Microalgae compound feed additive for relieving calf weaning stress and preparation method thereof

Abstract

This invention belongs to the field of feed additive technology, specifically relating to a microalgae compound feed additive for alleviating weaning stress in calves and its preparation method. The microalgae compound feed additive comprises the following raw materials in parts by weight: 5-10 parts microalgae powder, 2-6 parts unsaturated fatty acid / modified montmorillonite complex, 0.7-1.2 parts probiotics, and 0.3-0.5 parts walnut shell extract. This microalgae compound feed additive can improve the fragile intestinal barrier function and immunity of weaned calves, enhance their growth performance, and thus improve breeding efficiency.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a microalgae compound feed additive for alleviating weaning stress in calves and its preparation method. Background Technology

[0002] In modern dairy farming, the healthy rearing of calves is the cornerstone of ensuring the lifelong productivity of the herd and the economic benefits of the farm. Weaning is a crucial physiological and nutritional turning point in the growth and development of calves. During this stage, calves undergo a fundamental shift from a diet primarily composed of liquid milk or milk replacers to one primarily composed of solid feeds (such as starter feed and roughage). This abrupt change often triggers severe "weaning stress," posing a serious challenge to the calf's digestive system, immune status, and overall health, becoming a key bottleneck restricting the success rate of early calf rearing.

[0003] Weaning stress is the result of a combination of factors, with its core negative impacts manifesting in three main aspects: diarrhea, mucosal damage, and decreased immunity. These three factors are interconnected, forming a vicious cycle. First, diarrhea is the most common and readily apparent health problem during weaning. The digestive system of calves is not yet fully developed, and the complex plant proteins and carbohydrates (such as starch and non-starch polysaccharides) in the post-weaning diet place enormous pressure on their digestive enzyme system. Incompletely digested nutrients enter the hindgut, leading to a sharp imbalance in the intestinal flora, the proliferation of pathogenic microorganisms (such as E. coli and Salmonella), the production of toxins, and the onset of nutritional and bacterial diarrhea. Diarrhea not only causes nutrient absorption problems, growth retardation, and even weight loss, but in severe cases, it can lead to death due to dehydration and electrolyte imbalance, resulting in direct economic losses for farms.

[0004] Secondly, diarrhea is often accompanied by deeper intestinal mucosal damage. The intestinal mucosa is one of the most important barriers in the body, responsible for nutrient absorption and resisting the invasion of pathogens and toxins. Weaning stress damages the integrity of the mucosa through multiple pathways: (1) Physical and chemical stimulation: The physical structure of solid feed and the anti-nutritional factors it may contain directly stimulate the tender intestinal wall; (2) Inflammatory response: Dietary antigens and dysbiosis can activate excessive intestinal immune inflammatory response, release inflammatory mediators, and damage epithelial cells; (3) Nutritional deficiency: Stress-induced fluctuations in feed intake or poor digestion and absorption may cause intestinal epithelial cells to lack sufficient energy (such as glutamine, short-chain fatty acids) and key nutrients (such as vitamin A, zinc, etc.) to maintain their rapid renewal and repair. Mucosal damage leads to increased intestinal permeability (i.e., "leaky gut"), which further aggravates the translocation of pathogens and endotoxins and amplifies the systemic inflammatory response.

[0005] Furthermore, weaning stress profoundly affects the immune system function of calves. On the one hand, maternal antibody protection has largely faded at this stage, while the calf's own active immune system is still developing, creating an "immune window." On the other hand, weaning stress itself suppresses immune function through neuroendocrine pathways (such as activation of the hypothalamus-pituitary-adrenal axis and increased cortisol levels), reducing lymphocyte activity and antibody production capacity. Simultaneously, the systemic inflammatory response activated to address intestinal damage and potential infection consumes a large amount of nutrients and energy that should be used for growth, leading to an over-congestion of immune resources and creating a "competition between immunity and growth." This weakened immunity reduces the calf's resistance to pathogens, making it more susceptible to respiratory diseases and other secondary infections, further exacerbating its health condition.

