A kind of thermostable enteric-coated sustained-release composite probiotic micro-pellet and preparation method thereof

Abstract

The present invention belongs to the technical field of microbial coated pellet preparation process, and specifically relates to a high temperature resistant enteric sustained-release coated composite probiotic pellet and a preparation method thereof. The high temperature resistant enteric sustained-release coated composite probiotic pellet provided by the present invention comprises a pellet core and a coating material, wherein the pellet core comprises a probiotic dry bacterial powder and a composite protective agent in a mass ratio of 1:0.1 to 0.2; the coating material comprises ethyl cellulose, hydroxypropyl methylcellulose, polyvinyl phthalate and calcium stearate in a mass ratio of 18 to 22:0.5 to 2:0.5 to 2:1 to 5. The high temperature resistant enteric sustained-release coated composite probiotic pellet provided by the present invention can effectively protect probiotics, improve the physiological and biochemical and stability properties of probiotics, make them acid-resistant and bile-resistant, and can be sustained-released in the intestine through the stomach. Further, the use of coating technology is more conducive to its relatively long-term storage at room temperature.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial coated pellet preparation technology, and specifically relates to a high temperature resistant enteric-coated sustained-release coated composite probiotic pellet and a preparation method thereof. Background Art

[0002] How to improve product safety and quality while reducing costs has become a major challenge for livestock and poultry farmers and feed processing companies in the new situation. This also makes the selection and application of feed additives critical. Therefore, ecological preparations containing probiotics can be used in livestock and poultry feed to replace antibiotics as feed additives, which is expected to solve problems such as antibiotic residues and animal drug resistance, and has an immeasurable role in the development of animal husbandry.

[0003] Existing microecological preparations used as feed additives often contain not only one or several probiotics of the same genus, but mixed probiotic preparations containing multiple probiotics from different species or genera. At present, there are 35 species of microorganisms allowed to be added in the 2013 version of the "Catalogue of Feed Additives". Microecological preparations represented by Bacillus and lactic acid bacteria have the functions of maintaining the balance of animal intestinal microorganisms, preventing diseases, promoting growth, improving feed conversion rate, and improving the livestock and poultry breeding environment. They are increasingly becoming the focus of major feed processing companies and livestock and poultry breeding industries.

[0004] Since most lactic acid bacteria are anaerobic or facultative anaerobic, they do not form spores during their growth and have poor resistance. They are difficult to store for a long time in liquid conditions and are not conducive to transportation, storage and use. Lactic acid bacteria preparations must pass through the gastric environment to reach the intestines with a large number of surviving bacteria and colonize on the intestinal mucosa to exert their physiological effects. However, due to the bactericidal effects of gastric acid, bile acid, etc., the number of live bacteria in lactic acid bacteria preparations will drop significantly during this process. Therefore, how to make lactic acid bacteria preparations still be able to survive in large quantities (≥10 7 ) survival, and further germination, proliferation, and secretion of metabolites in the intestine has become an issue of increasing concern.

[0005] Using genetic engineering methods to improve the high temperature resistance (≥70℃) and high humidity resistance (≥20%) of lactic acid bacteria is one of the ways to solve the problem, and some progress has been made. However, this method has certain limitations: 1. High temperature resistance and fermentation efficiency of bacteria cannot be achieved at the same time; 2. High temperature resistance, physiological and biochemical characteristics, metabolites and their biological activity in animals cannot be achieved at the same time; 3. The safety of genetically modified bacteria cannot be guaranteed. Moreover, Europe has very strict requirements on the registration, verification and use of imported genetically modified feed probiotics, which makes it very difficult to sell genetically modified high temperature resistant lactic acid bacteria to the European market.

[0006] Therefore, how to improve the above properties of lactic acid bacteria by non-GMO methods becomes the key technical problem to be solved in the composite feed probiotic preparation of this application. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a high temperature resistant enteric-coated sustained-release composite probiotic micro-pellets and a preparation method thereof. The high temperature resistant enteric-coated sustained-release composite probiotic micro-pellets prepared by the present invention can effectively protect the probiotics through the added composite protection and special coating materials, and can improve the physiological and biochemical properties of the probiotics, such as high temperature resistance, acid resistance, and bile salt resistance, so that the survival rate is greatly improved during the high temperature granulation of the feed, and the animal body can resist acid and pass through the stomach, and only release in the intestine, and germinate spores to form a vegetative body, and grow and reproduce rapidly to play a biological function; at the same time, the use of coating technology is more conducive to its relatively long storage at room temperature.

[0008] The technical solution of the present invention is:

[0009] A high temperature resistant enteric sustained-release coated composite probiotic micropill comprises a pellet core and a coating material, wherein the pellet core comprises probiotic dry bacterial powder and a composite protective agent in a mass ratio of 1:0.1-0.2; and the coating material comprises ethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl phthalate and calcium stearate in a mass ratio of 18-22:0.5-2:0.5-2:1-5.

[0010] Furthermore, the coating material comprises ethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl phthalate and calcium stearate in a mass ratio of 20:1:1:3.

[0011] The coating material used in the present invention is composed of ethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl phthalate and calcium stearate in a specific ratio. The added ethyl cellulose is insoluble in water, soluble in organic solvents, has good film-forming properties, and the film is semi-permeable, which is an osmotic pump type controlled release water-insoluble film coating material; the added hydroxypropyl methyl cellulose (HPMC) has good film properties, and the film is transparent and tough, and is not easy to stick during production. Especially for composite probiotics that are easy to absorb moisture and change in quality, using this as a cut-off layer can greatly improve its invariance, and can also be used as a composite probiotic The auxiliary materials for the targeted release of probiotics are used in combination with polyvinyl phthalate (PVAP) and calcium stearate as the coating materials of the present invention, which can evenly form a dense waterproof film on the outer layer of the pellet core, effectively protect the activity of the probiotics in the pellet core from being harmed by the external environment, and effectively prolong the storage time of the probiotic micro-pills at room temperature; at the same time, it can also effectively improve the gastric acid resistance of the probiotic micro-pills, and can resist acid and pass through the stomach in animals, and only be released in the intestines, and germinate spores to form vegetative bodies, and grow and reproduce rapidly to exert biological functions.

[0012] Furthermore, the composite protective agent is composed of trehalose, sucrose and calcium alginate in a mass ratio of 0.3~2:0.5~3:0.5~2.

