A probiotic composition and a method for its preparation

Abstract

The present application relates to the technical field of microbial preparation, and specifically discloses a kind of compound probiotic preparation and preparation method thereof, and the preparation raw material includes targeting controlled-release live bacteria microspheres, antioxidant sugar compound, active peptide composite powder, composite mineral element, composite vitamin, moisture-proof coating auxiliary material and malt dextrin.The targeting controlled-release live bacteria microspheres are made of hydroxypropyl methyl cellulose, inulin, magnesium carbonate, sodium citrate and lactobacillus plantarum bacteria body;The antioxidant sugar compound is made of β-cyclodextrin, D-alloketose, vitamin C palmitate, d-alpha-tocopherol acetate and stachyose by cocrystallization.The preparation method is as follows: the premixed composite powder is obtained by mixing each raw material, then the coating liquid is atomized and sprayed, dried and cooled, and the compound probiotic preparation is obtained.By pH-enzyme double-triggered controlled release, microenvironment buffering and antioxidant cocrystallization synergy, it is beneficial to realize the colon targeting delivery and efficient colonization of probiotics, and improve the acid and oxygen tolerance stability of live bacteria, shelf life survival rate and intestinal mucosa repair capacity.

Description

Technical Field

[0001] This invention relates to the field of microbial preparation technology, specifically to a compound probiotic preparation and its preparation method. Background Technology

[0002] Probiotics, as a class of live microorganisms beneficial to the host, play an important role in regulating the balance of the intestinal microecology, repairing the intestinal mucosal barrier, and enhancing the body's immunity. The effective effects of functional strains such as Lactobacillus plantarum are highly dependent on the intestinal-targeted delivery and efficient colonization of live bacteria.

[0003] However, existing compound probiotic preparations still have significant defects in industrial preparation and clinical application: (1) Insufficient shelf-life stability: Traditional powders use physical and mechanical mixing of raw materials and excipients, and the bacteria are directly exposed to the external environment. There is a lack of an effective microenvironment activity protection system. Live bacteria are easily affected by moisture, oxygen and local pH fluctuations during storage and will be inactivated in large quantities; (2) Difficulty in penetrating the gastric acid barrier: Conventional preparations lack an effective gastric acid protection mechanism. Live bacteria are inactivated in large quantities under the strong acid environment of the stomach (pH 1.5-3.5) and the action of pepsin; (3) Difficulty in controlling the sequential release in the colon: Existing enteric coating technology mostly relies on conventional pH-responsive excipients. Although it can achieve a certain degree of intestinal targeting, it will dissolve explosively once it reaches the specific pH environment of the intestine. This kind of rapid release without time control leads to the probiotics being exposed too early and too quickly in the small intestine or the front end of the colon, and cannot achieve slow and sustained release that depends on enzyme degradation in the deep colon, which seriously affects the uniform colonization of the strain throughout the colon; (4) Low colonization efficiency: Existing formulations ignore the key influence of the integrity of the intestinal mucosal barrier on the colonization of the strain. In the microenvironment of mucosal damage or nutritional deficiency, the released probiotics lack adhesion anchoring points and proliferation substrates, making it difficult to achieve long-term colonization and function.

[0004] Based on the above statements, the present invention provides a compound probiotic preparation and its preparation method. Summary of the Invention

[0005] To address the shortcomings of existing compound probiotic preparations, such as the large differences in oxygen and acid resistance of live bacteria, high loss rate of live bacteria under gastric acid environment, disordered intestinal colonization sequence caused by multi-component compounding, easy oxidation and inactivation of heat-sensitive active ingredients and anaerobic bacteria in storage, transportation and gastrointestinal environment, and poor shelf stability, this invention provides a compound probiotic preparation and its preparation method.

[0006] In a first aspect, the present invention provides a compound probiotic preparation, which adopts the following technical solution:

[0007] A compound probiotic preparation comprises the following raw materials in parts by weight: 35-45 parts of targeted controlled-release live bacteria microspheres, 5-10 parts of antioxidant sugar complex, 10-20 parts of active peptide complex powder, 3-5 parts of complex mineral elements, 0.3-0.5 parts of complex vitamins, 5-10 parts of moisture-proof coating excipients, and 5-10 parts of maltodextrin.

[0008] Preferably, the method for preparing the targeted controlled-release live bacterial microspheres is as follows:

[0009] Hydroxypropyl methylcellulose and inulin were added to deionized water and stirred until homogeneous. Then, magnesium carbonate and sodium citrate were added, and the mixture was heated and stirred to obtain a sol matrix. After cooling, Lactobacillus plantarum cells were added to the sol matrix. After shearing emulsification, the mixture was spray-granulated to obtain targeted controlled-release live bacteria microspheres.

[0010] Preferably, the specific preparation method of the targeted controlled-release live bacterial microspheres is as follows:

[0011] Hydroxypropyl methylcellulose and inulin were added to deionized water at a mass ratio of 2-3:3-5:30-40 and stirred at 300-500 rpm for 20-30 min at room temperature to obtain mixture A. Magnesium carbonate and sodium citrate were added to mixture A at a mass ratio of 2-4:1-2:35-45 and heated to 75-80℃, then stirred at 300-500 rpm for 30-40 min to obtain a sol matrix. The sol matrix was cooled to 30-35℃, and Lactobacillus plantarum cells were added to the sol matrix at a mass ratio of 5-7:35-45. The mixture was emulsified at 5000-7000 rpm for 5-10 min. Finally, fluidized bed spray granulation was performed, with the inlet air temperature set at 45-50℃ and the outlet air temperature at 28-32℃. The spray granulation was carried out until the moisture content was ≤5% to obtain targeted controlled-release live bacteria microspheres.

