Preparation method of probiotic-metagen microcapsule, microcapsule and application thereof

Abstract

The invention belongs to the field of microbial preparations, and discloses a preparation method of a probiotic-metagen microcapsule, the microcapsule and application of the microcapsule. The preparation method comprises the following steps: S1, ultrahigh-pressure pretreatment: mixing probiotics and metabiotics, and adding water to prepare a mixed solution; performing ultrahigh pressure treatment on the mixed solution; s2, composite embedding: preparing a double-layer embedded microcapsule; k-carrageenan and whey protein isolate are used as embedding media to embed the probiotics and the metabiotics in the ultrahigh-pressure mixed liquid to serve as an inner layer of the microcapsule; embedding the inner layer of the microcapsule by taking pectin and chitosan as embedding agents and Ca < 2 + > as a cross-linking agent; and S3, drying and forming: performing freeze drying and forming on the product obtained in the step S2 to obtain the probiotic-metagen microcapsule. The microcapsule can be applied to pet food, the embedding rate of the probiotics can be effectively increased, and intestinal targeted synergistic release of the metabiotics and the probiotics is achieved.

Description

Technical Field

[0001] This invention belongs to the field of microbial preparations, specifically relating to a method for preparing probiotic-metogenic microcapsules, the microcapsules and their applications. Background Technology

[0002] With the development of the pet industry, pet health has received increasing attention. Digestive health is a crucial component of pet health, involving a pet's ability to digest and absorb food and the efficiency of nutrient utilization. A pet's digestive health not only affects its overall health but also directly relates to its lifespan and activity level. Dogs and cats, with their shorter gastrointestinal tracts, are prone to intestinal problems, which can seriously impact their health.

[0003] Probiotics have been proven to have various beneficial physiological effects on the animal gut, such as inhibiting the colonization of harmful bacteria, anti-oxidation, improving gut microbiota structure, enhancing immunity, improving the intestinal mechanical barrier, and reducing the expression of pro-inflammatory factors in the gut. Multiple studies have confirmed that single or mixed strains of probiotics can promote nutrition and health in pets. Currently, probiotics are used as feed additives in pet food. However, probiotics are easily inactivated by heat processing (such as baking and spray drying) and gastric acid, affecting their effectiveness in the gut. Therefore, some pet foods encapsulate probiotics to prevent inactivation; however, existing methods suffer from low encapsulation efficiency, resulting in unsatisfactory probiotic survival rates and failing to completely solve the problem of processing-induced or gastric acid-induced inactivation. Under conventional encapsulation processes, the survival rate of probiotics after exposure to gastric juice is typically <20%.

[0004] Metabiotics are a collective term for the metabolites of probiotics after processing, including the bacterial cells and metabolites. Metabiotics can regulate the intestinal flora and enhance the intestinal barrier function; therefore, they can be combined with probiotics in pet food to jointly protect the digestive health of pets. For example, Chinese invention patent application number CN202411473329.X, "A High-Activity Probiotic Pet Food and its Preparation Method," discloses the preparation of dry pet food by mixing probiotics and metabiotics. However, in existing products, metabiotics and probiotics are usually simply mixed, resulting in premature inactivation of the probiotics and failing to achieve targeted intestinal release of both. Summary of the Invention

[0005] To address the shortcomings of the existing technology, this invention provides a method for preparing probiotic-postbiotic microcapsules, the microcapsules and their applications, which can effectively improve the probiotic encapsulation rate, the double encapsulation layer degrades with pH gradient, and achieve targeted and synergistic release of postbiotics and probiotics into the intestine.

[0006] The specific technical solution is as follows:

[0007] One objective of this invention is to provide a method for preparing probiotic-metogenic microcapsules, characterized by comprising the following steps:

[0008] S1. Ultra-high pressure pretreatment: Mix probiotics and post-biotics and add water to prepare a mixture; subject the mixture to ultra-high pressure treatment at 300~450 MPa for 3~10 min to obtain an ultra-high pressure mixture.