[0013] Furthermore, the composite protective agent is composed of trehalose, sucrose and calcium alginate in a mass ratio of 1:1:1.

[0014] In addition to the probiotic dry powder, the core of the composite probiotic micropill prepared by the present invention also adds a composite protective agent, which is composed of trehalose, sucrose and calcium alginate. Trehalose is a typical stress metabolite, which can form a unique protective film on the cell surface under harsh environmental conditions such as high temperature, high cold, high osmotic pressure and drying and dehydration, effectively protecting the biological molecular structure from being destroyed, thereby maintaining the life process and biological characteristics of the living body. The composite protective agent composed of trehalose, sucrose and calcium alginate added by the present invention in a specific proportion is applied to the preparation process of the core of the present invention, and a "scaffold" for accommodating probiotics can be formed, which serves as a protective layer for probiotics, helps to stabilize the cellulose film of probiotics from high temperature during the drying process, and can also protect the cell integrity of probiotics, help protect probiotics from damage by harsh environments, and help protect probiotics from being decomposed by gastric acid before reaching the intestines, thereby greatly improving the stability of probiotics in the core of the ball, and the properties of high temperature resistance, acid resistance, and bile salt resistance.

[0015] Furthermore, in the thermostable enteric-coated sustained-release composite probiotic micropellets provided by the present invention, the probiotic dry bacterial powder is one of composite lactic acid bacteria powder, Bacillus coagulans powder and composite Bacillus powder.

[0016] Furthermore, the composite lactic acid bacteria powder is a mixed powder of enterococci and Lactobacillus plantarum, which is composed of a ratio of 1:10 in terms of viable bacteria count, and the total viable bacteria count of the composite lactic acid bacteria powder is ≥550×10 8 CFU / g;

[0017] The viable count of the Bacillus coagulans powder is ≥5×10 8 CFU / g;

[0018] The composite bacillus powder is a mixed bacterial powder of Clostridium butyricum, Bacillus licheniformis and Bacillus subtilis, which is composed of a living bacteria count ratio of 1:2:2, and the total living bacteria count of the composite bacillus powder is ≥25×10 8 CFU / g.

[0019] In addition, the present invention also provides a method for preparing thermostable enteric-coated sustained-release composite probiotic micropellets, comprising the following steps:

[0020] S1. Granulation:

[0021] (1) Preparation of pellet core:

[0022] After the probiotic dry powder and the composite protective agent are mixed evenly, they are put into the centrifugal turntable of the centrifugal pill making machine, and the fan and turntable are started. Deionized water is sprayed at the same time to form fluffy gravel-like particles. When the particle size reaches 0.2-0.6 mm, the turntable speed is increased to 400-500 rpm, which is conducive to rounding the loose pill cores. Then the spraying speed is reduced to 250-300 rpm to keep the particles rolling in a wet state, and the spraying is stopped;

[0023] The mass ratio of the probiotic dry powder, the composite protective agent and deionized water is 1:0.1-0.2:0.66-0.72.

[0024] (2) Drying and screening:

[0025] The rotating speed of the turntable is reduced to 150-250 rpm to keep the material obtained in step S1 (1) in a flowing state, and the heating device is turned on, and the material is dried and sieved to obtain pellet cores;

[0026] S2. Coating:

[0027] (1) Preparation of coating solution: deionized water is added to 95% ethanol to form a mixed solution, and the mixture is stirred evenly. Ethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl phthalate and calcium stearate in the coating materials are added thereto, and the mixture is stirred continuously. The mixture is filtered and the sieved coating solution is kept stirred to prepare a coating solution for standby use.

[0028] (2) Coating: preheat the fluidized bed, then place the pellet cores obtained in step S1 into the fluidized bed, start the fan and heating system, and spray the coating liquid;

[0029] (3) Drying and sieving: After spraying the coating liquid, reduce the air flow rate and maintain the convection state, so that the dry air flow dries the coated particles until the moisture content is less than 7%, and then sieve to obtain composite probiotic micropellets with a coating film thickness of 4-8%.

[0030] Furthermore, in step S1 (1), the rotation speed of the fan and the turntable when starting is 250-300 rpm, deionized water is sprayed, the mass ratio of the water spraying amount to the sum of the probiotic dry powder and the composite protective agent is 3:5, the water spraying speed is 4-8 g / min, and when the particle size reaches 0.2-0.6 mm, the turntable speed is increased to 400-500 rpm, and the liquid spraying speed is reduced to 1-3 g / min;

[0031] In step S1 (2), the rotating speed of the turntable is reduced to 250-300 rpm, the heating device is set to 36-37° C., and the drying time is 5-10 min.

[0032] Furthermore, in step S2 (1), the mass ratio of the ethanol with a mass concentration of 95% to deionized water is 7.4:1; the mass ratio of the coating material, ethyl cellulose, hydroxypropyl methylcellulose, polyvinyl phthalate and calcium stearate is 20:1:1:3; the mass ratio of the mixed solution of the ethanol and deionized water to the coating material is 1:2.4-2.5;

[0033] In the step S2 (1), the coating material is added and stirred for 30 minutes, and then filtered with a 60-mesh sieve.

[0034] Furthermore, in step S2 (2), the preheating time of the fluidized bed is 10 min, and the air flow rate of the fan is 3-10 m 3 / s, the air outlet temperature is 33~37℃, and the coating liquid spraying speed is 4~10g / min.

[0035] Furthermore, the air flow rate during drying in step S2 (3) is 5-10 m 3 / s; the particles with a particle size of 0.25-0.5 mm in the composite probiotic micropellets prepared in step S2 (3) account for 70% of the total mass, and the particles with a particle size of 0.6-0.8 mm account for 30%; the coating film thickness of the composite probiotic micropellets prepared in step S2 (3) is 4-8%, which is the percentage of the mass of ethyl cellulose to the total mass of the micropellets.

