A probiotic multilayered pellet and a nutritional combination and a method of preparation

Abstract

The present application belongs to the technical field of probiotic preparation, and particularly relates to a probiotic multilayer pellet preparation, a nutritional combination preparation and a preparation method. The probiotic multilayer pellet preparation is composed of a pellet core containing probiotics, an isolation layer wrapped outside the pellet core and an enteric coating layer wrapped outside the isolation layer. The isolation layer and the enteric coating layer can synergistically protect the probiotics, improve the survival rate of the probiotics in the shelf life and resist the damage of gastric acid. The nutritional combination preparation is made of the probiotic multilayer pellet preparation and nutritional particles containing nutritional substances, wherein the nutritional particles are prepared by wet granulation. The nutritional combination preparation can improve the mixing uniformity of the preparation, improve the loading consistency or the dose consistency.

Description

Technical Field

[0001] This invention belongs to the field of probiotic preparation technology, specifically relating to a multilayer probiotic microsphere formulation, a nutritional combination agent, and a preparation method. Background Technology

[0002] Probiotics are a class of live microorganisms that are beneficial to the host, can colonize the human body, and improve the host's microecological balance to produce definite health benefits. As a functional ingredient, probiotics have been widely used in the food, health food, and dietary supplement industries. Probiotic dietary supplements are becoming increasingly diverse in form, including capsules, powders, granules, tablets, and drops. Powdered probiotic dietary supplements account for the largest share of the market. Currently, most conventional probiotic supplements on the market are prepared by directly mixing probiotic powder with various nutrients and excipients. This dosage form is simple but does not consider the stability of the final probiotic product or the survival rate of the probiotics in the gastrointestinal tract.

[0003] When probiotics are combined with nutrients to form a compound, the addition of acidic components, salts, polyphenols, or other raw materials to the product system can damage the cell structure and biological activity of probiotics. Simultaneously, moisture migration occurs between different raw materials; highly hygroscopic excipients, or those with high moisture content and high water activity, can alter the humidity of the microenvironment in which probiotics reside, negatively impacting their stability and reducing the survival rate of live bacteria during the product's shelf life, resulting in poor storage stability. Furthermore, unencapsulated probiotics are susceptible to erosion by stomach acid and bile, making it difficult for them to successfully reach the intestines and colonize, significantly reducing the intestinal colonization rate and actual efficacy. Maximizing the in vitro stability of probiotics during storage and processing, and their in vivo intestinal survival rate after oral administration, is a significant challenge currently facing the production of probiotic formulations.

[0004] In recent years, in addition to researching different protective agents and carrier materials to protect probiotics from the influence of the external environment, this field has also developed new formulation technologies to improve the storage stability and activity of probiotics, such as the new encapsulation dosage form—probiotic microcapsules. Microcapsules (also known as pellets) are small spherical oral dosage forms with a diameter of approximately 1 mm, generally not exceeding 2.5 mm. In the pharmaceutical industry, microcapsules are often prepared between 500 and 1500 µm. Different formulations and preparation methods can also be used to formulate drugs into immediate-release, sustained-release, or other microcapsule formulations. However, while enteric-coated microcapsules can protect probiotics and reduce the harmful effects of gastric acid on them, the protective effect remains limited.

[0005] Conventional nutritional ingredients and excipients are mostly in powder form, which differs greatly from the particle size of probiotic microspheres. Direct mixing of the two can easily lead to stratification and segregation, resulting in substandard mixing uniformity and poor batch stability in the finished product. Summary of the Invention

[0006] To address the above problems, the purpose of this invention is to provide a probiotic multilayer microsphere formulation, a nutritional combination agent, and a preparation method. This invention includes the following technical solutions:

[0007] A probiotic multilayer microgranule, comprising a probiotic-containing core, an isolation layer surrounding the core, and an enteric coating layer surrounding the isolation layer; the core is prepared by wet granulation of probiotics and pharmaceutically acceptable excipients, followed by drying.