[0009] S2. Composite Encapsulation: Bilayer encapsulated microcapsules were prepared; the inner layer of the microcapsules was encapsulated using κ-carrageenan and whey protein isolate as encapsulating agents to encapsulate the probiotics and postbiotics in the ultra-high pressure mixture obtained in step S1, thus obtaining the inner layer mixture, which served as the inner layer of the microcapsules; the outer layer of the microcapsules was encapsulated using pectin and chitosan as encapsulating agents, Ca... 2+ As a cross-linking agent, it is used to encapsulate the inner layer of microcapsules;

[0010] S3. Drying and shaping: The product obtained in step S2 is freeze-dried and shaped to obtain probiotic-postbiotic microcapsules.

[0011] The mechanism of the above preparation method is as follows: Ultra-high pressure treatment leads to a pressure-osmotic synergistic effect. Ultra-high pressure temporarily permeates the bacterial cell membrane, promoting the adsorption of postbiotic components (such as bacteriocins) on the bacterial surface, forming a protective biofilm, causing the bacteria to enter a sub-dormant state, and forming a microporous structure, thus improving the subsequent encapsulation efficiency. The inner layer of the microcapsule, κ-carrageenan, forms a thermally reversible gel with whey protein isolate to encapsulate the mixture of probiotics and postbiotics, while the outer layer consists of pectin and chitosan. During digestion, the outer pectin-chitosan layer dissolves in gastric acid (pH 2-3), protecting the inner layer while releasing pectin. Pectin, as a prebiotic, enters the intestine together with the inner layer, while the inner κ-carrageenan layer dissolves in the intestine (pH 6-7), releasing the mixture of live bacteria and postbiotics, forming a double-layer protection, pH gradient release mechanism.

[0012] The term "postbiotic" refers to probiotics and their metabolites after processing, specifically inactivated probiotics.

[0013] Specifically, the probiotics are preferably at least one of Lactobacillus plantarum, Bifidobacterium Lactis, Lactobacillus acidophilus, Enterococcus faecalis, Lactobacillus delbrueckii, Saccharomyces cerevisiae, and Clostridium butyricum.

[0014] Specifically, the metabiotic is at least one of the following: inactivated Lactobacillus plantarum, inactivated Bifidobacterium Lactis, inactivated Lactobacillus acidophilus, inactivated Enterococcus faecalis, and inactivated Lactobacillus delbrueckii.

[0015] Furthermore, in step S1, the concentration of the mixture is preferably 3wt% to 10wt%.

[0016] In the mixture, the preferred mass ratio of probiotics to postbiotics is 1:(0.5~2).

[0017] The preferred live bacteria content of probiotics is 1×10⁻⁶. 8 ~1×10 10 CFU / g.

[0018] Specifically, in step S1: the mixture is sealed in a polyethylene bag and then placed in the high-pressure chamber of an ultra-high pressure device for ultra-high pressure treatment. In step S1, water is used as the pressure transmission medium, and the sample is completely immersed in water, causing the bacteria to enter a sub-dormant state and form a microporous structure.

[0019] Furthermore, in step S2: preferably, after the outer layer of the microcapsule is encapsulated, ultra-high pressure treatment is performed; the preferred ultra-high pressure treatment conditions are: ultra-high pressure treatment at 300~450 MPa for 4~12 min. Under ultra-high pressure treatment, the disulfide bonds between protein-polysaccharide molecules recombine, and the microcapsule bilayer membrane becomes denser, which can increase the membrane mechanical strength by up to 200% and reduce the rupture pressure to >1.5 MPa. This improvement in physical properties further enhances the encapsulation efficiency and the bioactivity of probiotics after encapsulation.

[0020] Furthermore, in step S2, the preferred mass ratio of κ-carrageenan to whey protein isolate in the inner layer of the microcapsule is 1:(1~6).

[0021] Furthermore, in step S2, the mass ratio of the total amount of probiotics and postbiotics to κ-carrageenan in the inner layer of the microcapsule is preferably 1:(0.5~4).

[0022] Specifically, in step S2, the microcapsule inner layer embedding step includes: mixing κ-carrageenan and whey protein isolate with water to prepare an inner layer embedding solution; in the inner layer embedding solution, the κ-carrageenan content is preferably 0.5wt%~1wt%, and the whey protein isolate content is preferably 1wt%~3wt%; mixing the inner layer embedding solution with the ultra-high pressure mixture obtained in step S1, preferably stirring and mixing at 35~40℃ and 4000~8000 r / min for 5~15 min to obtain the inner layer mixture; and then embedding the inner layer mixture into the outer layer of the capsule.