[0036] Compared with the prior art, the thermostable enteric-coated sustained-release composite probiotic pellets provided by the present invention have the following advantages:

[0037] The high-temperature-resistant enteric-coated sustained-release composite probiotic micro-pellets prepared by the present invention can effectively protect the probiotics through the added composite protection and special coating materials, and can improve the physiological and biochemical properties of the probiotics, such as high-temperature resistance, acid resistance, and bile salt resistance, so that the survival rate of the probiotics in the high-temperature granulation process of the feed is greatly improved, and the probiotics can resist acid and pass through the stomach in the animal body, and are only released in the intestine, and germinate spores to form vegetative bodies, and grow and reproduce rapidly to exert biological functions; at the same time, the storage time of the prepared probiotic micro-pellets at room temperature can also be effectively improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 In the first test example, when the ratio of dry bacterial powder to protective agent in the pellet core was 1:0.1 and 1:0.2, the composite lactic acid bacteria pellets with different coating thicknesses were heat treated (85 o C-5mins) after survival rate (%);

[0039] Figure 2 In the first test, when the coating thickness is 4%, the effect of different dry bacterial powder and protective agent dosage in the pellet core on the heat resistance of the composite lactic acid bacteria pellets (85 o C-5mins);

[0040] Figure 3 In the first test example, the coating thickness is 4%, and the ratio of dry bacterial powder to protective agent is 1:0.1.

[0041] and 1:0.2, different coating materials have an effect on the heat resistance of composite lactic acid bacteria pellets (85 o C-5mins);

[0042] Figure 4 In the first test example, when the ratio of dry bacterial powder to protective agent in the pellet core is 1:0.1 and 1:0.2 respectively,

[0043] Survival rate (%) of composite lactic acid bacteria micropellets with different coating thicknesses (3-9%) after acid treatment (pH 2.5-1hr);

[0044] Figure 5 In the first test example, when the coating thickness is 4%, the effect of different dry bacterial powder and protective agent dosage ratios in the pellet core on the acid resistance (pH2.5-1hr) of the composite lactic acid bacteria pellets;

[0045] Figure 6 In the first test example, when the coating thickness is 4%, the ratio of dry bacterial powder to protective agent is 1:0.1 and 1:0.2 respectively, the effect of different coating materials on the acid resistance (pH2.5-1hr) of the composite lactic acid bacteria micropellets;

[0046] Figure 7 In Experiment 1, when the ratio of dry bacterial powder to protective agent in the pellet core was 1:0.1 and 1:0.2 respectively, the survival rate (%) of composite lactic acid bacteria micropellets with different coating thicknesses (3-9%) after bile salt treatment (0.3%-1hr);

[0047] Figure 8 In Experimental Example 1, when the coating thickness is 4%, the effect of different dry bacterial powder and protective agent dosage ratios in the pellet core on the bile salt resistance (0.3%-1hr) of the composite lactic acid bacteria pellets;

[0048] Fig. 9 In the first test example, the coating thickness is 4%, and the ratio of dry bacterial powder to protective agent is 1:0.1.

[0049] and 1:0.2, the effect of different coating materials on the bile salt resistance of composite lactic acid bacteria micropellets (0.3%-1hr);

[0050] Fig.10 In the first test example, the ratio of dry bacterial powder to protective agent in the pellet core was 1:0.1 and 1:0.2 respectively.

[0051] When the composite lactic acid bacteria pellets with different coating thickness (3-9%) were coated at room temperature (≤30 o C) stable performance of storage;

[0052] Fig.11 In the first test, when the coating thickness is 4%, the dosage of different dry bacteria powder and protective agent in the pellet core has an effect on the composite lactic acid bacteria pellets at room temperature (≤30 o C) Effect of storage stability;

[0053] Fig.12 In the first test example, the coating thickness is 4%, and the ratio of dry bacterial powder to protective agent is 1:0.1.

[0054] and 1:0.2, different coating materials have different effects on the composite lactic acid bacteria pellets at room temperature (≤30 o C) Effect of storage stability;

[0055] Fig.13 In the first test example, when the ratio of dry bacterial powder to protective agent in the pellet core is 1:0.1 and 1:0.2 respectively,

[0056] Sustained release properties of composite lactic acid bacteria pellets with different coating thicknesses (3-9%);

[0057] Fig.14 In Experimental Example 1, when the coating thickness is 4%, the effect of different dry bacterial powder and protective agent dosage ratios in the pellet core on the sustained-release performance of the composite lactic acid bacteria pellets;

[0058] Fig.15In the first test example, the coating thickness is 4%, and the ratio of dry bacterial powder to protective agent is 1:0.1.

[0059] and 1:0.2, the effects of different coating materials on the sustained-release properties of compound lactic acid bacteria pellets;

[0060] Fig.16 In the second test example, when the ratio of dry bacterial powder to protective agent in the pellet core was 1:0.1 and 1:0.2, respectively, Bacillus coagulans pellets with different coating thicknesses (3-9%) were heat treated (85 o C-5mins) after survival;

[0061] Fig.17 In the second test example, when the coating thickness is 4%, the effect of different amounts of dry bacterial powder and protective agent in the pellet core on the heat resistance (85 o C-5mins);

[0062] Fig.18 In the second test, when the coating thickness was 4%, the ratio of dry bacterial powder to protective agent was 1:0.1 and 1:0.2 respectively, the effect of different coating materials in the pellet core on the heat resistance (85 o C-5mins);

[0063] Fig.19 In Experiment 2, the sustained-release performance of Bacillus coagulans micropellets with different coating thicknesses (3-9%) was shown when the ratio of dry bacterial powder to protective agent in the pellet core was 1:0.1 and 1:0.2 respectively;

[0064] Fig. 20 In Experimental Example 2, when the coating thickness is 4%, the effect of different dry bacterial powder and protective agent dosage ratios in the pellet core on the sustained-release performance of Bacillus coagulans pellets;

[0065] Fig.21 In the second test example, the coating thickness is 4%, and the ratio of dry bacterial powder to protective agent is 1:0.1.

[0066] and 1:0.2, the effects of different coating materials on the sustained-release properties of Bacillus coagulans pellets;

[0067] Fig. 22 In Experiment 3, the sustained-release performance of composite Bacillus micropellets with different coating thicknesses (3-9%) was shown when the ratio of dry bacterial powder to protective agent in the pellet core was 1:0.1 and 1:0.2 respectively;

[0068] Fig.23In Experimental Example 3, when the coating thickness is 4%, the effect of different dry bacterial powder and protective agent dosage ratios in the pellet core on the sustained-release performance of composite Bacillus micropellets;

[0069] Fig.24 In the third test example, the coating thickness is 4%, and the ratio of dry bacterial powder to protective agent is 1:0.1.

[0070] The effects of different coating materials on the sustained-release properties of composite Bacillus micropellets at a ratio of 1:0.0 and 1:0.2. DETAILED DESCRIPTION

[0071] The present invention is further illustrated below through the description of specific implementation methods, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not deviate from the basic idea of ​​the present invention, they are all within the protection scope of the present invention.