[0008] In a preferred embodiment, the pellet core is prepared from probiotic powder, microcrystalline cellulose, sugar or sugar alcohol, and a binder; the probiotic powder raw material is one or more strains listed in the "List of Strains that can be Used in Food" and the "List of Strains that can be Used in Infant Food" published by the People's Republic of China; the sugar or sugar alcohol includes one or more of erythritol, lactose, sorbitol, isomaltitol, D-mannitol, trehalose, sucrose, resistant dextrin, and stachyose. In a further preferred embodiment, the pellet core is prepared from probiotic powder, microcrystalline cellulose, isomaltitol, and a binder; wherein the weight ratio of probiotic powder, microcrystalline cellulose, and isomaltitol is 0.5~1.5:3~7:2~6. In another further preferred embodiment, the pellet core is prepared from probiotic powder, microcrystalline cellulose, trehalose and binder; wherein the weight ratio of probiotic powder, microcrystalline cellulose and trehalose is 0.5~1.5:3~7:2~6.

[0009] Preferably, the isolation layer uses an aqueous mixture of edible protein and / or edible polysaccharides as the coating solution. That is, the isolation layer coating solution can be an aqueous mixture of edible protein, an aqueous mixture of edible polysaccharides, or an aqueous mixture of edible protein and edible polysaccharides. More preferably, the edible protein in the isolation layer coating solution is selected from at least one of gelatin and soy protein; the edible polysaccharide in the isolation layer coating solution is selected from at least one of gum arabic, pullulan, guar gum, sodium carboxymethyl cellulose, starch, propylene glycol alginate, gellan gum, konjac gum, pectin, carrageenan, and xanthan gum. In a further preferred embodiment, the isolation layer coating solution is an aqueous mixture of gum arabic, wherein the mass concentration of gum arabic is 6% to 15%. In another further preferred embodiment, the isolation layer coating solution is an aqueous mixture of pullulan, wherein the mass concentration of pullulan is 1% to 15%. In another further preferred embodiment, the coating liquid of the isolation layer is a pectin aqueous solution, wherein the mass concentration of pectin is 0.5% to 5%.

[0010] Preferably, the enteric coating layer contains edible polysaccharides. More preferably, the edible polysaccharides in the enteric coating layer are selected from at least two of sodium alginate, hydroxypropyl methylcellulose, ethyl cellulose, and shellac. In a further preferred embodiment, the coating solution of the enteric coating layer is an alcoholic aqueous solution of sodium alginate, hydroxypropyl methylcellulose, and ethyl cellulose, wherein the total mass concentration of sodium alginate, hydroxypropyl methylcellulose, and ethyl cellulose is 5% to 25%. In another further preferred embodiment, the coating solution of the enteric coating layer is an alcoholic aqueous solution of shellac, hydroxypropyl methylcellulose, and magnesium stearate, wherein the total mass concentration of shellac, hydroxypropyl methylcellulose, and magnesium stearate is 5% to 25%.

[0011] Preferably, the weight of the isolation layer is 5% to 25% of the pellet core; the weight of the enteric coating layer is 5% to 25% of the pellet core.

[0012] A nutritional combination agent is made from the aforementioned probiotic multilayer microcapsules and nutrient granules containing nutrients, wherein the nutrient granules are obtained by wet granulation and drying.

[0013] A nutritional compound is made from the aforementioned probiotic multilayer microspheres and nutritional granules containing edible functional ingredients. The nutritional granules are prepared by wet granulation and drying. The edible functional ingredients include nutritional raw materials and / or edible raw materials with health-promoting functions.

[0014] Preferably, the raw materials of the nutritional granules include at least one of the following: vitamins, minerals, new food ingredients, food-medicine homologous ingredients, prebiotic dietary fiber, and enzymes. Vitamins include, but are not limited to, vitamins A, B, C, D, and E; minerals include, but are not limited to, calcium, iron, iodine, zinc, selenium, or their salts or complexes. New food ingredients are those officially released and dynamically updated by the National Health Commission (NHC) that have passed safety reviews and been approved for use. Food-medicine homologous ingredients include, but are not limited to, yam, goji berries, hawthorn, coix seed, and red dates, which are officially recognized as both food and medicine. Prebiotic dietary fiber includes, but is not limited to, dietary fiber that can be preferentially utilized by beneficial intestinal bacteria (such as bifidobacteria and lactic acid bacteria). Enzymes include, but are not limited to, coenzyme Q and other bioactive enzymes.

[0015] Preferably, the raw materials of the nutrient granules include at least one of the following: hawthorn powder, malt powder, yam powder, chicken gizzard powder, mixed fruit and vegetable enzyme powder, acerola cherry powder, and zinc gluconate.

