Liquid probiotic preparation for pets and preparation method thereof

Abstract

The invention relates to the technical field of probiotic preparations, in particular to a liquid probiotic preparation for pets and a preparation method of the liquid probiotic preparation. The liquid probiotic preparation comprises the following components in parts by weight: 30-50 parts of probiotics, 5-10 parts of a gel carrier material, 1-2 parts of a nutrition stabilizer, 1-2 parts of a multifunctional stabilizer, 2-4 parts of polyethylene glycol, 1-3 parts of natural vitamin E, 2-3 parts of calcium carbonate, 1-2 parts of sodium alginate, 5-10 parts of soybean oil and 3-7 parts of a sterile PBS buffer solution. According to the liquid probiotics for the pets, the synthesized gel carrier material is used as a carrier, and the probiotics are loaded and embedded into the gel carrier material in an adsorption and embedding mode, so that the contact between the probiotics and adverse factors in the external environment can be effectively reduced, and the survival rate of the probiotics in various environments is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic preparations, in particular to a pet liquid probiotic preparation and a preparation method thereof. Background Art

[0002] There are a large number of lactic acid bacteria in the human intestine. It is an essential flora in the human body with important physiological functions. Lactic acid bacteria can change the intestinal environment, inhibit the reproduction of harmful bacteria, adjust the balance of gastrointestinal flora, improve intestinal function, relieve or treat intestinal diseases to a large extent, and improve human immunity. Lactic acid bacteria also have anti-tumor, anti-aging, lipid-lowering, blood sugar-lowering, and blood pressure-lowering effects. At present, probiotic products on the market are divided into liquid dosage forms and solid dosage forms. Liquid dosage forms can quickly regulate the gastrointestinal microenvironment and have a fast effect because they contain rich metabolites. Liquid oral solutions also have the advantages of being easy to take, so they occupy a certain market share.

[0003] The biggest problem with liquid bacterial preparations is how to preserve a high amount of live bacteria. The number of live bacteria in live bacterial oral liquids is very important for their functionality. Only when the number of live bacteria reaches a certain level can probiotics play their beneficial role in the human body. The declaration and review of probiotic health foods (Document No. 202 of the State Food and Drug Administration

[2005] ) stipulates that the number of live bacteria in live probiotic health foods must not be less than 106 cfu / mL (g) during their shelf life, so improving the stability of probiotics is very important for the shelf life and efficacy of probiotic products. Summary of the invention

[0004] In view of the problems mentioned in the above background, the object of the present invention is to provide a pet liquid probiotic preparation and a preparation method thereof.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A pet liquid probiotic preparation, comprising the following components by weight: 30-50 parts of probiotics, 5-10 parts of gel carrier material, 1-2 parts of nutrient stabilizer, 1-2 parts of multifunctional stabilizer, 2-4 parts of polyethylene glycol, 1-3 parts of natural vitamin E, 2-3 parts of calcium carbonate, 1-2 parts of sodium alginate, 5-10 parts of soybean oil, and 3-7 parts of sterile PBS buffer;

[0007] The probiotics are obtained by mixing and fermenting equal amounts of Saccharomyces boulardii, Bifidobacterium animalis BB-12, and Lactobacillus plantarum;

[0008] The nutritional stabilizer is prepared by mixing sunflower oil, camellia oil, avocado oil and peony seed oil in a mass ratio of 1: (0.5-0.8): (0.2-0.4): (1.0-1.5);

[0009] The soybean oil contains glacial acetic acid, and the content of the glacial acetic acid is 2-3% of the mass of the soybean oil.

[0010] As a further preferred embodiment of the present invention, the preparation method of the gel carrier material is as follows:

[0011] 1) dissolving water-soluble citrus cellulose and sodium carboxymethyl cellulose in deionized water, gently stirring at room temperature until a clear solution is obtained, then adding citric acid to the solution, stirring until completely dissolved, drying the obtained product at 30-35° C. for 24-30 hours, and then storing it at 80-85° C. for 24-30 hours for cross-linking reaction to obtain a hydrogel;

[0012] 2) adding the composite nanomaterial into deionized water, fully dispersing to obtain a dispersion having a solid content of 3-6%, then adding the hydrogel into the dispersion, soaking for 24-30 hours under intermittent ultrasound at 100-150W, with an ultrasound interval of 10-20 minutes and an action time of 3-7 minutes. After the treatment is completed, centrifuging the product at 10000-15000 r / min for 15-25 minutes to obtain a gel carrier material.

[0013] As a further preferred embodiment of the present invention, in step 1), the usage ratio of the water-soluble citrus cellulose, sodium carboxymethyl cellulose, deionized water and citric acid is (15-30) g: (75-90) g: (300-500) mL: (0.4-0.8) g.

[0014] As a further preferred embodiment of the present invention, in step 2), the solid-liquid ratio of the hydrogel to the dispersion is 1 g: (15-30) mL.

