution of modified ascorbic acid immune-enhancing microparticles. 520g of pregelatinized corn starch and 200g of maltodextrin were added and stirred at 25℃ for 30min. Subsequently, conventional spray drying was performed with the inlet air temperature controlled at 110℃ and the outlet air temperature controlled at 60℃. After drying, the solution was further dried at 40℃ and a vacuum degree not exceeding -0.08MPa for 4h. The product was then granulated through a 40-mesh sieve to obtain a feed additive that enhances the immunity of ruminants.

[0061] The difference between Example 2 and Example 1 is as follows:

[0062] In step (1), 880g of purified water deoxygenated by bubbling under nitrogen for 30 minutes was added to a reaction vessel equipped with a mechanical stirrer, thermometer, and dropping device. 170g of L-ascorbic acid was added at 0-5℃, and the mixture was stirred in the dark until completely dissolved. Then, 80g of sodium bicarbonate was added in three portions, waiting for the bubbles to weaken significantly after each addition before continuing. After all additions were completed, stirring was continued for 18 minutes. Then, a 20% sodium hydroxide aqueous solution was added over 28 minutes to maintain the pH of the system at 8.8-9.2. Separately, 290g of purified water deoxygenated by bubbling under nitrogen for 30 minutes was added to 178g of sodium trimetaphosphate, and the mixture was stirred until completely dissolved to obtain a sodium trimetaphosphate aqueous solution. Under continuous nitrogen purging and in the dark, the sodium trimetaphosphate aqueous solution was added dropwise over 85 minutes until the ascorbate solution dissolved. During the dropwise addition, the temperature was maintained at 0-5℃ and the pH at 8.8-9.2. After the dropwise addition was completed, the reaction was continued at 0-5℃ for 2.5 hours, and then the temperature was raised to 24℃ for 6 hours. After the reaction was completed, 1 mol / L hydrochloric acid aqueous solution was added to adjust the pH of the system to 6.2-6.5. The inorganic small molecule salts were removed by conventional nanofiltration membrane with a molecular weight cutoff of 200 Da, and the solution was concentrated to a solid mass fraction of 38%. Then, 2500 g of 95% ethanol was added for precipitation. After filtration, the solution was dried at 42℃ and a vacuum degree not higher than -0.08 MPa for 7 hours to obtain ascorbic acid phosphate intermediate.

[0063] In step (II), 95g of ascorbic acid phosphate intermediate, 115g of lauric acid, 40g of 3Å molecular sieve activated at 250℃ for 4h and cooled to room temperature, 650g of tert-amyl alcohol, and 50g of dimethyl sulfoxide were added to a dry reaction vessel and stirred for 28min under nitrogen protection. Then, 17g of immobilized lipase was added, and the reaction was carried out at 43℃ for 9h, maintaining nitrogen protection during the reaction and controlling the moisture content of the system to be no higher than 1% by mass using the 3Å molecular sieve. After the reaction, the immobilized lipase and 3Å molecular sieve were removed by filtration, the filter cake was washed with tert-amyl alcohol, and the filtrates were combined and concentrated under reduced pressure below 45℃ until no obvious solvent distillation occurred. Adding... Add 290g of anhydrous ethanol and 190g of purified water, stir and disperse, then add 100g of calcium-magnesium alkaline slurry prepared in advance from 4g of calcium hydroxide, 2g of magnesium hydroxide and purified water, controlling the addition time to 28min, and continue stirring for 35min after addition to stabilize the pH of the system at 6.5-6.8; then add 950g of anhydrous ethanol, let stand at 4-10℃ for 2h for precipitation, filter and collect the precipitate, and wash it successively with 480g of 80% ethanol aqueous solution and 480g of anhydrous ethanol, and dry it at 42℃ and vacuum degree not higher than -0.08MPa for 9h to obtain phosphorylated-laurylated calcium magnesium ascorbate;

[0064] In step (iii), purified water, 1g of glacial acetic acid, and 14g of chitosan oligosaccharide lactate were added to a container and stirred at 25°C for 2 hours. Then, a 10% sodium hydroxide aqueous solution was added to stabilize the pH at 5.5-5.8, and purified water was added to bring the total mass to 500g, resulting in a short-chain chitosan dispersion. Separately, purified water, 3g of glacial acetic acid, and 9g of long-chain chitosan were added to a container and stirred at 25°C for 6 hours. Then, a 10% sodium hydroxide aqueous solution was added to stabilize the pH at 5.5-5.8, and purified water was added to bring the total mass to 500g, resulting in a long-chain chitosan dispersion. Then, purified water, 1g of glacial acetic acid, and 5g of sodium phytate hydrate were added to a container and stirred for 35 minutes until completely dissolved, resulting in 300g of sodium phytate segmented locking solution. This solution was then divided into 95g of the first sodium phytate segmented locking solution and 205g of the second sodium phytate segmented locking solution.

[0065] In step (iv), 940g of purified water and 500g of anhydrous ethanol were added to the dispersion container and stirred until homogeneous. Then, 240g of phosphorylated-laurylated calcium magnesium ascorbate was added, and the mixture was stirred for 18 minutes at 23°C and 580 rpm to form a uniform dispersion. Then, 500g of short-chain chitosan dispersion was added over 18 minutes, maintaining a stirring speed of 680 rpm and a pH of 5.5-5.8 during the addition. After the addition was complete, stirring was continued for 28 minutes. Next, 95g of the first sodium phytate fractional locking solution was added over 9 minutes, and stirring was continued for 12 minutes. Then, 500g of long-chain chitosan dispersion was added over 28 minutes, and stirring was continued for 38 minutes. Finally, 205g of the second sodium phytate fractional locking solution was added over 28 minutes, and stirring was continued for 45 minutes. The mixture was then stirred at 36°C and a vacuum degree not exceeding -0.06 MPa. Ethanol was removed under reduced pressure until no obvious ethanol was distilled off and the ethanol mass fraction in the initial dispersion was no higher than 0.5%. After removing ethanol under reduced pressure, the system was cooled to 25°C, and the pH was adjusted to 5.5-5.8 with a 1% acetic acid aqueous solution. Purified water was added, and the mixture was stirred at 950 rpm for 9 min to redisperse the particles evenly. The particles were then filtered through an 80-mesh sieve to remove a small amount of large particle agglomerates, and the dispersion under the sieve was collected. The dispersion under the sieve was centrifuged at 3800 rpm for 9 min, the supernatant was discarded, and the wet particles were collected. The wet particles were washed once with 480 g of purified water, and centrifuged again at 3800 rpm for 9 min, and the supernatant was discarded. The obtained wet particles were dehydrated at low speed for 18 min at 25°C and a vacuum degree no higher than -0.04 MPa to control the water content of the wet particles to 60%, thus obtaining modified ascorbic acid immune-enhancing particles.