[0016] The aforementioned method for preparing the nutrient combination agent includes the following steps:

[0017] Preparation of probiotic multilayer microcapsules: Probiotics and pharmaceutically acceptable excipients are wet-granulated and dried to obtain dried core pellets; an isolation layer and an enteric coating layer are sequentially coated on the outer layer of the dried core pellets, and then dried and sieved to obtain probiotic multilayer microcapsules;

[0018] Preparation of nutrient granules: Nutrient granules were prepared using a wet process, dried, and sieved.

[0019] Mixing: The probiotic multilayer microcapsules are mixed with nutrient granules to obtain a nutrient combination agent.

[0020] Preferably, the probiotic multilayer microspheres are microspheres that pass through an 18-mesh sieve but not a 24-mesh sieve; the nutrient granules are selected after drying and pass through an 18-24 mesh sieve.

[0021] Preferably, the wet granulation of the nutrient granules is performed using a rotary granulator or a swing granulator.

[0022] Preferably, the probiotic multilayer micro-pellets and nutrient granules are mixed using a three-dimensional mixer with a motor speed of 300 rpm to 1200 rpm and a mixing time of 5 min to 120 min.

[0023] Preferably, the weight ratio of the nutrient granules to the probiotic multilayer microcapsules in the mixture of probiotic multilayer microcapsules and nutrient granules is 10~20:1~9.

[0024] Beneficial effects:

[0025] The multilayer microcapsule of the present invention utilizes an isolation layer and an enteric coating to provide layered protection for the probiotics in the capsule core. The isolation layer and the enteric coating exhibit a synergistic protective effect on the probiotics, making the storage stability and gastric acid tolerance of the microcapsule far superior to that of enteric-coated microcapsules alone; and meeting the comprehensive requirements of resisting gastric acid digestion and being easily decomposed and releasing probiotics at intestinal pH.

[0026] The nutritional compound of this invention utilizes wet granulation to prepare nutritional granules, which are then mixed with micro-pellets. This effectively controls the mixing uniformity of the compound, reducing dosage variations and improving dosage consistency. The resulting compound can be further packaged using capsule shells, reducing the damage to micro-pellets and granules caused by environmental humidity. Attached Figure Description

[0027] Figure 1 This is a particle size distribution diagram of the combination agent in Experimental Example 5;

[0028] Figure 2 The particle size distribution diagram of the combination agent in Experimental Example 6;

[0029] Figure 3 The particle size distribution diagram of the combination agent in Experimental Example 7;

[0030] Figure 4This is a particle size distribution diagram of the combination agent in Comparative Example 3. Detailed Implementation

[0031] The technical solutions and effects of the present invention are shown and described below with reference to specific embodiments and accompanying drawings. These embodiments are for illustrative purposes only and are not intended to limit the scope of protection. In the following embodiments, the probiotic microspheres are preparations that protect probiotics, improve shelf-life survival rate and / or survival rate during processing, and improve gastrointestinal digestive tolerance. The *Lactobacillus plantarum* used is only a model strain (using *Lactobacillus plantarum* with the number ATCC8014 as the model probiotic). In practical applications, it can be replaced with other edible probiotics, including but not limited to other lactobacilli (such as *Lactobacillus acidophilus*, *Lactobacillus rhamnosus*), bifidobacteria, yeasts, and Bacillus.

[0032] The model probiotics can be commercially available freeze-dried bacterial powder, or they can be revived and cultured at 37℃ using MRS medium (medium ingredients: 10.0g casein, 8.0g beef powder, 4.0g yeast powder, 20.0g glucose, 0.2g magnesium sulfate, 5.0g sodium acetate, 2.0g triammonium citrate, 2.0g dipotassium hydrogen phosphate, 0.05g manganese sulfate, 1.0g Tween 80, 1000mL distilled water; pH 6.2±0.2) before being prepared into freeze-dried bacterial powder.

[0033] In the following examples, the term "coating weight gain" refers to the percentage weight gain relative to the dry pellet core.

[0034] Example 1: Preparation and protective effect study of different probiotic microspheres

[0035] This embodiment provides preparation methods and efficacy evaluation results for different probiotic coating formulations (microcapsules).

