Pet probiotic bilayer liposome as well as preparation method and application thereof

Abstract

The invention provides a probiotic bilayer liposome for pets and a preparation method thereof, probiotics and lactoferrin peptide are embedded in liposome, the liposome is modified by serine, and finally gellan gum is coated on the outer layer of the liposome to prepare the probiotic bilayer liposome. According to the invention, the probiotics and the lactoferrin peptide are jointly embedded in the lipidosome to synergistically exert an antibacterial effect; meanwhile, an amino group in serine is introduced to a phospholipid bilayer of the liposome, so that the damage of a gastric acid environment to the liposome is reduced, and finally, a layer of gellan gum is coated on the outer layer of the liposome, so that the embedding rate of the probiotics and the stability of the liposome are further improved, and the effect of regulating the intestinal health of pets is achieved after the liposome is applied to pet food.

Description

Technical Field

[0001] The invention belongs to the technical field of pet food, and in particular relates to a pet probiotic double-layer liposome and a preparation method and application thereof. Background Art

[0002] With the improvement of living standards, pet owners have more and more stringent requirements for pet food. Health, nutrition and safety have become the trend of pet owners in modern society to choose pet food. The cat food and dog food that pet cats and dogs have been eating for a long time are all processed foods, which are added with a large number of food additives such as pigments, flavors, preservatives, etc., and the widespread use of antibiotics, body resistance and drug residues have destroyed the balance of normal intestinal flora of pets, making pets prone to constipation, indigestion, gastroenteritis and other gastrointestinal diseases, which have brought serious harm to the health of pets. Probiotics are active microorganisms that work by improving the balance of the host's intestinal microbial flora. After a certain amount of oral administration, this active microorganism will improve the balance of microorganisms in the host's intestines, and then have a beneficial effect on the host's health. Pet wet food rich in probiotics can not only promote the intestinal health of pets and improve their ability to absorb and metabolize nutrients, but also enhance the resistance of pets and reduce the occurrence of immune diseases such as allergies. Currently, the probiotic products on the market will become inactivated due to environmental changes during processing, transportation, storage, digestion and absorption. The number of probiotics that can actually colonize in the intestine in a living state is insufficient to exert their probiotic effects. Liposomes have the advantages of protecting drug activity, reducing drug toxicity and side effects, and improving drug utilization, and are considered to be the most promising and mature nano-targeted drug delivery carriers. Encapsulating probiotics with liposomes can prevent them from adverse environments, protect them from gastric acid erosion, reduce the inactivation of probiotics, and increase their survival rate and colonization number in the intestine, thereby truly exerting their probiotic effects. However, during the preparation of liposomes, excessive or improper ultrasonic treatment may lead to a decrease in the activity of probiotics and the antibacterial activity of probiotic liposomes. In addition, liposomes will be degraded by digestive enzymes and bile acids in the gastrointestinal tract, and will be degraded or ruptured prematurely, resulting in the premature release of probiotics. Therefore, it is necessary to prepare a modified liposome to encapsulate probiotics, thereby improving the activity of probiotics and the stability of liposomes. Summary of the invention

[0003] Technical problems to be solved: In view of the above technical problems, the purpose of the present invention is to provide a pet probiotic bilayer liposome and a preparation method thereof, wherein probiotics and lactoferrin peptide are embedded in the liposome, and the liposome is modified by serine, and finally the outer layer of the liposome is coated with gellan gum. The present invention co-encapsulates probiotics and lactoferrin peptide in the liposome to synergistically exert an antibacterial effect; at the same time, the amino group in serine is introduced into the phospholipid bilayer of the liposome to reduce the damage of the gastric acid environment to the liposome, and finally a layer of gellan gum is coated on the outer layer of the liposome to further improve the embedding rate of probiotics and the stability of the liposome, and after being applied to pet food, it exerts the effect of regulating the intestinal health of pets.