[0012] Preferably, the viable count of the *Lactobacillus plantarum* cells is ≥1.0 × 10⁻⁶. 11 CFU / g.

[0013] Preferably, the method for preparing the antioxidant glycosaminoglycan complex is as follows:

[0014] β-Cyclodextrin and D-aloxose were added to deionized water, heated and stirred, cooled to 35-45℃, vitamin C palmitate and d-α-tocopherol acetate were added, and the mixture was sheared and emulsified under nitrogen protection. Stachyose was then added, stirred evenly, recrystallized, filtered, the precipitate was collected, washed, and freeze-dried to obtain the antioxidant sugar complex.

[0015] Preferably, the specific preparation method of the antioxidant glycosaminoglycan complex is as follows:

[0016] Add β-cyclodextrin and D-allulose to deionized water at a mass ratio of 1-2:3-5:30-40, heat to 60-70℃, and stir at 300-500 rpm for 25-35 min to obtain mixture B. After cooling to 35-45℃, add vitamin C palmitate and d-α-tocopherol acetate to mixture B at a mass ratio of 1-2:0.5-1:40-50. Under nitrogen protection, shear emulsify at 1500-2500 rpm for 20-30 min to obtain mixture C. Add stachyose to mixture C at a mass ratio of 2-4:40-50, stir at 300-500 rpm for 10-15 min at room temperature to obtain a homogeneous system. Cool the homogeneous system to -10℃ to -5℃ at a rate of 1-2℃ / min and maintain for 2-4 h for recrystallization. Filter, collect the precipitate, wash 2-3 times with anhydrous ethanol pre-cooled to 0-4℃, pre-freeze at -40℃ for 4-6 h, and then freeze-dry at 5-10 Pa and -50℃ to -60℃ for 24-36 h to obtain the antioxidant sugar complex.

[0017] Preferably, the active peptide compound powder is composed of fish collagen peptides, fructooligosaccharides, and L-arabinose in a mass ratio of 10-15:3-5:2-4.

[0018] Preferably, the complex mineral elements are selected from one or more of calcium citrate, zinc gluconate, and ferric pyrophosphate; the complex vitamins are selected from one or more of vitamin B1, vitamin B2, and vitamin B6.

[0019] Preferably, the composite mineral elements are composed of calcium citrate, zinc gluconate, and ferric pyrophosphate in a mass ratio of 30-40:10-15:6-8.

[0020] Preferably, the complex vitamin consists of vitamin B1, vitamin B2 and vitamin B6 in a mass ratio of 5-10:5-10:4-6.

[0021] Preferably, the moisture-proof coating material is composed of sodium octenyl succinate starch and gum arabic in a mass ratio of 2-3:1.

[0022] Secondly, the present invention provides a method for preparing a compound probiotic preparation, which adopts the following technical solution:

[0023] A method for preparing a compound probiotic preparation includes the following steps:

[0024] S1. Mix the targeted controlled-release live bacteria microspheres, antioxidant sugar complex and maltodextrin, stir evenly to obtain a premixed composite powder;

[0025] S2. Add the moisture-proof coating material to deionized water and stir well to obtain the moisture-proof coating solution;

[0026] S3. Atomize and spray the moisture-proof coating liquid onto the surface of the premixed composite powder to obtain the coating intermediate;

[0027] S4. The coating intermediate is dried by fluidized bed drying and cooled by fluidized bed cooling. Then it is mixed with active peptide compound powder, compound mineral elements and compound vitamins and stirred evenly to obtain a compound probiotic preparation.

[0028] Preferably, in step S1, the stirring temperature is 10-15℃, the stirring speed is 15-25 rpm, and the stirring time is 20-30 min.

[0029] Preferably, in step S2, the mass ratio of the moisture-proof coating material to deionized water is 1:6-9; the stirring speed is 400-600 rpm; and the stirring time is 30-40 min.

[0030] Preferably, the atomization spraying parameters in step S3 include: fluidized bed inlet air temperature of 35-38℃, outlet air temperature of 25-28℃, atomization pressure of 0.15-0.25MPa, and spraying rate of 15-25mL / min.

[0031] Preferably, the fluidized bed drying parameters in step S4 include: an inlet air temperature of 35-38℃, an outlet air temperature of 28-32℃, and drying to a moisture content ≤3%; the fluidized bed cooling parameters include: an inlet air temperature of 20-25℃, an outlet air temperature of 18-22℃, and a cooling time of 10-15 min; the stirring temperature is room temperature, the stirring speed is 200-400 rpm, and the stirring time is 15-25 min.

[0032] Thirdly, the present invention provides the application of the above-mentioned compound probiotic preparation in the preparation of health foods or medicines for assisting in enhancing the intestinal colonization ability of probiotics.