[0036] Probiotic microcapsules in Experiment Example 1:

[0037] S1 pellet core preparation: Take 8.0×10 11 100g of *Lactobacillus plantarum* (CFU / g), 500g of microcrystalline cellulose, and 400g of isomaltitol were mixed with 500g of pure water as a binder. The mixture was wet-granulated to prepare a soft material, which was then extruded and spheronized to obtain wet pellet cores. The pellet cores were then sieved through 20 and 24 mesh screens, and the wet pellet cores that passed through the 20 mesh screen but not the 24 mesh screen were collected. The wet pellet cores were then fluidized bed dried until the moisture content was ≤5% to obtain dried pellet cores.

[0038] Preparation of S2 isolation layer pellet core: Gum arabic was prepared with water to form an isolation layer coating solution with a gum arabic mass concentration of 10%. The dried pellet core was coated by bottom spraying in a fluidized bed with an atomization pressure of 2 bar and an inlet air temperature of 45°C. The coating weight gain was 10%. After drying, the isolation layer pellet core with the isolation layer was obtained.

[0039] Preparation of S3 enteric-coated pellet cores: 50g of sodium alginate, 70g of hydroxypropyl methylcellulose, and 30g of ethylcellulose were mixed and prepared with a 50% (v / v) ethanol solution to form an enteric coating solution with a total mass concentration of 15% (sodium alginate + hydroxypropyl methylcellulose + ethylcellulose). The separator pellet cores were coated by bottom spraying in a fluidized bed at an atomization pressure of 2 bar and an inlet air temperature of 45℃. The coating weight gain was 10%. After drying, the pellets were sieved sequentially through 18 and 24 mesh screens. Materials passing through the 18-mesh sieve but not the 24-mesh sieve were collected to form multi-layered enteric-coated probiotic microcapsules.

[0040] The probiotic microcapsules of Experiment 2 differed from those of Experiment 1 only in that the weight gain of the isolation layer coating was 15% and the weight gain of the enteric coating was 20%.

[0041] Probiotic microcapsules in Experiment Example 3:

[0042] S1 pellet core preparation: Take 8.0×10 11 100g of *Lactobacillus plantarum* (CFU / g), 650g of microcrystalline cellulose, and 250g of isomaltitol were mixed and 400g of a 1% sodium carboxymethyl cellulose aqueous solution was added as a binder. The mixture was wet-granulated to prepare a soft material, and then wet pellet cores were prepared by extrusion and spheronization. The pellet cores were then sieved through 20 and 24 mesh screens, and the wet pellet cores that passed through the 20 mesh screen but not the 24 mesh screen were collected. The wet pellet cores were then fluidized bed dried until the moisture content was ≤5% to obtain dried pellet cores.

[0043] Preparation of S2 isolation layer pellet core: Pullulan polysaccharide was prepared with water to form an isolation layer coating solution with a mass concentration of 5% pullulan polysaccharide. The dried pellet core was coated by bottom spraying in a fluidized bed with an atomization pressure of 2 bar and an inlet air temperature of 45°C. The coating weight gain was 15%. After drying, the isolation layer pellet core with an isolation layer was obtained.

[0044] Preparation of S3 enteric-coated pellet cores: 75g shellac, 22g hydroxypropyl methylcellulose, and 3g magnesium stearate were mixed and prepared with a 95% ethanol solution to form an enteric coating solution with a total mass concentration of 10% (shellac + hydroxypropyl methylcellulose + magnesium stearate). The separator-layer pellet cores were coated by bottom-spray coating in a fluidized bed at an atomization pressure of 2 bar and an inlet air temperature of 45℃. The coating weight gain was 20%. After drying, the pellets were sieved sequentially through 18 and 24 mesh screens. Materials passing through the 18-mesh sieve but not the 24-mesh sieve were collected to form multi-layered, encapsulated enteric probiotic microcapsules.

[0045] The probiotic microcapsules in Experiment 4 differed from those in Experiment 3 only in that the core material was 8.0 × 10⁻⁶. 11 100g of *Lactobacillus plantarum* (CFU / g), 650g of microcrystalline cellulose, and 250g of trehalose (adhesive and dosage remain unchanged). The isolation layer coating solution is a 1.5% pectin aqueous solution.

[0046] Comparative Example 1 probiotic preparation: 8.0 × 10 11 A mixed powder of 100g of CFU / g Lactobacillus plantarum, 500g of microcrystalline cellulose, and 400g of isomaltitol, without binders, isolation layer coating, enteric coating, or sieving.