[0015] As a further preferred embodiment of the present invention, the preparation method of the composite nanomaterial is as follows:

[0016] 1) dissolving konjac glucomannan in deionized water to obtain a konjac glucomannan solution, then mixing the konjac glucomannan solution with a sodium periodate solution in a volume ratio of 1:(0.3-0.7), stirring and reacting at 20-25° C. in the dark for 16-20 hours, after the reaction is completed, transferring the solution to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzing with a large amount of water, and lyophilizing the purified solution to obtain oxidized konjac glucomannan;

[0017] 2) dissolving oxidized konjac glucomannan, polyethylene oxide, polyethylene glycol octyl phenyl ether and dimethyl sulfoxide in water in a homogeneous manner, so that the polymer concentration is 3-5wt%, the mass ratio of oxidized konjac glucomannan to polyethylene oxide is (4-5):1, the concentration of polyethylene glycol octyl phenyl ether is 1-2wt%, and the concentration of dimethyl sulfoxide is 5-7wt%, and the prepared electrospinning solution is put into a syringe, and spinning is carried out at 25-30° C. and a humidity of 10-30% to obtain polysaccharide nanofibers;

[0018] 3) dissolving 0.8-0.9mmol cobalt nitrate, 0.1-0.2mmol manganese nitrate and 5-7mmol ammonium molybdate in 70-100mL deionized water, stirring thoroughly to obtain solution A, dissolving 2-3g sodium hydroxide in 50-70mL deionized water to obtain solution B, and then slowly dropping solution B into solution A under stirring in an oil bath until the pH value is 9.5-9.8, and nitrogen is passed throughout the process, stirring is continued for 20-30min, then transferred to a reactor, crystallized in an oven at 80-85°C, cooled to room temperature, centrifuged and repeatedly washed with deionized water to obtain hydrotalcite nanosheets;

[0019] 4) etching the hydrotalcite nanosheets in a phosphate buffer solution for 4-6 hours, then repeatedly washing with deionized water to obtain etched hydrotalcite nanosheets, then adding the etched hydrotalcite nanosheets into deionized water, and after uniform dispersion, adding polyethylene glycol, mixing and stirring for 12-15 hours, and then adding polysaccharide nanofibers, stirring at 500-800r / min for 3-5 hours under 200-300W ultrasonic action, and then centrifuging, repeatedly washing with deionized water, and drying to obtain a composite nanomaterial.

[0020] As a further preferred embodiment of the present invention, in step 1), the concentration of the konjac glucomannan solution is 0.5-0.8wt%;

[0021] The concentration of the sodium periodate solution is 10-13 wt %.

[0022] As a further preferred embodiment of the present invention, in step 2), the spinning conditions are: supply speed is 300-500 μL / min, voltage is 5-10 kV, and receiving distance is 20-30 cm.

[0023] As a further preferred embodiment of the present invention, in step 3), the temperature of the oil bath stirring is 80-85°C;

[0024] The crystallization time is 24-30h.

[0025] As a further preferred embodiment of the present invention, in step 4), the pH of the phosphate buffer solution is 4.0-4.5;

[0026] The usage ratio of the etched hydrotalcite nanosheets, deionized water, polyethylene glycol, and polysaccharide nanofibers is (5-10) g: (80-130) mL: (50-100) mg: (25-35) g.

[0027] A method for preparing a pet liquid probiotic preparation comprises the following steps:

[0028] 1) adding gel carrier material to probiotics according to weight proportions, and fully soaking for 24-30 hours to obtain a bacterial suspension;

[0029] 2) preparing a composite protective agent by mixing a nutritional stabilizer, a multifunctional stabilizer, polyethylene glycol and natural vitamin E;

[0030] 3) Dissolve calcium carbonate and sodium alginate in water, wherein the concentration of sodium alginate is 3-4 wt %, mix well, stand at room temperature for 12-15 h to fully swell, sterilize to obtain a mixed solution, and set aside;

[0031] 4) mixing the mixed solution with the bacterial suspension to prepare a bacterial gel mixture;

[0032] 5) Add soybean oil dropwise to the bacterial gel mixture, stir mechanically at 100-300 r / min for 30-50 min, add sterile PBS buffer, let stand for 1-3 h, remove the upper oil phase, centrifuge at 12000-15000 r / min for 20-30 min, collect the pre-embedded probiotic microcapsules, wash three times, and set aside;

[0033] 6) Add the collected probiotic microcapsules into the composite protective agent, mix and stir evenly, and then obtain a highly active liquid probiotic preparation.