[0023] The inner layer mixture can be pasteurized. As mentioned earlier, due to the ultra-high pressure, the bacteria are protected. Pasteurization can kill miscellaneous bacteria, but has no significant effect on probiotics.

[0024] Specifically, in step S2, the step of embedding the outer layer of the capsule includes:

[0025] The inner layer mixture is mixed with pectin solution and injected dropwise into a mixture of chitosan and soluble calcium salts, where a gel reaction occurs to form microcapsules.

[0026] The preferred volume ratio of the inner layer mixture to the pectin solution is 1:(15~30).

[0027] The concentration of the pectin solution is preferably 0.5wt% to 1.5wt%.

[0028] In the mixed solution of chitosan and soluble calcium salt, the concentration of chitosan is preferably 0.05~0.2 g / L, and the concentration of calcium salt (calculated as calcium ions) is preferably 0.05~0.15 mol / L. Specifically, the chitosan solution and the calcium salt solution can be prepared separately and mixed when needed.

[0029] The calcium salt is preferably calcium chloride.

[0030] Specifically, preferably 1-10 minutes after injection, the microcapsules are filtered and washed to remove excess embedding solution, resulting in moistened microcapsules. Washing is typically performed with water.

[0031] Specifically, the particle size of the microcapsules can be controlled by the size of the injection droplets. The droplet size is preferably controlled within the range of 1 to 4 mm.

[0032] Furthermore, in step S2: after the outer layer of the capsule is embedded, ultra-high pressure treatment is performed; the ultra-high pressure treatment conditions are: ultra-high pressure treatment at 300~450 MPa for 4~12 min.

[0033] The second objective of this invention is to provide a probiotic-postbiotic microcapsule obtained using the above preparation method.

[0034] A third objective of this invention is to provide the application of the aforementioned probiotic-metogenic microcapsules in pet food. These probiotic-metogenic microcapsules can be used in dry food, wet food, freeze-dried products, or nutritional supplements for dogs or cats.

[0035] The fourth objective of this invention is to provide a soft chewable pet snack containing the above-mentioned probiotic-postbiotic microcapsules, the raw materials of which include: probiotic-postbiotic microcapsules, enzymatically hydrolyzed meat powder, and cereal raw materials.

[0036] The above-mentioned soft chewable pet treats are suitable for dogs or cats.

[0037] Furthermore, the ingredients of the soft chewable pet snack, by weight, include: 1-10 parts of probiotic-postbiotic microcapsules, 30-40 parts of enzymatically hydrolyzed meat powder, and 1-10 parts of cereal ingredients.

[0038] Furthermore, the ingredients of the pet snacks may also include 1 to 10 parts of fruit and vegetable ingredients and / or 1 to 5 parts of nutrients by weight.

[0039] Specifically, the above-mentioned raw materials also include 20 to 40 parts of water.

[0040] The enzymatically hydrolyzed meat powder is preferably obtained by hydrolyzing at least one of chicken, duck, beef, pork, mutton, fish, and animal offal with a protease followed by drying. The drying method is preferably spray drying.

[0041] The cereal raw material is preferably at least one of soy protein isolate, pea protein, wheat gluten, rice flour, modified starch, corn starch, and wheat flour.

[0042] The preferred fruit and vegetable ingredients are at least one of the following: potatoes, pumpkins, purple sweet potatoes, sweet potatoes, carrots, beets, kale, tomatoes, peas, broccoli, konjac, chickpeas, zucchini, cabbage, apples, pears, blueberries, cranberries, and goji berries.

[0043] The preferred nutrient element is selected from at least one of the following: wheat oligopeptide, soybean oligopeptide, corn oligopeptide, rosemary extract, salmon oil, flaxseed oil, peanut oil, sunflower seed oil, seaweed powder, lecithin, calcium sulfate, phosphorus, potassium, magnesium, ferrous sulfate, copper sulfate, manganese sulfate, zinc sulfate, choline chloride, vitamin B1, vitamin B2, vitamin D, vitamin E, biotin, folic acid, and calcium phosphate.

[0044] Furthermore, the aforementioned soft chewable pet treats are prepared by hot air baking.