[0047] The probiotic preparation of Comparative Example 2 differs from that of Experimental Example 1 in that it does not undergo an isolation layer coating, but is directly coated with an enteric coating after the dried pellet core is prepared.

[0048] The probiotic preparations of Experimental Examples 1-4 and Comparative Examples 1 and 2 were subjected to room temperature storage stability tests and in vitro simulated digestion tests, respectively.

[0049] (1) To investigate the stability of probiotic preparations, the number of live bacteria was used as an indicator to conduct a room temperature stability test on the probiotic preparations. The preparations were placed at a temperature of 25℃±2℃ and a relative humidity of 60%±5% for 12 months. Samples were taken to detect the number of live bacteria and the survival rate of probiotics at room temperature for 12 months was calculated.

[0050] (2) To investigate the gastric acid resistance of the probiotic preparation, artificial gastric juice (containing 3.2 mg / ml pepsin and 0.2% (w / v) NaCl solution) was prepared according to the method of the Chinese Pharmacopoeia. The pH was 1.2. The probiotic preparation was placed in the simulated artificial gastric juice environment at 37°C for 2 hours, and the survival rate of the probiotics was tested.

[0051] (3) To investigate the intestinal digestion characteristics of probiotic preparations, the digestion state of the preparations was investigated by simulating the pH of the gastrointestinal tract: 2g of probiotic preparations were taken and 750 mL of 0.1mol / L hydrochloric acid solution was used as the dissolution medium (simulating the pH of gastric juice). The medium temperature was kept constant at 37℃±0.5℃ and the rotation speed was 100rpm. After 120min, the state of the probiotic preparations was observed. Then, 250 mL of 0.2mol / L sodium phosphate solution at a temperature of 37℃±0.5℃ was added to the above acid solution (if necessary, the pH value was adjusted to 6.8 with 2mol / L hydrochloric acid solution or 2mol / L sodium hydroxide solution to simulate the pH of intestinal juice). After stirring for 45min, the state of the probiotic preparations was observed.

[0052] The viable count determination of probiotic mixed powder (Comparative Example 1, without coating) was performed according to the method in GB 4789.35. The methods for determining the viable count of other coated probiotic preparations are as follows:

[0053] 1) Preparation of probiotic microsphere diluent (artificial intestinal fluid): Take 250 mL of 0.2 mol / L potassium dihydrogen phosphate solution, add 118 mL of 0.2 mol / L sodium hydroxide solution, 0.5 g of L-cysteine ​​hydrochloride, 0.5 g of agar, and dilute with water to 1000 mL. Heat to dissolve and then autoclave at 121℃ for 20 min.

[0054] 2) In a clean bench, take 225g of the sterilized probiotic microsphere dilution (artificial intestinal fluid) and pour it into a sterile homogenizing cup, then incubate at 40℃~50℃. Take 25g of probiotic microspheres (e.g., samples from different storage or digestion times) and add them to the sterile homogenizing cup containing 225g of microsphere dilution (artificial intestinal fluid), then place it in a 40℃~50℃ constant temperature water bath for 5 minutes. Homogenize at 5000rpm for 5 minutes.

[0055] 3) The counting and processing of lactic acid bacteria shall be performed in accordance with GB4789.35. The survival rate is the percentage of colony count after treatment relative to the colony count before treatment.

[0056] Table 1. Results of room temperature storage stability test and in vitro simulated digestion test of different probiotic preparations.

[0057] As shown in Table 1, in the room temperature storage stability test, the survival rate of probiotics in the preparations of Experiment Examples 1-4 was above 75% after 12 months of room temperature storage, which was significantly higher than that of Control Examples 1-2, and the long-term storage stability advantage was obvious.

[0058] After 2 hours of treatment with simulated gastric juice, the survival rate of probiotics in the preparations of Experiment Examples 1-4 was all above 88%. The multi-layer coating exhibited excellent acid resistance, and the enteric coating layer showed good resistance to gastric acid. Under intestinal pH conditions (pH up to 6.8), the enteric coating showed good digestibility, meeting the requirements for intestinal release of probiotic microspheres.

[0059] Comparative Example 1, without coating, exhibited the fastest viable bacterial attenuation and the lowest survival rate. Comparative Example 2, lacking an isolation layer, while showing some resistance to gastric acid, had significantly lower probiotic survival rates at room temperature for 12 months and after 2 hours of treatment with simulated gastric juice compared to experimental examples 1-4. This indicates that the isolation layer coating can prevent the probiotic core from contacting the outer enteric coating material or digestive fluid, avoiding long-term erosion of the bacteria by the coating components. Increasing the isolation layer coating can improve the stability and gastric acid resistance of the formulation.