[0066] In step (5), 760g of modified ascorbic acid immune-enhancing microparticles were added to 1700g of purified water and stirred for 18min at 25℃ and 950rpm to fully disperse the wet microparticles, resulting in a redispersible solution of modified ascorbic acid immune-enhancing microparticles. 510g of pregelatinized corn starch and 190g of maltodextrin were added and stirred at 25℃ for 28min. Subsequently, conventional spray drying was performed, with the inlet air temperature controlled at 105-108℃ and the outlet air temperature controlled at 58-60℃. After drying, the solution was further dried at 40℃ and under a vacuum degree not exceeding -0.08MPa for 4h. The product was then granulated through a 40-mesh sieve to obtain a feed additive that enhances the immunity of ruminants. The remaining conditions were the same as in Example 1.

[0067] The difference between Example 3 and Example 1 is as follows:

[0068] In step (1), 920g of purified water deoxygenated by bubbling under nitrogen for 30 min was added to a reaction vessel equipped with a mechanical stirrer, thermometer, and dropping device. 184g of L-ascorbic acid was added at 0-5℃, and the mixture was stirred in the dark until completely dissolved. Then, 88g of sodium bicarbonate was added in three portions, waiting for the bubbles to weaken significantly after each addition before continuing. After all additions were completed, stirring was continued for 22 min. Then, a 20% sodium hydroxide aqueous solution was added over 32 min to maintain the pH of the system at 8.8-9.2. Separately, 320g of purified water deoxygenated by bubbling under nitrogen for 30 min was added to 192g of sodium trimetaphosphate, and the mixture was stirred until completely dissolved to obtain a sodium trimetaphosphate aqueous solution. Under continuous nitrogen purging and in the dark, the sodium trimetaphosphate aqueous solution was added dropwise to the ascorbate solution over 95 min. During the dropwise addition, the temperature was maintained at 0-5℃ and the pH at 8.8-9.2. After the dropwise addition was completed, the reaction was continued at 0-5℃ for 2.5 hours, and then the temperature was raised to 26℃ for 6.5 hours. After the reaction was completed, 1 mol / L hydrochloric acid aqueous solution was added to adjust the pH of the system to 6.2-6.5. The inorganic small molecule salts were removed by conventional nanofiltration membrane with a molecular weight cutoff of 200 Da, and the mixture was concentrated to a solid mass fraction of 42%. Then, 2700 g of 95% ethanol was added for precipitation. After filtration, the mixture was dried at 46℃ and a vacuum degree not exceeding -0.08 MPa for 9 hours to obtain ascorbic acid phosphate intermediate.

[0069] In step (II), 105g of ascorbic acid phosphate intermediate, 130g of lauric acid, 50g of 3Å molecular sieve activated at 250℃ for 4h and cooled to room temperature, 680g of tert-amyl alcohol, and 70g of dimethyl sulfoxide were added to a dry reaction vessel and stirred for 32min under nitrogen protection. Then, 20g of immobilized lipase was added, and the reaction was carried out at 45℃ for 11h, maintaining nitrogen protection during the reaction and controlling the moisture content of the system to be no higher than 1% by mass using the 3Å molecular sieve. After the reaction, the immobilized lipase and 3Å molecular sieve were removed by filtration, the filter cake was washed with tert-amyl alcohol, and the filtrates were combined and concentrated under reduced pressure below 45℃ until no obvious solvent distillation occurred. Adding... Add 310g of anhydrous ethanol and 210g of purified water, stir and disperse, then add 100g of calcium-magnesium alkaline slurry prepared in advance from 6g of calcium hydroxide, 2g of magnesium hydroxide and purified water, controlling the addition time to 32min. After addition, continue stirring for 45min to stabilize the pH of the system at 6.5-6.8. Then add 1050g of anhydrous ethanol, let stand at 4-10℃ for 3h for precipitation, filter and collect the precipitate, and wash it successively with 520g of 80% ethanol aqueous solution and 520g of anhydrous ethanol. After washing, dry at 46℃ and vacuum degree not higher than -0.08MPa for 11h to obtain phosphorylated-laurylated calcium magnesium ascorbate.

[0070] In step (iii), purified water, 2g of glacial acetic acid, and 16g of chitosan oligosaccharide lactate were added to a container and stirred at 25°C for 2.5h. Then, a 10% sodium hydroxide aqueous solution was added to stabilize the pH at 5.5-5.8, and purified water was added to bring the total mass to 500g, resulting in a short-chain chitosan dispersion. Separately, purified water, 4g of glacial acetic acid, and 11g of long-chain chitosan were added to a container and stirred at 25°C for 7h. Then, a 10% sodium hydroxide aqueous solution was added to stabilize the pH at 5.5-5.8, and purified water was added to bring the total mass to 500g, resulting in a long-chain chitosan dispersion. Then, purified water, 1g of glacial acetic acid, and 6g of sodium phytate hydrate were added to a container and stirred for 45min until completely dissolved, resulting in 300g of sodium phytate segmented locking solution. This solution was then divided into 105g of the first sodium phytate segmented locking solution and 195g of the second sodium phytate segmented locking solution.

