Freeze-dried pet food capable of improving intestinal functions of pets and preparation method of freeze-dried pet food

Abstract

The invention provides freeze-dried pet food capable of improving intestinal functions of pets and a preparation method of the freeze-dried pet food. Firstly, lecithin and beta-sitosterol are used for constructing lipidosome embedded compound bacteria, and then lactoferrin is combined with embedded compound enzyme to form microspheres; chitosan oligosaccharide is used for connecting compound bacteria liposome and compound enzyme microspheres, the chitosan oligosaccharide is loaded in an alginate gel network to prepare a compound microcapsule, finally raw materials such as yoghourt, camel milk, milk mineral salt and galactooligosaccharide are mixed, and the freeze-dried pet food is prepared through vacuum freeze drying. The sodium alginate composite microcapsule prepared by the invention can greatly improve the stability of probiotics and enzymes, ensure the activity of the probiotics and the enzymes, and improve the palatability of pets to daily ration, increase the food consumption of the pets, promote the digestion and absorption of nutrient substances by the intestinal tracts of the pets and improve the immunity of the pets by virtue of targeted release of the intestinal tracts and synergistic effect of the prebiotics. The intestinal function of the pet is improved, and an efficient and safe solution is provided for the intestinal health of the pet.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and in particular relates to a freeze-dried pet food for improving the intestinal function of pets and a preparation method thereof. Background Art

[0002] The intestines are a key organ for nutrient absorption and digestion in pets, and their health plays a crucial role in their overall well-being. As a crucial site for nutrient digestion and absorption, intestinal dysfunction can lead to indigestion, insufficient absorption, and ultimately, malnutrition. Maintaining a balanced intestinal flora is a key factor in intestinal health. Beneficial bacteria inhibit the growth of harmful bacteria, promote nutrient absorption, enhance immunity, and even influence a pet's mood. Imbalanced flora can predispose pets to various illnesses, such as diarrhea, vomiting, and other intestinal ailments, and may also be associated with conditions like obesity and diabetes.

[0003] To improve pet intestinal health, existing research focuses on adding probiotics or enzyme preparations to pet food to enhance the pet's intestinal absorption capacity of nutrients and promote the proliferation of beneficial intestinal microorganisms. However, probiotics or enzyme preparations added alone are prone to losing their activity in the extreme environment of the pet's stomach, affecting the effectiveness of their beneficial effects. Therefore, bacterial strain immobilization or enzyme immobilization is often used to ensure their activity. Existing research mainly focuses on single-direction bacterial strain immobilization or enzyme immobilization. A single immobilized enzyme or immobilized bacterial strain sometimes struggles to fully activate a specific substrate. Therefore, co-immobilization technology can leverage the synergistic effects of enzymes and cells. However, research on bacterial and enzyme co-immobilization is still limited. Adsorption-encapsulation is usually used to achieve bacterial and enzyme co-immobilization. Since adsorption-encapsulation utilizes weak interactions between bacteria or enzymes and carrier materials, they are prone to detachment or leakage during the process, weakening the corresponding effect. In addition, existing research on bacterial and enzyme co-immobilization technology has mainly been applied to the brewing industry or pollutant treatment, and has been less widely used in pet food. Summary of the Invention

[0004] Technical problems to be solved: In response to the above technical problems, the purpose of the present invention is to provide a freeze-dried pet food that improves the intestinal function of pets and a preparation method thereof. First, lecithin and β-sitosterol are used to construct liposomes to embed composite bacteria, and then lactoferrin is used to bind and embed composite enzymes to form microspheres. Chitosan oligosaccharides are used to connect the composite bacteria liposomes and composite enzyme microspheres and load them into alginate gel networks to form composite microcapsules. Finally, yogurt, camel milk, milk mineral salts, oligogalactose and other raw materials are mixed and vacuum freeze-dried to form freeze-dried pet food. The sodium alginate composite microcapsules prepared by the present invention can greatly improve the stability of probiotics and enzymes, ensure the activity of probiotics and enzymes, and release them in an intestinal-targeted manner. With the synergistic effect of prebiotics, it improves the palatability of pets to the diet, increases the pet's food intake, promotes the digestion and absorption of nutrients by the pet's intestines, improves the intestinal function of pets, and provides an efficient and safe solution for the intestinal health of pets.

[0005] Technical solution: A freeze-dried pet food for improving the intestinal function of pets, characterized by comprising the following raw materials in parts by weight: 45-60 parts of yogurt, 15-20 parts of camel milk, 1-2.5 parts of milk mineral salts, 2-5 parts of galacto-oligosaccharides, and 1.5-3 parts of sodium alginate composite microcapsules.