[0033] In summary, the present invention has the following beneficial effects:

[0034] 1. Achieved precise, time-sequential, and targeted release of probiotics: This invention is based on a core-shell layered structure design of "outer active peptide complex powder for rapid gastric release - inner core live bacteria microspheres for sustained colonic release." Components such as fish collagen peptides in the outer active peptide complex powder dissolve and release rapidly in the stomach and upper small intestine, playing a leading role in intestinal mucosal pre-repair and nutritional preparation. The inner core live bacteria microspheres utilize the gastric acid swelling and inhibition properties of hydroxypropyl methylcellulose, combined with the enzymatic degradation properties of inulin, to form a pH-enzyme dual-trigger controlled-release mechanism. This effectively protects *Lactobacillus plantarum* from successfully passing through the gastric acid and bile salt environment, and allows for targeted disintegration and release in the colon, achieving precise, time-sequential, and segmented colonization of probiotics in the intestine.

[0035] 2. Significantly improves the shelf stability and acid / oxygen resistance of live bacteria in complex environments: This invention addresses the shortcomings of probiotics and heat-sensitive nutrients, such as easy oxidation and poor acid resistance, by constructing a dual protection mechanism of microenvironment buffering and external antioxidants. On the one hand, magnesium carbonate and sodium citrate are embedded inside the live bacteria microspheres as acid-base buffering stabilizers, creating a weakly acidic microenvironment within the microspheres to resist gastric acid invasion and maintain bacterial activity. On the other hand, through β-cyclodextrin molecular inclusion and low-temperature recrystallization of polysaccharides, vitamin C palmitate, d-α-tocopherol acetate, and stachyose are constructed into an antioxidant sugar complex with a stable structure. This complex acts as an external antioxidant barrier, effectively scavenging free radicals in the powder system and reducing oxidative damage to probiotics during storage, transportation, and gastrointestinal transit.

[0036] 3. A multi-prebiotic proliferation system with synergistic "carrier-carbon source" dual effects was constructed: This invention scientifically combines inulin, fructooligosaccharides, stachyose, L-arabinose, and D-alulose—a five-fold prebiotic combination—and spatially positions them according to the solubility characteristics and functional differences of each component: inulin is embedded in the microsphere shell as a colonic enzyme-responsive sustained-release framework; fructooligosaccharides and L-arabinose are distributed in the outer powder layer as a gastric fast-release carbon source; and stachyose and D-alulose are integrated into an antioxidant sugar complex as a long-acting sustained-release proliferation substrate. These prebiotics not only serve as auxiliary carriers for the formation of the controlled-release structure in the early stages, but also continue to be used as selective fermentation carbon sources by probiotics such as Lactobacillus plantarum after entering the intestine. The short-chain fatty acids (such as acetic acid, propionic acid, and butyric acid) produced by their metabolism can effectively reduce intestinal pH, inhibit pathogen colonization, and regulate the intestinal microecological balance, further contributing to the efficient colonization and proliferation of the core strains.

[0037] 4. Achieves a scientifically balanced and enhanced intake of trace elements and multidimensional nutrition: Based on the dietary nutritional deficiencies of modern populations, this invention scientifically combines complex mineral elements and complex vitamins, and incorporates small-molecule fish collagen peptides with an average molecular weight ≤1000 Da. The characteristic tripeptide Gly-Pro-Hyp in the fish collagen peptides promotes mineral bioavailability through chelation, while its mucosal repair function synergistically enhances the intestinal colonization of probiotics. The balanced ratio of nutrients not only repairs the intestinal mucosal barrier and regulates the intestinal microecology, but also synergistically improves the body's trace element metabolism and bone health, achieving precise multidimensional nutritional supplementation.

[0038] 5. The process is mild and stable, suitable for industrial mass production: In the final mixing and coating stage of the compound probiotic preparation, the present invention adopts low-temperature closed stepwise mixing and fluidized bed bottom spraying layering micro-encapsulation process, strictly limiting the production environment temperature, etc. This preparation process protects the number of live probiotics and heat-sensitive nutrient active ingredients to the greatest extent, while having the characteristics of simple steps, controllable parameters and small batch-to-batch differences. Detailed Implementation

[0039] The present invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0040] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0041] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0042] Hydroxypropyl methylcellulose was purchased from Xi'an Haotian Biotechnology Co., Ltd., CAS: 9004-65-3;

[0043] Inulin was purchased from Shaanxi Panier Biotechnology Co., Ltd.

[0044] Lactiplantibacillus plantarum was purchased from the China Industrial Microbial Culture Collection Center, strain number CICC 21794;

[0045] β-Cyclodextrin was purchased from Anhui Qianpin Biotechnology Co., Ltd., CAS: 7585-39-9;

[0046] D-allulose was purchased from Jiangsu Dongju Biotechnology Co., Ltd., CAS551-68-8;

[0047] Vitamin C palmitate was purchased from Jinan Yuncheng Biotechnology Co., Ltd.

[0048] d-α-tocopherol acetate was purchased from Shanxi Zhongnuo Biotechnology Co., Ltd.

[0049] Stachyose was purchased from Jinan Yuncheng Biotechnology Co., Ltd.

[0050] Fish collagen peptides were purchased from Xi'an Haotian Biotechnology Co., Ltd.