[0071] In step (iv), 1000g of purified water and 550g of anhydrous ethanol were added to the dispersion container and stirred until homogeneous. Then, 260g of phosphorylated-laurylated calcium magnesium ascorbate was added, and the mixture was stirred for 22 minutes at 24℃ and 620rpm to form a uniform dispersion. Then, 500g of short-chain chitosan dispersion was added over 22 minutes, maintaining a stirring speed of 720rpm and a pH of 5.5-5.8 during the addition. After the addition was complete, stirring was continued for 32 minutes. Next, 105g of the first sodium phytate fractional locking solution was added over 11 minutes, and stirring was continued for 18 minutes. Then, 500g of long-chain chitosan dispersion was added over 32 minutes, and stirring was continued for 42 minutes. Finally, 195g of the second sodium phytate fractional locking solution was added over 32 minutes, and stirring was continued for 48 minutes. The mixture was stirred at 38℃ and a vacuum degree not exceeding -0.06MPa. Ethanol was removed under reduced pressure until no obvious ethanol was distilled off and the ethanol mass fraction in the initial dispersion was no higher than 0.5%. After removing ethanol under reduced pressure, the system was cooled to 25°C, and the pH was adjusted to 5.5-5.8 with a 1% acetic acid aqueous solution. Purified water was added, and the mixture was stirred at 1050 rpm for 11 min to redisperse the particles evenly. The particles were then filtered through an 80-mesh sieve to remove a small amount of large particle agglomerates, and the dispersion under the sieve was collected. The dispersion under the sieve was centrifuged at 4200 rpm for 11 min, the supernatant was discarded, and the wet particles were collected. The wet particles were washed once with 520 g of purified water, and centrifuged again at 4200 rpm for 11 min, and the supernatant was discarded. The obtained wet particles were dehydrated at low speed for 22 min at 25°C and a vacuum degree no higher than -0.04 MPa to control the water content of the wet particles to 62%, thus obtaining modified ascorbic acid immune-enhancing particles.

[0072] In step (5), 840g of modified ascorbic acid immune-enhancing microparticles were added to 1900g of purified water and stirred for 22min at 25℃ and 1050rpm to fully disperse the wet microparticles, resulting in a redispersible solution of modified ascorbic acid immune-enhancing microparticles. 530g of pregelatinized corn starch and 210g of maltodextrin were added and stirred for 32min at 25℃. Subsequently, conventional spray drying was performed, with the inlet air temperature controlled at 108-110℃ and the outlet air temperature controlled at 60-62℃. After drying, the solution was further dried at 40℃ and a vacuum degree not exceeding -0.08MPa for 5h. The product was then granulated through a 40-mesh sieve to obtain a feed additive that enhances the immunity of ruminants. The remaining conditions were the same as in Example 1.

[0073] The difference between Example 4 and Example 1 is as follows:

[0074] In step (1), 850g of purified water deoxygenated by bubbling under nitrogen for 30 minutes was added to a reaction vessel equipped with a mechanical stirrer, thermometer, and dropping device. 160g of L-ascorbic acid was added at 0-5℃, and the mixture was stirred in the dark until completely dissolved. Then, 76g of sodium bicarbonate was added in three portions, waiting for the bubbles to weaken significantly after each addition before continuing. After all additions were completed, stirring was continued for 15 minutes. Then, a 20% sodium hydroxide aqueous solution was added over 25 minutes to maintain the pH of the system at 8.8-9.2. Separately, 280g of purified water deoxygenated by bubbling under nitrogen for 30 minutes was added to 170g of sodium trimetaphosphate, and the mixture was stirred until completely dissolved to obtain a sodium trimetaphosphate aqueous solution. Under continuous nitrogen purging and in the dark, the sodium trimetaphosphate aqueous solution was added dropwise to the ascorbate solution over 80 minutes. During the dropwise addition of the solution, the temperature was maintained at 0-5℃ and the pH at 8.8-9.2. After the dropwise addition was completed, the reaction was continued at 0-5℃ for 2 hours, and then the temperature was raised to 23℃ for 5 hours. After the reaction was completed, 1 mol / L hydrochloric acid aqueous solution was added to adjust the pH of the system to 6.2-6.5. The inorganic small molecule salts were removed by conventional nanofiltration membrane with a molecular weight cutoff of 200 Da, and the solution was concentrated to a solid mass fraction of 35%. Then, 2400 g of 95% ethanol was added for precipitation. After filtration, the solution was dried at 40℃ and a vacuum degree not exceeding -0.08 MPa for 6 hours to obtain ascorbic acid phosphate intermediate.

[0075] In step (II), 90g of ascorbic acid phosphate intermediate, 105g of lauric acid, 35g of 3Å molecular sieve activated at 250℃ for 4h and cooled to room temperature, 620g of tert-amyl alcohol, and 40g of dimethyl sulfoxide were added to a dry reaction vessel. The mixture was stirred for 25min under nitrogen protection to form a uniform dispersion of the ascorbic acid phosphate intermediate. Subsequently, 15g of immobilized lipase was added, and the reaction was carried out at 42℃ for 8h. During the reaction, nitrogen protection was maintained, and the moisture content of the system was controlled to be no more than 1% by mass using the 3Å molecular sieve. After the reaction, the immobilized lipase and 3Å molecular sieve were removed by filtration. The filter cake was washed with tert-amyl alcohol, and the filtrates were combined and concentrated under reduced pressure below 45℃ until no obvious solvent was detected. Distillation; Add 280g anhydrous ethanol and 180g purified water to the concentrated residue, stir to disperse, then add 100g of calcium magnesium alkaline slurry prepared in advance from 4g calcium hydroxide, 1g magnesium hydroxide and purified water, controlling the addition time to 25min, and continue stirring for 30min after addition to stabilize the pH of the system at 6.5-6.8; then add 900g anhydrous ethanol, let stand at 4℃ for 2h for precipitation, filter to collect the precipitate, and wash it successively with 450g of 80% ethanol aqueous solution and 450g of anhydrous ethanol, and dry it at 40℃ and vacuum degree not higher than -0.08MPa for 8h to obtain phosphorylated-laurylated calcium magnesium ascorbate;