[0045] Specifically, the preparation method of the above-mentioned soft chewable pet treats includes the following steps:

[0046] S1. Mixing: Mixing various raw materials, including water, to prepare a mixed liquid;

[0047] S2. Liquid molding: The mixed liquid is injected into the mold cavity and extruded from the mold to form the shape;

[0048] S3. Baking and shaping: Bake the extruded material at 55~60℃ with hot air for 15~30 minutes.

[0049] In step S2, after the mixed liquid is injected into the mold, it is preferable to remove air bubbles by gently tapping the mold or by using vacuum degassing to ensure that the product surface is smooth and free of defects.

[0050] The beneficial effects of this invention are as follows:

[0051] This invention uses ultra-high pressure treatment combined with postbiotic protection to induce the bacteria into a sub-dormant state and form a microporous structure, which effectively improves the encapsulation efficiency of subsequent probiotics and thus enhances the survival rate of probiotics during heat processing and gastric digestion.

[0052] Furthermore, through a pH gradient release design, a double-layer protection for probiotics is achieved. The outer layer of pectin-chitosan dissolves in gastric acid (pH 2~3), releasing pectin as a prebiotic, which enters the intestine together with the inner layer. The inner layer of κ-carrageenan dissolves in the intestine (pH 6~7), releasing live bacteria and postbiotics, thus achieving targeted and synergistic release of probiotics and postbiotics into the intestine.

[0053] In addition, ultra-high pressure treatment is performed after encapsulation to recombine the disulfide bonds between protein-polysaccharide molecules, thereby improving the mechanical strength of the membrane.

[0054] This invention utilizes an ultra-high pressure assisted composite coagulation method to prepare probiotic-metaseogenic microcapsules. The process is simple and easy to operate, with mild and easily controllable process parameters. This not only improves the density of the probiotic encapsulation structure but also addresses the issues of low survival rate and slow release rate of probiotics in dogs and cats during their short digestion time. The hydrophilic raw materials used in the encapsulation wall can be rapidly degraded by the host's own enzyme system and microbial community in the gastrointestinal tract of dogs and cats.

[0055] The soft chewable snack provided by this invention is made by low-temperature casting, which protects heat-sensitive components and provides a new dosage form and processing solution for functional pet snacks containing probiotics. Detailed Implementation

[0056] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0057] Example 1

[0058] (a) Preparation of probiotic-metogenic microcapsules, the method is as follows:

[0059] S1. Ultra-high pressure pretreatment: At room temperature, probiotics (Lactobacillus plantarum, content 1×10⁻⁶) at a mass ratio of 1:1 are added. 9 The mixture of CFU / g and post-biotic (inactivated Lactobacillus plantarum) was prepared with water to form a total concentration of 6wt%. The mixture was sealed in a polyethylene bag and then placed in the high-pressure chamber of an ultra-high pressure equipment for ultra-high pressure treatment. The treatment pressure was set to 400MPa and the treatment time was 5 min to obtain the ultra-high pressure mixture.

[0060] S2. Composite encapsulation: Preparation of bilayer encapsulated microcapsules; the bilayer encapsulated microcapsules include an inner microcapsule layer and an outer microcapsule layer;

[0061] Inner layer encapsulation of microcapsules: κ-carrageenan and whey protein isolate were mixed with water to prepare the inner layer encapsulation solution; the inner layer encapsulation solution contained 0.5 wt% κ-carrageenan and 1 wt% whey protein isolate; the ultra-high pressure mixture obtained in step S1 was mixed with the inner layer encapsulation solution at a mass ratio of 1:10 (the mass ratio of total probiotics and postbiotics to κ-carrageenan was 1:0.83), and stirred at 37℃ and 6000 r / min for 10 min; pasteurized in an 80℃ water bath for 5 min; the inner layer mixture was obtained and ready for outer layer encapsulation of microcapsules.

[0062] For microcapsule outer layer encapsulation: the inner layer mixture was mixed with a 1 wt% pectin solution at a volume ratio of 1:20, and then injected dropwise into a mixed solution containing 0.08 g / L chitosan and 0.1 mol / L calcium chloride. A gel reaction occurred to form microcapsules, and the size of the injection droplets was controlled between 1 and 4 mm. Five minutes after injection, the mixture was filtered and washed with water to remove excess encapsulation solution, resulting in moist microcapsules.