[0072] In the following examples and comparative examples, reagents not otherwise specified are conventional reagents and can be purchased from conventional reagent production and sales companies.

[0073] Example 1 High temperature resistant enteric-coated sustained-release composite probiotic micropellets and preparation method thereof

[0074] The thermostable enteric-coated sustained-release composite probiotic micropellets include a pellet core and a coating material;

[0075] The pellet core is composed of probiotic dry bacterial powder and a composite protective agent, wherein the composite protective agent is composed of trehalose, sucrose and calcium alginate in a mass ratio of 1:1:1; the coating material is composed of ethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl phthalate and calcium stearate in a mass ratio of 20:1:1:3.

[0076] The probiotic dry bacteria powder is respectively compound lactic acid bacteria powder or bacillus coagulans powder or compound bacillus powder.

[0077] The probiotic dry bacteria powder is a composite lactic acid bacteria powder, which is composed of enterococci and Lactobacillus plantarum in a ratio of 1:10 in terms of live bacteria count, and the total number of lactic acid bacteria is ≥550×10 8 CFU / g;

[0078] The probiotic dry bacteria powder is Bacillus coagulans powder, and the number of live bacteria is ≥5×10 8 CFU / g;

[0079] The probiotic dry bacteria powder is a composite bacillus powder, which is composed of Clostridium butyricum, Bacillus licheniformis and Bacillus subtilis in a ratio of 1:2:2 in terms of live bacteria count, and the total number of bacillus is ≥25×10 8 CFU / g.

[0080] The following is an example of producing 100 kg of composite probiotic pellets with coating film thicknesses of 4%, 6% and 8% (the percentage is the percentage of the mass of ethyl cellulose to the total mass of the pellet core). The pellets are divided into two groups, A and B, according to different mass ratios of dry bacterial powder to protective agent in the pellet core (1:0.1 and 1:0.2), to prepare coated composite lactic acid bacteria pellets, coated Bacillus coagulans pellets and coated composite Bacillus pellets, respectively. The ratios and amounts of various raw materials in each group are listed in Tables 1 and 2.

[0081] Table 1 Raw material formula group A (dry bacteria powder: protective agent = 1:0.1, coating film thickness 4~8%)

[0082]

[0083] Table 2 Raw material formula group B (dry bacteria powder: protective agent = 1:0.2, coating film thickness 4~8%)

[0084]

[0085] The preparation method of the thermostable enteric-coated sustained-release composite probiotic micropellets comprises the following steps:

[0086] S1. Granulation:

[0087] (1) Preparation of pellet core:

[0088] The probiotic powder and the composite protective agent are mixed evenly and then put into the centrifugal turntable of the centrifugal pill making machine, and the fan and the turntable are started. The turntable speed is 300 rpm, and deionized water is sprayed at the same time. The spraying speed is 6 g / min (the mass ratio of the spraying amount to the sum of the probiotic dry powder and the composite protective agent is 3:5) to form fluffy gravel-like particles. When the particle size reaches 0.2-0.6 mm, the turntable speed is increased to 450 rpm, and the spraying speed is reduced to 2 g / min, so that the particles are kept in a wet state and rounded, and the spraying is stopped;

[0089] (2) Drying and screening:

[0090] The rotating speed of the turntable is reduced to 280 rpm again to keep the material obtained in step S1 (1) in a flowing state, the heating device is turned on, and the material is dried at 36-37°C for 10 min, and sieved to obtain pellet cores;

[0091] S2. Coating:

[0092] Preparation of coating solution: deionized water is added to 95% ethanol to form a mixed solution (the mass ratio of the ethanol solution to water is 7.4:1), and stirred evenly. Ethyl cellulose, hydroxypropyl methylcellulose, polyvinyl phthalate and calcium stearate are added to the mixed solution of ethanol and water in a mass ratio of 20:1:1:3, and stirring is continued for 30 minutes. Then, it is filtered with a 60-mesh sieve, and the sieved coating solution is kept stirred to obtain a coating solution for stand-alone use.

[0093] Coating: Preheat the fluidized bed for 10 minutes, then put the pellets prepared in step S1 into the fluidized bed, start the fan and heating system, and the air volume is 8m 3 / s, the air outlet temperature is 35℃, and the coating liquid is sprayed at a spray rate of 8g / min.

[0094] Drying and sieving: After spraying the coating liquid, reduce the air flow to 5m 3 / s, maintaining the convection state, so that the dry air flow dries the coated particles to a moisture content of <7%, and sieves to obtain coated composite lactic acid bacteria pellets, coated coagulant bacillus pellets, and coated composite bacillus pellets with a coating film thickness of 4-8%, respectively. The composite probiotic pellets prepared in step S2 (3) have a particle size of 0.25-0.5 mm accounting for 70% of the total mass, and particles with a particle size of 0.6-0.8 mm accounting for 30%.

[0095] Other process parameters involved in the granulation and coating process are shown in Table 3:

[0096] Table 3

[0097]

[0098] The drying process parameters are shown in Table 4:

[0099] Table 4

[0100]

[0101] Comparative Example 1 High temperature resistant enteric-coated sustained-release composite probiotic micropellets and preparation method thereof

[0102] Compared with Example 1, the difference of Comparative Example 1 is that the coating film thickness of the coated composite probiotic powder micropellets of Group C and Group D is 3% and 9%, respectively, and the other parameters and operations are the same as those of Example 1. The specific component contents are shown in Tables 5 and 6.

[0103] Table 5 Raw material formula C (dry bacteria powder: protective agent = 1:0.1, coating film thickness 3% and 9%)

[0104]

[0105] Table 6 Raw material formula D (dry bacteria powder: protective agent = 1:0.2, coating film thickness 3% and 9%)

[0106]

[0107] Comparative Example 2 High temperature resistant enteric-coated sustained-release composite probiotic micropellets and preparation method thereof

[0108] Compared with Example 1, the difference of Comparative Example 2 is that in the pellet cores of the coated composite probiotic powder micropellets of Group E and Group F, the mass ratios of dry bacterial powder and protective agent are 1:0.05 and 1:0.3, respectively, and the coating film thickness is 4%. Other parameters and operations are the same as those of Example 1. The specific component contents are shown in Table 7.

