A probiotic microcapsule, its preparation method, and its application in the preparation of products for the prevention and treatment of calf diarrhea.

Abstract

This invention belongs to the field of microbial technology, specifically disclosing a probiotic microcapsule, its preparation method, and its application in the preparation of products for preventing and treating calf diarrhea. Targeting the digestive physiology and pathogenesis of diarrhea in calves, this invention prepares A-type and B-type microcapsules with different probiotics, targeting different segments of the digestive tract for release, and then coats them together to obtain probiotic microcapsules. Through the combination of microalgae, strains, and formulation design, the probiotic microcapsules prepared by this invention not only exhibit excellent acid resistance and superior small intestinal epithelial barrier repair function, but also can deliver probiotics in a gastrointestinal zone with targeted delivery, effectively inhibiting the proliferation of pathogenic bacteria and thus better preventing and treating calf diarrhea.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a probiotic microcapsule, its preparation method, and its application in the preparation of products for the prevention and treatment of calf diarrhea. Background Technology

[0002] Diarrhea in newborn calves is the leading cause of death. Traditional clinical treatments primarily rely on antibiotics, but long-term or improper use can easily lead to bacterial resistance, intestinal flora imbalance, and drug residues, hindering the healthy and sustainable development of the livestock industry. Probiotics have received widespread attention and application as a green and safe alternative, but current probiotic-related technologies still face several bottlenecks: First, poor strain compatibility. Most commercially available probiotic strains are isolated from humans or adult ruminants, resulting in poor compatibility with the intestinal microecological environment of newborn calves, weak colonization ability, and easy rejection by the host's inherent intestinal flora, making it difficult to colonize long-term and maintain their effects. Second, poor stability and preservation of live bacteria. Probiotics are sensitive to temperature, humidity, gastric acid, and bile salt environments, and significant loss of live bacteria occurs during feed processing, storage, transportation, and calf digestion, making it difficult to reach the effective threshold for the number of live bacteria reaching the target intestinal region. Third, insufficient functional targeting. Current probiotic products are mostly general-purpose compound strains, without targeted screening for the main pathogens of calf diarrhea and the intestinal development characteristics of calves at different physiological stages, leading to unstable actual prevention and treatment effects and significant individual differences.

[0003] To address the issues of poor stability and insufficient tolerance of probiotics, microencapsulation has become a commonly used protective strategy. However, current microencapsulation technologies mostly focus on single targets, such as improving the strain's tolerance to gastric acid or achieving colon-targeted release, making it difficult to achieve full-process protection and precise controlled release in the complex gastrointestinal environment of calves. Therefore, developing a probiotic microcapsule formulation capable of precise regional delivery and programmed release is of significant practical importance for improving the survival rate and colonization efficiency of probiotics in calves and enhancing the prevention and treatment of calf diarrhea. Summary of the Invention

[0004] In view of the above-mentioned problems existing in the prior art, the present invention provides a probiotic microcapsule, its preparation method, and its application in the preparation of products for the prevention and treatment of calf diarrhea. Targeting the digestive physiology and pathogenesis of diarrhea in calves, the present invention provides a microcapsule with good acid resistance, excellent small intestinal epithelial barrier repair function, and the ability to deliver probiotics in a targeted manner, thereby enabling the microcapsule to better exert its effect in preventing and treating calf diarrhea.

[0005] To achieve the above-mentioned objectives, the embodiments of the present invention employ the following technical solutions: In a first aspect, the present invention provides a method for preparing probiotic microcapsules, comprising the following steps: S1. After dispersing Lactobacillus plantarum, Lactobacillus paracasei, Chlorella CV-5 and intestinal function regulator in a polysaccharide gel substrate solution, the microspheres were solidified by drop casting in a coagulation bath, collected and dried to obtain type A microcapsules. S2. Disperse Bifidobacterium in a Chlorella polysaccharide-dispersant solution, dry, and obtain type B microcapsules; S3. Mix the type A microcapsules and type B microcapsules, and coat them to obtain probiotic microcapsules; The Chlorella CV-5, with accession number CGMCC No. 15206, has the Latin name [missing information]. Chlorella pyrenoidosa; The Chlorella polysaccharide-dispersant solution contains Chlorella CV-5 polysaccharide and protein dispersants.

[0006] The Chlorella cumulus CV-5 provided by this invention was isolated from a water source in Shijiazhuang City, Hebei Province, and its Latin name is [missing information]. Chlorella pyrenoidosa, This algae was deposited on January 12, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.15206.

[0007] This invention controls the release of two types of microcapsules in different segments of the digestive tract, allowing strains with different functions to exert their maximum efficacy in the most suitable physiological environment, forming a seamless synergy of rapid symptom control and long-term ecological restoration. The enzyme-triggered colonic release mechanism of the type B microcapsule enables synchronous and targeted delivery of probiotics and prebiotics, greatly improving the colonization success rate and persistence of exogenous bacteria. This invention, by separately preparing type A and type B microcapsules and then mixing them for coating, significantly enhances the product's stability during processing, storage, and in the gastric environment through the use of different protective layers.

[0008] Preferably, the *Lactobacillus plantarum* includes *Lactobacillus plantarum* JMCC0017.