[0063] The material obtained from the outer layer encapsulation is subjected to ultra-high pressure treatment again: the material obtained from the outer layer encapsulation is sealed and placed into a polyethylene bag, and then placed in the high-pressure chamber of the ultra-high pressure equipment for ultra-high pressure treatment. The treatment pressure is set to 400 MPa and the treatment time is 6 minutes.

[0064] S3. Drying and shaping: The product obtained in step S2 is freeze-dried and shaped; the resulting dried powder is vacuum-sealed in an opaque aluminum foil bag and placed in a desiccator for later use, which is the probiotic-postbiotic microcapsule.

[0065] (ii) Using the probiotic-metasein microcapsules obtained by the aforementioned method, soft chewable pet treats were prepared. The raw materials, by weight, are as follows:

[0066] Two portions of probiotic-postbiotic microcapsules, 40 portions of enzymatically hydrolyzed chicken breast powder, 5 portions of soy protein isolate, 5 portions of wheat gluten, 2 portions of broccoli, 2 portions of cabbage, 2 portions of multivitamins, 2 portions of multiminerals, and 40 portions of water.

[0067] The preparation method of the above probiotic-postbiotic microcapsules is as follows:

[0068] S1. Mixing: Mixing various raw materials, including water, to prepare a mixed liquid;

[0069] S2. Liquid molding: The mixed liquid obtained in step S1 is injected into the mold cavity through an injection pump; the pouring speed is controlled to avoid the generation of air bubbles; in addition, the liquid filling amount is strictly controlled to avoid overflow or insufficient filling, and to ensure the uniformity of each mold cavity; after the liquid is injected into the mold, the mold is gently tapped to remove air bubbles in the liquid, ensuring that the product surface is smooth and without defects; extrusion molding; the granules obtained after extrusion molding from the mold are spread evenly on the mesh frame;

[0070] S3. Baking and shaping: Bake at a constant temperature of 60℃ with hot air for 20 minutes, then remove from the mesh and cool to obtain the finished product.

[0071] Example 2

[0072] Referring to Example 1, the difference from Example 1 is that in the preparation process of probiotic-post-biotic microcapsules, in step S2: the material obtained by outer layer encapsulation was not subjected to ultra-high pressure treatment again;

[0073] Other technical features are the same as in Example 1.

[0074] Example 3

[0075] The method for preparing probiotic-metagorogens microcapsules is as follows:

[0076] S1. Ultra-high pressure pretreatment: At room temperature, probiotics (Lactobacillus plantarum, content 1×10⁻⁶) at a mass ratio of 1:1 are added. 9 The mixture of CFU / g and post-biotic (inactivated Lactobacillus plantarum) was prepared with water to form a total concentration of 3wt%. The mixture was sealed in a polyethylene bag and then placed in the high-pressure chamber of an ultra-high pressure equipment for ultra-high pressure treatment. The treatment pressure was set to 450MPa and the treatment time was 3 min to obtain the ultra-high pressure mixture.

[0077] S2. Composite encapsulation: Preparation of bilayer encapsulated microcapsules; the bilayer encapsulated microcapsules include an inner microcapsule layer and an outer microcapsule layer;

[0078] Microcapsule inner layer encapsulation: κ-carrageenan and whey protein isolate were mixed with water to prepare the inner layer encapsulation solution; the inner layer encapsulation solution contained 1 wt% κ-carrageenan and 1 wt% whey protein isolate; the ultra-high pressure mixture obtained in step S1 was mixed with the inner layer encapsulation solution at a mass ratio of 1:12 (the mass ratio of total probiotics and postbiotics to κ-carrageenan was 1:4), and stirred at 37℃ and 6000 r / min for 10 min; pasteurized in an 80℃ water bath for 5 min; the inner layer mixture was obtained and ready for microcapsule outer layer encapsulation;

[0079] For microcapsule outer layer encapsulation: the inner layer mixture was mixed with a 1 wt% pectin solution at a volume ratio of 1:20, and then injected dropwise into a mixed solution containing 0.08 g / L chitosan and 0.1 mol / L calcium chloride. A gel reaction occurred to form microcapsules, and the size of the injection droplets was controlled between 1 and 4 mm. Five minutes after injection, the mixture was filtered and washed with water to remove excess encapsulation solution, resulting in moist microcapsules.