[0006] Furthermore, the preparation method of the sodium alginate composite microcapsules comprises the following steps: Step 1. activating Lactobacillus plantarum, Bacillus coagulans and Pediococcus pentosaceus to obtain Lactobacillus plantarum bacterial solution, Bacillus coagulans bacterial solution and Pediococcus pentosaceus bacterial solution respectively, and mixing them to prepare a composite bacterial solution; (1) Activation of Lactobacillus plantarum: freeze-dried Lactobacillus plantarum was inoculated into MRS liquid culture medium, cultured at 37°C for 24 hours, and then inoculated at an inoculum volume of 1-4% of the culture medium, cultured at 37°C for 24 hours, and the obtained bacterial solution was centrifuged at 4000 r / min for 10 minutes, washed twice with sterile phosphate buffer, and centrifuged to discard the supernatant to obtain a bacterial precipitate. Sterile physiological saline was added to the bacterial precipitate to obtain a Lactobacillus plantarum bacterial solution; (2) Activation of Bacillus coagulans: freeze-dried Bacillus coagulans was inoculated into PCA liquid culture medium, cultured at 37°C for 18 hours, and then inoculated at an inoculum rate of 1-5% of the culture medium, cultured at 37°C for 24 hours, and the resulting bacterial solution was centrifuged at 4000 rpm for 10 minutes, washed twice with sterile phosphate buffer, and centrifuged to discard the supernatant to obtain a bacterial precipitate. Sterile physiological saline was added to the bacterial precipitate to obtain a Bacillus coagulans bacterial solution; (3) Activation of Pediococcus pentosaceus: freeze-dried Pediococcus pentosaceus was inoculated into MRS liquid culture medium, cultured at 37°C for 24 hours, and then inoculated at an inoculum volume of 1-4% of the culture medium. The culture was cultured at 37°C for 24 hours, and the obtained bacterial solution was centrifuged at 4000 r / min for 10 minutes. The solution was washed twice with sterile phosphate buffer and centrifuged to discard the supernatant to obtain a bacterial precipitate. Sterile physiological saline was added to the bacterial precipitate to obtain a Pediococcus pentosaceus bacterial solution. Step 2. preparing lysozyme, phytase and xylanase into lysozyme solution, phytase solution and xylanase solution respectively, and mixing them to prepare a composite enzyme solution; Step 3. Phosphatidylcholine and β-sitosterol are mixed uniformly in a mass ratio of (6-10): (1-3) and dissolved in anhydrous ethanol, and subjected to rotary evaporation at 40-50°C and vacuum drying for 4-8 hours to obtain a phospholipid film; the composite bacterial solution and the phospholipid film are mixed and ultrasonically treated, filtered, and hydrated to obtain composite bacterial liposomes; Step 4. Lactoferrin is dissolved in water to prepare a lactoferrin solution with a concentration of 20-35 mg / mL and the pH is adjusted to 5.0-6.5. The complex enzyme solution is uniformly added dropwise to the lactoferrin solution and ultrasonically treated for 5-10 minutes, followed by drying to obtain complex enzyme microspheres. Step 5. The composite bacterial liposomes, chitosan oligosaccharides and composite enzyme microspheres are blended to obtain a bacterial enzyme complex, and then the bacterial enzyme complex is evenly mixed with a sodium alginate solution at a volume ratio of 1: (3-6), and the mixture is dropwise added into a calcium lactate solution with a mass fraction of 2-4.5% and cured at 350-500 r / min for 30-60 min, and dried to obtain sodium alginate composite microcapsules.

[0007] Furthermore, in step 1, the volume ratio of the Lactobacillus plantarum solution, the Bacillus coagulans solution and the Pediococcus pentosaceus solution is (1-3): (1.5-3): (1-4), and the concentration of the solution is (1.5-4)×10 8 CFU / mL.

[0008] Furthermore, in step 2, the volume ratio of the lysozyme solution, the phytase solution and the xylanase solution is (2-4.5): (1-2): (1-3).

[0009] Furthermore, in step 3, the ratio of the composite bacterial solution to the phospholipid film is 1 mL: (1-3.5) g; the ultrasonic treatment conditions are ultrasonic power 200-300 W and ultrasonic time 10-20 min; and the hydration treatment conditions are hydration temperature 40-50° C. and hydration time 1-3 h.

[0010] Furthermore, in step 4, the volume ratio of the complex enzyme solution to the lactoferrin solution is 1:(1-4); and in step 5, the addition ratio of the complex bacterial liposomes, chitosan oligosaccharides and complex enzyme microspheres is (1-2.5) mL:(1-2) g:(1-2) g.

[0011] A method for preparing freeze-dried pet food for improving intestinal function of pets, characterized by comprising the following steps: S1. yogurt, camel milk, milk mineral salts, galacto-oligosaccharides, and sodium alginate composite microcapsules are blended to prepare a blend; S2. The blended material is vacuum freeze-dried, shaped, and packaged to obtain freeze-dried pet food.

[0012] 9. The method for preparing freeze-dried pet food for improving intestinal function of pets according to claim 7, characterized in that the specific conditions for vacuum freeze drying in S2 are as follows: ① pre-freezing stage: pre-freezing temperature -45--35°C, pre-freezing time ≥ 3 hours; ② sublimation drying stage: temperature 40-60°C, drying time 10-12 hours, vacuum degree 50-70 Pa; ③ desorption drying stage: temperature 40-55°C, drying time 6-8 hours, vacuum degree 50-70 Pa; the cold trap temperature of the vacuum freeze drying is -50--45°C.