[0109] Table 7 Raw material formula E, F group (dry bacteria powder: protective agent = 1:0.05 and 1:0.3, coating film thickness 4%)

[0110]

[0111] Comparative Example 3 High temperature resistant enteric-coated sustained-release composite probiotic micropellets and preparation method thereof

[0112] Compared with Example 1, the difference of Comparative Example 3 is that in the coating film of the coated composite probiotic powder micropellets of Group G and Group H, the main material combination of the coating film is replaced with (enteric-coated sustained-release) acrylic resin II, acrylic resin III, polyethylene glycol and triethyl citrate, with a mass ratio of 20:1:1:3, and the coating film thickness is 4% (expressed as the percentage of the mass of acrylic resin II to the total mass of the powdery substance in the pill core). Other parameters and operations are the same as those of Example 1, and the specific component contents are shown in Table 8.

[0113] Table 8 Raw material formula G and H (dry bacteria powder: protective agent = 1: 0.1 and 1: 0.2, coating film thickness 4%)

[0114]

[0115] Test Example 1: Performance test of the thermostable enteric-coated sustained-release composite lactic acid bacteria pellets prepared by the present invention

[0116] Test objects: coated composite lactic acid bacteria pellets A-4, A-6, A-8, B-4, B-6 and B-8 prepared in Example 1 of the present invention; coated composite lactic acid bacteria pellets C-3, C-9, D-3 and D-9 prepared in Comparative Example 1; coated composite lactic acid bacteria pellets E-4 and F-4 prepared in Comparative Example 2; coated composite lactic acid bacteria pellets G-4 and H-4 prepared in Comparative Example 3. Test methods: (1) High temperature resistance test

[0117] Take 10g of each of the coated composite lactic acid bacteria pellets prepared in Example 1 of the present invention and Comparative Examples 1, 2 and 3, and divide them into two equal parts. One part is placed in a test tube at room temperature without heat treatment; the other part is placed in a test tube preheated to 85 o C in a test tube, put 85 o After the heat treatment is completed, the test tube is placed in a cold water bath and cooled to room temperature.

[0118] Prepare MRS liquid culture medium (pH 6.5): 10.0 g / L peptone, 5.0 g / L beef extract powder, 4.0 g / L yeast extract powder, 20.0 g / L glucose, 1.0 g / L Tween-80, 2.0 g / L potassium dihydrogen phosphate heptahydrate, 5.0 g / L sodium acetate·3H2O, 2.0 g / L triammonium citrate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate tetrahydrate and 15.0 g / L agar powder, sterilize (121°C, 15 min), and cool after sterilization for use. Take appropriate amount of heat-treated and unheat-treated pellets and place them in 250ml stoppered conical flasks, add 100ml MRS liquid culture medium (pH6.5) respectively, stir magnetically until the pellet particles are completely crushed into powder, place in a constant temperature (37℃) incubator for anaerobic culture for 3 hours, and finally determine the number of viable bacteria T by plate count method (48 hours). h and T nh .

[0119] Heat resistance of coated composite lactic acid bacteria pellets (%) = (T h / T nh ) × 100%. T h is the number of viable bacteria in the pellets after heat treatment, T nh is the number of viable bacteria in the pellets without heat treatment. Each group of counts requires three parallel groups. The test results are as follows Figure 1 , 2 and 3.

[0120] (2) Acid resistance test

[0121] Prepare MRS liquid culture medium (pH 6.5) (as shown above), adjust the pH of the MRS liquid culture medium to 2.5 with 2 mol / L HCl solution as an acidic liquid culture medium, sterilize (121°C, 15 min), and cool after sterilization for use.

[0122] 10 g of each of the coated composite lactic acid bacteria pellets prepared in Example 1 of the present invention, Comparative Examples 1, 2 and 3 were taken and divided into two equal parts. One part was placed in a 250 ml stoppered conical flask, and 100 ml of unacidified MRS liquid culture medium (pH 6.5) was added. The pellet particles were magnetically stirred until they were completely crushed into powder. The pellets were then placed in a constant temperature (37° C.) incubator for anaerobic culture for 3 hours, and the number of viable bacteria was determined by the plate count method (48 hours).na The other part was placed in a small nylon bag (400 mesh), sealed, and placed in a 250 ml stoppered conical flask, 100 ml of acidic liquid culture medium was added, and a constant temperature (37 o C) Treat with acid in an incubator for 1 hour, remove the nylon bag, wash the pellets in the nylon bag with 100 ml of unacidified MRS liquid culture medium (pH 6.5) into a new 250 ml stoppered conical flask, stir magnetically until the pellets are completely crushed into powder, place in a constant temperature (37°C) incubator for anaerobic culture for 3 hours, and determine the number of viable bacteria T by plate count method (48 hours) a .

[0123] Acid resistance of coated composite lactic acid bacteria pellets (%) = (T a / T na )×100%. T a is the number of viable bacteria in the pellets after acid treatment, T na is the number of viable bacteria in the pellets that have not been treated with acid. Each group of counts requires three parallel groups. The test results are as follows Figure 4 , 5 and 6.

[0124] (3) Bile salt resistance test

[0125] Prepare MRS liquid culture medium (pH 6.5) (as shown above), add 0.3% (w / v) porcine bile salt into the MRS liquid culture medium as bile salt liquid culture medium, sterilize (121°C, 15 min), and cool after sterilization for use.

[0126] 10 g of the coated composite lactic acid bacteria pellets prepared in Example 1 of the present invention, Comparative Examples 1, 2, and 3 were taken in equal amounts and divided into two portions. One portion was placed in a 250 ml stoppered conical flask, 100 ml of bile salt-free MRS liquid culture medium (pH 6.5) was added, and magnetic stirring was performed until the pellet particles were completely crushed into powder. The pellets were then placed in a constant temperature (37° C.) incubator for anaerobic culture for 3 hours, and the number of viable bacteria was determined by the plate count method (48 hours). nb The other part was placed in a small nylon bag (400 mesh), sealed, and placed in a 250 ml stoppered conical flask, 100 ml of bile salt liquid medium was added, and a constant temperature (37 o C) Treat with bile salts in the incubator for 1 hour, take out the two nylon bags, wash the pellets in the nylon bags into a new 250ml stoppered conical flask with 100ml bile salt-free MRS liquid culture medium (pH 6.5), stir magnetically until the pellets are completely crushed into powder, place in a constant temperature (37°C) incubator for anaerobic culture for 3 hours, and determine the number of viable bacteria T by plate count method (48 hours) b .