[0080] The material obtained from the outer layer encapsulation is subjected to ultra-high pressure treatment again: the material obtained from the outer layer encapsulation is sealed and placed into a polyethylene bag, and then placed in the high-pressure chamber of the ultra-high pressure equipment for ultra-high pressure treatment. The treatment pressure is set to 300 MPa and the treatment time is 12 minutes.

[0081] S3. Drying and shaping: The product obtained in step S2 is freeze-dried and shaped; the resulting dried powder is vacuum-sealed in an opaque aluminum foil bag and placed in a desiccator for later use, which is the probiotic-postbiotic microcapsule.

[0082] Example 4

[0083] The method for preparing probiotic-metagorogens microcapsules is as follows:

[0084] S1. Ultra-high pressure pretreatment: At room temperature, probiotics (Lactobacillus plantarum, content 1×10⁻⁶) at a mass ratio of 1:1 are added. 9 The mixture of CFU / g and post-biotic (inactivated Lactobacillus plantarum) was prepared with water to form a total concentration of 10wt%. The mixture was sealed in a polyethylene bag and then placed in the high-pressure chamber of an ultra-high pressure equipment for ultra-high pressure treatment. The treatment pressure was set to 300MPa and the treatment time was 10 min to obtain the ultra-high pressure mixture.

[0085] S2. Composite encapsulation: Preparation of bilayer encapsulated microcapsules; the bilayer encapsulated microcapsules include an inner microcapsule layer and an outer microcapsule layer;

[0086] Inner layer encapsulation of microcapsules: κ-carrageenan and whey protein isolate were mixed with water to prepare the inner layer encapsulation solution; the inner layer encapsulation solution contained 0.5 wt% κ-carrageenan and 3 wt% whey protein isolate; the ultra-high pressure mixture obtained in step S1 was mixed with the inner layer encapsulation solution at a mass ratio of 1:10 (the mass ratio of total probiotics and postbiotics to κ-carrageenan was 1:0.5), and stirred at 37℃ and 6000 r / min for 10 min; pasteurized in an 80℃ water bath for 5 min; the inner layer mixture was obtained and ready for outer layer encapsulation of microcapsules;

[0087] For the outer layer encapsulation of microcapsules: the inner layer mixture was mixed with a 1 wt% pectin solution at a volume ratio of 1:20, and then injected dropwise into a mixed solution containing 0.08 g / L chitosan and 0.1 mol / L calcium chloride. A gel reaction occurred to form microcapsules, and the size of the injection droplets was controlled between 1 and 4 mm. Five minutes after injection, the mixture was filtered and washed with water to remove excess encapsulation solution, resulting in moist microcapsules.

[0088] The material obtained from the outer layer encapsulation is subjected to ultra-high pressure treatment again: the material obtained from the outer layer encapsulation is sealed and placed into a polyethylene bag, and then placed in the high-pressure chamber of the ultra-high pressure equipment for ultra-high pressure treatment. The treatment pressure is set to 450 MPa and the treatment time is 4 minutes.

[0089] S3. Drying and shaping: The product obtained in step S2 is freeze-dried and shaped; the resulting dried powder is vacuum-sealed in an opaque aluminum foil bag and placed in a desiccator for later use, which is the probiotic-postbiotic microcapsule.

[0090] Comparative Example 1

[0091] Referring to Example 2, the difference from Example 2 is that in step S1: the mixture was not subjected to ultra-high pressure treatment, and the mixture was directly composite-embedded.

[0092] Comparative Example 2

[0093] Referring to Example 1, the difference from Example 1 is that in step S1: the mixture was not subjected to ultra-high pressure treatment, and the mixture was directly composite-embedded.

[0094] Comparative Example 3

[0095] Referring to Example 1, the difference from Example 1 is that: in step S1, no postbiotic was added, and 3wt% of probiotics (Lactobacillus plantarum, content 1×10) were directly added. 9 The mixture of CFU / g was subjected to ultra-high pressure treatment.