[0004] Technical solution: A pet probiotic bilayer liposome, wherein the pet probiotic bilayer liposome is a bilayer structure, wherein the inner layer is a modified liposome encapsulating probiotics and lactoferrin peptide, and the outer layer is gellan gum; The modified liposomes are modified with serine. Furthermore, the probiotics are a mixture of Lactobacillus acidophilus, Bifidobacterium and Bacillus subtilis. Further, the following steps are included: S1. Dissolve Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide in phosphate buffer solution and mix well to obtain a mixed bacterial solution; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and then serine was added, stirred, rotary evaporated, and vacuum dried to obtain a modified lipid film; S3. ultrasonically hydrating and filtering the modified lipid film and the mixed bacterial solution to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir to dissolve, to obtain a gellan gum solution, then immerse the modified probiotic liposomes in the gellan gum solution, stir evenly, filter, and obtain pet probiotic bilayer liposomes. Furthermore, in step S1, the mass ratio of Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide is (2-4):(3-5):(1-3):(1-1.5), and the concentration of the mixed bacterial solution is 2-3 mg / mL. Furthermore, in step S2, the mass ratio of soybean lecithin, cholesterol and serine is (6-7):(0.8-1.2):(2-3). Furthermore, in step S3, the mass volume ratio of the modified lipid film to the mixed bacterial solution is (2-3):1; the ultrasonic power is 100-200W, and the ultrasonic time is 10-15min; the hydration temperature is 40-50°C, and the hydration time is 1.5-2h. Furthermore, in step S4, the gellan gum is heated and dissolved at a temperature of 70-80° C., and the heating time is 15-30 min; and the gellan gum solution concentration is 0.5-1.5 wt.%. Furthermore, in step S4, the mass volume ratio of the modified probiotic liposome to the gellan gum solution is 1:(1-2). Application of the pet probiotic bilayer liposome prepared by the above preparation method in pet wet food. Beneficial effects: 1. The present invention prepares liposomes to embed probiotics, thereby avoiding the problem that probiotics are difficult to tolerate gastric acid environment and activity decreases. Liposomes contain phospholipid bilayers, and the surface of probiotic cells has high hydrophobicity, making it easier for them to combine with the hydrophobic end of the phospholipid bilayer; lactoferrin peptide is added and dissolved in the probiotic solution, and then embedded in the liposomes together. Since lactoferrin peptide is hydrophilic, it can combine with the hydrophilic end of the phospholipid bilayer, so that the probiotics and lactoferrin peptide are co-embedded in the liposomes, thereby improving the antibacterial effect and avoiding the problem that the liposomes are affected by digestive enzymes and bile acids in the gastrointestinal tract, and are prematurely degraded or ruptured, resulting in the early release of probiotics and lactoferrin peptides and the decrease of antibacterial activity; 2. The present invention grafts serine on the surface of liposomes to prepare modified liposomes. The amino groups in serine form hydrogen bonds with the phosphate groups on the phospholipid bilayer of the liposomes to enhance the intermolecular forces in the head region. The introduced amino groups are protonated (forming -NH3 + ), the repulsive force between the negative charge of the phosphate group changes the arrangement of the lipid bilayer, making the head region more compact and reducing H + The permeation damage to the lipid bilayer improves the acid stability of liposomes and protects the activity of probiotics and lactoferrin peptide inside; 3. The present invention coats a layer of gellan gum on the outer layer of the modified liposome, which is beneficial to improving the encapsulation rate of probiotics. In addition, the gel network of gellan gum can achieve directional release of probiotics and lactoferrin peptides through pH responsiveness. In the gastric acid environment, the liposomes coated with gellan gum can remain stable; while in the intestinal environment, the gellan gum network will dissolve or swell due to pH changes, thereby releasing the internal probiotics and lactoferrin peptides. This mechanism ensures that the probiotics can reach and play a role in the intestine instead of being destroyed in the gastric acid environment. DETAILED DESCRIPTION The present invention will be further described below in conjunction with embodiments, which are explanations of the present invention and are not limited to the following embodiments: Example 1 A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide were dissolved in a phosphate buffer solution in a mass ratio of 2:3:1:1, mixed evenly, and a mixed bacterial solution with a concentration of 2 mg / mL was obtained; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and serine was added in a mass ratio of 6:1:2, stirred evenly, rotary evaporated, and vacuum dried to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at 150 W for 15 min at a mass volume ratio of 2:1, hydrated at 45 ° C for 2 h, and filtered to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir at 70°C for 20 minutes to obtain a gellan gum solution with a concentration of 1wt.