[0013] Beneficial effects: 1. The present invention uses liposomes made of lecithin and β-sitosterol to embed probiotics. The phospholipid bilayer formed by lecithin-β-sitosterol can isolate the influence of the external environment on the activity of probiotics. At the same time, lactoferrin has a strong positive charge under a neutral environment, directly electrostatically binds to negatively charged phytase and xylanase, and uses a hydrophobic core to embed positively charged lysozyme. Under ultrasound-induced disulfide cross-linking in lactoferrin forms a stable three-dimensional network microsphere. In addition, the linear glycoside long chain of chitosan oligosaccharide contains amino acids at both ends. The amino group at one end is statically cross-linked with the phosphate group in the head group of lecithin in the composite bacterial liposome to form an ionic bond, and its hydroxyl group forms a hydrogen bond with the hydroxyl group of β-sitosterol in the composite bacterial liposome; the amino group at the other end is combined with the carboxyl group of lactoferrin in the composite enzyme microspheres through an ionic bond, and its hydroxyl group forms a hydrogen bond with the carbonyl / hydroxyl group of lactoferrin in the composite enzyme microspheres, thereby improving the binding stability of the linear glycoside chain of chitosan oligosaccharide with the composite bacterial liposomes and composite enzyme microspheres, forming a "dumbbell structure" unit.

[0014] 2. The present invention uses sodium alginate to embed "dumbbell structure" units to prepare sodium alginate microcapsules with pH response. When the bacterial enzyme composite microcapsules enter the pet's stomach, the acidic environment of the stomach will keep the carboxyl groups of the sodium alginate in a protonated state, and the intermolecular hydrogen bonds will be strengthened, causing the gel network to shrink and form a dense barrier, thereby protecting the activity of probiotics and enzyme preparations; when the bacterial enzyme composite microcapsules enter the pet's intestines, the slightly neutral environment of the intestines will deprotonate the carboxyl groups of the sodium alginate (negatively charged), resulting in increased electrostatic repulsion, and the gel structure absorbs water and swells, thereby releasing the "dumbbell structure" units; the amino groups (positive charge) in the long linear glycoside chain of the chitosan oligosaccharide connecting the bridge of the "dumbbell structure" unit produce electrostatic interactions with the intestinal mucosa (negative charge). At the same time, the three-dimensional structure of lactoferrin enables it to bind to specific cell receptors in the pet's intestines, causing it to colonize in the intestinal mucosa, thereby stably and long-term releasing probiotics and enzyme preparations in the pet's intestines.

[0015] 3. The present invention adopts the synergistic effect of compound probiotics + compound enzyme preparations to improve the intestinal function of pets and maintain the intestinal health of pets. The compound bacteria (Lactobacillus plantarum / Bacillus coagulans / Pediococcus pentosaceus) and compound enzymes (lysozyme / phytase / xylanase) can maintain good activity in the pet's intestines (intestinal temperature 37.5-39°C). On the one hand, the probiotics composed of Lactobacillus plantarum + Bacillus coagulans + Pediococcus pentosaceus: ① can increase the biodensity of beneficial bacteria in the intestine, regulate the structure of intestinal flora and produce lactic acid, moderately lower the intestinal pH value, thereby inhibiting the proliferation of harmful bacteria and promoting the growth and colonization of beneficial bacteria; ② promote the tight junction of intestinal epithelial cells, enhance the intestinal barrier function, and at the same time induce the immune system to produce anti-inflammatory cytokines, reduce intestinal inflammatory response, thereby protecting the intestinal barrier and improving the intestinal barrier function, thereby achieving the purpose of improving the intestinal function of pets. On the other hand, the enzyme preparation composed of lysozyme / phytase / xylanase: ① Lysozyme can destroy the peptidoglycan scaffold of the cell wall of harmful bacteria and destroy the cell wall, thereby reducing the number of harmful bacteria in the pet's intestines; at the same time, phytase increases the level of available phosphorus, regulates the development of intestinal microbial flora, and promotes the growth of beneficial bacteria; xylanase can hydrolyze the indigestible cellulose components in the pet's complete diet, and the oligoxylose obtained by hydrolysis can serve as a prebiotic, which can promote the proliferation of beneficial intestinal bacteria (such as lactobacilli) and improve the structure of intestinal flora; ② Reduce the secretion of mucus in the intestine, reduce the nutritional source of pathogens, and enhance the intestinal barrier function to prevent the invasion of harmful bacteria, thereby reducing the occurrence of intestinal inflammation and disease and protecting the intestinal health of pets. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The gastrointestinal stability of the sodium alginate composite microcapsules prepared in Example 1 and Comparative Examples 1-3; Figure 2 The short-chain fatty acid content in the feces of cats in Example 9, Example 10, and Comparative Examples 4-7. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with examples, which are provided to explain the present invention and are not limited to the following examples: The activation of Lactobacillus plantarum, Bacillus coagulans and Pediococcus pentosaceus in the preparation methods described in the following Examples 1-8 and Comparative Examples 1-3 is specific as follows: 1. activation of Lactobacillus plantarum: the lyophilized Lactobacillus plantarum was inoculated in MRS liquid culture medium, and after incubation at 37°C for 24h, an inoculum size of 1% of the culture solution was inoculated, and incubated at 37°C for 24h, the resulting bacterial liquid was centrifuged at 4000r / min for 10min, washed twice with sterile phosphate buffered saline, and the supernatant was discarded after centrifugation to obtain a bacterial sediment, and sterile physiological saline was added to the bacterial sediment to obtain a Lactobacillus plantarum bacterial liquid; ② The lyophilized Bacillus coagulans was inoculated into PCA liquid medium, cultured at 37°C for 18 hours, and then inoculated at an inoculum size of 2% of the culture medium. The culture was incubated at 37°C for 24 hours, and the resulting bacterial solution was centrifuged at 4000 r / min for 10 minutes. The solution was washed twice with sterile phosphate buffer and centrifuged to discard the supernatant to obtain a bacterial precipitate. Sterile physiological saline was added to the bacterial precipitate to obtain a Bacillus coagulans bacterial solution. ③ Activation of Pediococcus pentosaceus: The lyophilized Pediococcus pentosaceus was inoculated into MRS liquid medium, cultured at 37°C for 24 hours, and then inoculated at an inoculum size of 1.5% of the culture medium. The culture was incubated at 37°C for 24 hours, and the resulting bacterial solution was centrifuged at 4000 r / min for 10 minutes. The solution was washed twice with sterile phosphate buffer and centrifuged to discard the supernatant to obtain a bacterial precipitate. Sterile physiological saline was added to the bacterial precipitate to obtain a Pediococcus pentosaceus bacterial solution.