[0006] Currently, conventional management strategies for weaning stress in calves mainly focus on gradual ration transition, optimizing the feeding environment, and using feed additives. Among these, feed additives such as probiotics, prebiotics, acidifiers, and enzymes have shown some effectiveness in regulating gut microbiota and promoting digestion. However, given the complex and multifaceted physiological challenges induced by weaning stress (especially the profound needs for mucosal repair, antioxidant activity, and immune regulation), single-component additives often have limited effectiveness. Against this backdrop, this invention provides a microalgae compound feed additive for alleviating weaning stress in calves. Summary of the Invention

[0007] In order to overcome the shortcomings of the prior art, the first objective of this invention is to provide a microalgae compound feed additive that can alleviate weaning stress in calves, improve the fragile intestinal barrier function and immunity of weaned calves, enhance calf growth performance, and thus improve breeding efficiency.

[0008] The second objective of this invention is to provide a method for preparing a microalgae compound feed additive that alleviates weaning stress in calves, which is simple in process.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A microalgae compound feed additive for relieving weaning stress in calves comprises the following raw materials in parts by weight: 5-10 parts microalgae powder, 2-6 parts unsaturated fatty acid / modified montmorillonite compound, 0.7-1.2 parts probiotics, and 0.3-0.5 parts walnut shell extract.

[0011] Preferably, the preparation process of the unsaturated fatty acid / modified montmorillonite complex is as follows:

[0012] (1) Add montmorillonite to an aqueous ethanol solution, then add a silane coupling agent, heat to react, and purify after the reaction is complete to obtain silane coupling agent modified montmorillonite;

[0013] (2) The silane coupling agent modified montmorillonite and oxypullan polysaccharide from step (1) were added to deionized water to react and obtain modified montmorillonite.

[0014] (3) The modified montmorillonite from step (2) is added to deionized water, and then unsaturated fatty acids and emulsifiers are added. After shearing, homogenization and freeze drying, the unsaturated fatty acid / modified montmorillonite complex is obtained.

[0015] Preferably, in step (1), the ratio of montmorillonite, silane coupling agent, and ethanol aqueous solution is 1g:(0.05-0.1)g:(10-15)mL; the concentration of the ethanol aqueous solution is 75wt%; the silane coupling agent is KH-550; and the heating reaction temperature is 50-60℃ and the time is 3-4h.

[0016] Preferably, in step (2), the mass ratio of silane coupling agent-modified montmorillonite to oxidized pullulan polysaccharide is 1:(1-4); the concentration of oxidized pullulan polysaccharide in deionized water is 2-4 wt%; and the reaction time is 5-8 h.

[0017] Preferably, the method for preparing the oxidized pullulan polysaccharide is as follows:

[0018] Pullulan polysaccharide is added to deionized water, followed by sodium periodate. After stirring in the dark, ethylene glycol is added to terminate the reaction. The product is then obtained by dialysis and freeze-drying.

[0019] Preferably, the mass ratio of pullulan, sodium periodate, and deionized water is 1 g: (0.2-0.6) g: (150-200) mL; and the stirring time in the dark is 24-36 h.

[0020] Preferably, the mass ratio of modified montmorillonite, unsaturated fatty acid, emulsifier, and deionized water in step (3) is 5:(20-25):(3-7):(15-20); the unsaturated fatty acid is selected from α-linolenic acid, γ-linolenic acid, and arachidonic acid; and the emulsifier is Tween-80.

[0021] Preferably, the method for preparing the walnut shell extract is as follows:

[0022] Walnut shells were crushed and added to a 60-70% ethanol aqueous solution at a solid-liquid ratio of 1:(30-40). The mixture was heated at 70-80℃ for 1-3 hours, filtered, concentrated, and freeze-dried to obtain walnut shell extract.

[0023] Preferably, the microalgae powder is Haematococcus pluvialis powder; the probiotic is Lactobacillus plantarum.

[0024] The preparation method of the above-mentioned microalgae compound feed additive for alleviating weaning stress in calves includes the following steps:

[0025] Mix the ingredients thoroughly according to the stated weight proportions.