%, then immerse the modified probiotic liposomes in the gellan gum solution with a mass-to-volume ratio of 1:1, stir evenly, and filter to obtain pet probiotic bilayer liposomes. Example 2 A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide were dissolved in a phosphate buffer solution in a mass ratio of 2:3:1:1.5 and mixed to obtain a mixed bacterial solution with a concentration of 2 mg / mL; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and serine was added in a mass ratio of 6:1:2, stirred evenly, rotary evaporated, and vacuum dried to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at 150 W for 15 min at a mass volume ratio of 2:1, hydrated at 45 ° C for 2 h, and filtered to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir at 70°C for 20 minutes to obtain a gellan gum solution with a concentration of 1wt.%, then immerse the modified probiotic liposomes in the gellan gum solution with a mass-to-volume ratio of 1:1, stir evenly, and filter to obtain pet probiotic bilayer liposomes. Example 3 A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide were dissolved in a phosphate buffer solution in a mass ratio of 2:3:1:1, mixed evenly, and a mixed bacterial solution with a concentration of 2 mg / mL was obtained; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and serine was added in a mass ratio of 6:1:2.5, stirred evenly, rotary evaporated and vacuum dried to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at 150 W for 15 min at a mass volume ratio of 2:1, hydrated at 45 ° C for 2 h, and filtered to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir at 70°C for 20 minutes to obtain a gellan gum solution with a concentration of 1wt.%, then immerse the modified probiotic liposomes in the gellan gum solution with a mass-to-volume ratio of 1:1, stir evenly, and filter to obtain pet probiotic bilayer liposomes. Example 4 A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide were dissolved in a phosphate buffer solution in a mass ratio of 2:3:1:1, mixed evenly, and a mixed bacterial solution with a concentration of 2 mg / mL was obtained; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and serine was added in a mass ratio of 6:1:3, stirred evenly, rotary evaporated, and vacuum dried to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at 150 W for 15 min at a mass volume ratio of 2:1, hydrated at 45 ° C for 2 h, and filtered to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir at 70°C for 20 minutes to obtain a gellan gum solution with a concentration of 1wt.%, then immerse the modified probiotic liposomes in the gellan gum solution with a mass-to-volume ratio of 1:1, stir evenly, and filter to obtain pet probiotic bilayer liposomes. Example 5 A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide were dissolved in a phosphate buffer solution in a mass ratio of 2:3:1:1, mixed evenly, and a mixed bacterial solution with a concentration of 2 mg / mL was obtained; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and serine was added in a mass ratio of 6:1:2, stirred evenly, rotary evaporated, and vacuum dried to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at 150 W for 15 min at a mass volume ratio of 2:1, hydrated at 45 ° C for 2 h, and filtered to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir at 70°C for 20 minutes to obtain a gellan gum solution with a concentration of 1wt.%, then immerse the modified probiotic liposomes in the gellan gum solution with a mass volume ratio of 1:1.5, stir evenly, and filter to obtain pet probiotic bilayer liposomes. Example 6 A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide were dissolved in a phosphate buffer solution in a mass ratio of 2:3:1:1, mixed evenly, and a mixed bacterial solution with a concentration of 2 mg / mL was obtained; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and serine was added in a mass ratio of 6:1:2, stirred evenly, rotary evaporated, and vacuum dried to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at 150 W for 15 min at a mass volume ratio of 2:1, hydrated at 45 ° C for 2 h, and filtered to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir at 70°C for 20 minutes to obtain a gellan gum solution with a concentration of 1wt.