[0076] In step (iii), purified water, 1g of glacial acetic acid, and 12g of chitosan oligosaccharide lactate were added to a container and stirred at 25°C for 1.5h. Then, a 10% sodium hydroxide aqueous solution was added to stabilize the pH at 5.5-5.8, and purified water was added to bring the total mass to 500g, resulting in a short-chain chitosan dispersion. Separately, purified water, 2g of glacial acetic acid, and 8g of long-chain chitosan were added to a container and stirred at 25°C for 5h. Then, a 10% sodium hydroxide aqueous solution was added to stabilize the pH at 5.5-5.8, and purified water was added to bring the total mass to 500g, resulting in a long-chain chitosan dispersion. Then, purified water, 1g of glacial acetic acid, and 5g of sodium phytate hydrate were added to a container and stirred for 30min until completely dissolved, resulting in 300g of sodium phytate segmented locking solution. This solution was then divided into 90g of the first sodium phytate segmented locking solution and 210g of the second sodium phytate segmented locking solution.

[0077] In step (iv), 900g of purified water and 475g of anhydrous ethanol were added to the dispersion container and stirred until homogeneous. Then, 225g of phosphorylated-laurylated calcium magnesium ascorbate was added, and the mixture was stirred for 15 minutes at 22℃ and 550rpm to form a uniform dispersion. Then, 500g of short-chain chitosan dispersion was added over 15 minutes, maintaining a stirring speed of 650rpm and a pH of 5.5-5.8 during the addition. After the addition was complete, stirring was continued for 25 minutes. Next, 90g of the first sodium phytate fractional locking solution was added over 8 minutes, and stirring was continued for 10 minutes. Then, 500g of long-chain chitosan dispersion was added over 25 minutes, and stirring was continued for 35 minutes. Finally, 210g of the second sodium phytate fractional locking solution was added over 25 minutes, and stirring was continued for 40 minutes. The mixture was then stirred at 35℃ and a vacuum degree not exceeding -0.06MPa. Ethanol was removed under reduced pressure until no obvious ethanol was distilled off and the ethanol mass fraction in the initial dispersion was no higher than 0.5%. After removing ethanol under reduced pressure, the system was cooled to 25°C, and the pH was adjusted to 5.5-5.8 with a 1% acetic acid aqueous solution. Purified water was added, and the mixture was stirred at 900 rpm for 8 min to redisperse the particles evenly. The particles were then filtered through an 80-mesh sieve to remove a small amount of large particle agglomerates, and the dispersion under the sieve was collected. The dispersion under the sieve was centrifuged at 3500 rpm for 8 min, the supernatant was discarded, and the wet particles were collected. The wet particles were washed once with 450 g of purified water, and centrifuged again at 3500 rpm for 8 min, and the supernatant was discarded. The obtained wet particles were dehydrated at low speed for 15 min at 25°C and a vacuum degree no higher than -0.04 MPa to control the water content of the wet particles to 55%, thus obtaining modified ascorbic acid immune-enhancing particles.

[0078] In step (5), 720g of modified ascorbic acid immune-enhancing microparticles were added to 1600g of purified water and stirred for 15min at 25℃ and 900rpm to fully disperse the wet microparticles, resulting in a redispersible solution of modified ascorbic acid immune-enhancing microparticles. 500g of pregelatinized corn starch and 180g of maltodextrin were added and stirred for 25min at 25℃. Subsequently, conventional spray drying was performed, with the inlet air temperature controlled at 105℃ and the outlet air temperature controlled at 58℃. After drying, the solution was further dried at 40℃ and a vacuum degree not exceeding -0.08MPa for 3h. The product was then granulated through a 40-mesh sieve to obtain a feed additive that enhances the immunity of ruminants.

[0079] The difference between Example 5 and Example 1 is as follows: In step (i), 950g of purified water deoxygenated by bubbling under nitrogen for 30 minutes was added to a reaction vessel equipped with a mechanical stirrer, thermometer, and dropping device. 190g of L-ascorbic acid was added at 0-5℃, and the mixture was stirred in the dark until completely dissolved. Then, 92g of sodium bicarbonate was added in three portions, with the addition continuing only after the bubbles significantly weakened. After all additions were completed, stirring continued for 25 minutes. Then, a 20% sodium hydroxide aqueous solution was added over 35 minutes to maintain the pH of the system at 8.8-9.2. Separately, 330g of purified water deoxygenated by bubbling under nitrogen for 30 minutes was added to 200g of sodium trimetaphosphate, and the mixture was stirred until completely dissolved to obtain a sodium trimetaphosphate aqueous solution. Under continuous nitrogen purging and in the dark, the sodium trimetaphosphate aqueous solution was added dropwise to the ascorbate solution over 100 minutes. During the dropwise addition of the solution, the temperature was maintained at 0-5℃ and the pH at 8.8-9.2. After the dropwise addition was completed, the reaction was continued at 0-5℃ for 3 hours, and then the temperature was raised to 27℃ for 7 hours. After the reaction was completed, 1 mol / L hydrochloric acid aqueous solution was added to adjust the pH of the system to 6.2-6.5. The inorganic small molecule salts were removed by conventional nanofiltration membrane with a molecular weight cutoff of 200 Da, and the solution was concentrated to a solid mass fraction of 45%. Then, 2800 g of 95% ethanol was added for precipitation. After filtration, the solution was dried at 48℃ and a vacuum degree not higher than -0.08 MPa for 10 hours to obtain ascorbic acid phosphate intermediate.