[0018] Example 1 A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 70 g of lecithin and 30 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 20 g of the phospholipid film were mixed, ultrasonicated at 250 W for 15 min, and hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 25 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 5. 20 mL of composite bacterial liposomes, 20 g of chitosan oligosaccharide and 20 g of composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0019] Example 2 A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 40 mL of Bacillus coagulans and 20 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.15 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 70 g of lecithin and 30 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 20 g of the phospholipid film were mixed, ultrasonicated at 250 W for 15 min, and hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 25 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 5. 20 mL of composite bacterial liposomes, 20 g of chitosan oligosaccharide and 20 g of composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0020] Example 3 A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 30 mL of lysozyme solution, 10 mL of neutral phytase solution, and 10 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 70 g of lecithin and 30 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 20 g of the phospholipid film were mixed, ultrasonicated at 250 W for 15 min, and hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 25 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 5. 20 mL of composite bacterial liposomes, 20 g of chitosan oligosaccharide and 20 g of composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0021] Example 4 A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 70 g of lecithin and 30 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 40 g of the phospholipid film were mixed and ultrasonicated at 250 W for 15 min, and then hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 25 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 5. 20 mL of composite bacterial liposomes, 20 g of chitosan oligosaccharide and 20 g of composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0022] Example 5 A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 70 g of lecithin and 30 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 20 g of the phospholipid film were mixed, ultrasonicated at 250 W for 15 min, and hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 40 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 5. 20 mL of composite bacterial liposomes, 20 g of chitosan oligosaccharide and 20 g of composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0023] Example 6 A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 70 g of lecithin and 30 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 20 g of the phospholipid film were mixed, ultrasonicated at 250 W for 15 min, and hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 25 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 5. 20 mL of composite bacterial liposomes, 10 g of chitosan oligosaccharide and 20 g of composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0024] Example 7 A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 70 g of lecithin and 30 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 20 g of the phospholipid film were mixed, ultrasonicated at 250 W for 15 min, and hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 25 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 5. 20 mL of composite bacterial liposomes, 20 g of chitosan oligosaccharide and 20 g of composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 80 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0025] Example 8 A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 90 g of lecithin and 10 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 20 g of the phospholipid film were mixed and ultrasonicated at 250 W for 15 min, and then hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 25 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 5. 20 mL of composite bacterial liposomes, 20 g of chitosan oligosaccharide and 20 g of composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0026] Comparative Example 1 The difference between this comparative example and Example 1 is that chitosan oligosaccharide and lactoferrin are not added.

[0027] A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 70 g of lecithin and 30 g of β-sitosterol were mixed and dissolved in 400 mL of anhydrous ethanol, rotary evaporated at 45°C and vacuum dried for 6 h to obtain a phospholipid film; 20 mL of the composite bacterial solution and 20 g of the phospholipid film were mixed, ultrasonicated at 250 W for 15 min, and hydrated at 50°C for 2 h to obtain composite bacterial liposomes; Step 4. 20 mL of composite bacterial liposomes and 20 mL of composite enzyme solution were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was dropped into 200 mL of 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0028] Comparative Example 2 The difference between this comparative example and Example 1 is that lecithin, β-sitosterol and chitosan oligosaccharide are not added.

[0029] A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. Lactoferrin was dissolved in water to prepare a lactoferrin solution with a concentration of 25 mg / mL and the pH was adjusted to 6.0. 10 mL of the complex enzyme solution was uniformly added dropwise to 25 mL of the lactoferrin solution and ultrasonicated for 10 min. The complex enzyme microspheres were then dried. Step 4. 20 mL of the composite bacterial solution and 20 g of the composite enzyme microspheres were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of a 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0030] Comparative Example 3 The difference between this comparative example and Example 1 is that only sodium alginate is used.

[0031] A method for preparing sodium alginate composite microcapsules comprises the following steps: Step 1. Mix 20 mL of Lactobacillus plantarum, 20 mL of Bacillus coagulans and 30 mL of Pediococcus pentosaceus to prepare a composite bacterial solution (with a bacterial concentration of 3.0 × 10 8 CFU / mL); Step 2. Dissolve lysozyme, neutral phytase, and β-xylosidase in sterile phosphate buffer to prepare 15 mg / mL lysozyme solution, 15 mg / mL neutral phytase solution, and 15 mg / mL β-xylosidase solution, respectively; then mix 20 mL of lysozyme solution, 20 mL of neutral phytase solution, and 20 mL of β-xylosidase solution to prepare a composite enzyme solution; Step 3. 20 mL of the composite bacterial solution and 20 mL of the composite enzyme solution were blended at room temperature for 2 h to obtain a bacterial enzyme complex. Then, 20 mL of the bacterial enzyme complex was evenly mixed with 50 mL of sodium alginate solution, and the mixture was added dropwise to 200 mL of a 2.5% calcium lactate solution and cured at 400 r / min for 45 min. The mixture was dried to obtain sodium alginate composite microcapsules.