[0034] The beneficial effects of the present invention are:

[0035] In the present invention, periodate is used to oxidize polysaccharides into aldehyde polysaccharides, polysaccharide nanofibers are obtained by electrostatic spinning, and hydrotalcite nanosheets are synthesized by a simple single-drop hydrothermal method, and then etched by simple acid treatment to obtain etched hydrotalcite nanosheets rich in hole defects, and then the etched hydrotalcite nanosheets are surface-modified with polyethylene glycol to help improve their stability, and the polysaccharide nanofibers are embedded in the hole defects of the etched hydrotalcite nanosheets by ultrasound and mechanical stirring, so that the two are physically combined to form a composite nanomaterial; and then citric acid is used as a cross-linking agent to react with soluble citrus cellulose and Sodium carboxymethyl cellulose is reacted to obtain a hydrogel with a three-dimensional network structure. In order to increase the cross-linking density of the internal structure of the hydrogel and better load probiotics, intermittent ultrasonic action and high-speed mechanical stirring are used. Under the capillary action of the hydrogel, the penetration of the composite nanomaterial into the hydrogel can be accelerated, thereby constructing a highly cross-linked mesh structure in the three-dimensional network structure inside the hydrogel, increasing the cross-linking density inside the hydrogel, and because the composite nanomaterial contains nanosheets, a winding channel structure can be constructed inside the hydrogel, thereby delaying the rate of material flow and achieving a better confinement effect.

[0036] The pet liquid probiotics in the present invention use a synthetic gel carrier material as a carrier, and the probiotics are loaded and embedded in the gel carrier material by adsorption and embedding, thereby effectively reducing the contact of the probiotics with adverse factors in the external environment, thereby significantly improving its survival rate in various environments; and, introducing a new type of stable nutrient can improve the nutritional absorption and health level of pets, and the introduced multifunctional stabilizer can make the probiotics enter a dormant state, isolating the external environment from affecting the activity of the probiotics, thereby ensuring the activity of the probiotics. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] In the present invention, the multifunctional stabilizer comprises the following components in parts by weight: 7 parts of sunflower oil, 5 parts of camellia oil, 2 parts of avocado oil, 3 parts of peony seed oil, 2 parts of polyethylene glycol, 7 parts of soybean oil, 1.5 parts of sodium alginate, 1.5 parts of calcium carbonate, 0.8 parts of natural vitamin E, and 1.5 parts of antioxidant microcapsules;

[0039] The preparation method of the multifunctional stabilizer specifically comprises the following steps:

[0040] 1) According to weight percentage, sunflower oil, camellia oil, avocado oil, peony seed oil, soybean oil, polyethylene glycol, and natural vitamin E are mixed in a certain proportion to form a composite protective agent;

[0041] 2) Dissolve calcium carbonate and sodium alginate in water, with the concentration of sodium alginate being 3 wt %, mix thoroughly, let stand at room temperature for 13 h to fully swell, and then sterilize for later use;

[0042] 3) The sodium alginate calcium carbonate mixture and the antioxidant microcapsules are sequentially added to the composite protective agent, and after mixing and stirring evenly, a multifunctional stabilizer is obtained.

[0043] The preparation method of the antioxidant microcapsules is as follows:

[0044] 1) Lactobacillus acidophilus and Bifidobacterium are prepared in a mass ratio of 2.5:1, and then the above strains are added to a stock solution in a mass ratio of 1:120, wherein the stock solution is mixed with the following components in parts by weight: 7 parts of brown sugar, 3 parts of phosphate, and 95 parts of purified water, and then put into a fermentation tank and stirred evenly, and then cultured in a semi-closed manner at 31° C. for 2 days, and then cultured in a closed manner, and continued to culture for 15 days after the pH value is less than 4.0, to obtain a composite culture solution;

[0045] 2) taking lavender, jasmine and rosemary, washing and drying them, crushing them, and then mixing the composite culture liquid with the raw materials in a mass ratio of 1:50, wherein the raw materials are composed of the following components in parts by weight: 40 parts of lavender powder, 15 parts of jasmine powder, 15 parts of rosemary powder, 3 parts of brown sugar, 3 parts of phosphate, and 60 parts of purified water, and then putting them into a fermentation tank and stirring them evenly, and then culturing them in a semi-closed manner at 31° C. for 2 days, and then culturing them in a closed manner for 15 days, and then adding water to the fermentation tank to make the solid-liquid ratio reach 1:4, soaking for 2 days, and centrifuging to obtain a fermentation liquid;

[0046] 3) According to the mass ratio of 1:40, the fermentation broth was placed in MRS liquid medium for overnight culture, 1.5 mL of the culture broth was collected by centrifugation at 5000 r / min for 20 min, washed three times with a 0.82 wt% sodium chloride solution, resuspended in an equal amount of physiological saline, and then added to an equal amount of 0.25 mmol / LDPPH solution. After mixing evenly, the mixture was placed in a dark place at 31° C. and incubated for 40 min, and then centrifuged at 4500 r / min for 15 min to obtain an antioxidant strain;

[0047] 4) dissolving skim milk powder in distilled water to prepare a skim milk solution with a concentration of 32 wt%, stirring the solution at 5°C overnight, then adding 2.5 g of an antioxidant strain and 500 μL of rennet to 30 g of the skim milk solution, stirring the solution at 5°C at 400 r / min for 2 h, and obtaining a bacterial-milk mixture;

[0048] 5) 13 g of the bacterial-emulsion mixture was added with 1.6 g of calcium carbonate and mixed evenly, then added to 130 mL of sunflower oil, stirred and reacted for 18 min under the action of a magnetic stirrer, and the mixture was dispersed into the oil phase to form a uniform water-in-oil emulsion, 130 μL of glacial acetic acid was added, and the mixture was allowed to stand for 40 min, and then centrifuged at 1300 r / min for 2 min. After washing, the water was drained to obtain antioxidant microcapsules.