[0080] In step (II), 110g of ascorbic acid phosphate intermediate, 135g of lauric acid, 55g of 3Å molecular sieve activated at 250℃ for 4h and cooled to room temperature, 700g of tert-amyl alcohol, and 80g of dimethyl sulfoxide were added to a dry reaction vessel. The mixture was stirred for 35min under nitrogen protection to form a uniform dispersion of the ascorbic acid phosphate intermediate. Subsequently, 22g of immobilized lipase was added, and the reaction was carried out at 46℃ for 12h. During the reaction, nitrogen protection was maintained, and the moisture content of the system was controlled to be no more than 1% by mass using the 3Å molecular sieve. After the reaction, the immobilized lipase and 3Å molecular sieve were removed by filtration. The filter cake was washed with tert-amyl alcohol, and the filtrates were combined and concentrated under reduced pressure below 45℃ until no obvious solvent was detected. Distillation; Add 320g anhydrous ethanol and 220g purified water to the concentrated residue, stir and disperse, then add 100g of calcium magnesium alkaline slurry prepared in advance from 6g calcium hydroxide, 3g magnesium hydroxide and purified water, controlling the addition time to 35min, and continue stirring for 50min after addition to stabilize the pH of the system at 6.5-6.8; then add 1100g anhydrous ethanol, let stand at 10℃ for 3h for precipitation, filter and collect the precipitate, and wash it successively with 550g of 80% ethanol aqueous solution and 550g of anhydrous ethanol, and dry it for 12h at 48℃ and a vacuum degree not higher than -0.08MPa to obtain phosphorylated-laurylated calcium magnesium ascorbate;

[0081] In step (iii), purified water, 2g of glacial acetic acid, and 18g of chitosan oligosaccharide lactate were added to a container and stirred at 25°C for 2.5h. Then, a 10% sodium hydroxide aqueous solution was added to stabilize the pH at 5.5-5.8, and purified water was added to bring the total mass to 500g, resulting in a short-chain chitosan dispersion. Separately, purified water, 4g of glacial acetic acid, and 12g of long-chain chitosan were added to a container and stirred at 25°C for 7h. Then, a 10% sodium hydroxide aqueous solution was added to stabilize the pH at 5.5-5.8, and purified water was added to bring the total mass to 500g, resulting in a long-chain chitosan dispersion. Then, purified water, 1g of glacial acetic acid, and 7g of sodium phytate hydrate were added to a container and stirred for 50min until completely dissolved, resulting in 300g of sodium phytate segmented locking solution, which was then divided into 110g of the first sodium phytate segmented locking solution and 190g of the second sodium phytate segmented locking solution.

[0082] In step (iv), 1050g of purified water and 575g of anhydrous ethanol were added to the dispersion container and stirred until homogeneous. Then, 275g of phosphorylated-laurylated calcium magnesium ascorbate was added, and the mixture was stirred for 25 minutes at 25°C and 650 rpm to form a uniform dispersion. Then, 500g of short-chain chitosan dispersion was added over 25 minutes, maintaining a stirring speed of 750 rpm and a pH of 5.5-5.8 during the addition. After the addition was complete, stirring was continued for 35 minutes. Next, 110g of the first sodium phytate fractional locking solution was added over 12 minutes, and stirring was continued for 20 minutes. Then, 500g of long-chain chitosan dispersion was added over 35 minutes, and stirring was continued for 45 minutes. Finally, 190g of the second sodium phytate fractional locking solution was added over 35 minutes, and stirring was continued for 50 minutes. The mixture was stirred at 40°C and a vacuum degree not exceeding -0.06 MPa. Ethanol was removed under reduced pressure until no obvious ethanol was distilled off and the ethanol mass fraction in the initial dispersion was no higher than 0.5%. After removing ethanol under reduced pressure, the system was cooled to 25°C, and the pH was adjusted to 5.5-5.8 with a 1% acetic acid aqueous solution. Purified water was added, and the system was stirred at 1100 rpm for 12 min to redisperse the particles evenly. The particles were then filtered through an 80-mesh sieve to remove a small amount of large particle agglomerates, and the dispersion under the sieve was collected. The dispersion under the sieve was centrifuged at 4500 rpm for 12 min, the supernatant was discarded, and the wet particles were collected. The wet particles were washed once with 550 g of purified water, and centrifuged again at 4500 rpm for 12 min, and the supernatant was discarded. The obtained wet particles were dehydrated at low speed at 25°C and a vacuum degree no higher than -0.04 MPa for 25 min to control the water content of the wet particles to 70%, thus obtaining modified ascorbic acid immune-enhancing particles.

[0083] In step (5), 880g of modified ascorbic acid immune-enhancing microparticles were added to 2000g of purified water and stirred for 25min at 25℃ and 1100rpm to fully disperse the wet microparticles, resulting in a redispersible solution of modified ascorbic acid immune-enhancing microparticles. 540g of pregelatinized corn starch and 220g of maltodextrin were added and stirred for 35min at 25℃. Subsequently, conventional spray drying was performed, with the inlet air temperature controlled at 110℃ and the outlet air temperature controlled at 62℃. After drying, the solution was further dried at 40℃ and a vacuum degree not exceeding -0.08MPa for 5h. The product was then granulated through a 40-mesh sieve to obtain a feed additive that enhances the immunity of ruminants.

[0084] The difference between Comparative Example 1 and Example 1 is that: the preparation of the ascorbic acid phosphate intermediate described in step (i) is not performed; in step (ii), the ascorbic acid phosphate intermediate is replaced with an equal amount of L-ascorbic acid; in step (iv), the short-chain chitosan dispersion, the first sodium phytate segmented locking solution, the long-chain chitosan dispersion, and the second sodium phytate segmented locking solution are added in the same order as in Example 1; the remaining conditions are the same as in Example 1.