[0127] Bile salt resistance of coated composite lactic acid bacteria (%) = (Tb / T nb )×100%. T b is the number of viable bacteria in the pellets after bile salt treatment, T nb is the number of viable bacteria in the pellets without bile salt treatment. Each group of counts requires three parallel groups. The test results are as follows Figure 7 , 8 and 9.

[0128] (4) Stability performance test

[0129] Prepare MRS liquid culture medium (pH 6.5) (as shown above), sterilize (121°C, 15 min), and cool after sterilization for later use.

[0130] 10 g of each of the coated composite lactic acid bacteria pellets prepared in Example 1 of the present invention and Comparative Examples 1, 2, and 3, which were freshly prepared and stored at room temperature for 1, 3, and 6 months, were placed in four 250 ml stoppered conical flasks, 100 ml of MRS liquid culture medium (pH 6.5) was added to each of the flasks, and magnetic stirring was performed until the pellet particles were completely crushed into powder. The pellets were then placed in a constant temperature (37°C) incubator for anaerobic culture for 3 hours, and the viable counts T0 and T1 were determined by the plate count method (48 hours). 1~6 .

[0131] Stability of coated composite lactic acid bacteria (%) = (T 1~6 / T0)×100%. The number of viable bacteria in the pellets after storage for different time periods T 1~6 , the number of viable bacteria in the freshly prepared pellets is T0. Each group of counts requires three parallel groups, and the test results are as follows Fig.10 , 11 and 12.

[0132] (5) Sustained release performance test

[0133] Prepare MRS liquid culture medium (pH 6.5) (as shown above), sterilize (121°C, 15 min), and cool after sterilization for later use.

[0134] 10 g of each of the coated composite lactic acid bacteria pellets prepared in Example 1 of the present invention, Comparative Examples 1, 2, and 3 were taken and divided into four equal portions, and placed in four 250 ml stoppered conical flasks, respectively. 100 ml of MRS liquid culture medium (pH 6.5) was added, and one portion was magnetically stirred until the pellet particles were completely crushed into powder, and then placed in a constant temperature (37°C) incubator for anaerobic culture for 3 hours, and the number of viable bacteria T was determined by the plate count method (48 hours). cr The other three samples were placed in a constant temperature (37°C) incubator for anaerobic culture for 1, 2 and 3 hours, and the number of viable bacteria was determined by plate count method (48 hours). r1~3 .

[0135] Release rate of coated composite lactic acid bacteria (%) = (T r1~3 / T cr ) × 100%. (Under neutral conditions) The number of viable bacteria released from the pellets in the liquid culture medium at different time periods T r1~3 , (under neutral conditions) the number of viable bacteria completely released from the pellets in the liquid culture medium T cr Each group of counts requires three parallel groups, and the test results are as follows Fig.13 , 14 and 15.

[0136] from Figures 1 to 15 It can be seen that the coating thickness of the coated composite lactic acid bacteria micropellets prepared by the present invention is in a positive linear relationship with its stress resistance and stability. When the coating thickness reaches more than 4%, the heat resistance, acid resistance and bile salt resistance of the implementation groups A and B are greatly improved, and more than 70% of the live bacteria survive. At the same time, the storage stability is good. After 6 months, more than 80% of the live bacteria are still alive ( Figure 1 , 4 , 7 and 10); the coated composite lactic acid bacteria pellets are sustained-release dosage forms, and the coating thickness is inversely linearly related to the release performance. When the coating thickness is ≤8%, under neutral (simulated intestinal) conditions, more than 90% of the live bacteria are basically released after 3 hours, while when the coating thickness is >8%, the release performance is reduced. Under the same conditions, after 3 hours, about 15% of the live bacteria cannot be released ( Fig.13 ); Under the condition of the same coating film thickness, the smaller the mass ratio of dry bacterial powder / protective agent in the pellet core, that is, the higher the content of trehalose + sucrose + calcium alginate, the better the stress resistance and stability of the pellets, but the lower the release performance. For example, in the implementation group, when the mass ratio of dry bacterial powder / protective agent was 5 to 10 times, the comprehensive performance (stress resistance, stability and release performance) was the best ( Figure 2 , 5 , 8, 11 and 14).

[0137] If the coating thickness of the micropellets is 4%, the material and mass ratio of the dry bacterial powder and protective agent in the pellet core are the same as those of the implementation group, but the main material combination of the coating film is changed to (enteric-coated sustained-release type) acrylic resin II, acrylic resin III, polyethylene glycol and triethyl citrate (mass ratio is 20:1:1:3), the stress resistance and stability of the micropellets are not as good as the micropellets with the same coating thickness in the implementation group; at the same time, the micropellets are released faster. Under neutral (simulated intestinal) conditions, more than 90% of the live bacteria are basically released after 2 hours, and the sustained-release effect is not as good as the micropellets with the same coating thickness in the implementation group ( Figure 3 , 6 and 9).

[0138] Test Example 2: Performance test of the thermostable enteric-coated sustained-release Bacillus coagulans micropellets prepared by the present invention

[0139] Test objects: Coated Bacillus coagulans pellets A-4, A-6, A-8, B-4, B-6, B-8 prepared in Example 1 of the present invention; coated composite Bacillus pellets C-3, C-9, D-3, D-9 prepared in Comparative Example 1; coated composite Bacillus pellets E-4 and F-4 prepared in Comparative Example 2; coated composite Bacillus pellets G-4 and H-4 prepared in Comparative Example 3. Test method: (1) High temperature resistance test (under acidic conditions)

[0140] Prepare MRS-CaCO3 liquid culture medium (pH 5.7): 10.0 g / L casein digest, 10 g / L beef extract powder, 4.0 g / L yeast extract powder, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate heptahydrate, 0.05 g / L manganese sulfate tetrahydrate, 2.0 g / L potassium hydrogen phosphate heptahydrate, 20.0 g / L glucose, 1.08 g / L Tween-80, 2.5 g / L calcium carbonate, 20 g / L agar. Adjust the pH of MRS-CaCO3 liquid culture medium to 2.5 with 2 mol / L HCl solution as an acidic liquid culture medium, sterilize it (121°C, 15 min), and cool it for use after sterilization.