[0049] In the present invention, the probiotics are obtained by mixing and fermenting equal masses of Saccharomyces boulardii, Bifidobacterium animalis BB-12, and Lactobacillus plantarum;

[0050] The nutritional stabilizer is prepared by mixing sunflower oil, camellia oil, avocado oil and peony seed oil in a mass ratio of 1:0.5:0.4:1.5;

[0051] Soybean oil contains glacial acetic acid, and the content of glacial acetic acid is 2.5% of the mass of soybean oil.

[0052] Example 1

[0053] A pet liquid probiotic preparation, comprising the following components by weight: 30 parts of probiotics, 5 parts of gel carrier material, 1 part of nutrient stabilizer, 1 part of multifunctional stabilizer, 2 parts of polyethylene glycol, 1 part of natural vitamin E, 2 parts of calcium carbonate, 1 part of sodium alginate, 5 parts of soybean oil, and 3 parts of sterile PBS buffer;

[0054] The preparation method of the liquid probiotic preparation specifically comprises the following steps:

[0055] 1) adding gel carrier material to probiotics according to weight proportions, and fully soaking for 24 hours to obtain a bacterial suspension;

[0056] 2) preparing a composite protective agent by mixing a nutritional stabilizer, a multifunctional stabilizer, polyethylene glycol and natural vitamin E;

[0057] 3) Dissolve calcium carbonate and sodium alginate in water, wherein the concentration of sodium alginate is 3 wt %, mix well, let stand at room temperature for 12 h to fully swell, sterilize to obtain a mixed solution, and set aside;

[0058] 4) mixing the mixed solution with the bacterial suspension to prepare a bacterial gel mixture;

[0059] 5) Add soybean oil dropwise to the bacterial gel mixture, stir mechanically at 100 r / min for 30 min, add sterile PBS buffer, let stand for 1 h, remove the upper oil phase, centrifuge at 12000 r / min for 20 min, collect the pre-embedded probiotic microcapsules, wash three times, and set aside;

[0060] 6) Add the collected probiotic microcapsules into the composite protective agent, mix and stir evenly, and then obtain a highly active liquid probiotic preparation.

[0061] Wherein, the preparation method of gel carrier material is as follows:

[0062] 1) dissolving konjac glucomannan in deionized water to obtain a 0.5wt% konjac glucomannan solution, then mixing the konjac glucomannan solution with a 10wt% sodium periodate solution in a volume ratio of 1:0.3, stirring and reacting at 20°C in the dark for 16h, after the reaction is completed, transferring the solution to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzing with a large amount of water, and freeze-drying the purified solution to obtain oxidized konjac glucomannan;

[0063] 2) dissolving oxidized konjac glucomannan, polyethylene oxide, polyethylene glycol octyl phenyl ether and dimethyl sulfoxide in water in a homogeneous manner, so that the polymer concentration is 3wt%, the mass ratio of oxidized konjac glucomannan to polyethylene oxide is 4:1, the concentration of polyethylene glycol octyl phenyl ether is 1wt%, and the concentration of dimethyl sulfoxide is 5wt%, putting the prepared electrospinning solution into a syringe, controlling the supply speed to 300μL / min, the voltage to 5kV, the receiving distance to 20cm, spinning at 25°C and a humidity of 10% to obtain polysaccharide nanofibers;

[0064] 3) 0.8 mmol of cobalt nitrate, 0.1 mmol of manganese nitrate and 5 mmol of ammonium molybdate were dissolved in 70 mL of deionized water and stirred thoroughly to obtain solution A. 2 g of sodium hydroxide was dissolved in 50 mL of deionized water to obtain solution B. Solution B was then slowly added dropwise to solution A under stirring in an oil bath at 80° C. until the pH value reached 9.5, and nitrogen was passed throughout the process. Stirring was continued for 20 min, and then the mixture was transferred to a reactor, crystallized in an oven at 80° C. for 24 h, cooled to room temperature, centrifuged and repeatedly washed with deionized water to obtain hydrotalcite nanosheets;

[0065] 4) etching the hydrotalcite nanosheets in a phosphate buffer solution at pH 4.0 for 4 h, then repeatedly washing with deionized water to obtain etched hydrotalcite nanosheets, then adding 5 g of the etched hydrotalcite nanosheets to 80 mL of deionized water, and after uniform dispersion, adding 50 mg of polyethylene glycol, mixing and stirring for 12 h, and then adding 25 g of polysaccharide nanofibers, stirring at 500 r / min for 3 h under 200 W ultrasound, and then centrifuging, repeatedly washing with deionized water, and drying to obtain a composite nanomaterial;