%, then immerse the modified probiotic liposomes in the gellan gum solution with a mass volume ratio of 1:2, stir evenly, and filter to obtain pet probiotic bilayer liposomes. Comparative Example 1 The difference between this comparative example and Example 3 is that lactoferrin peptide is not added, specifically as follows: A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, and Bacillus subtilis were dissolved in a phosphate buffer solution at a mass ratio of 2:3:1, mixed evenly, and a mixed bacterial solution with a concentration of 2 mg / mL was obtained; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and serine was added in a mass ratio of 6:1:2.5, stirred evenly, rotary evaporated and vacuum dried to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at 150 W for 15 min at a mass volume ratio of 2:1, hydrated at 45 ° C for 2 h, and filtered to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir at 70°C for 20 minutes to obtain a gellan gum solution with a concentration of 1wt.%, then immerse the modified probiotic liposomes in the gellan gum solution with a mass-to-volume ratio of 1:1, stir evenly, and filter to obtain pet probiotic bilayer liposomes. Comparative Example 2 The difference between this comparative example and Example 3 is that no serine is added, as follows: A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide were dissolved in a phosphate buffer solution in a mass ratio of 2:3:1:1, mixed evenly, and a mixed bacterial solution with a concentration of 2 mg / mL was obtained; S2. Dissolve soybean lecithin and cholesterol in anhydrous ethanol in a mass ratio of 6:1, stir evenly, rotary evaporate, and vacuum dry to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at 150 W for 15 min at a mass volume ratio of 2:1, hydrated at 45 ° C for 2 h, and filtered to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir at 70°C for 20 minutes to obtain a gellan gum solution with a concentration of 1wt.%, then immerse the modified probiotic liposomes in the gellan gum solution with a mass-to-volume ratio of 1:1, stir evenly, and filter to obtain pet probiotic bilayer liposomes. Comparative Example 3 The difference between this comparative example and Example 3 is that gellan gum is not added, and the details are as follows: A method for preparing a pet probiotic bilayer liposome is as follows: S1. Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide were dissolved in a phosphate buffer solution in a mass ratio of 2:3:1:1, mixed evenly, and a mixed bacterial solution with a concentration of 2 mg / mL was obtained; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and serine was added in a mass ratio of 6:1:2.5, stirred evenly, rotary evaporated and vacuum dried to obtain a modified lipid film; S3. The modified lipid film and the mixed bacterial solution were ultrasonicated at a power of 150 W for 15 minutes in a mass-to-volume ratio of 2:1, hydrated at 45°C for 2 hours, and filtered to obtain modified probiotic liposomes. Performance Test: 1. Particle size and encapsulation efficiency The prepared probiotic bilayer liposomes were diluted with PBS buffer, and the average particle size was measured by a dynamic light scattering particle size analyzer; an appropriate amount of the prepared probiotic bilayer liposomes and an empty liposome suspension without probiotics were taken, and the encapsulation efficiency of the probiotic bilayer liposomes was measured by the centrifugal viable count method. The results are shown in Table 1 below: Table 1 Particle size and encapsulation efficiency of probiotic bilayer liposomes prepared in Examples 1-6 and Comparative Examples 1-3 As can be seen from Table 1, the particle size of the probiotic bilayer liposomes prepared in Examples 1-6 is 5.88-6.23 μm, and the encapsulation efficiency is 82.54-86.63%, while the particle size and encapsulation efficiency of the probiotic bilayer liposomes prepared in Comparative Example 1 without adding lactoferrin peptide are not greatly affected, and the particle size of Comparative Example 2 without adding serine modified liposomes does not change much, while the encapsulation efficiency is slightly decreased to 75.12%; Comparative Example 3 does not add gellan gum coating, the particle size is 5.13 μm, and the encapsulation efficiency is 71.26%. It can be seen that serine modified liposomes and coating the outer layer of the liposome with a layer of gellan gum are beneficial to improving the encapsulation efficiency. 