[0026] The beneficial technical effects of this invention are as follows:

[0027] 1. The microalgae compound feed additive of the present invention comprises the following raw materials: microalgae powder, unsaturated fatty acid / modified montmorillonite compound, probiotics, and walnut shell extract. The raw materials are used in combination to improve the fragile intestinal barrier function and immunity of weaned calves, improve the growth performance of calves, and thus improve breeding efficiency.

[0028] 2. This invention effectively reduces the risk of diarrhea in calves by adding a modified montmorillonite complex. Further analysis reveals that montmorillonite, after being modified with a silane coupling agent to introduce amino groups, undergoes a Schiff base reaction with the aldehyde groups on oxidized pullulan polysaccharide, thus modifying the surface of montmorillonite to obtain modified montmorillonite. This modified montmorillonite not only adsorbs intestinal endotoxins, promoting the colonization of probiotics and reducing the risk of stress-induced diarrhea, but also, through the pullulan polysaccharide coating, protects the intestinal mucosa, repairs stress-induced mucosal damage, and uses modified montmorillonite as a carrier to load unsaturated fatty acids, improving the stability of these fatty acids and enabling their slow release in the intestine, thereby enhancing the calf's immunity. Attached Figure Description

[0029] Figure 1 This is a SEM image of the unsaturated fatty acid / modified montmorillonite composite obtained in Example 4 of this invention. Detailed Implementation

[0030] The following is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0031] (a) Preparation example

[0032] Preparation Example 1

[0033] This preparation example provides an oxidized pullulan polysaccharide, and the preparation process is as follows:

[0034] Pullulan, sodium periodate, and deionized water were mixed in a mass ratio of 1 g: 0.4 g: 180 mL. Pullulan was added to deionized water, followed by sodium periodate. The mixture was stirred in the dark for 30 h, and then ethylene glycol was added to terminate the reaction. The mixture was dialyzed for 3 days using a dialysis bag with a molecular cutoff of 3500, and then freeze-dried at -45℃ for 28 h to obtain oxidized pullulan.

[0035] Preparation Example 2

[0036] This preparation example provides an oxidized pullulan polysaccharide, and the preparation process is as follows:

[0037] Pullulan, sodium periodate, and deionized water were mixed in a mass ratio of 1 g: 0.2 g: 150 mL. Pullulan was added to deionized water, followed by sodium periodate. The mixture was stirred in the dark for 24 h, and then ethylene glycol was added to terminate the reaction. The mixture was dialyzed for 3 days using a dialysis bag with a molecular cutoff of 3500, and then freeze-dried at -45°C for 28 h to obtain oxidized pullulan.

[0038] Preparation Example 3

[0039] This preparation example provides an oxidized pullulan polysaccharide, and the preparation process is as follows:

[0040] Pullulan, sodium periodate, and deionized water were mixed in a mass ratio of 1 g: 0.6 g: 200 mL. Pullulan was added to deionized water, followed by sodium periodate. The mixture was stirred in the dark for 36 h, and then ethylene glycol was added to terminate the reaction. The mixture was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 3500, and then freeze-dried at -45°C for 28 h to obtain oxidized pullulan.

[0041] Preparation Example 4

[0042] This preparation example provides an unsaturated fatty acid / modified montmorillonite complex, and the preparation process is as follows:

[0043] (1) The ratio of montmorillonite, KH-550 and ethanol aqueous solution was 1g:0.08g:14mL; montmorillonite was added to 75wt% ethanol aqueous solution, and then KH-550 (γ-aminopropyltriethoxysilane) was added. The reaction was carried out at 55℃ for 3.5h. After the reaction was completed, the reaction solution was cooled to room temperature, centrifuged, washed twice with ethanol and twice with deionized water, and dried under vacuum to obtain silane coupling agent modified montmorillonite.