[0051] Soybean oligopeptides were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0052] The fructooligosaccharides were purchased from Henan Qinuo Food Ingredients Co., Ltd.

[0053] L-arabinose was purchased from Jinan Yuncheng Biotechnology Co., Ltd.

[0054] Sodium octenyl succinate starch was purchased from Anhui Youtai Bioengineering Co., Ltd., CAS: 66829-29-6;

[0055] The gum arabic was purchased from Jiangsu Feiqing Biotechnology Co., Ltd., CAS: 9000-07-1.

[0056] Example 1

[0057] A compound probiotic preparation comprises the following raw materials in parts by weight: 35 parts targeted controlled-release live bacteria microspheres, 5 parts antioxidant sugar complex, 10 parts active peptide complex powder, 3 parts compound mineral elements, 0.3 parts compound vitamins, 5 parts moisture-proof coating excipients, and 5 parts maltodextrin.

[0058] The active peptide compound powder is composed of fish collagen peptides, fructooligosaccharides, and L-arabinose in a mass ratio of 10:3:2.

[0059] The complex mineral elements consist of calcium citrate, zinc gluconate, and iron pyrophosphate in a mass ratio of 30:10:6.

[0060] A multivitamin consists of vitamin B1, vitamin B2, and vitamin B6 in a mass ratio of 5:5:4.

[0061] The moisture-proof coating material is composed of sodium octenyl succinate starch and gum arabic in a mass ratio of 2:1.

[0062] The specific preparation method of targeted controlled-release live bacterial microspheres is as follows:

[0063] Hydroxypropyl methylcellulose and inulin were added to deionized water at a mass ratio of 2:3:30 and stirred at 300 rpm for 20 min at room temperature to obtain mixture A. Magnesium carbonate and sodium citrate were added to mixture A at a mass ratio of 2:1:35, and the mixture was heated to 75°C and stirred at 300 rpm for 30 min to obtain a sol matrix. The sol matrix was cooled to 30°C, and *Lactobacillus plantarum* cells (viable count 1.0 × 10⁻⁵) were added at a mass ratio of 5:35. 11 CFU / g) was added to the sol matrix and emulsified at high speed of 5000 rpm for 5 min; finally, fluidized bed spray granulation was performed, with the inlet air temperature set at 45℃ and the outlet air temperature at 28℃, and spray granulation was carried out until the moisture content was 5%, to obtain targeted controlled-release live bacteria microspheres.

[0064] The specific preparation method of the antioxidant glycoside complex is as follows:

[0065] β-Cyclodextrin and D-alulose were added to deionized water at a mass ratio of 1:3:30, heated to 60°C, and stirred at 300 rpm for 25 min to obtain mixture B. After cooling to 35°C, vitamin C palmitate and d-α-tocopherol acetate were added to mixture B at a mass ratio of 1:0.5:40. Under nitrogen protection, the mixture was sheared and emulsified at 1500 rpm for 20 min to obtain mixture C. Stachyose was added to mixture C at a mass ratio of 2:40, and stirred at 300 rpm for 10 min at room temperature to obtain a homogeneous system. The homogeneous system was cooled to -10°C at a rate of 1°C / min and kept for 2 h for recrystallization. The precipitate was filtered, collected, washed twice with anhydrous ethanol pre-cooled to 0°C, pre-frozen at -40°C for 4 h, and then freeze-dried at 5 Pa and -50°C for 24 h to obtain the antioxidant sugar complex.

[0066] A method for preparing a compound probiotic preparation includes the following steps:

[0067] S1. Mix the targeted controlled-release live bacteria microspheres, antioxidant sugar complex and maltodextrin, and stir at 10℃ and 15rpm for 20min to obtain a premixed composite powder.

[0068] S2. Add the moisture-proof coating material to deionized water at a mass ratio of 1:6, and stir at 400 rpm for 30 minutes at room temperature to obtain the moisture-proof coating solution.

[0069] S3. Set the fluidized bed inlet air temperature to 35℃, outlet air temperature to 25℃, atomization pressure to 0.15MPa, and spraying rate to 15mL / min. Atomize and spray the moisture-proof coating liquid onto the surface of the premixed composite powder to obtain the coating intermediate.

[0070] S4. Set the inlet air temperature to 35℃ and the outlet air temperature to 28℃. Fluidize and dry the coating intermediate to a moisture content of 3%. Then set the inlet air temperature to 20℃ and the outlet air temperature to 18℃. Fluidize and cool for 10 minutes. Then mix with active peptide compound powder, compound mineral elements and compound vitamins. Stir at 200 rpm for 15 minutes at room temperature to obtain a compound probiotic preparation.

[0071] Example 2

[0072] A compound probiotic preparation comprises the following raw materials in parts by weight: 40 parts targeted controlled-release live bacteria microspheres, 7.5 parts antioxidant sugar complex, 15 parts active peptide complex powder, 4 parts compound mineral elements, 0.4 parts compound vitamins, 7.5 parts moisture-proof coating excipients, and 7.5 parts maltodextrin.

[0073] The active peptide compound powder is composed of fish collagen peptides, fructooligosaccharides, and L-arabinose in a mass ratio of 12:4:3.

[0074] The complex mineral elements consist of calcium citrate, zinc gluconate, and iron pyrophosphate in a mass ratio of 30:10:7.