[0096] Test 1

[0097] The encapsulation efficiency (EE) of the microcapsules obtained in Examples 1-4 and Comparative Examples 1-3 was tested. The test method is as follows: 1 g of microcapsule was placed in 9 mL of simulated intestinal fluid, centrifuged at 20000 r / min for 10 min, and then decapsulated. The capsules were then plated and counted.

[0098] The method for preparing simulated intestinal fluid is as follows: Dissolve 6.8 g of potassium dihydrogen phosphate in 500 mL of distilled water, adjust the pH to 6.8 with 0.1 mol / L NaOH solution, and sterilize at 120℃ for 20 min. Separately, dissolve 10 g of trypsin in water, mix and dilute the two solutions to 1000 mL, filter through a 0.22 μm sterile filter membrane, and use immediately after preparation.

[0099] The encapsulation rate is calculated using the formula: EE = N / N0 × 100%;

[0100] In the formula: N is the number of live bacteria after probiotic encapsulation (CFU / mL); N0 is the number of live bacteria before probiotic encapsulation (CFU / mL).

[0101] The test results are shown in Table 1.

[0102] Table 1 Comparison of microcapsule encapsulation rates

[0103]

[0104] Referring to Table 1: Comparing the encapsulation rates of Example 1 (two ultra-high pressure treatments), Example 2 (ultra-high pressure pretreatment only), Comparative Example 1 (no ultra-high pressure treatment), and Comparative Example 2 (ultra-high pressure treatment only after encapsulation), it can be seen that both ultra-high pressure treatments of the present invention can significantly improve the encapsulation rate of probiotics. Meanwhile, comparing Comparative Example 3 with Example 1, it shows the effect of adding postbiotics on improving the encapsulation rate.

[0105] Test 2

[0106] Simulated digestion tests were conducted on the microcapsules obtained in Examples 1-4 and Comparative Examples 1-3, using the following methods:

[0107] 1. Preparation of experimental materials:

[0108] (1) Preparation of simulated gastric juice: Add 0.32 g pepsin and 0.2 g sodium chloride to every 100 mL of deionized water. Adjust the pH to 2.5 with 1M HCl (to simulate the gastric acid environment of dogs and cats on an empty stomach).

[0109] (2) Preparation of simulated intestinal fluid: Prepare phosphate buffer with deionized water, adjust the pH to 6.8 with 1M NaOH (simulating the small intestinal environment), and add 0.1 g trypsin and 0.08 g porcine bile salt to every 100 mL of phosphate buffer.

[0110] 2. Simulating stomach digestion:

[0111] (1) Preparation: Preheat the simulated gastric juice to 39°C (dog and cat body temperature).

[0112] (2) Inoculation: Weigh 1.0 g of microcapsule sample and free probiotic sample respectively, and place them in a centrifuge tube containing 10 mL of simulated gastric fluid to ensure full suspension.

[0113] (3) Digestion: The centrifuge tubes were placed in a 39°C constant temperature shaker and shaken at 100 rpm for 2 hours to simulate the peristalsis of the stomach.

[0114] (4) Sampling: At 0 hours (initial), 0.5 hours, 1 hour and 2 hours of simulated gastric digestion, 1 mL of sample was taken and immediately adjusted to neutral with phosphate buffer solution and placed in an ice water bath to inactivate enzymes. Then viable bacteria count was performed to assess the survival rate of bacteria during gastric digestion.

[0115] 3. Simulating intestinal digestion:

[0116] (1) pH adjustment: The pH of the remaining sample mixture after 2 hours of gastric digestion was slowly adjusted to 6.8 using 1M NaHCO3 solution.

[0117] (2) Convert digestive fluid: Add an equal volume of simulated intestinal fluid preheated to 39°C to the above system.

[0118] (3) Continue digestion: The mixture was placed in a constant temperature shaker at 39°C and shaken at 100 rpm for 4 hours to simulate the digestion process in the small intestine.

[0119] (4) Sampling: 1 mL samples were taken at 1, 2 and 4 hours after the start of intestinal digestion to count live bacteria and assess the release and survival of probiotics in the intestine.

[0120] 4. Detection and Analysis:

[0121] Viable bacteria were counted using the plate count method. The samples were serially diluted with sterile physiological saline, plated onto MRS broth, and incubated for 32 hours under suitable conditions. The viable count was then calculated, and the viable count was expressed as colony-forming units (CFU).