[0044] (2) The mass ratio of silane coupling agent-modified montmorillonite and oxidized pullulan polysaccharide is 1:3. The silane coupling agent-modified montmorillonite from step (1) and the oxidized pullulan polysaccharide from Preparation Example 1 are added to deionized water. The concentration of oxidized pullulan polysaccharide in deionized water is 3wt%. The reaction is carried out at room temperature for 6 hours and then dried to obtain modified montmorillonite.

[0045] (3) With a mass ratio of modified montmorillonite, α-linolenic acid, Tween-80, and deionized water of 5:22:6:17, the modified montmorillonite from step (2) was added to deionized water, followed by the addition of α-linolenic acid and Tween-80. After shearing, homogenization, and freeze-drying, an unsaturated fatty acid / modified montmorillonite complex was obtained. The SEM image of the unsaturated fatty acid / modified montmorillonite complex can be found in [reference needed]. Figure 1 .

[0046] Preparation Example 5

[0047] This preparation example provides an unsaturated fatty acid / modified montmorillonite complex, and the preparation process is as follows:

[0048] (1) The ratio of montmorillonite, KH-550 and ethanol aqueous solution was 1g:0.05g:10mL; montmorillonite was added to 75wt% ethanol aqueous solution, and then KH-550 was added. The reaction was carried out at 50℃ for 4h. After the reaction was completed, the reaction solution was cooled to room temperature, centrifuged, washed twice with ethanol and deionized water, and vacuum dried to obtain silane coupling agent modified montmorillonite.

[0049] (2) The mass ratio of silane coupling agent-modified montmorillonite and oxidized pullulan polysaccharide is 1:1. The silane coupling agent-modified montmorillonite from step (1) and the oxidized pullulan polysaccharide from Preparation Example 2 are added to deionized water. The concentration of oxidized pullulan polysaccharide in deionized water is 2wt%. The reaction is carried out at room temperature for 5h and then dried to obtain modified montmorillonite.

[0050] (3) With the mass ratio of modified montmorillonite, γ-linolenic acid, Tween-80 and deionized water being 5:20:3:15, the modified montmorillonite from step (2) was added to deionized water, followed by the addition of γ-linolenic acid and Tween-80. After shearing, homogenization and freeze-drying, an unsaturated fatty acid / modified montmorillonite complex was obtained.

[0051] Preparation Example 6

[0052] This preparation example provides an unsaturated fatty acid / modified montmorillonite complex, and the preparation process is as follows:

[0053] (1) The ratio of montmorillonite, KH-550 and ethanol aqueous solution was 1g:0.1g:10mL; montmorillonite was added to 75wt% ethanol aqueous solution, and then KH-550 was added. The reaction was carried out at 60℃ for 3h. After the reaction was completed, the reaction solution was cooled to room temperature, centrifuged, washed twice with ethanol and deionized water, and vacuum dried to obtain silane coupling agent modified montmorillonite.

[0054] (2) The mass ratio of silane coupling agent-modified montmorillonite and oxidized pullulan polysaccharide is 1:4. The silane coupling agent-modified montmorillonite from step (1) and the oxidized pullulan polysaccharide from Preparation Example 3 are added to deionized water. The concentration of oxidized pullulan polysaccharide in deionized water is 4wt%. The reaction is carried out at room temperature for 8 hours and then dried to obtain modified montmorillonite.

[0055] (3) With the mass ratio of modified montmorillonite, arachidonic acid, Tween-80 and deionized water being 5:25:7:20, the modified montmorillonite from step (2) was added to deionized water, followed by arachidonic acid and Tween-80. After shearing, homogenization and freeze-drying, an unsaturated fatty acid / modified montmorillonite complex was obtained.

[0056] Preparation Example 7

[0057] The difference between Preparation Example 7 and Preparation Example 4 is that step (2) is omitted.

[0058] Preparation Example 8

[0059] This preparation example provides a walnut shell extract, prepared by the following method:

[0060] Walnut shells were crushed and added to a 65% ethanol aqueous solution at a solid-liquid ratio of 1:34. The mixture was heated at 75°C for 2 hours, filtered, concentrated, and freeze-dried to obtain walnut shell extract.