[0009] Preferably, the Lactobacillus paracasei includes Lactobacillus paracasei N1115.

[0010] Lactobacillus plantarum JMCC0017 and Lactobacillus paracasei N1115 have strong colonization and activity, ensuring that they can play a role in regulating the intestinal flora, repairing the intestinal tract, and reducing inflammation.

[0011] Preferably, the intestinal function regulator includes at least one of glutamine, zinc glycinate, or fructooligosaccharides.

[0012] For example, the mass ratio of glutamine, zinc glycine, and fructooligosaccharides is 3.5-6.5:0.6-1.2:2-4.

[0013] Preferably, the polysaccharide gel substrate comprises sodium alginate.

[0014] Preferably, the coagulation solution used for solidification includes calcium salts and chitosan.

[0015] Preferably, in step S1, the drying includes freeze drying.

[0016] In this invention, Chlorella vulgaris CV-5 is added in the form of algal powder during the preparation of type A microcapsules. The specific function of this algal powder is as follows: 1. Encapsulating Lactobacillus plantarum and Lactobacillus paracasei in their cell wall matrix forms a double physical barrier with calcium alginate gel, which significantly enhances the probiotics' tolerance to gastric acid. In addition, the algae's rich protein, polysaccharides and minerals provide a continuous carbon and nitrogen source for the probiotics, promoting their survival and proliferation. 2. Synergistic effect with prebiotics: The complex polysaccharides in the cell wall of Chlorella CV-5 can act as a slow-release prebiotic, which, together with prebiotics such as fructooligosaccharides, prolongs the colonization time of probiotics in the intestine and continuously produces short-chain fatty acids to inhibit pathogens. 3. Chlorella CV-5 provides comprehensive nutrients that work synergistically with the main energy source of intestinal epithelium (glutamine) and repair enzyme cofactor (zinc glycine) to promote the regeneration of damaged intestinal mucosa from multiple aspects, including energy supply, raw material replenishment and enzyme activation. 4. Anti-inflammatory synergy: The carotenoids, chlorophyll, and other natural antioxidants in Chlorella powder work synergistically with probiotic metabolites to eliminate free radicals, creating a favorable microenvironment for intestinal repair.

[0017] Preferably, the Bifidobacterium includes Bifidobacterium i771.

[0018] Preferably, the protein dispersant comprises sodium caseinate.

[0019] Preferably, in step S2, the drying includes spray drying.

[0020] Preferably, the coating agent used for coating includes a 3%-5% Eudragit L100 isopropanol solution.

[0021] Preferably, the ratio of viable Lactobacillus plantarum to Lactobacillus paracasei is 8-12:10.

[0022] Preferably, the ratio of the viable count of *Lactobacillus plantarum* to the mass of *Chlorella pyrenoidosa* CV-5 is (1 × 10⁻⁶). 9 -8×10 10 CFU: (1.5-3)g.

[0023] Preferably, the mass ratio of the protein-nucleated Chlorella CV-5 to the intestinal function regulator is 1.5-3:7-12.

[0024] Preferably, in step S3, the mass ratio of type A microcapsules to type B microcapsules during mixing is 0.8-1.2:1.

[0025] More preferably, the preparation method of the Chlorella CV-5 polysaccharide includes the following steps: Chlorella CV-5 with protein nucleus was mixed with phosphate-citric acid buffer, and enzymatically hydrolyzed with a complex cellulose degrading enzyme. Chlorella extract was then obtained by a programmed pressure- and temperature-controlled cyclic extraction method. The Chlorella extract was precipitated with alcohol and purified to obtain Chlorella proteolyticus CV-5 polysaccharide.

[0026] For example, the complex cellulase includes β-glucanase and β-glucosidase.

[0027] Preferably, the mass-to-volume ratio of the Chlorella CV-5 protein nucleus to the phosphate-citric acid buffer is 1 g: (15-40) mL.

[0028] Preferably, the programmed pressure and temperature controlled cyclic extraction method includes: maintaining the pressure at 0.25MPa-0.35MPa and 100℃-110℃ for 4min-6min, then depressurizing to atmospheric pressure for 25s-35s and maintaining it at 90℃-98℃ for 2.5min-3.5min; repeating this program for 4-6 cycles.

[0029] For example, the extraction solution in this invention is described using a disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.2-5.8.

[0030] Preferably, the Chlorella extract is evaporated and concentrated, precipitated with alcohol, and the solid and liquid are separated to obtain crude polysaccharide of Chlorella CV-5 with protein nucleus. The crude polysaccharide of Chlorella pulvinata CV-5 was reconstituted, and after removing the protein by adding Sevage reagent, it was washed with ethanol and acetone and dried to obtain Chlorella pulvinata CV-5 polysaccharide.

[0031] The method for preparing polysaccharides from Chlorella CV-5 provided by this invention can not only significantly improve the polysaccharide extraction yield, but also more effectively promote the growth of Bifidobacterium bifidum i771.

[0032] Secondly, the present invention provides a probiotic microcapsule prepared by the above-mentioned method for preparing probiotic microcapsules.

[0033] In response to the digestive physiology and pathogenesis of diarrhea in calves, this invention provides probiotic microcapsules with a single-shell, dual-core structure through the combination of microalgae, bacterial strains, and formulation design.