[0032] Performance Testing (1) Average particle size The particle size of the sodium alginate composite microcapsules prepared in Examples 1-8 and Comparative Examples 1-3 was measured using a particle size analyzer. Specifically, 1.0 g of the sample was placed in a sample holder containing 1 L of distilled water and mixed evenly (diluted 1000 times), and then measured using the particle size analyzer.

[0033] (2) Embedment rate The embedding efficiency of the sodium alginate composite microcapsules prepared in Examples 1-8 and Comparative Examples 1-3 was analyzed. Specific procedures: ① Weigh 1.0 g of sodium alginate composite microcapsules and add them to 9.0 mL of sterile sodium citrate solution (0.05 M). Vigorously shake at 700 rpm for 20 minutes and ultrasonically break the emulsion at 400 W for 10 minutes. Centrifuge at 8000 rpm for 20 minutes, and discard the supernatant to obtain a precipitate. ② Add 10 mL of phosphate buffer solution to the precipitate, add a certain amount of lysozyme, and enzymatically hydrolyze at 40°C for 60 minutes to break the probiotic cell walls. Then, pass through 50 ③ Place 1 mL of the enzymatic hydrolyzate in a test tube, add 5 mL of Coomassie Brilliant Blue G-250 reagent, mix well, and let stand for 2 minutes. Measure the absorbance at 595 nm using a UV spectrophotometer, and set a blank control. ④ Calculate the encapsulation efficiency (EE) of the sodium alginate composite microcapsules: EE (%) = W1 / W × 100%, where W1 is the protein content of the sodium alginate composite probiotics (minus the amount of lysozyme added), and W is the sample mass.

[0034] Table 1 Average particle size and embedding rate of Examples 1-8 and Comparative Examples 1-3

[0035] As can be seen from Table 1, the average particle size of the sodium alginate composite microcapsules prepared in Examples 1-8 is smaller than that in Comparative Examples 1-3, while the embedding rates are all higher than those in Comparative Examples 1-3, indicating that one end of the chitosan oligosaccharide is connected to the lecithin-β-sitosterol liposome-embedded composite bacteria (Lactobacillus plantarum, Bacillus coagulans and Pediococcus pentosaceus), and the other end is connected to the lactoferrin microspheres-embedded composite enzymes (lysozyme, neutral phytase and β-xylosidase), and then embedded in the gel network of sodium alginate to form a microcapsule structure, which can reduce the influence of the mutual movement between the bacteria and the enzyme molecules on the instability of the sodium alginate composite microcapsule structure, thereby achieving tight connection and efficient and stable loading.

[0036] (3) Gastrointestinal digestion stability The gastrointestinal digestion stability of the sodium alginate composite microcapsules prepared in Example 1 and Comparative Examples 1-3 was analyzed by an in vitro simulated digestion test, as follows: ① Preparation of artificial simulated gastric fluid: Dissolve 1.64 mL of 0.5 mol / L diluted HCl in 90 mL of sterile distilled water, adjust the pH to 2.0, add 1.00 g of pepsin, shake thoroughly, dilute to 100 mL with sterile water, and store in a 4°C refrigerator until ready for use. ② Preparation of artificial simulated intestinal fluid: Measure an appropriate amount of PBS buffer solution with a pH of 7 into a beaker, add 1.00 g of trypsin, shake thoroughly, dilute to 100 mL with sterile water, and place in a 4°C refrigerator until ready for use; ③ Determination of digestive stability: Weigh 1.0 g of the prepared probiotic microcapsules into a sterile centrifuge tube, add them to 9 mL of artificial simulated gastric fluid, shake thoroughly for 15 seconds, and shake at 180 r / min at 37°C for 0, 1, 2, and 3 hours, respectively. Samples were taken and the release rate of the sodium alginate composite microcapsules in the simulated gastric digestion stage was calculated according to the treatment (2); the samples treated with artificial simulated gastric fluid were centrifuged at 4 000 r / min for 5 minutes, the supernatant was discarded, the precipitate was taken, and added to 30 mL of artificial simulated intestinal fluid, shake thoroughly (15 seconds), and shake at 180 r / min at 37°C for 0, 1, 2, and 3 hours, respectively. Samples were taken and the release rate of the sodium alginate composite microcapsules in the simulated intestinal digestion stage was calculated according to the treatment (2).

[0037] Depend on Figure 1 It can be seen that there is no significant difference in the cumulative release rate of the sodium alginate composite microcapsules prepared in Example 1 and Comparative Examples 1-3 in the simulated gastric digestion stage, but they are rapidly released in the simulated intestinal digestion stage, indicating that the sodium alginate gel structure of the composite microcapsule wall material has pH responsiveness, which enables the composite bacterial enzyme to exist stably in the gastric juice in the body, and is efficiently released and exerts its effect when it reaches the intestine of the body.