[0075] A multivitamin consists of vitamin B1, vitamin B2, and vitamin B6 in a mass ratio of 5:5:5.

[0076] The moisture-proof coating material is composed of sodium octenyl succinate starch and gum arabic in a mass ratio of 2.5:1.

[0077] The specific preparation method of targeted controlled-release live bacterial microspheres is as follows:

[0078] Hydroxypropyl methylcellulose and inulin were added to deionized water at a mass ratio of 2.5:4:35 and stirred at 400 rpm for 25 min at room temperature to obtain mixture A. Magnesium carbonate and sodium citrate were added to mixture A at a mass ratio of 3:1.5:40, and the mixture was heated to 78°C and stirred at 400 rpm for 35 min to obtain a sol matrix. The sol matrix was cooled to 32°C, and *Lactobacillus plantarum* cells (viable count 1.0 × 10⁻⁶) were added at a mass ratio of 6:40. 11 CFU / g) was added to the sol matrix and emulsified at high speed of 6000 rpm for 7.5 min; finally, fluidized bed spray granulation was carried out, with the inlet air temperature set at 47℃ and the outlet air temperature at 30℃, and spray granulation was carried out until the moisture content was 5%, to obtain targeted controlled-release live bacteria microspheres.

[0079] The specific preparation method of the antioxidant glycoside complex is as follows:

[0080] β-Cyclodextrin and D-alulose were added to deionized water at a mass ratio of 1.5:4:35, heated to 65°C, and stirred at 400 rpm for 30 min to obtain mixture B. After cooling to 40°C, vitamin C palmitate and d-α-tocopherol acetate were added to mixture B at a mass ratio of 1.5:0.75:45. Under nitrogen protection, the mixture was sheared and emulsified at 2000 rpm for 25 min to obtain mixture C. Stachyose was added to mixture C at a mass ratio of 3:45, and stirred at 400 rpm for 12 min at room temperature to obtain a homogeneous system. The homogeneous system was cooled to -8°C at a rate of 1.5°C / min and kept for 3 h for recrystallization. The precipitate was filtered, collected, washed twice with anhydrous ethanol pre-cooled to 2°C, pre-frozen at -40°C for 5 h, and then freeze-dried at 8 Pa and -55°C for 30 h to obtain the antioxidant sugar complex.

[0081] A method for preparing a compound probiotic preparation includes the following steps:

[0082] S1. Mix the targeted controlled-release live bacteria microspheres, antioxidant sugar complex and maltodextrin, and stir at 12℃ and 20rpm for 25min to obtain a premixed composite powder.

[0083] S2. Add the moisture-proof coating material to deionized water at a mass ratio of 1:8, and stir at 500 rpm for 35 minutes at room temperature to obtain the moisture-proof coating solution.

[0084] S3. Set the fluidized bed inlet air temperature to 37℃, outlet air temperature to 26℃, atomization pressure to 0.20MPa, and spraying rate to 20mL / min. Atomize and spray the moisture-proof coating liquid onto the surface of the premixed composite powder to obtain the coating intermediate.

[0085] S4. Set the inlet air temperature to 37℃ and the outlet air temperature to 30℃. Fluidize and dry the coating intermediate to a moisture content of 3%. Then set the inlet air temperature to 22℃ and the outlet air temperature to 20℃. Fluidize and cool for 12 minutes. Then mix with active peptide compound powder, compound mineral elements and compound vitamins. Stir at 300 rpm for 20 minutes at room temperature to obtain a compound probiotic preparation.

[0086] Example 3

[0087] A compound probiotic preparation comprises the following raw materials in parts by weight: 45 parts targeted controlled-release live bacteria microspheres, 10 parts antioxidant sugar complex, 20 parts active peptide complex powder, 5 parts compound mineral elements, 0.5 parts compound vitamins, 10 parts moisture-proof coating excipients, and 10 parts maltodextrin.

[0088] The active peptide compound powder is composed of fish collagen peptides, fructooligosaccharides, and L-arabinose in a mass ratio of 15:5:4.

[0089] The complex mineral elements consist of calcium citrate, zinc gluconate, and iron pyrophosphate in a mass ratio of 30:10:8.

[0090] A multivitamin consists of vitamin B1, vitamin B2, and vitamin B6 in a mass ratio of 5:5:6.

[0091] The moisture-proof coating material is composed of sodium octenyl succinate starch and gum arabic in a mass ratio of 3:1.

[0092] The specific preparation method of targeted controlled-release live bacterial microspheres is as follows:

[0093] Hydroxypropyl methylcellulose and inulin were added to deionized water at a mass ratio of 3:5:40 and stirred at 500 rpm for 30 min at room temperature to obtain mixture A. Magnesium carbonate and sodium citrate were added to mixture A at a mass ratio of 4:2:45, and the mixture was heated to 80°C and stirred at 500 rpm for 40 min to obtain a sol matrix. The sol matrix was cooled to 35°C, and *Lactobacillus plantarum* cells (viable count 1.0 × 10⁻⁵) were added at a mass ratio of 7:45. 11CFU / g) was added to the sol matrix and emulsified at high speed of 7000 rpm for 10 min; finally, fluidized bed spray granulation was performed, with the inlet air temperature set at 50℃ and the outlet air temperature at 32℃, and spray granulation was carried out until the moisture content was 5%, to obtain targeted controlled-release live bacteria microspheres.