2. Acid and bile salt resistance test 3 g of the probiotic bilayer liposomes prepared in Examples 1-6 and Comparative Examples 1-3 were added to 27 mL of simulated gastric juice preheated to 37° C., vortexed for 5 s, the simulated gastric juice was adjusted to a pH of 2.0 with 1 M HCl, pepsin and gastric lipase were added, and digested for 2 h; after simulated gastric digestion, the simulated gastric juice was transferred to simulated intestinal juice containing bile salts and pancreatic digestive enzymes, and the simulated gastric juice was adjusted to a pH of 7.4 with 1 M NaOH, and digested for 2 h; samples were taken every 1 h, 10 mL of 0.1 mol / L phosphate buffer was added, vortexed for 5 min to completely dissolve the probiotic bilayer liposomes, 1 mL of the sample solution was taken for gradient dilution, and the number of viable bacteria was measured according to the plate count method; at the same time, probiotics without liposome encapsulation were used as a control, and the results are shown in Table 2 below: Table 2 Survival rates of probiotic bilayer liposomes prepared in Examples 1-6 and Comparative Examples 1-3 in artificial simulated gastric juice and intestinal juice As can be seen from Table 2, the probiotic bilayer liposomes prepared in Examples 1-6 have a probiotic survival rate of 82.83-85.12% after digestion in simulated artificial gastric juice for 2 hours, and a probiotic survival rate of 84.91-90.21% after digestion in simulated artificial intestinal juice for 2 hours; the probiotic bilayer liposomes prepared in Comparative Example 1 without the addition of lactoferrin peptide have little effect on the survival rate; Comparative Example 2 does not add serine-modified liposomes, and the survival rate decreases slightly; Comparative Example 3 does not add gellan gum coating, which has a greater effect on the survival rate. It can be seen that serine-modified liposomes and gellan gum-coated liposomes are beneficial to improving the acid stability of liposomes and improving the survival rate of internal probiotics. 3. Antibacterial Experiment The antibacterial effect of the probiotic bilayer liposomes prepared in Examples 1-6 and Comparative Examples 1-3 was determined by the agar diffusion method. MRS agar medium (20 mL) cooled to 50° C. after sterilization was mixed with 200 μL of intestinal pathogenic bacteria solution (10 6cfu / mL) were poured into a plate and mixed. After the MRS agar medium with intestinal pathogenic bacteria solution was cooled and solidified, a hole with a diameter of 6-7 μm was punched on the plate using a hole puncher. The probiotic bilayer liposomes prepared in Examples 1-6 and Comparative Examples 1-3 were added to each hole, respectively. After diffusion in a 4°C refrigerator for 12 hours, the probiotic bilayer liposomes were cultured at 37°C for 48 hours. The size of the inhibition zone was observed, and the diameter of the inhibition zone was measured using a vernier caliper (retaining two significant figures). The test results are shown in Table 3: Table 3 Antibacterial properties of probiotic bilayer liposomes prepared in Examples 1-6 and Comparative Examples 1-3 Escherichia coli (mm) Salmonella enterica serovar Mutica (mm) Example 1 26.05 33.12 Example 2 26.12 33.28 Example 3 26.47 33.55 Example 4 25.78 32.68 Example 5 26.63 33.07 Example 6 25.85 33.54 Comparative Example 1 22.08 29.53 Comparative Example 2 24.27 32.15 Comparative Example 3 24.06 32.13 It can be seen from Table 3 that the diameters of the inhibition zones of the probiotic bilayer liposomes prepared in Examples 1-6 are all larger than the diameter of the inhibition zone of the probiotic bilayer liposomes prepared in Comparative Example 1. This shows that simultaneously encapsulating probiotics and lactoferrin peptide into the liposomes is beneficial to improving the antibacterial effect of the liposomes. In summary, the probiotic bilayer liposomes prepared in Example 3 were selected for the subsequent preparation of pet wet food. Example 7 A method for preparing pet wet food, comprising the following steps: Step 1. Pretreatment: chop 20 kg of chicken breast meat, 8 kg of chicken skeleton, and 8 kg of chicken liver into meat paste to obtain mixed meat paste; then chop 8 kg of carrots and 8 kg of pumpkin into 5 mm cubes respectively and mix them with 8 kg of green peas to obtain mixed vegetable auxiliary materials; Step 2. Pulping treatment: put 8 kg corn starch, 1 kg potassium, and 0.02 kg zinc into 45 kg water, stir at 1000-1200 rpm for 2 min, and mix well to obtain a nutritious soup; Step 3. Initial emulsification: Put the mixed meat paste and nutrient soup into the emulsifier for high-speed emulsification to ensure that the materials are fully mixed and evenly mixed; Step 4. Ripening treatment: the material obtained after the primary emulsification treatment in step (3) is boiled and cooked, and the mixed vegetable auxiliary material is steamed, wherein the boiling and cooking treatment is to keep it at a rolling