[0038] Example 9 A method for preparing freeze-dried pet food for improving intestinal function of pets, characterized by comprising the following steps: S1. 60 parts of yogurt, 15 parts of camel milk, 2 parts of milk mineral salts, 4 parts of galacto-oligosaccharides, and 3 parts of the sodium alginate composite microcapsules prepared in Example 1 were blended to obtain a blend; S2. The blend is pre-frozen at -40°C for 3 hours to form ice crystals. Sublimation drying is then performed during vacuum freeze-drying (the temperature is raised to 40°C within 30 minutes and maintained for 2 hours, the temperature is raised to 50°C within 30 minutes and maintained for 6 hours, and the temperature is raised to 60°C within 30 minutes and maintained for 4 hours). Desorption drying is then performed during vacuum freeze-drying (the temperature is lowered to 55°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 50°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 45°C within 30 minutes and maintained for 2 hours, and the temperature is lowered to 40°C within 30 minutes). The freeze-dried pet food is then shaped and packaged.

[0039] Example 10 A method for preparing freeze-dried pet food for improving intestinal function of pets, characterized by comprising the following steps: S1. 45 parts of yogurt, 20 parts of camel milk, 1 part of milk mineral salt, 4 parts of galacto-oligosaccharide, 3 parts of sodium alginate composite microcapsules prepared in Example 1 were blended to obtain a blend; S2. The blend is pre-frozen at -40°C for 3 hours to form ice crystals. Sublimation drying is then performed during vacuum freeze-drying (the temperature is raised to 40°C within 30 minutes and maintained for 2 hours, the temperature is raised to 50°C within 30 minutes and maintained for 6 hours, and the temperature is raised to 60°C within 30 minutes and maintained for 4 hours). Desorption drying is then performed during vacuum freeze-drying (the temperature is lowered to 55°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 50°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 45°C within 30 minutes and maintained for 2 hours, and the temperature is lowered to 40°C within 30 minutes). The freeze-dried pet food is then shaped and packaged.

[0040] Comparative Example 4 The difference between this comparative example and Example 9 is that the sodium alginate composite microcapsules prepared in Comparative Example 1 were used.

[0041] A method for preparing freeze-dried pet food for improving intestinal function of pets, characterized by comprising the following steps: S1. 60 parts of yogurt, 15 parts of camel milk, 2 parts of milk mineral salts, 4 parts of galacto-oligosaccharides, and 3 parts of sodium alginate composite microcapsules prepared in Comparative Example 1 were blended to obtain a blend; S2. The blend is pre-frozen at -40°C for 3 hours to form ice crystals. Sublimation drying is then performed during vacuum freeze-drying (the temperature is raised to 40°C within 30 minutes and maintained for 2 hours, the temperature is raised to 50°C within 30 minutes and maintained for 6 hours, and the temperature is raised to 60°C within 30 minutes and maintained for 4 hours). Desorption drying is then performed during vacuum freeze-drying (the temperature is lowered to 55°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 50°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 45°C within 30 minutes and maintained for 2 hours, and the temperature is lowered to 40°C within 30 minutes). The freeze-dried pet food is then shaped and packaged.

[0042] Comparative Example 5 The difference between this comparative example and Example 9 is that the sodium alginate composite microcapsules prepared in Comparative Example 2 were used.

[0043] A method for preparing freeze-dried pet food for improving intestinal function of pets, characterized by comprising the following steps: S1. 60 parts of yogurt, 15 parts of camel milk, 2 parts of milk mineral salts, 4 parts of galacto-oligosaccharides, and 3 parts of sodium alginate composite microcapsules prepared in Comparative Example 2 were blended to obtain a blend; S2. The blend is pre-frozen at -40°C for 3 hours to form ice crystals. Sublimation drying is then performed during vacuum freeze-drying (the temperature is raised to 40°C within 30 minutes and maintained for 2 hours, the temperature is raised to 50°C within 30 minutes and maintained for 6 hours, and the temperature is raised to 60°C within 30 minutes and maintained for 4 hours). Desorption drying is then performed during vacuum freeze-drying (the temperature is lowered to 55°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 50°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 45°C within 30 minutes and maintained for 2 hours, and the temperature is lowered to 40°C within 30 minutes). The freeze-dried pet food is then shaped and packaged.

[0044] Comparative Example 6 The difference between this comparative example and Example 9 is that the sodium alginate composite microcapsules prepared in Comparative Example 3 were used.

[0045] A method for preparing freeze-dried pet food for improving intestinal function of pets, characterized by comprising the following steps: S1. 60 parts of yogurt, 15 parts of camel milk, 2 parts of milk mineral salts, 4 parts of galacto-oligosaccharides, and 3 parts of sodium alginate composite microcapsules prepared in Comparative Example 3 were blended to obtain a blend; S2. The blend is pre-frozen at -40°C for 3 hours to form ice crystals. Sublimation drying is then performed during vacuum freeze-drying (the temperature is raised to 40°C within 30 minutes and maintained for 2 hours, the temperature is raised to 50°C within 30 minutes and maintained for 6 hours, and the temperature is raised to 60°C within 30 minutes and maintained for 4 hours). Desorption drying is then performed during vacuum freeze-drying (the temperature is lowered to 55°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 50°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 45°C within 30 minutes and maintained for 2 hours, and the temperature is lowered to 40°C within 30 minutes). The freeze-dried pet food is then shaped and packaged.

[0046] Comparative Example 7 The difference between this comparative example and Example 9 is that a bacterial enzyme mixture consisting of a commercially available composite probiotic and a commercially available composite enzyme preparation in a mass ratio of 1:1 is used.