[0085] The difference between Comparative Example 2 and Example 1 is that lauroylation is not performed in step (II). 100g of the ascorbic acid phosphate intermediate obtained in step (I) is directly added to 300g of anhydrous ethanol and 200g of purified water for dispersion. Then, a calcium-magnesium alkaline slurry prepared in advance from 5g of calcium hydroxide, 2g of magnesium hydroxide, and 100g of purified water is added, with the addition time controlled at 30min. After addition, stirring continues for 40min to stabilize the pH of the system at 6.5-6.8. Subsequently, 1000g of anhydrous ethanol is added. Alcohol was allowed to stand for 2 hours for precipitation. The precipitate was collected by filtration and washed successively with 500g of 80% ethanol aqueous solution and 500g of anhydrous ethanol. After washing, it was dried at 45°C and a vacuum degree not higher than -0.08MPa for 10 hours to obtain unlaurylated calcium magnesium ascorbate phosphate. In step (iv), 250g of phosphorylated-laurylated calcium magnesium ascorbate was replaced with an equal amount of 250g of unlaurylated calcium magnesium ascorbate phosphate. The remaining conditions were the same as in Example 1.

[0086] The difference between Comparative Example 3 and Example 1 is that in step (iii), instead of preparing short-chain chitosan dispersion and long-chain chitosan dispersion separately, 1000g of single long-chain chitosan dispersion is prepared. The preparation method of the single long-chain chitosan dispersion is as follows: add purified water, 4g of glacial acetic acid and 25g of long-chain chitosan to a container, stir at 25°C for 6h, then add 10% sodium hydroxide aqueous solution to stabilize the pH at 5.5-5.8, and add purified water to make the total mass of the system 1000g, thus obtaining 1000g of single long-chain chitosan dispersion. In step (iv), add 500g of single long-chain chitosan dispersion at the same position where 500g of short-chain chitosan dispersion was originally added, and add 500g of single long-chain chitosan dispersion at the same position where 500g of long-chain chitosan dispersion was originally added. The other conditions are the same as in Example 1.

[0087] The difference between Comparative Example 4 and Example 1 is as follows: In step (iii), 300g of sodium phytate segmented locking solution is still prepared, but it is not divided into a first sodium phytate segmented locking solution and a second sodium phytate segmented locking solution; In step (iv), after adding 500g of short-chain chitosan dispersion and continuing to stir for 30min, 300g of sodium phytate segmented locking solution is added all at once within 30min. After the addition is completed, stirring is continued for 15min, then 500g of long-chain chitosan dispersion is added and stirring is continued for 40min. No second sodium phytate segmented locking solution is added afterward; the other conditions are the same as in Example 1.

[0088] The difference between Comparative Example 5 and Example 1 is that the order of adding chitosan and sodium phytate in step (iv) is changed. Specifically, after a uniform dispersion is formed, 500g of long-chain chitosan dispersion is added within 30 minutes, and stirring is continued for 40 minutes after the addition is completed; then 300g of sodium phytate segmented locking solution is added at once within 30 minutes, and stirring is continued for 45 minutes after the addition is completed; finally, 500g of short-chain chitosan dispersion is added within 20 minutes, and stirring is continued for 30 minutes after the addition is completed; the other conditions are the same as in Example 1.

[0089] The difference between Comparative Example 6 and Example 1 is as follows: In step (iii), instead of using 6g of sodium phytate hydrate, 300g of purified water is used in place of the 300g sodium phytate fractional locking solution; in step (iv), 100g of purified water is added at the original position where 100g of the first sodium phytate fractional locking solution was added, and 200g of purified water is added at the original position where 200g of the second sodium phytate fractional locking solution was added, to maintain a consistent total amount of liquid added. All other conditions are the same as in Example 1.

[0090] Performance testing

[0091] The feed additives for enhancing the immunity of ruminants obtained in Examples 1 to 5 and Comparative Examples 1 to 6 were used as test samples, and three independent batches were prepared for each sample. Before testing, sampling and sample preparation were carried out in accordance with GB / T 14699-2023 "Feed Sampling" and GB / T 20195-2024 "Preparation of Animal Feed Samples". At least 500g of sample was taken from each independent batch, and after being reduced by quartering, it was used for testing moisture, particle size, residual solvent, ascorbic acid equivalent content, pelleting stability, simulated rumen stability, post-release, and neutrophil phagocytic activity. The test results were recorded as the average of the three independent batches, and the allowable error for judging the feed test results was in accordance with GB / T 18823-2010 "Allowable Error for Judging Feed Test Results".

[0092] Moisture and particle size distribution tests: Moisture content was tested according to GB / T 6435-2014 "Determination of Moisture in Feed"; particle size distribution was tested according to GB / T 19077-2024 "Particle Size Analysis by Laser Diffraction". For moisture testing, 5g of each sample was taken, dried to constant weight according to the standard, and the moisture mass fraction was calculated. For particle size testing, 0.2g of each sample was added to 200mL of purified water, stirred at 500rpm for 3min at 25℃, then ultrasonically dispersed for 60s, and laser particle size analysis was performed immediately, and the D50 was recorded.

[0093] Total residual solvent test: The test was conducted according to the headspace gas chromatography principle of GB 5009.262-2016 "National Food Safety Standard - Determination of Residual Solvents in Food". 1 g of each sample was placed in a 20 mL headspace vial, and 5 mL of N,N-dimethylacetamide was added as the solvent. After sealing, the vial was equilibrated at 80℃ for 30 min. Ethanol, tert-amyl alcohol, and dimethyl sulfoxide were detected using headspace gas chromatography. External standard curves were established using ethanol, tert-amyl alcohol, and dimethyl sulfoxide standards, respectively, and the total residual solvent content was calculated.