[0034] Thirdly, the present invention provides the application of the above-mentioned probiotic microcapsules in the preparation of products for the prevention and treatment of calf diarrhea.

[0035] The probiotic microcapsules provided by this invention not only have excellent acid resistance and superior repair function of the small intestinal epithelial barrier, but also can deliver probiotics in a targeted manner in the gastrointestinal region, which can effectively inhibit the reproduction of pathogenic bacteria, thereby better achieving the effect of preventing and treating calf diarrhea. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 The results of relative TEER values ​​of probiotic microcapsule release solutions in different groups in Example 3 of the present invention; Figure 2 The results of measuring the relative abundance of enterotoxigenic Escherichia coli in the feces of diarrheal calves in different groups before and after intervention with probiotic microcapsules in Example 4 of the present invention; Figure 3 The results of measuring the relative abundance of Clostridium perfringens in feces of different groups of diarrheal calves before and after intervention with probiotic microcapsules in Example 4 of the present invention; Figure 4 The results of measuring the relative abundance of Salmonella in feces of different groups of diarrheal calves before and after intervention with probiotic microcapsules in Example 4 of the present invention; Figure 5 The results of measuring the relative abundance of Campylobacter in feces of diarrheal calves in different groups before and after intervention with probiotic microcapsules in Example 4 of the present invention are shown. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] The algal strain CV-5 in this invention was isolated from a water source in Luquan District, Shijiazhuang City, Hebei Province. After isolation, purification, and expansion, morphological and molecular biological identification confirmed that the algal strain is *Chlorella pyrenoidosa*. Chlorella pyrenoidosa .

[0040] The 18S rDNA gene sequence was sequenced and is shown in SEQ ID NO:1. Algal strain CV-5 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 15206.

[0041] Example 1 First, this embodiment of the invention prepares a polysaccharide from Chlorella vulgaris CV-5, the preparation method of which includes the following steps: Step 1: Take 10g of Chlorella CV-5 algal powder that has passed through a 60-mesh sieve, suspend it in 400mL of pH 5.5 disodium hydrogen phosphate-citric acid buffer, add 0.25g of compound cellulose degrading enzyme, and treat with shaking at 50℃ and 120rpm for 2.5h. The complex cellulose-degrading enzyme consists of β-glucanase and β-glucosidase in a mass ratio of 2:1; the enzyme activity of β-glucanase is 20,000 U / g; and the enzyme activity of β-glucosidase is 50,000 U / g.

[0042] Step 2: Transfer the enzymatically hydrolyzed mixture to a programmable pressure- and temperature-controlled reactor to extract the extract. The reactor is set with the following cycle program: maintain at 0.3 MPa and 105℃ for 5 min, then depressurize to atmospheric pressure within 30 s and maintain at 95℃ for 3 min. Repeat this cycle 5 times.

[0043] Step 3: Cool the extract, centrifuge at 8000 rpm and 4℃ for 20 min, and collect the supernatant; The supernatant was concentrated to 1 / 3 of its original volume by rotary evaporation at 60℃. 480 mL of pre-cooled anhydrous ethanol was slowly added while stirring. The mixture was allowed to stand in a 4℃ refrigerator for 12 h, centrifuged at 8000 rpm and 4℃ for 20 min, and the precipitate was collected to obtain crude polysaccharide of Chlorella CV-5. The obtained crude polysaccharide was reconstituted with 100 mL of deionized water, and 25 mL of Sevage reagent (chloroform:n-butanol volume ratio 4:1, freshly prepared) was added. After vigorous shaking for 20 min, the mixture was transferred to a separatory funnel and allowed to stand for 30 min until separation. The upper aqueous phase (containing polysaccharide) was collected, and the lower organic phase and the intermediate denatured protein layer were discarded. The above operation was repeated 3 times until no obvious protein precipitation was observed in the intermediate layer. The precipitate was washed once with anhydrous ethanol, once with acetone, and once with anhydrous ethanol, centrifuged after each wash, and the washed precipitate was spread evenly in a petri dish, vacuum dried, ground into a fine powder, and passed through an 80-mesh sieve to obtain 0.85 g of Chlorella vulgaris CV-5 polysaccharide.

[0044] This invention also provides a probiotic microcapsule, the preparation method of which includes the following steps: S1. Mix 2.5g of Lactobacillus plantarum JMCC0017 powder, 2.5g of Lactobacillus paracasei N1115 powder, 2g of Chlorella proteoglycans CV-5 algal powder (passed through 80 mesh), 5g of glutamine, 1g of zinc glycine and 3g of fructooligosaccharides evenly, and disperse them in 100mL of 3% sodium alginate solution to obtain a mixed solution; The above mixture was dropped into a coagulation bath containing 1.5% calcium chloride and 1.0% chitosan using an electrostatic dripping device. After solidification for 30 minutes, the gel microspheres were collected and freeze-dried to obtain type A microcapsules. S2. Disperse 10g of Bifidobacterium bifidum i771 bacterial powder in 200mL of an aqueous solution containing 15% Chlorella cum CV-5 polysaccharide and 10% sodium caseinate, and spray dry (inlet temperature 140℃, outlet temperature 75℃) to obtain type B microcapsules. S3. Mix the type A microcapsules and type B microcapsules at a mass ratio of 1:1, place them in a fluidized bed coating machine, use a 5% Eudragit L100 isopropanol solution as the coating liquid, and use a bottom spraying process to coat until the coating weight gain reaches 30% of the total particle weight, thus obtaining probiotic microcapsules.