[0066] 5) dissolving 15 g of water-soluble citrus cellulose and 75 g of sodium carboxymethyl cellulose in 300 mL of deionized water, gently stirring at room temperature until a clear solution is obtained, then adding 0.4 g of citric acid to the solution, stirring until completely dissolved, and drying the obtained product at 30° C. for 24 h, and then storing it at 80° C. for 24 h for cross-linking reaction to obtain a hydrogel;

[0067] 6) The composite nanomaterial is added to deionized water and fully dispersed to obtain a dispersion with a solid content of 3%. Then, the hydrogel is added to the dispersion at a solid-liquid ratio of 1 g:15 mL, and immersed in 100 W intermittent ultrasound for 24 hours, with an ultrasound interval of 10 minutes and an action time of 3 minutes. After the treatment is completed, the product is centrifuged at 10,000 r / min for 15 minutes to obtain a gel carrier material.

[0068] Example 2

[0069] A pet liquid probiotic preparation, comprising the following components by weight: 40 parts of probiotics, 7 parts of gel carrier material, 2 parts of nutritional stabilizer, 2 parts of multifunctional stabilizer, 3 parts of polyethylene glycol, 2 parts of natural vitamin E, 3 parts of calcium carbonate, 1 part of sodium alginate, 7 parts of soybean oil, and 5 parts of sterile PBS buffer;

[0070] The preparation method of the liquid probiotic preparation specifically comprises the following steps:

[0071] 1) adding gel carrier material to probiotics according to weight proportions, and fully soaking for 28 hours to obtain a bacterial suspension;

[0072] 2) preparing a composite protective agent by mixing a nutritional stabilizer, a multifunctional stabilizer, polyethylene glycol and natural vitamin E;

[0073] 3) Dissolve calcium carbonate and sodium alginate in water, wherein the concentration of sodium alginate is 3.5 wt %, mix well, let stand at room temperature for 13 h to fully swell, sterilize to obtain a mixed solution, and set aside;

[0074] 4) mixing the mixed solution with the bacterial suspension to prepare a bacterial gel mixture;

[0075] 5) Add soybean oil dropwise to the bacterial gel mixture, stir mechanically at 200 r / min for 40 min, add sterile PBS buffer, let stand for 2 h, remove the upper oil phase, centrifuge at 13000 r / min for 25 min, collect the pre-embedded probiotic microcapsules, wash three times, and set aside;

[0076] 6) Add the collected probiotic microcapsules into the composite protective agent, mix and stir evenly, and then obtain a highly active liquid probiotic preparation.

[0077] Wherein, the preparation method of gel carrier material is as follows:

[0078] 1) dissolving konjac glucomannan in deionized water to obtain a 0.7wt% konjac glucomannan solution, then mixing the konjac glucomannan solution with a 12wt% sodium periodate solution in a volume ratio of 1:0.5, stirring and reacting at 23°C in the dark for 18h, after the reaction is completed, transferring the solution to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzing with a large amount of water, and lyophilizing the purified solution to obtain oxidized konjac glucomannan;

[0079] 2) dissolving oxidized konjac glucomannan, polyethylene oxide, polyethylene glycol octyl phenyl ether and dimethyl sulfoxide in water in a homogeneous manner, so that the polymer concentration is 4wt%, the mass ratio of oxidized konjac glucomannan to polyethylene oxide is 4.5:1, the concentration of polyethylene glycol octyl phenyl ether is 1.5wt%, and the concentration of dimethyl sulfoxide is 6wt%. Putting the prepared electrospinning solution into a syringe, controlling the supply speed to 400μL / min, the voltage to 8kV, the receiving distance to 25cm, spinning at 28°C and a humidity of 20% to obtain polysaccharide nanofibers;

[0080] 3) 0.9 mmol of cobalt nitrate, 0.2 mmol of manganese nitrate and 6 mmol of ammonium molybdate were dissolved in 80 mL of deionized water and stirred thoroughly to obtain solution A. 2.5 g of sodium hydroxide was dissolved in 60 mL of deionized water to obtain solution B. Solution B was then slowly added dropwise to solution A under stirring in an oil bath at 82° C. until the pH value reached 9.6, and nitrogen was passed throughout the process. Stirring was continued for 25 min, and then the mixture was transferred to a reactor, crystallized in an oven at 82° C. for 28 h, cooled to room temperature, centrifuged, and repeatedly washed with deionized water to obtain hydrotalcite nanosheets;

[0081] 4) etching the hydrotalcite nanosheets in a phosphate buffer solution at pH 4.5 for 5 h, then repeatedly washing with deionized water to obtain etched hydrotalcite nanosheets, then adding 7 g of the etched hydrotalcite nanosheets to 100 mL of deionized water, and after uniform dispersion, adding 80 mg of polyethylene glycol, mixing and stirring for 13 h, and then adding 30 g of polysaccharide nanofibers, stirring at 700 r / min for 4 h under 250 W ultrasound, and then centrifuging, repeatedly washing with deionized water, and drying to obtain a composite nanomaterial;