[0060] Example 2: Preparation of a combination of probiotic microcapsules and nutrient granules

[0061] This embodiment further prepares a combination of probiotic microcapsules and nutrient granules based on Example 1, and presents the results of its formulation investigation.

[0062] The combination of reagents in Experimental Example 5:

[0063] Preparation of S4 nutrient granules: The materials for the nutrient granules are 0.72g of zinc gluconate (which can be used to supplement trace elements), 1.5g of vitamin C (which can be used to supplement vitamin C), 10g of yeast β-glucan, 100g of resistant dextrin, 50g of acerola cherry powder (acerola cherry powder is rich in vitamin C and other substances and has antioxidant effects; commercially available freeze-dried acerola cherry powder is used here), 837.78g of erythritol, and 35g of a 2% sodium carboxymethyl cellulose aqueous solution (binder). The materials are mixed and wet granulated to prepare soft material, and then wet granules are prepared using a 20-mesh rotary granulator. The wet granules are dried in an oven (55℃ for 16h) and passed through a 20-mesh sieve to obtain nutrient granules.

[0064] Preparation of S5 combination agent: The nutrient particles obtained in step S4 are mixed with the probiotic microspheres containing the isolation layer and enteric coating obtained in Example 2 of Example 1, wherein the weight ratio of nutrient particles to probiotic microspheres is 85:15 (i.e. 17:3). The mixture is prepared by mixing with a three-dimensional mixer at a motor speed of 600 rpm for 30 min to obtain the combination agent.

[0065] The combination agent of Experimental Example 6 differs from that of Experimental Example 5 only in that step S4 uses a gyratory granulator (using a 20-mesh sieve) to prepare wet granules of nutrient granules.

[0066] The combination agent of Comparative Example 3 differs from that of Experimental Example 5 only in that step S4 does not use a binder (sodium carboxymethyl cellulose aqueous solution) and directly uses materials other than the binder to mix the powder without granulation and sieving, and mixes it with the probiotic microcapsules containing the isolation layer and enteric coating obtained in Example 1 and Experimental Example 2.

[0067] The combination of agents in Experiment Example 7:

[0068] Preparation of S4 Nutritional Granules: The materials for the nutritional granules are 50g hawthorn powder, 30g malt powder, 30g yam powder, 20g chicken gizzard powder, 20g mixed fruit and vegetable enzyme powder (a commercially available powdered nutritional product made from a variety of fresh fruits and vegetables through careful fermentation, extraction, and concentration processes, rich in various enzymes, vitamins, minerals, and amino acids), 50g galactooligosaccharides, 8g citric acid, 50g maltodextrin, 50g microcrystalline cellulose, 692g lactose, and 40g purified water (binder). The materials are mixed and wet granulated to prepare a soft mass. Then, a 20-mesh rotary granulator is used to prepare wet granules. The obtained wet granules are then subjected to fluidized bed drying with an inlet air temperature set at 75℃ until the moisture content is ≤5%. After passing through a 20-mesh sieve, the nutritional granules are obtained.

[0069] Preparation of S5 combination agent: The nutrient particles obtained in step S4 are mixed with the probiotic microspheres containing the isolation layer and enteric coating obtained in Example 2 of Example 1, wherein the weight ratio of nutrient particles to probiotic microspheres is 65:35 (i.e. 13:7). The mixture is mixed using a three-dimensional mixer with a motor speed of 600 rpm and a mixing time of 30 min to obtain the combination agent.

[0070] The formulation mixing uniformity of the combination machine in Experimental Examples 5-7 and Comparative Example 3 was verified respectively.

[0071] (1) Immediately after the preparation of the combination agent, take 50g of each sample and place them in a small beaker. Let them stand for 30 minutes to observe whether there is stratification.

[0072] (2) Immediately after preparation, extract the combination agent sample from the top, middle and bottom of the three-dimensional mixer, measure the number of viable bacteria according to the method of Example 1, calculate the number of viable bacteria in samples at different distribution positions of the same preparation, calculate the relative standard deviation (RSD%), and further verify the mixing uniformity.