[0045] Among them, the live bacteria content of Lactobacillus plantarum JMCC0017 powder and Lactobacillus paracasei N1115 powder was 2×10⁻⁶. 10 CFU / g; The viable bacteria content of Bifidobacterium bifidum i771 powder is 1×10⁻⁶. 10 CFU / g.

[0046] Example 2 First, this embodiment of the invention prepares a polysaccharide from Chlorella vulgaris CV-5, the preparation method of which includes the following steps: Step 1: Take 10g of Chlorella CV-5 algal powder that has passed through a 60-mesh sieve, suspend it in 200mL of pH 5.5 disodium hydrogen phosphate-citric acid buffer, add 0.25g of compound cellulose degrading enzyme, and treat with shaking at 50℃ and 120rpm for 2.5h. The complex cellulose-degrading enzyme consists of β-glucanase and β-glucosidase in a mass ratio of 2:1; the enzyme activity of β-glucanase is 20,000 U / g; and the enzyme activity of β-glucosidase is 50,000 U / g.

[0047] Step 2: Transfer the enzymatically hydrolyzed mixture to a programmable pressure- and temperature-controlled reactor to extract the extract. The reactor is set with the following cycle program: maintain at 0.35 MPa and 110°C for 4 min, then depressurize to atmospheric pressure within 35 s and maintain at 90°C for 3.5 min. Repeat this cycle 4 times.

[0048] Step 3: Cool the extract, centrifuge at 8000 rpm and 4℃ for 20 min, and collect the supernatant; The supernatant was concentrated to 1 / 3 of its original volume by rotary evaporation at 60℃. 480 mL of pre-cooled anhydrous ethanol was slowly added while stirring. The mixture was allowed to stand in a 4℃ refrigerator for 12 h, centrifuged at 8000 rpm and 4℃ for 20 min, and the precipitate was collected to obtain crude polysaccharide of Chlorella CV-5. The crude polysaccharide was reconstituted with 100 mL of deionized water, and 25 mL of Sevage reagent (chloroform:n-butanol volume ratio 4:1, freshly prepared) was added. After vigorous shaking for 20 min, the mixture was transferred to a separatory funnel and allowed to stand for 30 min until separation. The upper aqueous phase (containing polysaccharide) was collected, and the lower organic phase and the intermediate denatured protein layer were discarded. The above operation was repeated 4 times until no obvious protein precipitation was observed in the intermediate layer. The precipitate was washed once with anhydrous ethanol, once with acetone, and once with anhydrous ethanol, centrifuged after each wash, and the washed precipitate was spread evenly in a petri dish, vacuum dried, ground into a fine powder, and passed through an 80-mesh sieve to obtain 0.82 g of Chlorella CV-5 polysaccharide.

[0049] This invention also provides a probiotic microcapsule, the preparation method of which includes the following steps: S1. Mix 2.2g of Lactobacillus plantarum JMCC0017 powder, 2.8g of Lactobacillus paracasei N1115 powder, 3g of Chlorella cumulus CV-5 algal powder (passed through an 80-mesh sieve), 3.5g of glutamine, 1.2g of zinc glycine, and 4g of fructooligosaccharides evenly and disperse them in 100mL of 3% sodium alginate solution to obtain a mixed solution. The above mixture was dropped into a coagulation bath containing 1.5% calcium chloride and 1.0% chitosan using an electrostatic dripping device. After solidification for 30 minutes, the gel microspheres were collected and freeze-dried to obtain type A microcapsules. S2. Disperse 10g of Bifidobacterium bifidum i771 in 200mL of an aqueous solution containing 18% Chlorella cum CV-5 polysaccharide and 8% sodium caseinate, and spray dry (inlet temperature 140℃, outlet temperature 75℃) to obtain type B microcapsules. S3. Mix the type A microcapsules and type B microcapsules at a mass ratio of 8:10, place them in a fluidized bed coating machine, use a 4% Eudragit L100 isopropanol solution as the coating liquid, and use a bottom spraying process to coat until the coating weight gain reaches 32% of the total particle weight, thus obtaining probiotic microcapsules.

[0050] Among them, the live bacteria content of Lactobacillus plantarum JMCC0017 powder and Lactobacillus paracasei N1115 powder was 5×10⁻⁶. 9 CFU / g; The viable bacteria content of Bifidobacterium bifidum i771 powder is 8×10⁻⁶. 9 CFU / g.

[0051] Example 3 First, this embodiment of the invention prepares a polysaccharide from Chlorella vulgaris CV-5, the preparation method of which includes the following steps: Step 1: Take 10g of Chlorella CV-5 algal powder that has passed through a 60-mesh sieve, suspend it in 300mL of pH 5.5 disodium hydrogen phosphate-citric acid buffer, add 0.25g of compound cellulose degrading enzyme, and treat with shaking at 50℃ and 120rpm for 2.5h. The complex cellulose-degrading enzyme consists of β-glucanase and β-glucosidase in a mass ratio of 2:1; the enzyme activity of β-glucanase is 20,000 U / g; and the enzyme activity of β-glucosidase is 50,000 U / g.