[0082] 5) dissolving 25 g of water-soluble citrus cellulose and 85 g of sodium carboxymethyl cellulose in 400 mL of deionized water, gently stirring at room temperature until a clear solution is obtained, then adding 0.6 g of citric acid to the solution, stirring until completely dissolved, and drying the obtained product at 32° C. for 28 h, and then storing it at 82° C. for 28 h for cross-linking reaction to obtain a hydrogel;

[0083] 6) The composite nanomaterial is added to deionized water and fully dispersed to obtain a dispersion with a solid content of 5%. Then, the hydrogel is added to the dispersion at a solid-liquid ratio of 1 g:25 mL, and immersed in 120 W intermittent ultrasound for 28 hours, with an ultrasound interval of 15 minutes and an action time of 5 minutes. After the treatment is completed, the product is centrifuged at 13000 r / min for 20 minutes to obtain a gel carrier material.

[0084] Example 3

[0085] A pet liquid probiotic preparation, comprising the following components by weight: 50 parts of probiotics, 10 parts of gel carrier material, 2 parts of nutrient stabilizer, 2 parts of multifunctional stabilizer, 4 parts of polyethylene glycol, 3 parts of natural vitamin E, 3 parts of calcium carbonate, 2 parts of sodium alginate, 10 parts of soybean oil, and 7 parts of sterile PBS buffer;

[0086] The preparation method of the liquid probiotic preparation specifically comprises the following steps:

[0087] 1) adding gel carrier material to probiotics according to weight proportions, and fully soaking for 30 hours to obtain a bacterial suspension;

[0088] 2) preparing a composite protective agent by mixing a nutritional stabilizer, a multifunctional stabilizer, polyethylene glycol and natural vitamin E;

[0089] 3) Dissolve calcium carbonate and sodium alginate in water, wherein the concentration of sodium alginate is 4 wt %, mix well, let stand at room temperature for 15 h to fully swell, sterilize to obtain a mixed solution, and set aside;

[0090] 4) mixing the mixed solution with the bacterial suspension to prepare a bacterial gel mixture;

[0091] 5) Add soybean oil dropwise to the bacterial gel mixture, stir mechanically at 300 r / min for 50 min, add sterile PBS buffer, let stand for 3 h, remove the upper oil phase, centrifuge at 15000 r / min for 30 min, collect the pre-embedded probiotic microcapsules, wash three times and set aside;

[0092] 6) Add the collected probiotic microcapsules into the composite protective agent, mix and stir evenly, and then obtain a highly active liquid probiotic preparation.

[0093] Wherein, the preparation method of gel carrier material is as follows:

[0094] 1) dissolving konjac glucomannan in deionized water to obtain a 0.8wt% konjac glucomannan solution, then mixing the konjac glucomannan solution with a 13wt% sodium periodate solution at a volume ratio of 1:0.7, stirring and reacting at 25°C in the dark for 20h, after the reaction is completed, transferring the solution to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzing with a large amount of water, and freeze-drying the purified solution to obtain oxidized konjac glucomannan;

[0095] 2) dissolving oxidized konjac glucomannan, polyethylene oxide, polyethylene glycol octyl phenyl ether and dimethyl sulfoxide in water in a homogeneous manner, so that the polymer concentration is 5wt%, the mass ratio of oxidized konjac glucomannan to polyethylene oxide is 5:1, the concentration of polyethylene glycol octyl phenyl ether is 2wt%, and the concentration of dimethyl sulfoxide is 7wt%, and the prepared electrospinning solution is put into a syringe, and the supply speed is controlled to be 500μL / min, the voltage is 10kV, the receiving distance is 30cm, and spinning is performed at 30°C and a humidity of 30% to obtain polysaccharide nanofibers;

[0096] 3) 0.9 mmol of cobalt nitrate, 0.2 mmol of manganese nitrate and 7 mmol of ammonium molybdate were dissolved in 100 mL of deionized water and stirred thoroughly to obtain solution A, and then 3 g of sodium hydroxide was dissolved in 70 mL of deionized water to obtain solution B, and then solution B was slowly added dropwise to solution A under stirring in an oil bath at 85° C. until the pH value reached 9.8, and nitrogen was passed throughout the process. After stirring for 30 min, the mixture was transferred to a reactor, crystallized in an oven at 85° C. for 30 h, cooled to room temperature, centrifuged and repeatedly washed with deionized water to obtain hydrotalcite nanosheets;