[0094] Ascorbic acid equivalent content test: The test was conducted according to the liquid chromatography method in GB 5009.86-2025 "National Food Safety Standard - Determination of Ascorbic Acid in Food", with an added enzymatic release step in the pretreatment stage. Take 0.500 g of each sample, add 25 mL of pH 7.4 phosphate buffer, 1000 U alkaline phosphatase, and 1000 U carboxylesterase, and enzymatically hydrolyze at 37℃ and 100 rpm for 2 h in the dark to release the phosphate ester bond and lauroyl ester bond into measurable ascorbic acid. Then, add 25 mL of 3% (w / w) metaphosphoric acid aqueous solution to terminate the reaction and make up to volume. Centrifuge at 12000 rpm for 10 min, and filter the supernatant through a 0.22 μm filter membrane before injection for detection. The liquid chromatography conditions were: octadecylsilane-bonded silica gel column, column temperature 30℃, mobile phase 0.1% (w / w) oxalic acid aqueous solution, flow rate 1.0 mL / min, detection wavelength 245 nm, and injection volume 10 μL.

[0095] Granulation stability test: Samples obtained from Examples 1 to 5 and Comparative Examples 1 to 6 were added to the basic pelleted feed material for weaned calves at an ascorbic acid equivalent of 100 mg / kg. The mixture was mixed at 60 rpm for 8 minutes using a horizontal mixer. The mixture was then steam-conditioned at 75°C for 90 seconds, followed by pelleting using a ring die pellet mill with a 3 mm aperture and a compression ratio of 1:8. After cooling to room temperature, the pellets were sieved to remove powder. The ascorbic acid equivalent content in the mixture before pelleting and in the pellets after pelleting were measured. The retention rate after pelleting was calculated as "ascorbic acid equivalent content in the pellets after pelleting / ascorbic acid equivalent content in the mixture before pelleting × 100%".

[0096] Simulated rumen stability and post-flank release test: 1.000 g of each sample obtained from Examples 1 to 5 and Comparative Examples 1 to 6 were placed in nylon bags with a pore size of 45 μm. Simulated rumen buffer was prepared, containing 9.8 g sodium bicarbonate, 7.0 g disodium hydrogen phosphate dodecahydrate, 0.57 g potassium chloride, 0.47 g sodium chloride, 0.12 g magnesium sulfate heptahydrate, and 0.04 g calcium chloride per 1000 mL of simulated rumen buffer. The buffer was then purged with carbon dioxide for 20 min, and the pH was adjusted to 6.8. The nylon bags containing the samples were added to 100 mL of simulated rumen buffer and shaken anaerobically at 39 °C and 120 rpm for 8 h. After removing the nylon bags, the ascorbic acid equivalent in the residual samples was measured, and the 8-h rumen retention rate was calculated. The sample, after being treated with a simulated rumen, was then transferred to 100 mL of simulated abomasum fluid, which was an aqueous solution of hydrochloric acid containing 3 g / L pepsin at pH 2.5, and treated at 39 °C and 120 rpm for 2 h. Next, it was transferred to 100 mL of simulated small intestinal fluid, which was a phosphate buffer containing 10 g / L pancreatin and 3 g / L bile salts at pH 6.8, and treated at 39 °C and 120 rpm for 4 h. The ascorbic acid equivalent in the released fluid was measured, and the cumulative release rate from the simulated abomasum to the small intestine was calculated.

[0097] Neutrophil phagocytic activity assay: Anticoagulated peripheral blood was collected from six healthy weaned Holstein calves. Neutrophils were isolated using bovine peripheral blood neutrophil separation medium and adjusted to a cell concentration of 1×10⁻⁶ cells using RPMI 1640 medium. 6 The simulated abomasum-small intestine release solution was filtered through a 0.22 μm filter membrane and then added to the cell suspension at the same volume fraction as the simulated digestion release solution obtained from the same initial sample mass. The mixture was incubated at 37°C and 5% CO2 for 30 min. Fluorescently labeled *E. coli* particles were then added to achieve a bacterial particle to neutrophil ratio of 10:1, and incubation continued for another 30 min. 0.2% trypan blue solution was added to quench the exogenous fluorescence of unphagocytosed particles, and flow cytometry was used to detect at least 10,000 neutrophils per sample. The phagocytic index of neutrophils in each sample group was calculated, with the phagocytic index of the blank culture group recorded as 100%.

[0098] Table 1 Performance Test Results

[0099]

[0100] Data Analysis: Table 1 shows that Comparative Example 1, which did not undergo ascorbic acid phosphorylation, still exhibited a certain degree of granulation stability after lauroylation and subsequent chitosan and sodium phytate treatment. However, its retention rate after granulation, 8-hour retention rate in the simulated rumen, and neutrophil phagocytic index were all lower than those of Example 1. This indicates that without phosphate groups, ascorbic acid is more easily oxidized or diffused in the granulation hot pressing and rumen environment, and it is difficult to form inner-layer positioning sites for preferential adsorption of short-chain chitosan. Comparative Example 2, which did not undergo lauroylation, retained phosphate ester characteristics, but its rumen retention was insufficient, and its later-stage release was... The high release rate indicates that simple phosphorylation is prone to premature release along the aqueous phase channel. Comparative Example 3 uses a single long-chain chitosan, Comparative Example 4 adds sodium phytate all at once, Comparative Example 5 changes the addition order, and Comparative Example 6 removes sodium phytate hydrate. All of these results in a decrease in particle size, thermal stability, retention rate after granulation, retention rate in simulated rumen, or neutrophil phagocytic activity. This indicates that the sequential structure of short-chain chitosan first positioning, internal locking in the first sodium phytate segmented locking solution, subsequent film formation of long-chain chitosan, and external locking in the second sodium phytate segmented locking solution cannot be replaced by simple coating or one-time crosslinking.

[0101] Therefore, the feed additive obtained by this invention is suitable for use in the diets of weaned calves, periparturient dairy cows, transport-stressed beef cattle, and high-temperature heat-stressed sheep. It can improve the effective retention of active ingredients after passing through the pelleting and rumen stages at a lower ascorbic acid equivalent addition level, reduce the waste caused by premature oxidation or release of ordinary ascorbic acid, and help improve the nutritional regulation stability of ruminants during the immune stress period.