[0052] Step 2: Transfer the enzymatically hydrolyzed mixture to a programmable pressure- and temperature-controlled reactor to extract the extract. The reactor is set with the following cycle program: maintain at 0.25 MPa and 100°C for 6 min, then depressurize to atmospheric pressure within 25 s and maintain at 98°C for 2.5 min. Repeat this cycle 6 times.

[0053] Step 3: Cool the extract, centrifuge at 8000 rpm and 4℃ for 20 min, and collect the supernatant; The supernatant was concentrated to 1 / 3 of its original volume by rotary evaporation at 60℃. 480 mL of pre-cooled anhydrous ethanol was slowly added while stirring. The mixture was allowed to stand in a 4℃ refrigerator for 12 h, centrifuged at 8000 rpm and 4℃ for 20 min, and the precipitate was collected to obtain crude polysaccharide of Chlorella CV-5. The crude polysaccharide was reconstituted with 100 mL of deionized water, and 25 mL of Sevage reagent (chloroform:n-butanol volume ratio 4:1, freshly prepared) was added. After vigorous shaking for 20 min, the mixture was transferred to a separatory funnel and allowed to stand for 30 min until separation. The upper aqueous phase (containing polysaccharide) was collected, and the lower organic phase and the intermediate denatured protein layer were discarded. The above operation was repeated 4 times until no obvious protein precipitation was observed in the intermediate layer. The precipitate was washed once with anhydrous ethanol, once with acetone, and once with anhydrous ethanol, centrifuged after each wash, and the washed precipitate was spread evenly in a petri dish, vacuum dried, ground into a fine powder, and passed through an 80-mesh sieve to obtain 0.87 g of Chlorella CV-5 polysaccharide.

[0054] This invention also provides a probiotic microcapsule, the preparation method of which includes the following steps: S1. Mix 2.7g of Lactobacillus plantarum JMCC0017 powder, 2.3g of Lactobacillus paracasei N1115 powder, 2.5g of Chlorella cumulus CV-5 algal powder (passed through an 80-mesh sieve), 6.4g of glutamine, 0.6g of zinc glycine, and 2g of fructooligosaccharides evenly and disperse them in 100mL of 3% sodium alginate solution to obtain a mixed solution; The above mixture was dropped into a coagulation bath containing 1.5% calcium chloride and 1.0% chitosan using an electrostatic dripping device. After solidification for 30 minutes, the gel microspheres were collected and freeze-dried to obtain type A microcapsules. S2. Disperse 10g of Bifidobacterium bifidum i771 in 200mL of an aqueous solution containing 15% Chlorella cum CV-5 polysaccharide and 10% sodium caseinate, and spray dry (inlet temperature 140℃, outlet temperature 75℃) to obtain type B microcapsules. S3. Mix the type A microcapsules and type B microcapsules at a mass ratio of 12:10, place them in a fluidized bed coating machine, use a 5% Eudragit L100 isopropanol solution as the coating liquid, and use a bottom spraying process to coat until the coating weight gain reaches 30% of the total particle weight, thus obtaining probiotic microcapsules.

[0055] Among them, the live bacteria content of Lactobacillus plantarum JMCC0017 powder and Lactobacillus paracasei N1115 powder was 1×10⁻⁶. 10 CFU / g; The viable bacteria content of Bifidobacterium bifidum i771 powder is 5×10⁻⁶ CFU / g. 9 CFU / g.

[0056] Comparative Example 1 This comparative example provides a polysaccharide from Chlorella vulgaris CV-5, the preparation method of which includes the following steps: Step 1: Take 10g of Chlorella CV-5 algal powder and suspend it in a 10% NaCl solution at a ratio of 1:20 (w / v). Add 0.25g of compound cellulase, shake to mix, and extract by ultrasonication at 500W for 30min. Centrifuge at 8500rpm for 15min to obtain the extract. The complex cellulase consists of β-glucanase and β-glucosidase in a mass ratio of 2:1; the enzyme activity of β-glucanase is 20,000 U / g; and the enzyme activity of β-glucosidase is 50,000 U / g.

[0057] Step 2: Cool the extract, centrifuge at 8000 rpm and 4℃ for 20 min to collect the supernatant, then concentrate the supernatant to 1 / 3 of its original volume by rotary evaporation at 60℃, slowly add 480 mL of pre-cooled anhydrous ethanol while stirring, let stand in a 4℃ refrigerator for 12 h, centrifuge at 8000 rpm and 4℃ for 20 min, collect the precipitate, and obtain crude polysaccharide of Chlorella CV-5. The crude polysaccharide was reconstituted with 100 mL of deionized water, and 25 mL of Sevage reagent (chloroform:n-butanol volume ratio 4:1, freshly prepared) was added. After vigorous shaking for 20 min, the mixture was transferred to a separatory funnel and allowed to stand for 30 min until separation. The upper aqueous phase (containing polysaccharide) was collected, and the lower organic phase and the intermediate denatured protein layer were discarded. The above operation was repeated 3 times until no obvious protein precipitation was observed in the intermediate layer. The precipitate was washed once with anhydrous ethanol and once with acetone, centrifuged after each wash, and the washed precipitate was spread evenly in a petri dish, vacuum dried, ground into a fine powder, and passed through an 80-mesh sieve to obtain 0.42 g of Chlorella vulgaris CV-5 polysaccharide.