[0094] The specific preparation method of the antioxidant glycoside complex is as follows:

[0095] β-Cyclodextrin and D-alulose were added to deionized water at a mass ratio of 2:5:40, heated to 70°C, and stirred at 500 rpm for 35 min to obtain mixture B. After cooling to 45°C, vitamin C palmitate and d-α-tocopherol acetate were added to mixture B at a mass ratio of 2:1:50. Under nitrogen protection, the mixture was sheared and emulsified at 2500 rpm for 30 min to obtain mixture C. Stachyose was added to mixture C at a mass ratio of 4:50, and stirred at 500 rpm for 15 min at room temperature to obtain a homogeneous system. The homogeneous system was cooled to -5°C at a rate of 2°C / min and kept for 4 h for recrystallization. The precipitate was filtered, collected, washed three times with anhydrous ethanol pre-cooled to 4°C, pre-frozen at -40°C for 6 h, and then freeze-dried at 10 Pa and -60°C for 36 h to obtain the antioxidant sugar complex.

[0096] A method for preparing a compound probiotic preparation includes the following steps:

[0097] S1. Mix the targeted controlled-release live bacteria microspheres, antioxidant sugar complex and maltodextrin, and stir at 15℃ and 25rpm for 30min to obtain a premixed composite powder.

[0098] S2. Add the moisture-proof coating material to deionized water at a mass ratio of 1:9, and stir at 600 rpm for 40 minutes at room temperature to obtain the moisture-proof coating solution.

[0099] S3. Set the fluidized bed inlet air temperature to 38℃, outlet air temperature to 28℃, atomization pressure to 0.25MPa, and spraying rate to 25mL / min. Atomize and spray the moisture-proof coating liquid onto the surface of the premixed composite powder to obtain the coating intermediate.

[0100] S4. Set the inlet air temperature to 38℃ and the outlet air temperature to 32℃. Fluidize and dry the coating intermediate to a moisture content of 3%. Then set the inlet air temperature to 25℃ and the outlet air temperature to 22℃. Fluidize and cool for 15 minutes. Then mix with active peptide compound powder, compound mineral elements and compound vitamins. Stir at 400 rpm for 25 minutes at room temperature to obtain a compound probiotic preparation.

[0101] Comparative Example 1

[0102] This comparative example provides a compound probiotic preparation and its preparation method. The only difference from Example 2 is that in the preparation of the targeted controlled-release live bacteria microspheres, inulin is replaced with an equal mass of maltodextrin. The other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0103] Specifically:

[0104] The specific preparation method of targeted controlled-release live bacterial microspheres is as follows:

[0105] Hydroxypropyl methylcellulose and maltodextrin were added to deionized water at a mass ratio of 2.5:4:35 and stirred at 400 rpm for 25 min at room temperature to obtain mixture A. Magnesium carbonate and sodium citrate were added to mixture A at a mass ratio of 3:1.5:40, and the mixture was heated to 78°C and stirred at 400 rpm for 35 min to obtain a sol matrix. The sol matrix was cooled to 32°C, and *Lactobacillus plantarum* cells (viable count 1.0 × 10⁻⁶) were added at a mass ratio of 6:40. 11 CFU / g) was added to the sol matrix and emulsified at high speed of 6000 rpm for 7.5 min; finally, fluidized bed spray granulation was carried out, with the inlet air temperature set at 47℃ and the outlet air temperature at 30℃, and spray granulation was carried out until the moisture content was 5%, to obtain targeted controlled-release live bacteria microspheres.

[0106] Comparative Example 2

[0107] This comparative example provides a compound probiotic preparation and its preparation method. The only difference from Example 2 is that magnesium carbonate and sodium citrate are removed in the preparation of the targeted controlled-release live bacteria microspheres. The other raw material types, dosages and preparation process parameters are completely consistent with Example 2.

[0108] Specifically:

[0109] The specific preparation method of targeted controlled-release live bacterial microspheres is as follows:

[0110] Hydroxypropyl methylcellulose and inulin were added to deionized water at a mass ratio of 2.5:4:35 and stirred at 400 rpm for 25 min at room temperature to obtain mixture A. This mixture was then heated to 78°C and stirred at 400 rpm for 35 min to obtain a sol matrix. The sol matrix was cooled to 32°C, and *Lactobacillus plantarum* cells (viable count 1.0 × 10⁻⁶) were added at a mass ratio of 6:40. 11 CFU / g) was added to the sol matrix and emulsified at high speed of 6000 rpm for 7.5 min; finally, fluidized bed spray granulation was carried out, with the inlet air temperature set at 47℃ and the outlet air temperature at 30℃, and spray granulation was carried out until the moisture content was 5%, to obtain targeted controlled-release live bacteria microspheres.

[0111] Comparative Example 3

[0112] This comparative example provides a compound probiotic preparation and its preparation method. The only difference from Example 2 is that the antioxidant sugar complex is replaced by a simple physical mixture of antioxidants. The other raw material types, amounts, and preparation process parameters are completely consistent with Example 2.