[0102] Those skilled in the art should understand that the discussion of any of the above embodiments is merely exemplary and is not intended to imply that the scope of the invention is limited to these examples; within the framework of the invention, the technical features of the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations of the different aspects of the invention as described above, which are not provided in detail for the sake of brevity.

Claims

1. A method for preparing a feed additive that enhances the immunity of ruminants, characterized in that, Includes the following steps: S1: L-ascorbic acid is reacted with sodium trimetaphosphate under alkaline, light-protected and nitrogen-protected conditions to obtain ascorbic acid phosphate intermediate; S2: The ascorbate phosphate intermediate is esterified with lauric acid in the presence of immobilized lipase, and then salinated with calcium magnesium alkaline slurry to obtain phosphorylated-laurylated calcium magnesium ascorbate. S3: Prepare short-chain chitosan dispersion, long-chain chitosan dispersion and sodium phytate segmented locking liquid respectively, and divide the sodium phytate segmented locking liquid into a first sodium phytate segmented locking liquid and a second sodium phytate segmented locking liquid; S4: The phosphorylated-laurylated calcium magnesium ascorbate salt is redispersed in the mixture, and then the short-chain chitosan dispersion, the first sodium phytate segmented locking solution, the long-chain chitosan dispersion and the second sodium phytate segmented locking solution are added in sequence to obtain modified ascorbic acid immune-enhancing microparticles. S5: The modified ascorbic acid immune-enhancing microparticles are mixed with pregelatinized corn starch and maltodextrin and then spray-dried to obtain a feed additive that enhances the immunity of ruminants. The ratio of L-ascorbic acid to sodium trimetaphosphate in step S1 is 160-190:170-200 by weight. By weight, the ratio of ascorbic acid phosphate intermediate, lauric acid, immobilized lipase and calcium-magnesium alkaline slurry in step S2 is 90-110:105-135:15-22:100; the calcium-magnesium alkaline slurry, by weight, is prepared from 4-6 parts calcium hydroxide, 1-3 parts magnesium hydroxide and the remainder purified water, with a weight of 100 parts. The ratio of phosphorylated-laurylated calcium magnesium ascorbate, short-chain chitosan dispersion, first sodium phytate segmented locking solution, long-chain chitosan dispersion and second sodium phytate segmented locking solution in step S4 is 225-275:500:90-110:500:190-210 by weight.

2. The preparation method according to claim 1, characterized in that, The preparation of the ascorbic acid phosphate intermediate in step S1 includes: adding L-ascorbic acid to purified water and adjusting the pH of the system to 8.8-9.2, then adding an aqueous solution of sodium trimetaphosphate dropwise to the system; after the sodium trimetaphosphate aqueous solution is added, the reaction continues at 0-5℃ for 2-3 hours, then the temperature is raised to 23-27℃ for 5-7 hours, and the pH is adjusted to 6.2-6.

5. The ascorbic acid phosphate intermediate is obtained by filtration, concentration, and precipitation.

3. The preparation method according to claim 1, characterized in that, The esterification reaction described in step S2 is carried out under a nitrogen atmosphere at a temperature of 42-46°C for 8-12 hours.

4. The preparation method according to claim 1, characterized in that, The calcium-magnesium alkaline slurry salinization in step S2 includes: adding the calcium-magnesium alkaline slurry to a dispersion system formed by the concentrated residue obtained from the esterification reaction, anhydrous ethanol, and purified water, stabilizing the pH of the system at 6.5-6.8, then adding anhydrous ethanol for precipitation, and finally obtaining phosphorylated-laurylated calcium magnesium ascorbate after filtration, washing, and drying.

5. The preparation method according to claim 1, characterized in that, The short-chain chitosan dispersion in step S3 is prepared in 500 parts by weight, consisting of 1-2 parts glacial acetic acid, 12-18 parts chitosan oligosaccharide lactate, and the remainder purified water, with the pH adjusted to 5.5-5.8 using sodium hydroxide aqueous solution; the long-chain chitosan dispersion is prepared in 500 parts by weight, consisting of 2-4 parts glacial acetic acid, 8-12 parts long-chain chitosan, and the remainder purified water, with the pH adjusted to 5.5-5.8 using sodium hydroxide aqueous solution.

6. The preparation method according to claim 1, characterized in that, The chitosan oligosaccharide lactate in step S3 has a number-average molecular weight of 4000 Da-6000 Da and a degree of deacetylation greater than 90%; the long-chain chitosan has an average weight-average molecular weight of 50 kDa.

7. The preparation method according to claim 1, characterized in that, The sodium phytate segmented locking solution in step S3 is prepared in 300 parts by weight, consisting of 1 part glacial acetic acid, 5-7 parts sodium phytate saline, and the remainder purified water. The sodium phytate segmented locking solution includes a first sodium phytate segmented locking solution and a second sodium phytate segmented locking solution. The weight ratio of the first sodium phytate segmented locking solution to the second sodium phytate segmented locking solution is 90-110:190-210.

8. The preparation method according to claim 1, characterized in that, The moisture content of the modified ascorbic acid immune-enhancing microparticles described in step S4 is controlled to be 55%-70%.

9. The preparation method according to claim 1, characterized in that, The inlet air temperature of the spray drying in step S5 is 105-110℃, the outlet air temperature is 58-62℃, and after spray drying, it is dried at 40℃ and vacuum degree not higher than -0.08MPa for 3-5 hours, and then granulated through a 40-mesh sieve.

10. A feed additive for enhancing the immunity of ruminants, characterized in that, The feed additive for enhancing the immunity of ruminants, prepared according to any one of claims 1-9, has a moisture content of no more than 6%, an effective ingredient content of 12% to 16% based on ascorbic acid equivalent, and a median particle size of 25-55 μm.

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