[0058] Comparative Example 2 This comparative example provides a probiotic microcapsule, the preparation method of which is basically the same as that of Example 1, except that Chlorella nigra CV-5 is replaced with an equal amount of commercial food-grade Chlorella nigra, denoted as Chlorella nigra JY. The preparation method of Chlorella nigra JY polysaccharide is the same as that of Example 1. 10g of Chlorella nigra JY finally yields 0.66g of Chlorella nigra JY polysaccharide.

[0059] In this case, following the method described in Example 1, Chlorella CV-5 and its polysaccharide were replaced with an equal amount of Chlorella JY and its polysaccharide, and probiotic microcapsules were finally prepared.

[0060] Example 1 This invention investigated the effects of Chlorella CV-5 polysaccharide prepared in Example 1 and Comparative Example 1, and Chlorella JY polysaccharide prepared in Comparative Example 2, on increasing the biomass of Bifidobacterium bifidum i771. The specific details are as follows: Take the Chlorella CV-5 polysaccharide prepared in Example 1 or Comparative Example 1, and the Chlorella JY polysaccharide prepared in Comparative Example 2, add water to prepare polysaccharide solutions with a concentration of 10 mg / mL.

[0061] Take 0.5 mL of each of the three polysaccharide solutions mentioned above and add them to 8.5 mL of MRS medium. Then add 1 mL of Bifidobacterium bifidum i771 culture solution cultured to the logarithmic growth phase (the viable count of the culture solution is 1 × 10⁻⁶). 7 The cells were incubated at 37℃ for 18 hours (CFU / mL). After incubation, the viable count of i771 cells in different groups was measured, expressed as 1g (CFU / mL). Each group had three replicates. MRS medium without added polysaccharides was used as a control. The results are shown in Table 1.

[0062] Table 1

[0063] The experimental results show that the Chlorella CV-5 polysaccharide prepared in Example 1 of this invention can more significantly promote the proliferation of Bifidobacterium bifidum i771.

[0064] Example 2 This example examines the gastric acid resistance of the probiotic microcapsules provided in Example 1 of the present invention. Specifically, 1g of the probiotic microcapsules prepared in Example 1 were placed in 50mL of calf abomasal gastric juice simulation solution (pH 2.5, pepsin content 3.2mg / mL), and shaken in a water bath at 37℃ for 2h. The number of viable bacteria was then detected.

[0065] The results showed that the survival rate of probiotics in the probiotic microcapsules reached 96.21%.

[0066] Example 3 This invention evaluates the probiotic microcapsules prepared in different embodiments on the small intestinal epithelial barrier repair function, the details of which are as follows: A bovine small intestinal epithelial cell monolayer barrier model was constructed using the Transwell system, and hydrogen peroxide was used to induce barrier damage. Calf small intestinal epithelial cells were harvested and cultured in DMEM / F12 complete medium containing 10% fetal bovine serum, 5 μg / L epidermal growth factor, and 10 μg / L insulin-transferrin-selenium at 39°C in a 5% CO2 incubator. Logarithmic growth phase cells were harvested, digested, and then adjusted to a density of 5 × 10⁶ cells / year. 5 Cells / mL were seeded in the upper chamber of a 6-well Transwell chamber (0.4 μm pore size, nested membrane growth area 4.67 cm²), with 1 mL of cell suspension (1 × 10⁻⁶ cells / mL) per well. 5 Add 1.5 mL of complete culture medium to the lower chamber of each cell / well. Incubate at 39°C in a 5% CO2 incubator, changing the culture medium in both chambers every 2 days. Starting from day 3 post-inoculation, measure the transmembrane resistance (TEER) daily using a cell resistance meter. When the TEER value continuously increases and stabilizes at ≥1000 Ω·cm² (usually 7-10 days post-inoculation), and the fluctuation is less than 10% for 3 consecutive days, the cells are considered to have formed a dense, tight junction monolayer, and the model is considered successfully established and can be used for subsequent experiments.

[0067] Three groups were set up: a control group, experimental group 1, and experimental group 2. The control group consisted of a cell model with 2 mL of 0.8 mM H2O2 culture medium added to the upper chamber. Experimental group 1 had 2 mL of culture medium containing 0.8 mM H2O2 and 1 mL of probiotic microcapsule release solution prepared in Example 1 (collected after simulated small intestinal digestion) added to the upper chamber. Experimental group 2 had 2 mL of culture medium containing 0.8 mM H2O2 and 1 mL of probiotic microcapsule release solution prepared in Comparative Example 2 (collected after simulated small intestinal digestion) added to the upper chamber. After mixing, the cultures were incubated at 39°C in a 5% CO2 incubator for 2, 4, and 6 hours, at which time the relative transmembrane resistance (i.e., relative TEER value) was measured. The preparation method of the microcapsule release solution after simulating small intestinal digestion is as follows: First, prepare a simulated small intestinal fluid containing 6.8 g / L KH2PO4, 10 g / L pancreatic enzymes, and 3 g / L bile salts, and adjust the pH to 6.8 with NaOH. Take 2.0 g of probiotic microcapsules and add them to 20 mL of simulated small intestinal fluid. Shake at 39°C and 100 rpm for 4 hours, and collect the microcapsule release fluid after simulated small intestinal digestion. The formula for calculating the relative transmembrane resistance is as follows:

[0068] In the formula, n represents the detection time point, which is 2h, 4h and 6h respectively.