boil for 25 minutes, and the steaming treatment is to steam it at a rolling boil for 7 minutes; Step 5. Secondary emulsification treatment: the cooled boiled and cooked material is mixed with 1 kg of vitamin K and then put into an emulsifier for high-speed emulsification to ensure that the material is fully mixed, and then mixed with the cooled steamed material and 2 kg of the probiotic bilayer liposomes prepared in Example 3; Step 6. Canning and sterilization: Use a vacuum sealer to vacuum seal the cans, and then send them into an irradiation sterilization device for sterilization at a dose of 10kGy to obtain pet wet food. Comparative Example 4 The difference between this comparative example and Example 7 is that the probiotic bilayer liposomes prepared in Comparative Example 1 are selected. Comparative Example 5 The difference between this comparative example and Example 7 is that the probiotic bilayer liposomes prepared in Comparative Example 2 are selected. Comparative Example 6 The difference between this comparative example and Example 7 is that the probiotic bilayer liposomes prepared in Comparative Example 3 are selected. Comparative Example 7 The difference between this comparative example and Example 7 is that probiotics are directly added, as follows: A method for preparing pet wet food, comprising the following steps: Step 1. Pretreatment: chop 20 kg of chicken breast meat, 8 kg of chicken skeleton, and 8 kg of chicken liver into meat paste to obtain mixed meat paste; then chop 8 kg of carrots and 8 kg of pumpkin into 5 mm cubes respectively and mix them with 8 kg of green peas to obtain mixed vegetable auxiliary materials; Step 2. Pulping treatment: put 8 kg corn starch, 1 kg potassium, and 0.02 kg zinc into 45 kg water, stir at 1000-1200 rpm for 2 min, and mix well to obtain a nutritious soup; Step 3. Initial emulsification: Put the mixed meat paste and nutrient soup into the emulsifier for high-speed emulsification to ensure that the materials are fully mixed and evenly mixed; Step 4. Ripening treatment: the material obtained after the primary emulsification treatment in step (3) is boiled and cooked, and the mixed vegetable auxiliary material is steamed, wherein the boiling and cooking treatment is to keep it at a rolling boil for 25 minutes, and the steaming treatment is to steam it at a rolling boil for 7 minutes; Step 5. Secondary emulsification treatment: the cooled boiled and cooked material is mixed with 1 kg of vitamin K and then put into an emulsifier for high-speed emulsification to ensure that the material is fully mixed, and then mixed with the cooled steamed material and the mixed bacterial solution with the same addition amount as in Example 7; Step 6. Canning and sterilization: Use a vacuum sealer to vacuum seal the cans, and then send them into an irradiation sterilization device for sterilization at a dose of 10kGy to obtain pet wet food. Intestinal regulation effect detection: 25 pet dogs with moderate diarrhea were selected from a pet hospital and fed with the pet wet food containing probiotic bilayer liposomes prepared in Example 7 and Comparative Examples 4-7. The diarrhea of ​​the pets was observed after 10 days, 30 days and 60 days of feeding. The experimental results are shown in Table 4 below: Table 4 Diarrhea of ​​pets fed with wet food prepared in Example 7 and Comparative Examples 4-7 As shown in Table 4, after feeding the pet wet food prepared in Example 7, the diarrhea situation is significantly improved, while Comparative Example 4 uses the probiotic bilayer liposomes prepared in Comparative Example 1 (without lactoferrin peptide), and the antibacterial effect is reduced; Comparative Examples 5 and 6 respectively use the probiotic bilayer liposomes prepared in Comparative Examples 2 and 3 (without serine modification and gellan gum), and the diarrhea situation is worse than that in Example 7; Comparative Example 7 directly adds probiotics when preparing pet wet food, and the probiotics are not protected by liposomes, so the probiotic activity in the pet wet food is significantly reduced, and the pet diarrhea rate is high. Therefore, it can be seen that the probiotic bilayer liposomes prepared by the present invention can achieve the protective effect on probiotics, so that the antibacterial effect of pet wet food is better, which can effectively reduce the incidence of diarrhea and improve the intestinal health of pets. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A pet probiotic bilayer liposome, characterized in that: The pet probiotic bilayer liposome is a bilayer structure, the inner layer is a modified liposome encapsulating probiotics and lactoferrin peptide, and the outer layer is gellan gum; The modified liposomes are modified with serine.