[0097] 4) etching the hydrotalcite nanosheets in a phosphate buffer solution at pH 4.5 for 6 h, then repeatedly washing with deionized water to obtain etched hydrotalcite nanosheets, then adding 10 g of the etched hydrotalcite nanosheets to 130 mL of deionized water, and after uniform dispersion, adding 100 mg of polyethylene glycol, mixing and stirring for 15 h, and then adding 35 g of polysaccharide nanofibers, stirring at 800 r / min for 5 h under 300 W ultrasonic action, and then centrifuging, repeatedly washing with deionized water, and drying to obtain a composite nanomaterial;

[0098] 5) dissolving 30 g of water-soluble citrus cellulose and 90 g of sodium carboxymethyl cellulose in 500 mL of deionized water, gently stirring at room temperature until a clear solution is obtained, then adding 0.8 g of citric acid to the solution, stirring until completely dissolved, and drying the obtained product at 35° C. for 30 h, and then storing it at 85° C. for 30 h for cross-linking reaction to obtain a hydrogel;

[0099] 6) The composite nanomaterial is added to deionized water and fully dispersed to obtain a dispersion with a solid content of 6%. Then, the hydrogel is added to the dispersion at a solid-liquid ratio of 1 g:30 mL, and immersed in 150 W intermittent ultrasound for 30 hours, with an ultrasound interval of 20 minutes and an action time of 7 minutes. After the treatment is completed, the product is centrifuged at 15000 r / min for 25 minutes to obtain a gel carrier material.

[0100] Comparative Example 1: This comparative example is substantially the same as Example 1, except that it does not contain a nutritional stabilizer.

[0101] Comparative Example 1: This comparative example is basically the same as Example 1, except that it does not contain a multifunctional stabilizer.

[0102] Comparative Example 3: This comparative example is basically the same as Example 1, except that it does not contain the gel carrier material.

[0103] Comparative Example 4: This comparative example is basically the same as Example 1, except that 1)-2) are omitted in the preparation of the gel carrier material.

[0104] Comparative Example 5: This comparative example is basically the same as Example 1, except that 3)-4) are omitted in the preparation of the gel carrier material.

[0105] Test experiment:

[0106] The methods provided in Examples 1-3 and Comparative Examples 1-5 were used to obtain liquid probiotic preparation samples, respectively, and then stored at 4°C for 30 days. After storage, the survival rate of the probiotics in each sample was detected, wherein the detection method of the effective viable count: the boulardii Saccharomyces cerevisiae detection method was performed in accordance with the requirements of GB4789.15-2016 National Food Safety Standard Food Microbiology Inspection Yeast Inspection, the animal Bifidobacterium BB-12 detection method was performed in accordance with the requirements of GB4789.34-2016 National Food Safety Standard Food Microbiology Inspection Bifidobacterium Inspection, and the plant Lactobacillus detection method was performed in accordance with the requirements of GB7300.502-2023 National Food Safety Standard Food Microbiology Inspection Lactobacillus Inspection. The survival rate was calculated as follows: (total number of effective viable bacteria after 30 days / initial total number of effective viable bacteria) × 100%; the results are shown in Table 1.

[0107] Table 1

[0108]

[0109]

[0110] It can be seen from Table 1 that the liquid probiotic preparation of the present invention has good stability and a high survival rate of probiotics.

[0111] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A pet liquid probiotic preparation, characterized in that: The liquid probiotic preparation comprises the following components by weight: 30-50 parts of probiotics, 5-10 parts of gel carrier material, 1-2 parts of nutrient stabilizer, 1-2 parts of multifunctional stabilizer, 2-4 parts of polyethylene glycol, 1-3 parts of natural vitamin E, 2-3 parts of calcium carbonate, 1-2 parts of sodium alginate, 5-10 parts of soybean oil, and 3-7 parts of sterile PBS buffer; The probiotics are obtained by mixing and fermenting equal amounts of Saccharomyces boulardii, Bifidobacterium animalis BB-12, and Lactobacillus plantarum; The nutritional stabilizer is prepared by mixing sunflower oil, camellia oil, avocado oil and peony seed oil in a mass ratio of 1: (0.5-0.8): (0.2-0.4): (1.0-1.5); The soybean oil contains glacial acetic acid, and the content of the glacial acetic acid is 2-3% of the mass of the soybean oil.

2. A pet liquid probiotic preparation according to claim 1, characterized in that: The preparation method of the gel carrier material is as follows: 1) dissolving water-soluble citrus cellulose and sodium carboxymethyl cellulose in deionized water, gently stirring at room temperature until a clear solution is obtained, then adding citric acid to the solution, stirring until completely dissolved, drying the obtained product at 30-35° C. for 24-30 hours, and then storing it at 80-85° C. for 24-30 hours for cross-linking reaction to obtain a hydrogel; 2) adding the composite nanomaterial into deionized water, fully dispersing to obtain a dispersion having a solid content of 3-6%, then adding the hydrogel into the dispersion, soaking for 24-30 hours under intermittent ultrasound at 100-150W, with an ultrasound interval of 10-20 minutes and an action time of 3-7 minutes. After the treatment is completed, centrifuging the product at 10000-15000 r / min for 15-25 minutes to obtain a gel carrier material.