[0061] Preparation Example 9

[0062] This preparation example provides a walnut shell extract, prepared by the following method:

[0063] Walnut shells were crushed and added to a 60% ethanol aqueous solution at a solid-liquid ratio of 1:30. The mixture was heated at 70°C for 3 hours, filtered, concentrated, and freeze-dried to obtain walnut shell extract.

[0064] Preparation Example 10

[0065] This preparation example provides a walnut shell extract, prepared by the following method:

[0066] Walnut shells were crushed and added to a 70% ethanol aqueous solution at a solid-liquid ratio of 1:40. The mixture was heated at 80°C for 1 hour, filtered, concentrated, and freeze-dried to obtain walnut shell extract.

[0067] (II) Implementation Examples

[0068] Example 1

[0069] This embodiment provides a microalgae compound feed additive to alleviate weaning stress in calves, comprising the following raw materials in parts by weight: 7 parts microalgae powder, 5 parts unsaturated fatty acid / modified montmorillonite compound of Preparation Example 4, 1 part probiotic, and 0.4 parts walnut shell extract of Preparation Example 8.

[0070] This embodiment also provides a method for preparing the above-mentioned microalgae compound feed additive for alleviating weaning stress in calves, including the following steps:

[0071] Mix the ingredients thoroughly according to the stated weight proportions.

[0072] Example 2

[0073] This embodiment provides a microalgae compound feed additive to alleviate weaning stress in calves, comprising the following raw materials in parts by weight: 5 parts microalgae powder, 2 parts unsaturated fatty acid / modified montmorillonite compound of Preparation Example 5, 0.7 parts probiotics, and 0.3 parts walnut shell extract of Preparation Example 9.

[0074] This embodiment also provides a method for preparing the above-mentioned microalgae compound feed additive for alleviating weaning stress in calves, including the following steps:

[0075] Mix the ingredients thoroughly according to the stated weight proportions.

[0076] Example 3

[0077] This embodiment provides a microalgae compound feed additive to alleviate weaning stress in calves, comprising the following raw materials in parts by weight: 10 parts microalgae powder, 6 parts unsaturated fatty acid / modified montmorillonite compound of Preparation Example 6, 1.2 parts probiotics, and 0.5 parts walnut shell extract of Preparation Example 10.

[0078] This embodiment also provides a method for preparing the above-mentioned microalgae compound feed additive for alleviating weaning stress in calves, including the following steps:

[0079] Mix the ingredients thoroughly according to the stated weight proportions.

[0080] (III) Comparative Example

[0081] Comparative Example 1

[0082] The difference between this comparative example and Example 1 is that a mixture of α-linolenic acid, montmorillonite, and pullulan was used instead of the unsaturated fatty acid / modified montmorillonite complex in Preparation Example 4, wherein the ratio of α-linolenic acid, montmorillonite, and pullulan in the mixture is the same as in Preparation Example 4.

[0083] Comparative Example 2

[0084] The difference between this comparative example and Example 1 is that the product of Preparation Example 7 is used instead of the unsaturated fatty acid / modified montmorillonite complex of Preparation Example 4.

[0085] Comparative Example 3

[0086] The difference between this comparative example and Example 1 is that the walnut shell extract of Example 8 is omitted.

[0087] (iv) Examples of Results

[0088] 1. Laboratory animals and experimental design

[0089] Seventy weaned calves (Simmental cattle) with an average weight of (65±5) kg were selected and randomly divided into seven groups: Example 1-3 groups, Comparative Examples 1-3 groups, and a control group, with 10 calves in each group. Example 1-3 groups and Comparative Examples 1-3 groups were fed pelleted feed with free access. 2g / calves / day of each feed additive was dissolved in 500mL of water, and water was provided normally after the calves finished drinking to ensure free access to water. The control group was fed pelleted feed. The experiment lasted for 28 days. The main nutritional components of the calf pelleted feed are shown in Table 1.