[0141] 10 g of each of the coated Bacillus coagulans pellets prepared in Example 1 of the present invention, Comparative Examples 1, 2 and 3 were taken and divided into two equal parts. One part was put into a small nylon bag (400 mesh), and after the nylon bag was sealed, it was placed in a 250 ml stoppered conical flask, 100 ml of acidic liquid culture medium was added, and it was placed at room temperature for 5 minutes. The nylon bag was taken out, and the pellets in the nylon bag were washed into a new 250 ml stoppered conical flask with 100 ml of MRS-CaCO3 liquid culture medium (PH5.7), and magnetic stirring was performed until the particles were completely crushed into powder, and the pellets were placed in a constant temperature (37°C) incubator for anaerobically cultured for 3 hours, and the number of viable bacteria T was determined by the plate count method (48 hours). anh The other part was placed in a small nylon bag (400 mesh), sealed, and placed in a 250ml stoppered conical flask, 100ml of acidic liquid culture medium was added, and 85 o C oven for 5 min, take out the nylon bag, wash the pellets in the nylon bag into a new 250 ml stoppered conical flask with 100 ml MRS-CaCO3 liquid culture medium (pH 5.7), stir magnetically until the particles are completely crushed into powder, place in a constant temperature (37 ° C) incubator for anaerobic culture for 3 hours, and determine the number of viable bacteria T by plate count method (48 hours) ah .

[0142] Heat resistance of coated Bacillus coagulans (under acidic conditions) (%) = (T ha / T hna )×100%. T ahis the number of viable bacteria in the pellets after heat treatment (under acidic conditions), T anh It is the number of viable bacteria in the pellets without heat treatment (under acidic conditions). Each group of counts requires three parallel groups. The test results are as follows Fig.16 , 17 and 18.

[0143] (2) Sustained release performance test

[0144] The detection method is the same as that for determining the sustained-release performance of coated composite lactic acid bacteria pellets. Three parallel groups are required for each group of counting. The test results are as follows: Fig.19 , 20 and 21.

[0145] from Figures 16 to 21 It can be seen that the coating thickness of the coated Bacillus coagulans prepared by the present invention is in a positive linear relationship with its stress resistance. When the coating thickness reaches more than 4%, the heat resistance of the implementation groups A and B is greatly improved, and more than 70% of the live bacteria survive ( Fig.16 ); Coated Bacillus coagulans pellets are sustained-release dosage forms. The coating thickness is inversely linearly related to the release performance. When the coating thickness is ≤8%, under neutral (simulated intestinal) conditions, more than 90% of the live bacteria are basically released after 3 hours. When the coating thickness is >8%, the release performance is reduced. Under the same conditions, after 3 hours, about 15% of the live bacteria cannot be released ( Fig.19 ); Under the condition of the same coating film thickness, the smaller the mass ratio of dry bacterial powder / protective agent in the pellet core, that is, the higher the content of trehalose + sucrose + calcium alginate, the better the heat resistance of the pellets, but the lower the release performance. For example, in the implementation group, when the mass ratio of dry bacterial powder / protective agent was 5 to 10 times, the comprehensive performance (heat resistance and sustained release performance) was the best ( Fig.17 and 20 ).

[0146] If the coating thickness of the micropellets is 4%, the material and mass ratio of the dry bacterial powder and protective agent in the pellet core are the same as those in the implementation group, but the main material combination of the coating film is changed to (enteric-coated sustained-release type) acrylic resin II, acrylic resin III, polyethylene glycol and triethyl citrate (mass ratio is 20:1:1:3), the heat resistance of the micropellets is not as good as that of the micropellets with the same coating thickness in the implementation group ( Fig.18 ); At the same time, its microcapsules released faster. Under neutral (simulated intestinal) conditions, more than 90% of the live bacteria were basically released after 2 hours. The sustained-release effect was not as good as the microcapsules with the same coating thickness in the implementation group ( Fig.21 ).

[0147] Test Example 3: Performance test of the thermostable enteric-coated sustained-release composite Bacillus micropellets prepared by the present invention

[0148] Test objects: Coated composite bacillus micropellets A-4, A-6, A-8, B-4, B-6, B-8 prepared in Example 1 of the present invention; coated composite bacillus micropellets C-3, C-9, D-3, D-9 prepared in Comparative Example 1; coated composite bacillus micropellets E-4 and F-4 prepared in Comparative Example 2; coated composite bacillus micropellets G-4 and H-4 prepared in Comparative Example 3. Detection method: The sustained-release performance detection method is the same as that for determining the sustained-release performance of coated composite lactic acid bacteria micropellets. Three parallel groups are required for each group count, and the test results are as follows: Fig. 22 , 23 and 24.

[0149] from Figures 22-24 It can be seen from the figure that the coated composite bacillus micropellets prepared by the present invention are sustained-release dosage forms, and the coating thickness is inversely linearly related to the release performance. When the coating thickness is ≤8%, under neutral (simulated intestinal) conditions, more than 90% of the live bacteria are basically released after 3 hours, while when the coating thickness is >8%, the release performance is reduced. Under the same conditions, after 3 hours, about 15% of the live bacteria cannot be released ( Fig. 22 ). When the coating film thickness is the same, the smaller the mass ratio of dry bacterial powder to protective agent in the pellet core, that is, the higher the content of trehalose + sucrose + calcium alginate, the lower the release performance of the pellet. For example, in the implementation group, when the mass ratio of dry bacterial powder to protective agent is in the range of 5 to 10 times, its sustained release performance is the best ( Fig.23 ).

[0150] If the coating thickness of the micropellets is 4%, the material and mass ratio of the dry bacterial powder and protective agent in the pellet core are the same as those in the implementation group, but the main material combination of the coating film is changed to (enteric-coated sustained-release type) acrylic resin II, acrylic resin III, polyethylene glycol and triethyl citrate (mass ratio is 20:1:1:3), the micropellets are released faster. Under neutral (simulated intestinal) conditions, more than 90% of the live bacteria are basically released after 2 hours. The sustained-release effect is not as good as the micropellets with the same coating thickness in the implementation group ( Fig.24 ).