[0113] Specifically:

[0114] The specific preparation method of antioxidants is as follows:

[0115] According to the amounts of β-cyclodextrin, D-allulose, vitamin C palmitate, d-α-tocopherol acetate, and stachyose used in Example 2, the β-cyclodextrin, D-allulose, vitamin C palmitate, d-α-tocopherol acetate, and stachyose were mixed and stirred at 400 rpm for 20 min at room temperature to obtain an antioxidant.

[0116] Comparative Example 4

[0117] This comparative example provides a compound probiotic preparation and its preparation method. The only difference from Example 2 is that the antioxidant sugar complex is replaced with an acyclodextrin antioxidant sugar complex of equal mass. The other raw material types, amounts and preparation process parameters are completely consistent with Example 2.

[0118] Specifically:

[0119] The specific preparation method of the acyclodextrin antioxidant sugar complex is as follows:

[0120] D-alulose was added to deionized water at a mass ratio of 5.5:35, heated to 65°C, and stirred at 400 rpm for 30 min to obtain mixture B. After cooling to 40°C, vitamin C palmitate and d-α-tocopherol acetate were added to mixture B at a mass ratio of 1.5:0.75:45. Under nitrogen protection, the mixture was sheared and emulsified at 2000 rpm for 25 min to obtain mixture C. Stachyose was added to mixture C at a mass ratio of 3:45, and stirred at 400 rpm for 12 min at room temperature to obtain a homogeneous system. The homogeneous system was cooled to -8°C at a rate of 1.5°C / min and kept for 3 h for recrystallization. The precipitate was filtered, collected, washed twice with anhydrous ethanol pre-cooled to 2°C, pre-frozen at -40°C for 5 h, and then freeze-dried at 8 Pa and -55°C for 30 h to obtain the acyclodextrin antioxidant sugar complex.

[0121] Comparative Example 5

[0122] This comparative example provides a compound probiotic preparation and its preparation method. The only difference from Example 2 is that the fish collagen peptide in the active peptide compound powder is replaced with soybean oligopeptides. The other raw material types, dosages and preparation process parameters are completely consistent with Example 2.

[0123] Performance testing

[0124] 1. Detection Object

[0125] The compound probiotic preparations prepared in Examples 1-3 and Comparative Examples 1-5 of this invention.

[0126] 2. Testing Items and Methods

[0127] (1) Determination of the survival rate of simulated gastrointestinal bacteria

[0128] Artificial gastric juice tolerance test: Weigh 1.0g of each group of samples and put them into 100mL of artificial gastric juice (containing 0.32% pepsin, and the pH was adjusted to 2.0 with hydrochloric acid). Digest at 37℃ and 100rpm for 2 hours.

[0129] Artificial intestinal fluid release and survival rate determination: The pH of the above mixture after gastric juice treatment was adjusted back to 6.8, and an equal volume of artificial intestinal fluid (containing 1% trypsin and 0.3% porcine bile salts) was added. The mixture was then further digested at 37°C and 100 rpm for 3 hours with shaking.

[0130] Sampling and Cell Wall Disruption Counting: After 3 hours of intestinal digestion, the digestion solution containing residual microspheres was placed in a sterile homogenizing bag and subjected to thorough physical cell wall disruption using a tapping homogenizer to completely release the surviving bacteria protected by the microspheres. The homogenized sample was immediately serially diluted with physiological saline, anaerobically cultured on MRS agar at 37°C for 48 hours, and viable cell counts were performed.

[0131] Calculation formula: Survival rate of gastrointestinal bacteria (%) = (Number of viable bacteria after digestion and homogenization / Initial number of viable bacteria in the sample before digestion) × 100%.

[0132] (2) Determination of viable bacteria retention rate and antioxidant component retention rate during high-temperature accelerated storage shelf life

[0133] High-temperature accelerated viable cell retention rate (%): The viable cell count of each group of samples was determined after 6 months of accelerated storage at 40℃. Immediately after sampling, samples were serially diluted with physiological saline, anaerobically cultured on MRS agar medium at 37℃ for 48 hours, and viable cell counts were performed.

[0134] Calculation formula: High temperature accelerated viable bacteria retention rate (%) = (Number of viable bacteria measured after 6 months of accelerated storage / Number of viable bacteria measured on the day the sample was prepared) × 100%.

[0135] High-temperature accelerated vitamin C retention (%): The total L-ascorbic acid content (converted from vitamin C palmitate) in the sample after 6 months of accelerated storage was determined by high-performance liquid chromatography (HPLC). Chromatographic conditions: C18 reversed-phase column (4.6 mm × 250 mm, 5 μm), mobile phase: methanol-phosphate buffer (pH 3.0, 75:25, v / v), detection wavelength: 254 nm, flow rate: 1.0 mL / min, column temperature: 30 °C, injection volume: 20 μL.

[0136] Calculation formula: High temperature accelerated VC retention rate (%) = (Total VC content measured after 6 months of accelerated storage / Total VC content measured on the day the sample was prepared) × 100%.

[0137] (3) Determination of the adsorption and cushioning repair properties of simulated intestinal mucosa (in vitro cell model)

[0138] An in vitro monolayer cell model was constructed using human colon adenocarcinoma cells (Caco-2). Caco-2 cells were seeded into 12-well cell culture plates (1 × 10⁶ cells per well). 5 (100 cells) were cultured at 37°C and 5% CO2 until a dense monolayer was formed. Each group of samples was dissolved in serum-free DMEM medium (final concentration 1 mg / mL) and added to cell well plates for co-incubation.