[0090] Table 1

[0091]

[0092] 3. Experimental Procedure and Index Detection

[0093] 3.1 Growth Indicators

[0094] Each calf was weighed at 1 and 28 days of age before morning feeding, and the average daily weight gain was calculated.

[0095] Record the weight of the starter feed given to each calf on the first day and the weight of the leftover feed at the same time the following day. Calculate the calf's daily feed intake and feed conversion ratio using the following method:

[0096] Feed conversion ratio (%) = [feed intake (kg) / average daily weight gain (kg)] × 100%, see Table 2 for results.

[0097] 3.2 Diarrhea status

[0098] Calf feces were observed and scored every morning during feeding, and the results were recorded. The scoring criteria were as shown in Table 3, using a 4-point fecal scoring method. A score of ≥3 was recorded as diarrhea, and the results are shown in Table 4.

[0099] 3.3 Intestinal Microbiome Detection

[0100] Before morning feeding on day 28, five healthy calves from each group were sampled, and 5g of fecal sample was scraped from the rectum of each calf using a fecal sampler and stored in a sterile cryovial at -80°C for intestinal flora detection. Genomic DNA was extracted from the samples using the cetyltrimethylammonium bromide (CTAB) method, followed by PCR amplification. The PCR products were then co-cultured and purified. The entire library was prepared through steps including end repair, A-tailing, sequencing adapter addition, and purification. Finally, sequencing was performed to analyze the effects of each group's calf gut microbiota. The results are shown in Tables 5-6.

[0101] 3.4 Serum markers

[0102] Before morning feeding on day 28, fasting blood samples were collected from calves to detect the levels of IgA, IgG, and IgM in their serum. The results are shown in Table 7.

[0103] Table 2

[0104]

[0105] Table 3

[0106]

[0107] Table 4

[0108]

[0109] Table 5

[0110]

[0111] Table 6

[0112]

[0113] Table 7

[0114]

[0115] Table 2 shows that, compared with the control group, the growth performance of calves fed with the additives in Examples 1-3 and Comparative Examples 1-3 was significantly improved. However, compared with Example 1, the effect of Comparative Examples 1-3 was not as good. These results indicate that the combined use of microalgae powder, unsaturated fatty acid / modified montmorillonite complex, probiotics, and walnut shell extract improves the growth performance of calves, thereby increasing breeding efficiency.

[0116] Table 4 shows that, compared with the control group, the diarrhea rate of calves fed with the additives of Examples 1-3 and Comparative Examples 1-3 was significantly reduced. However, compared with Example 1, the effect of Comparative Examples 1-2 was less effective. These results indicate that the modified montmorillonite complex of the present invention can effectively reduce the risk of diarrhea in calves. Further analysis revealed that montmorillonite, after being modified with a silane coupling agent to introduce amino groups, undergoes a Schiff base reaction with the aldehyde groups on oxidized pullulan polysaccharide to modify the surface of montmorillonite, thus preparing modified montmorillonite. This modified montmorillonite not only adsorbs intestinal endotoxins, which is beneficial for the colonization of probiotics and reduces the risk of stress-induced diarrhea, but also, through the pullulan polysaccharide coating, protects the intestinal mucosa, repairs stress-induced mucosal damage, and uses modified montmorillonite as a carrier to load unsaturated fatty acids, improving the stability of unsaturated fatty acids and achieving slow release of unsaturated fatty acids in the intestine, thereby enhancing the immunity of calves.

[0117] Tables 5-6 show that the dominant intestinal flora of calves consisted of Bacteroidetes, Proteobacteria, Fusobacteria, and Firmicutes, while the dominant genera were Bacteroidetes, Faecalibacterium, Micrococcus, Prevotella, and Clostridia_UCG-014. Compared to the control group, the relative abundance of Bacteroidetes, Proteobacteria, Fusobacteria, and Bacteroidetes decreased after feeding the additives in Examples 1-3 and Comparative Examples 1-3, while the relative abundance of Firmicutes, Micrococcus, Faecalibacterium, Prevotella, and Clostridia_UCG-014 significantly increased. The effects of Comparative Examples 1-3 were less effective than in Example 1. These results indicate that the combined use of microalgae powder, unsaturated fatty acid / modified montmorillonite complex, probiotics, and walnut shell extract can effectively improve the fragile intestinal barrier function of weaned calves.