3. A pet liquid probiotic preparation according to claim 2, characterized in that: In step 1), the usage ratio of the water-soluble citrus cellulose, sodium carboxymethyl cellulose, deionized water and citric acid is (15-30) g: (75-90) g: (300-500) mL: (0.4-0.8) g.

4. A pet liquid probiotic preparation according to claim 2, characterized in that: In step 2), the solid-liquid ratio of the hydrogel to the dispersion is 1 g: (15-30) mL.

5. A pet liquid probiotic preparation according to claim 2, characterized in that: The preparation method of the composite nano material is as follows: 1) dissolving konjac glucomannan in deionized water to obtain a konjac glucomannan solution, then mixing the konjac glucomannan solution with a sodium periodate solution in a volume ratio of 1:(0.3-0.7), stirring and reacting at 20-25° C. in the dark for 16-20 hours, after the reaction is completed, transferring the solution to a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzing with a large amount of water, and lyophilizing the purified solution to obtain oxidized konjac glucomannan; 2) dissolving oxidized konjac glucomannan, polyethylene oxide, polyethylene glycol octyl phenyl ether and dimethyl sulfoxide in water in a homogeneous manner, so that the polymer concentration is 3-5wt%, the mass ratio of oxidized konjac glucomannan to polyethylene oxide is (4-5):1, the concentration of polyethylene glycol octyl phenyl ether is 1-2wt%, and the concentration of dimethyl sulfoxide is 5-7wt%, and the prepared electrospinning solution is put into a syringe, and spinning is carried out at 25-30° C. and a humidity of 10-30% to obtain polysaccharide nanofibers; 3) dissolving 0.8-0.9mmol cobalt nitrate, 0.1-0.2mmol manganese nitrate and 5-7mmol ammonium molybdate in 70-100mL deionized water, stirring thoroughly to obtain solution A, dissolving 2-3g sodium hydroxide in 50-70mL deionized water to obtain solution B, and then slowly dropping solution B into solution A under stirring in an oil bath until the pH value is 9.5-9.8, and nitrogen is passed throughout the process, stirring is continued for 20-30min, then transferred to a reactor, crystallized in an oven at 80-85°C, cooled to room temperature, centrifuged and repeatedly washed with deionized water to obtain hydrotalcite nanosheets; 4) etching the hydrotalcite nanosheets in a phosphate buffer solution for 4-6 hours, then repeatedly washing with deionized water to obtain etched hydrotalcite nanosheets, then adding the etched hydrotalcite nanosheets into deionized water, and after uniform dispersion, adding polyethylene glycol, mixing and stirring for 12-15 hours, and then adding polysaccharide nanofibers, stirring at 500-800r / min for 3-5 hours under 200-300W ultrasonic action, and then centrifuging, repeatedly washing with deionized water, and drying to obtain a composite nanomaterial.

6. A pet liquid probiotic preparation according to claim 5, characterized in that: In step 1), the concentration of the konjac glucomannan solution is 0.5-0.8wt%; The concentration of the sodium periodate solution is 10-13 wt %.

7. The pet liquid probiotic preparation according to claim 5, characterized in that: In step 2), the spinning conditions are: supply speed is 300-500 μL / min, voltage is 5-10 kV, and receiving distance is 20-30 cm.

8. The pet liquid probiotic preparation according to claim 5, characterized in that: In step 3), the temperature of the oil bath stirring is 80-85°C; The crystallization time is 24-30h.

9. The pet liquid probiotic preparation according to claim 5, characterized in that: In step 4), the pH of the phosphate buffer solution is 4.0-4.5; The usage ratio of the etched hydrotalcite nanosheets, deionized water, polyethylene glycol, and polysaccharide nanofibers is (5-10) g: (80-130) mL: (50-100) mg: (25-35) g.

10. The method for preparing a pet liquid probiotic preparation according to any one of claims 1 to 9, characterized in that: The specific steps include: 1) adding gel carrier material to probiotics according to weight proportions, and fully soaking for 24-30 hours to obtain a bacterial suspension; 2) preparing a composite protective agent by mixing a nutritional stabilizer, a multifunctional stabilizer, polyethylene glycol and natural vitamin E; 3) Dissolve calcium carbonate and sodium alginate in water, wherein the concentration of sodium alginate is 3-4 wt %, mix well, stand at room temperature for 12-15 h to fully swell, sterilize to obtain a mixed solution, and set aside; 4) mixing the mixed solution with the bacterial suspension to prepare a bacterial gel mixture; 5) Add soybean oil dropwise to the bacterial gel mixture, stir mechanically at 100-300 r / min for 30-50 min, add sterile PBS buffer, let stand for 1-3 h, remove the upper oil phase, centrifuge at 12000-15000 r / min for 20-30 min, collect the pre-embedded probiotic microcapsules, wash three times, and set aside; 6) Add the collected probiotic microcapsules into the composite protective agent, mix and stir evenly, and then obtain a highly active liquid probiotic preparation.

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