[0151] In summary, coated composite lactic acid bacteria pellets, coated Bacillus coagulans pellets and coated composite Bacillus micropellets are sustained-release dosage forms. At the same time, the stress resistance of the above three coated pellets is positively linearly related to the thickness of their coating film, that is, the thicker the film, the better the stress resistance, while their release performance is inversely linearly related to the thickness of their coating film, that is, the thicker the film, the worse the release performance; when the coating thickness is in the range of 4%~8% and the mass ratio of dry bacterial powder / protective agent (trehalose+sucrose+calcium alginate) is in the range of 5~10 times, the comprehensive performance is best: the heat resistance, acid resistance and bile salt resistance are greatly improved, and more than 70% of the live bacteria can be guaranteed to survive, and the release degree after 3 hours can be guaranteed to be more than 95%. After being coated, the lactic acid bacteria pellets have greatly improved their resistance to high temperatures, acid, choline and stability, and can pass through the stomach and slowly release and colonize in the intestines; after being coated, the heat resistance of Bacillus coagulans pellets under acidic conditions (pH 2.5) has been greatly improved, solving the problem of Bacillus coagulans being intolerant to heat when a large amount of acidifiers are added to the feed; at the same time, after being coated, the Bacillus pellets can be slowly released and colonized in the intestines, improving the utilization rate of Bacillus in the feed.

[0152] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A thermostable enteric-coated sustained-release composite probiotic pellet, characterized in that: The invention comprises a pellet core and a coating material, wherein the pellet core comprises probiotic dry bacteria powder and a composite protective agent in a mass ratio of 1:0.1-0.2, wherein the composite protective agent comprises trehalose, sucrose and calcium alginate in a mass ratio of 0.3-2:0.5-3:0.5-2; the coating material comprises ethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl phthalate and calcium stearate in a mass ratio of 18-22:0.5-2:0.5-2:1-5, and the coating film thickness of the composite probiotic micro-pellet is 4-8%, and the thickness is expressed as the percentage of the mass of ethyl cellulose to the total mass of the pellet core.

2. The thermostable enteric-coated sustained-release composite probiotic pellets according to claim 1, characterized in that: The coating material is ethyl cellulose, hydroxypropyl methyl cellulose, polyvinyl phthalate and calcium stearate in a mass ratio of 20:1:1:

3.

3. The thermostable enteric-coated sustained-release composite probiotic pellets according to claim 1, characterized in that: The composite protective agent is composed of trehalose, sucrose and calcium alginate in a mass ratio of 1:1:

1.

4. The thermostable enteric-coated sustained-release composite probiotic pellets according to claim 1, characterized in that: The probiotic dry bacteria powder is one of compound lactic acid bacteria powder, Bacillus coagulans powder and compound Bacillus powder. The compound lactic acid bacteria powder is composed of enterococci and Lactobacillus plantarum in a ratio of 1:10 in terms of live bacteria count, and the total number of lactic acid bacteria is ≥550×10 8 CFU / g; The number of viable bacteria of Bacillus coagulans powder is ≥5×10 8 CFU / g; The composite bacillus powder is composed of Clostridium butyricum, Bacillus licheniformis and Bacillus subtilis in a ratio of 1:2:2 in terms of live bacteria count, and the total number of bacillus is ≥25×10 8 CFU / g.

5. A method for preparing a high temperature resistant enteric sustained-release coated composite probiotic micropellets according to any one of claims 1 to 4, characterized in that: The steps include: S1. Granulation: (1) Preparation of pellet core: Mix the probiotic dry powder and the composite protective agent evenly and put them on the centrifugal turntable of the centrifugal pill making machine. Start the fan and turntable and spray deionized water at the same time to form fluffy gravel-like particles. When the particle size reaches 0.2-0.6mm, increase the turntable speed and reduce the spraying speed to keep the particles in a wet state and round them, then stop spraying. (2) Drying and screening: The rotating speed of the rotating disk is reduced to keep the material obtained in step S1 (1) in a flowing state, and the heating device is turned on to dry the material, and the material is sieved to obtain a pellet core; S2. Coating: (1) Preparation of coating solution: Deionized water is added to 95% ethanol to form a mixed solution, and the mixture is stirred evenly. Ethyl cellulose, hydroxypropyl methylcellulose, polyvinyl phthalate and calcium stearate in the coating material are added thereto, and the mixture is stirred continuously and filtered. The sieved coating solution is kept stirred to prepare a coating solution for standby use. (2) Coating: First, preheat the fluidized bed, then put the pellet cores obtained in step S1 into the fluidized bed, start the fan and heating system, and spray the coating liquid; (3) Drying and screening: After spraying the coating liquid, reduce the air flow rate and maintain the convection state, so that the dry air flow dries the coated particles until the moisture content is less than 7%, and then sieve to obtain the composite probiotic micro-pellets.

6. The method for preparing a thermostable enteric-coated sustained-release composite probiotic micropellet according to claim 5, characterized in that: In the step S1 (1), the rotation speed of the fan and the turntable when starting is 250-300 rpm, deionized water is sprayed, the mass ratio of the water spray amount to the sum of the probiotic dry powder and the composite protective agent is 3:5, the water spray speed is 4-8 g / min, and when the particle size reaches 0.2-0.6 mm, the turntable speed is increased to 400-500 rpm, and the spray speed is reduced to 1-3 g / min; in the step S1 (2), the turntable speed is reduced to 250-300 rpm, the heating device is set to 36-37° C., and the drying time is 5-10 min.

7. The method for preparing a thermostable enteric-coated sustained-release composite probiotic micropellet according to claim 5, characterized in that: In the step S2 (1), the mass ratio of the 95% ethanol solution to deionized water is 7.4:1; the mass ratio of the mixed solution of ethanol and deionized water to the coating material is 1:2.4-2.5; in the step S2 (1), after adding the coating material, stirring is carried out for 30 minutes, and then filtering is carried out with a 60-mesh sieve.

8. The method for preparing a thermostable enteric-coated sustained-release composite probiotic micropellet according to claim 5, characterized in that: In step S2 (2), the preheating time of the fluidized bed is 10 minutes, and the air flow rate of the fan is 3-10m 3 / s, the air outlet temperature is 33~37℃, and the coating liquid spraying speed is 4~10g / min.

9. The method for preparing a thermostable enteric-coated sustained-release composite probiotic micropellet according to claim 5, characterized in that: The air flow rate during drying in step S2 (3) is 5-10 m 3 / s; the particles of the composite probiotic micropellets prepared in step S2 (3) have a particle size of 0.25-0.5 mm accounting for 70% of the total mass, and the particles of 0.6-0.8 mm accounting for 30%; the coating film thickness of the composite probiotic micropellets prepared in step S2 (3) is 4-8%, and the thickness is expressed as the percentage of the mass of ethyl cellulose to the total mass of the pellet core.

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