[0118] Table 7 shows that, compared with the control group, the levels of IgA, IgG, and IgM in the serum of calves increased after feeding them the additives from Examples 1-3 and Comparative Examples 1-3. However, the effects of Comparative Examples 1-3 were less pronounced than those of Example 1. These results indicate that the combined use of microalgae powder, unsaturated fatty acid / modified montmorillonite complex, probiotics, and walnut shell extract can enhance the immunity of calves.

[0119] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A microalgal composite feed additive for alleviating weaning stress in calves, characterized in that, The ingredients include the following parts by weight: 5-10 parts microalgae powder, 2-6 parts unsaturated fatty acid / modified montmorillonite complex, 0.7-1.2 parts probiotics, and 0.3-0.5 parts walnut shell extract; The preparation process of the unsaturated fatty acid / modified montmorillonite complex is as follows: (1) Add montmorillonite to an ethanol aqueous solution, then add silane coupling agent, heat to react, and after the reaction is completed, cool the reaction solution to room temperature, centrifuge, wash twice with ethanol and deionized water respectively, and vacuum dry to obtain silane coupling agent modified montmorillonite. (2) The silane coupling agent modified montmorillonite and oxypullan polysaccharide from step (1) were added to deionized water to react and obtain modified montmorillonite. (3) The modified montmorillonite from step (2) was added to deionized water, and then unsaturated fatty acids and emulsifiers were added. After shearing, homogenization and freeze drying, unsaturated fatty acid / modified montmorillonite composite was obtained. In step (1), the ratio of montmorillonite, silane coupling agent, and ethanol aqueous solution is 1g:(0.05-0.1)g:(10-14)mL; the concentration of the ethanol aqueous solution is 75wt%; the silane coupling agent is KH-550; the heating reaction temperature is 50-60℃ and the time is 3-4h. In step (2), the mass ratio of silane coupling agent-modified montmorillonite to oxidized pullulan is 1:(1-4); the concentration of oxidized pullulan in deionized water is 2-4 wt%; and the reaction time is 5-8 h. In step (3), the mass ratio of modified montmorillonite, unsaturated fatty acid, emulsifier, and deionized water is 5:(20-25):(3-7):(15-20); the unsaturated fatty acid is selected from α-linolenic acid, γ-linolenic acid, and arachidonic acid; the emulsifier is Tween-80.

2. The microalgal composite feed additive for alleviating weaning stress in calves according to claim 1, characterized in that, The preparation method of the oxidized pullulan polysaccharide is as follows: Pullulan polysaccharide is added to deionized water, followed by sodium periodate. After stirring in the dark, ethylene glycol is added to terminate the reaction. The product is then obtained by dialysis and freeze-drying.

3. The microalgae compound feed additive for alleviating weaning stress in calves according to claim 2, characterized in that, The ratio of pullulan polysaccharide, sodium periodate, and deionized water is 1g:(0.2-0.6)g:(150-200)mL; the stirring time in the dark is 24-36h.

4. The microalgae compound feed additive for alleviating weaning stress in calves according to claim 1, characterized in that, The preparation method of the walnut shell extract is as follows: Walnut shells were crushed and added to a 60-70% ethanol aqueous solution at a solid-liquid ratio of 1:(30-40). The mixture was heated at 70-80℃ for 1-3 hours, filtered, concentrated, and freeze-dried to obtain walnut shell extract.

5. The microalgae compound feed additive for alleviating weaning stress in calves according to claim 1, characterized in that, The microalgae powder is Haematococcus pluvialis powder; the probiotic is Lactobacillus plantarum.

6. A method for preparing a microalgae compound feed additive for alleviating weaning stress in calves as described in any one of claims 1-5, characterized in that, Includes the following steps: Mix the ingredients thoroughly according to the stated weight proportions.

Images