[0047] A method for preparing freeze-dried pet food for improving intestinal function of pets, characterized by comprising the following steps: S1. 60 parts of yogurt, 15 parts of camel milk, 2 parts of milk mineral salts, 4 parts of galacto-oligosaccharides, and 3 parts of a bacterial enzyme mixture were prepared to obtain a blend; S2. The blend is pre-frozen at -40°C for 3 hours to form ice crystals. Sublimation drying is then performed during vacuum freeze-drying (the temperature is raised to 40°C within 30 minutes and maintained for 2 hours, the temperature is raised to 50°C within 30 minutes and maintained for 6 hours, and the temperature is raised to 60°C within 30 minutes and maintained for 4 hours). Desorption drying is then performed during vacuum freeze-drying (the temperature is lowered to 55°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 50°C within 30 minutes and maintained for 2 hours, the temperature is lowered to 45°C within 30 minutes and maintained for 2 hours, and the temperature is lowered to 40°C within 30 minutes). The freeze-dried pet food is then shaped and packaged.

[0048] Performance Testing (1) Pet cat feeding test Twenty-four British shorthair cats (half male and half female, aged 1-2 years, with an average weight of 4.0 ± 0.50kg), deworming and immunization were performed before the test, and the cats were not in special stages such as pregnancy, lactation, or disease treatment. Before the test, 24 cats were kept in the same environment for at least 45 days, and the test period was 60 days; the 24 cats were randomly divided into 6 groups, 4 cats in each group (half male and half female), and set as the control group, Example 9 group, Comparative Example 4 group, Comparative Example 5 group, Comparative Example 6 group, and Comparative Example 7 group; the control group was fed with commercially available basic cat food, Example 9 group was commercially available basic cat food + 5% freeze-dried pet food prepared in Example 9, Comparative Example 4 group was commercially available basic cat food + 5% freeze-dried pet food prepared in Comparative Example 4, Comparative Example 5 group was commercially available basic cat food + 5% freeze-dried pet food prepared in Comparative Example 5, Comparative Example 6 group was commercially available basic cat food + 5% freeze-dried pet food prepared in Comparative Example 6, and Comparative Example 7 group was commercially available basic cat food + 5% freeze-dried pet food prepared in Comparative Example 7, and the feeding amount was 120 kg per cat per day. g. Each cat was housed in a single cage, and the cattery was disinfected regularly to ensure cleanliness and tidiness of the experiment. The amount of food and the amount of food left over were weighed and recorded daily, and the fecal status was recorded and scored. The cats were allowed free access to food and water throughout the experiment.

[0049] (I) Feed intake, body weight change, and fecal scores The cats were weighed on an empty stomach on the first and 60th day, and the food intake of the cats in each group was recorded at 08:00 every morning. The feces were observed and scored according to the pet fecal scoring standard (Table 2).

[0050] Table 2 Pet feces scoring criteria

[0051] (II) Nutrient Apparent Digestibility Fecal samples (100.0 g) were collected from each group of cats and immediately treated with 10% hydrochloric acid for nitrogen fixation. The samples were then dried at 60°C to constant weight, crushed through a 60-mesh sieve, and refrigerated for later use. The apparent digestibility of nutrients in each group was determined using the acid-insoluble ash method, as described in the standard "Determination of Hydrochloric Acid-Insoluble Ash in Feeds." Crude ash content was determined by calcination at 550°C, dry matter content by direct drying, crude protein content by an automatic Kjeldahl nitrogen analyzer, and crude fat content by Soxhlet extraction. The apparent digestibility of nutrients (%) was calculated as [1-(A / B × C / D)] × 100%, where A is the content of a nutrient in feces (%), B is the content of the corresponding nutrient in the diet (%), C is the acid-insoluble ash content in the diet, and D is the acid-insoluble ash content in feces.

[0052] Table 3 Feed intake, weight changes and fecal scores of each group of pet cats in the feeding experiment

[0053] As can be seen from Table 3, the food intake, weight change, fecal score and apparent digestibility of nutrients of the cats in the feeding test of the pet cats in the Example 9 group are better than those of the control group and the comparative examples 4-7 groups, indicating that the freeze-dried pet food prepared by the sodium alginate composite microcapsules prepared in Example 1, compounded with yogurt, camel milk and oligosaccharides, when combined with the pet diet, can increase the food intake of the pet cats, so that the pet cats gain the most weight during the feeding test, the feces are in good condition and the soft stool is reduced, indicating that under the action of the composite bacterial enzyme, the intestinal function of the pet cats is greatly improved, the digestion and utilization rate of nutrients is improved, and the intestinal function of the pets is ensured to be in good condition.

[0054] (III) Short-chain fatty acids (SCFAs) in feces Accurately weigh 0.2 g of wet feces into a 2 mL centrifuge tube, add 1 mL of ultrapure water, vortex mix for 5 minutes, sonicate in an ice bath for 10 minutes, and then centrifuge at 13,000 r / min at 4°C for 10 minutes. Pour all the supernatant into a new 2 mL centrifuge tube, add 20 µL of 25% metaphosphoric acid and 0.25 g of anhydrous sodium sulfate, vortex mix for 1 minute for acidification and salting out, then add 1 mL of methyl tert-butyl ether, vortex mix for 5 minutes, and centrifuge at 13,000 r / min at 4°C for 10 minutes. The upper methyl tert-butyl ether extract is collected, filtered through a 0.22 µm microporous filter membrane, and added to an injection bottle with an inner tube for analysis using an LC-MS platform.