[0073] (3) Immediately after the preparation of the compound, random samples are taken and the particle size distribution of the particulate material is detected by sieving.

[0074] Test results are shown Figures 1-4 And Table 2.

[0075] Table 2. Results of static stratification observation and relative standard deviation of viable cell count for different combinations of agents.

[0076] The experimental results show that the combined agents in Examples 5-7 have a high particle size concentration, with over 80% concentrated in the 700-1000 μm range. Wet granulation can reduce the particle size difference between the nutrient materials and the probiotic microspheres, effectively suppressing the stratification phenomenon after mixing. Among them, the nutrient particles prepared by rotary extrusion granulation have a concentrated particle size and low fine powder content, and the RSD of viable bacteria after three-dimensional mixing is ≤5.70%, with the mixing uniformity meeting the formulation requirements. The particles prepared by rocking granulation are loose, with more fine powder, and the mixing uniformity is slightly worse. The ungranulated powder mixture is prone to stratification, with an RSD as high as 19.79%, and the dosage consistency cannot be guaranteed. The preferred process is a combination of wet granulation and rotary extrusion granulation.

[0077] The composition of probiotic microspheres and nutrient granules of the present invention maintains good mixing uniformity by controlling the consistency between the particle size of the nutrient granules and the particle size of the probiotic microspheres through granulation. The process is stable, the finished product has good uniformity, and it is suitable for industrial production.

[0078] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention, and such modifications and alterations are also within the protection scope of the present invention.

Claims

1. A probiotic multilayer micro-pellet formulation, characterized in that, The probiotic multilayer micro-pellets consist of a probiotic-containing pellet core, an isolation layer surrounding the pellet core, and an enteric coating layer surrounding the isolation layer. The pellet core is prepared by wet granulation of probiotics and pharmaceutically acceptable excipients, followed by drying.

2. The probiotic multilayer micro-pellet formulation according to claim 1, characterized in that, The isolation layer uses an aqueous mixture of edible protein and / or edible polysaccharides as the coating solution.

3. The probiotic multilayer micro-pellet formulation according to claim 2, characterized in that, The edible protein in the isolation layer coating solution is selected from at least one of gelatin and soy protein; the edible polysaccharide in the isolation layer coating solution is selected from at least one of gum arabic, pullulan, guar gum, sodium carboxymethyl cellulose, starch, propylene glycol alginate, gellan gum, konjac gum, pectin, carrageenan, and xanthan gum.

4. The probiotic multilayer micro-pellet formulation according to claim 1, characterized in that, The enteric coating layer contains edible polysaccharides.

5. The probiotic multilayer micro-pellet formulation according to claim 4, characterized in that, The edible polysaccharide in the enteric coating layer is selected from at least two of sodium alginate, hydroxypropyl methylcellulose, ethylcellulose, and shellac.

6. The probiotic multilayer micro-pellet formulation according to claim 1, characterized in that, The weight of the isolation layer is 5% to 25% of the pellet core; the weight of the enteric coating layer is 5% to 25% of the pellet core.

7. A nutritional combination agent, characterized in that, It is made from the probiotic multilayer microcapsules and nutrient granules as described in any one of claims 1 to 6, wherein the nutrient granules are prepared by wet granulation and drying.

8. A nutritional compound according to claim 7, characterized in that, The raw materials of the nutritional granules include at least one of the following: vitamins, minerals, new food ingredients, food and medicine homology, prebiotic dietary fiber, and enzymes.

9. The method for preparing the nutrient compound according to claim 7, characterized in that, The preparation method includes the following steps: Preparation of probiotic multilayer microcapsules: Probiotics and pharmaceutically acceptable excipients are wet-granulated and dried to obtain dried core pellets; an isolation layer and an enteric coating layer are sequentially coated on the outer layer of the dried core pellets, and then dried and sieved to obtain probiotic multilayer microcapsules; Preparation of nutrient granules: Nutrient granules were prepared using a wet process, dried, and sieved. Mixing: The probiotic multilayer microcapsules are mixed with nutrient granules to obtain a nutrient combination agent.

10. The method for preparing the nutrient compound according to claim 9, characterized in that, The probiotic multilayer micro-pellets are micro-pellets that pass through an 18-mesh sieve but not a 24-mesh sieve; the nutrient granules are selected after drying and pass through an 18-24 mesh sieve.

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