[0122] The survival rate is calculated using the following formula: Simulated digestion survival rate = N1 / N0 × 100%;

[0123] In the formula: N1 is the number of live bacteria after simulated digestion of probiotics (CFU / mL); N0 is the number of live bacteria before encapsulation of probiotics (CFU / mL).

[0124] The experimental results of intestinal digestion for 4 hours are shown in Table 2.

[0125] Table 2 Survival rate of microcapsules after simulated digestion

[0126]

[0127] Under near-neutral pH and bile salt conditions, the microcapsules begin to swell, degrade, and continuously release surviving probiotics. As shown in Table 2, the capsules of Example 1 maintained a high level of viable bacteria count after 4 hours of intestinal digestion, with a survival rate of 86.17%. This indicates that the microcapsule wall material effectively blocks the invasion of gastric acid and enzymes into the internal probiotics, not only protecting the probiotics as they pass through the stomach but also enabling targeted release in the intestines, ensuring a sufficient number of live bacteria reach the site of action.

[0128] 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, improvements, etc., 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-metogenic microcapsules, characterized in that, Includes the following steps: S1. Ultra-high pressure pretreatment: Mix probiotics and post-biotics and add water to prepare a mixture; subject the mixture to ultra-high pressure treatment at 300~450MPa for 3~10 min to obtain an ultra-high pressure mixture. S2. Composite Encapsulation: Bilayer encapsulated microcapsules were prepared; the inner layer of the microcapsules was encapsulated using κ-carrageenan and whey protein isolate as encapsulating agents to encapsulate the probiotics and postbiotics in the ultra-high pressure mixture obtained in step S1, thus obtaining the inner layer mixture, which served as the inner layer of the microcapsules; the outer layer of the microcapsules was encapsulated using pectin and chitosan as encapsulating agents, Ca... 2+ As a cross-linking agent, it is used to encapsulate the inner layer of microcapsules; S3. Drying and shaping: The product obtained in step S2 is freeze-dried and shaped to obtain probiotic-postbiotic microcapsules.

2. The preparation method according to claim 1, characterized in that, The probiotics mentioned are at least one of Lactobacillus plantarum, Bifidobacterium lactis, Lactobacillus acidophilus, Enterococcus faecalis, Lactobacillus delbrueckii, Saccharomyces cerevisiae, and Clostridium butyricum. The metabiotic is at least one of inactivated Lactobacillus plantarum, inactivated Bifidobacterium lactis, inactivated Lactobacillus acidophilus, inactivated Enterococcus faecalis, and inactivated Lactobacillus delbrueckii.

3. The preparation method according to claim 1 or 2, characterized in that, In step S1: the concentration of the mixture is 3wt%~10wt%.

4. The preparation method according to claim 1 or 2, characterized in that, In step S2: After the outer layer of the capsule is embedded, ultra-high pressure treatment is performed; the ultra-high pressure treatment conditions are: ultra-high pressure treatment at 300~450 MPa for 4~12 minutes.

5. The preparation method according to claim 1 or 2, characterized in that, In step S2, during the inner layer embedding of the capsule: The mass ratio of κ-carrageenan to whey protein isolate was 1:(1~6); The total amount of probiotics and postbiotics was in a mass ratio of 1:(0.5~4) to κ-carrageenan.

6. The preparation method according to claim 1 or 2, characterized in that, Step S2, the step of embedding the outer layer of the capsule, includes: The inner layer mixture is mixed with pectin solution and injected dropwise into a mixture of chitosan and soluble calcium salts, where a gel reaction occurs to form microcapsules.

7. A probiotic-postbiotic microcapsule, obtained using the preparation method described in any one of claims 1 to 6.

8. The application of the probiotic-metogenic microcapsule as described in claim 7 in pet food.

9. A soft-chewable pet treat comprising the probiotic-metaseogenic microcapsules as described in claim 7, characterized in that, Raw materials include: Probiotic-postbiotic microcapsules, enzymatically hydrolyzed meat powder, and cereal ingredients.

10. The soft chewable pet treat according to claim 9, characterized in that, It is prepared by hot air baking.

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