[0055] Depend on Figure 2 It can be seen that compared with the control group and comparative example groups 4-7, the cat feces of Example 9 and Example 10 have higher contents of short-chain fatty acids, among which the propionic acid content is significantly increased and the butyric acid content is on a downward trend. In addition, the study found that the proportion of propionic acid in the intestines of cats with chronic intestinal disease is low and the proportion of butyric acid is high. Feeding the freeze-dried pet food prepared by Example 9 and Example 10 in combination with the basic diet can not only promote the decomposition and metabolism of dietary fiber in the intestines of pet cats, but also increase the propionic acid content and reduce the butyric acid content in the feces of pet cats, indicating that the content of short-chain fatty acids (SCFAs) in the intestines of pet cats can be significantly improved, the intestinal barrier function can be enhanced, the immune system can be regulated and inflammation can be reduced, and it has a positive effect on the intestinal health of pet cats.

[0056] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, make many possible changes and modifications to the technical solution of the present invention using the methods and technical contents disclosed above, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A freeze-dried pet food for improving the intestinal function of pets, characterized in that: The invention comprises the following raw materials in parts by weight: 45-60 parts of yogurt, 15-20 parts of camel milk, 1-2.5 parts of milk mineral salt, 2-5 parts of oligosaccharide and 1.5-3 parts of sodium alginate composite microcapsules.

2. The freeze-dried pet food for improving pet intestinal function according to claim 1, characterized in that: The preparation method of the sodium alginate composite microcapsules comprises the following steps: Step 1. activating Lactobacillus plantarum, Bacillus coagulans and Pediococcus pentosaceus to obtain Lactobacillus plantarum bacterial solution, Bacillus coagulans bacterial solution and Pediococcus pentosaceus bacterial solution respectively, and mixing them to prepare a composite bacterial solution; Step 2. preparing lysozyme, phytase and xylanase into lysozyme solution, phytase solution and xylanase solution respectively, and mixing them to prepare a composite enzyme solution; Step 3. Phosphatidylcholine and β-sitosterol are mixed uniformly in a mass ratio of (6-10): (1-3) and dissolved in anhydrous ethanol, and subjected to rotary evaporation at 40-50°C and vacuum drying for 4-8 hours to obtain a phospholipid film; the composite bacterial solution and the phospholipid film are mixed and ultrasonically treated, filtered, and hydrated to obtain composite bacterial liposomes; Step 4. Lactoferrin is dissolved in water to prepare a lactoferrin solution with a concentration of 20-35 mg / mL and the pH is adjusted to 5.0-6.

5. The complex enzyme solution is uniformly added dropwise to the lactoferrin solution and ultrasonically treated for 5-10 minutes, followed by drying to obtain complex enzyme microspheres. Step 5. The composite bacterial liposomes, chitosan oligosaccharides and composite enzyme microspheres are blended to obtain a bacterial enzyme complex, and then the bacterial enzyme complex is evenly mixed with a sodium alginate solution at a volume ratio of 1: (3-6), and the mixture is dropwise added into a calcium lactate solution with a mass fraction of 2-4.5% and cured at 350-500 r / min for 30-60 min, and dried to obtain sodium alginate composite microcapsules.

3. The freeze-dried pet food for improving pet intestinal function according to claim 2, characterized in that: In step 1, the volume ratio of the Lactobacillus plantarum solution, the Bacillus coagulans solution and the Pediococcus pentosaceus solution is (1-3): (1.5-3): (1-4), and the concentration of the solution is (1.5-4)×10 8 CFU / mL.

4. The freeze-dried pet food for improving pet intestinal function according to claim 2, characterized in that: In step 2, the volume ratio of the lysozyme solution, the phytase solution and the xylanase solution is (2-4.5): (1-2): (1-3).

5. The freeze-dried pet food for improving pet intestinal function according to claim 2, characterized in that: In step 3, the ratio of the composite bacterial solution to the phospholipid film is 1 mL: (1-3.5) g; the ultrasonic treatment conditions are an ultrasonic power of 200-300 W and an ultrasonic time of 10-20 min; and the hydration treatment conditions are a hydration temperature of 40-50° C. and a hydration time of 1-3 h.

6. The freeze-dried pet food for improving pet intestinal function according to claim 2, characterized in that: The volume ratio of the complex enzyme solution to the lactoferrin solution in step 4 is 1:(1-4); the addition ratio of the complex bacterial liposomes, chitosan oligosaccharides and complex enzyme microspheres in step 5 is (1-2.5) mL:(1-2) g:(1-2) g.

7. The method for preparing freeze-dried pet food for improving intestinal function of pets according to claim 1, characterized in that: The following steps are involved: S1. yogurt, camel milk, milk mineral salts, galacto-oligosaccharides, and sodium alginate composite microcapsules are blended to prepare a blend; S2. The blended material is vacuum freeze-dried, shaped, and packaged to obtain freeze-dried pet food.

8. The method for preparing freeze-dried pet food for improving intestinal function of pets according to claim 7, characterized in that: The specific conditions for vacuum freeze drying in S2 are as follows: ① pre-freezing stage: pre-freezing temperature -45--35°C, pre-freezing time ≥3h; ② sublimation drying stage: temperature 40-60°C, drying time 10-12h, vacuum degree 50-70Pa; ③ desorption drying stage: temperature 40-55°C, drying time 6-8h, vacuum degree 50-70Pa; the cold trap temperature of the vacuum freeze drying is -50--45°C.

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