[0069] The relative TEER values ​​of different groups of probiotic microcapsule release solutions were measured as follows: Figure 1 As shown.

[0070] Depend on Figure 1 It is known that H2O2 causes varying degrees of oxidative damage to cells, disrupting the tight junctions between cells and consequently reducing the membrane resistance of monolayer cell cultures. Compared to the control group, the addition of probiotic microcapsules provided in Example 1 of this invention has a certain alleviating effect on the disruption of damaged tight junctions between cells, and the effect is significantly better than that of the probiotic microcapsules provided in Comparative Example 2. The above results indicate that the contents released from the small intestine by the probiotic microcapsules provided by this invention can effectively repair the intestinal epithelial barrier damaged by pathogens.

[0071] Example of effect 4 This invention examines the effects of different probiotic microcapsules on pathogenic bacteria in the feces of diarrheal calves, evaluating the impact of different probiotic microcapsules on the intestinal flora of calves. The specific details are as follows: Calf aged 15-35 days exhibiting diarrhea symptoms were selected from a pasture as experimental subjects. Individuals that had recently received antibiotics, probiotics, or other medications were excluded, as were those that were weak or had irregular feeding habits. Fecal samples were collected from 10 calves with diarrhea and placed in disposable sterile fecal cups (approximately 50g each). After collection, the samples were immediately placed on dry ice for preservation. Finally, the fecal samples were frozen at -80°C for later use.

[0072] The selected fecal samples from diarrheal calves were thoroughly mixed, and 1.5 g of feces was accurately weighed and added to sterile saline to prepare a 10% (w / v) fecal bacterial suspension. After centrifugation at 3000 rpm for 15 seconds to remove impurities, the fecal bacterial suspension was collected using a sterile syringe. Nine aliquots (0.5 mL each) of the fecal bacterial suspension were randomly divided into three groups, with three replicates for each group.

[0073] Each fecal bacterial suspension was mixed with 0.1 g of the probiotic microcapsule release solution provided in Example 1 (i.e., Experimental Group 1 in Effect Example 3) and Comparative Example 2 (i.e., Experimental Group 2 in Effect Example 3), and added to 5 mL of YCFA medium. These were designated as Experimental Group 1 and Experimental Group 2 in this Effect Example, respectively, and cultured at 37°C under anaerobic conditions for 24 h. Simultaneously, a culture medium containing only the fecal bacterial suspension and no sample was cultured under the same conditions as a negative control (designated as the control group). After culture, the pathogenic enterotoxigenic Escherichia coli (EEC) in the samples was detected by qPCR. Enterotoxigenic Escherichia coli ETEC), Clostridium perfringens ( Clostridium perfringens ),salmonella( Salmonella ) and Campylobacter (Campylobacter) The relative abundance of ).

[0074] The specific methods for determining the relative abundance of the four pathogenic bacteria are as follows: Standard plasmids were constructed targeting specific fragments of the 16S rDNA of the four pathogenic bacteria. Based on the known concentration of the standard plasmid DNA, the initial molecular copy number was calculated. Subsequently, the standard plasmids were serially diluted, and qPCR amplification was performed on the plasmid standards from each dilution. The resulting Ct values ​​were fitted to the corresponding copy numbers to construct a standard curve. After the standard curve was established, sample DNA was subjected to qPCR. The Ct values ​​of the sample DNA were substituted into the standard curve to calculate the cell copy number of the target pathogenic bacteria. Finally, the relative abundance of the target pathogenic bacteria in each experimental group was calculated.

[0075] The specific method for determining total bacterial count is as follows: A standard plasmid is constructed using the universal sequence of the V4 variable region of 16S rDNA as a template. Based on the known concentration of the standard plasmid DNA, its initial molecular copy number is calculated. Subsequently, the standard plasmid is serially diluted, and plasmid standards from each dilution are subjected to qPCR amplification. The resulting Ct values ​​are fitted to the corresponding copy numbers to construct a standard curve. After establishing the standard curve, sample DNA is subjected to qPCR. The Ct values ​​of the sample DNA are substituted into the standard curve to calculate the cell copy number of the target pathogenic bacteria.

[0076] The relative abundance of enterotoxigenic Escherichia coli in feces of diarrheal calves in different groups before and after intervention with probiotic microcapsules is as follows: Figure 2 As shown in the figure. The relative abundance of Clostridium perfringens in feces of diarrheal calves in different groups before and after intervention with probiotic microcapsules is as follows. Figure 3 As shown in the figure. The relative abundance of Salmonella in feces of diarrheal calves in different groups before and after intervention with probiotic microcapsules is as follows. Figure 4 As shown in the figure. The relative abundance of Campylobacter in feces of diarrheal calves in different groups before and after intervention with probiotic microcapsules is as follows. Figure 5 As shown.