2. The pet probiotic bilayer liposome according to claim 1, characterized in that: The probiotics are mixed bacteria of Lactobacillus acidophilus, Bifidobacterium and Bacillus subtilis.

3. The method for preparing a pet probiotic bilayer liposome according to claim 2, characterized in that: The following steps are involved: S1. Dissolve Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide in phosphate buffer solution and mix well to obtain a mixed bacterial solution; S2. The soybean lecithin and cholesterol were dissolved in anhydrous ethanol, and then serine was added, stirred, rotary evaporated, and vacuum dried to obtain a modified lipid film; S3. ultrasonically hydrating and filtering the modified lipid film and the mixed bacterial solution to obtain modified probiotic liposomes; S4. Dissolve gellan gum in water, heat and stir to dissolve, to obtain a gellan gum solution, then immerse the modified probiotic liposomes in the gellan gum solution, stir evenly, filter, and obtain pet probiotic bilayer liposomes.

4. The method for preparing a pet probiotic bilayer liposome according to claim 3, characterized in that: In step S1, the mass ratio of Lactobacillus acidophilus, Bifidobacterium, Bacillus subtilis and lactoferrin peptide is (2-4):(3-5):(1-3):(1-1.5), and the concentration of the mixed bacterial solution is 2-3 mg / mL.

5. The method for preparing a pet probiotic bilayer liposome according to claim 3, characterized in that: The mass ratio of soybean lecithin, cholesterol and serine in step S2 is (6-7):(0.8-1.2):(2-3).

6. The method for preparing a pet probiotic bilayer liposome according to claim 3, characterized in that: In the step S3, the mass volume ratio of the modified lipid film to the mixed bacterial solution is (2-3):1; the ultrasonic power is 100-200W, and the ultrasonic time is 10-15min; the hydration temperature is 40-50°C, and the hydration time is 1.5-2h.

7. The method for preparing a pet probiotic bilayer liposome according to claim 3, characterized in that: In step S4, the gellan gum is heated and dissolved at a temperature of 70-80° C. for a heating time of 15-30 min. The gellan gum solution concentration is 0.5-1.5 wt.%.

8. The method for preparing a pet probiotic bilayer liposome according to claim 3, characterized in that: In the step S4, the mass volume ratio of the modified probiotic liposome to the gellan gum solution is 1:(1-2).

9. Use of the pet probiotic bilayer liposome according to any one of claims 1 to 2 in pet wet food.

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