[0077] Depend on Figure 2-5 It can be seen that after in vitro intervention with probiotic microcapsules in the feces of diarrheal calves, the relative abundance of the four pathogenic bacteria generally showed a decreasing trend. Compared with the probiotic microcapsules prepared in Comparative Example 2, the probiotic microcapsules prepared in Example 1 of this invention have a more significant inhibitory effect on the growth of pathogenic bacteria in the feces of diarrheal calves, especially on enterotoxigenic Escherichia coli and Campylobacter, which are significantly better than those in Comparative Example 2.

[0078] In summary, the probiotic microcapsules provided in this invention can effectively improve the intestinal flora structure of calves and inhibit the growth and reproduction of pathogenic bacteria. Therefore, they can be used to prepare veterinary drugs, feeds or feed additives that improve the intestinal health of calves, especially products related to the prevention and treatment of calf diarrhea.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing probiotic microcapsules, characterized in that: Includes the following steps: S1. After dispersing Lactobacillus plantarum, Lactobacillus paracasei, Chlorella CV-5 and intestinal function regulator in a polysaccharide gel substrate solution, solidifying, collecting gel microspheres, and drying, type A microcapsules are obtained. S2. Disperse Bifidobacterium in a Chlorella polysaccharide-dispersant solution, dry, and obtain type B microcapsules; S3. Mix the type A microcapsules and type B microcapsules, and coat them to obtain probiotic microcapsules; The Chlorella CV-5, with accession number CGMCC No. 15206, has the Latin name [missing information]. Chlorella pyrenoidosa ; The Chlorella polysaccharide-dispersant solution contains Chlorella CV-5 polysaccharide and protein dispersants.

2. The method for preparing probiotic microcapsules as described in claim 1, characterized in that: The *Lactobacillus plantarum* includes *Lactobacillus plantarum* JMCC0017; and / or The *Lactobacillus paracasei* includes *Lactobacillus paracasei* N1115; and / or The intestinal function regulator includes at least one of glutamine, zinc glycine, or fructooligosaccharides; and / or The polysaccharide gel substrate includes sodium alginate; and / or The coagulation solution used for solidification includes calcium salts and chitosan; and / or In step S1, the drying includes freeze drying.

3. The method for preparing probiotic microcapsules as described in claim 1, characterized in that: The Bifidobacteria include Bifidobacterium i771; and / or The protein dispersant includes sodium caseinate; and / or In step S2, the drying includes spray drying.

4. The method for preparing probiotic microcapsules as described in claim 1, characterized in that: The coating agent used includes a 3%-5% Eudragit L100 isopropanol solution.

5. The method for preparing probiotic microcapsules as described in claim 1, characterized in that: The ratio of viable Lactobacillus plantarum to Lactobacillus paracasei is 8-12:10; and / or The ratio of the viable count of *Lactobacillus plantarum* to the mass of *Chlorella pulmonata* CV-5 is (1 × 10⁻⁶). 9 -8×10 10 CFU: (1.5-3) g; and / or The mass ratio of the protein-nucleated Chlorella CV-5 to the intestinal function regulator is 1.5-3:7-12; In step S3, the mass ratio of type A microcapsules to type B microcapsules is 0.8-1.2:

1.

6. The method for preparing probiotic microcapsules according to any one of claims 1-5, characterized in that: The preparation method of the Chlorella CV-5 polysaccharide includes the following steps: Chlorella CV-5 with protein nucleus was mixed with phosphate-citric acid buffer, and enzymatically hydrolyzed with a complex cellulose degrading enzyme. Chlorella extract was then obtained by a programmed pressure- and temperature-controlled cyclic extraction method. The Chlorella extract was precipitated with alcohol and purified to obtain Chlorella proteolyticus CV-5 polysaccharide.

7. The method for preparing probiotic microcapsules as described in claim 6, characterized in that: The mass-to-volume ratio of Chlorella CV-5 to phosphate-citrate buffer is 1 g:(15-40) mL; and / or The programmed pressure and temperature controlled cyclic extraction method includes: maintaining the pressure at 0.25MPa-0.35MPa and 100℃-110℃ for 4min-6min, then depressurizing to atmospheric pressure for 25s-35s and maintaining it at 90℃-98℃ for 2.5min-3.5min; repeating this program for 4-6 cycles.

8. The method for preparing probiotic microcapsules as described in claim 6, characterized in that: The Chlorella extract was evaporated and concentrated, precipitated with alcohol, and the solid and liquid were separated. The precipitate was taken to obtain crude polysaccharide of Chlorella CV-5 with protein nucleus. The crude polysaccharide of Chlorella pulvinata CV-5 was reconstituted, and after removing the protein by adding Sevage reagent, it was washed with ethanol and acetone and dried to obtain Chlorella pulvinata CV-5 polysaccharide.

9. A probiotic microcapsule, characterized in that: It is prepared by the method of preparing probiotic microcapsules according to any one of claims 1-8.

10. The use of the probiotic microcapsules according to claim 9 in the preparation of products for the prevention and treatment of calf diarrhea.

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