[0139] Peptide mucosal coverage during release (%): 30 min after the start of simulated digestion, cell culture supernatant was aspirated, and the content of free small molecule peptides was determined using the o-phthalaldehyde (OPA) method to calculate the peptide release rate. Simultaneously, bioactive peptides were labeled using FITC fluorescence labeling, and the percentage of the surface area of ​​fluorescently labeled peptides on the surface of Caco-2 cell monolayers was observed and quantitatively analyzed using laser confocal microscopy (5 fields of view were randomly selected from each well, and the average value was taken). A higher percentage of the attached area indicates better mucosal coating and repair performance of the bioactive peptides.

[0140] Probiotic cell adhesion rate (%): After simulated digestion, the unadsorbed bacterial solution was discarded, and the cell layer was gently washed three times with sterile PBS buffer (pH 7.2) to remove unadhered free cells. Cells were lysed with 0.5% Triton X-100, and the lysate was collected, serially diluted, and anaerobically cultured on MRS agar medium at 37°C for 48 h. The number of viable bacteria adhering to the cell surface was counted.

[0141] Calculation formula: Probiotic cell adhesion rate (%) = (number of live bacteria adsorbed on the cell surface after washing / total number of live bacteria added) × 100%.

[0142] 3. Test Results

[0143] The specific test results are shown in Table 1.

[0144] Table 1 Performance test results of compound probiotic preparations

[0145] Group Survival rate of gastrointestinal bacteria (%) High temperature accelerates the retention rate of live bacteria (%) High temperature accelerates VC retention (%) Release phase peptide mucosal coverage (%) Probiotic cell adhesion rate (%) Example 1 85.5 80.4 87.2 90.5 13.5 Example 2 89.6 85.1 89.4 94.2 15.6 Example 3 87.9 83.3 88.6 91.0 14.8 Comparative Example 1 42.1 75.3 78.5 81.3 6.2 Comparative Example 2 58.7 62.4 79.1 82.0 11.4 Comparative Example 3 84.1 43.5 45.1 83.2 12.1 Comparative Example 4 85.3 59.2 55.7 84.6 12.3 Comparative Example 5 80.9 77.1 86.7 83.5 9.2

[0146] As shown in Table 1, the compound probiotic preparations prepared in Examples 1-3 of this invention outperformed Comparative Examples 1-5 in all five core performance indicators. This fully demonstrates that the organic combination of the core inventive points of this invention achieves an unexpected synergistic effect between the components and the process, significantly improving the acid and oxygen resistance and intestinal colonization and repair activity of the probiotic preparations. Among them, Example 2 showed the best overall effect.

[0147] Compared to Example 2, in Comparative Example 1, replacing inulin with maltodextrin in the microsphere preparation resulted in a significant decrease in both the survival rate of simulated gastrointestinal bacteria and the final cell adhesion rate. This indicates that maltodextrin readily dissolves in gastric juice and the upper small intestine, completely lacking the colon-specific degradation response characteristics. This leads to premature exposure of the internal probiotics to gastric acid and bile salts, resulting in significant inactivation. This directly demonstrates that inulin, as a specific response framework, makes an irreplaceable technical contribution to ensuring targeted colonization of bacterial strains.

[0148] Compared to Example 2, Comparative Example 2 showed a significant deterioration in gastrointestinal viable bacteria survival rate and high-temperature accelerated viable bacteria retention rate after removing magnesium carbonate and sodium citrate from the targeted controlled-release microspheres. This strongly demonstrates that the acid-base buffer stabilizer embedded in the microspheres can spontaneously construct a microenvironment that resists gastric acid invasion, thereby deeply locking in the activity of viable bacteria throughout the digestive chain.

[0149] Compared to Example 2, Comparative Examples 3 and 4 showed a common deterioration in long-term storage stability. In Comparative Example 3, replacing the antioxidant glycoconjugate with a physically mixed antioxidant resulted in the lowest retention rate of viable bacteria and vitamin C during shelf life. In Comparative Example 4, replacing the antioxidant glycoconjugate with an acyclodextrin antioxidant glycoconjugate also significantly reduced the antioxidant and viability retention effects. This demonstrates that the "inclusion-eutectic bistable structure" constructed in this invention is not a simple accumulation of raw materials, but rather forms a dense physical antioxidant shield, effectively blocking external oxygen from damaging viable bacteria and heat-sensitive components.

[0150] Compared to Example 2, in Comparative Example 5, although both are small molecule peptides and their peptide segment mucosal coverage during the release phase is acceptable, the final probiotic cell adhesion rate still decreased significantly after replacing the small molecule fish collagen peptide with soybean oligopeptides. This indicates that the physical properties of a single small molecule are insufficient to construct an ideal colonization environment. The fish collagen peptide (Gly-Pro-Hyp) specified in this invention possesses specific mucosal repair and biocompatibility lacking in soybean oligopeptides, and can form a unique synergistic effect with probiotics, constructing an irreplaceable microecological environment for the subsequent efficient colonization of probiotics.

[0151] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A compound probiotic preparation, characterized in that, The ingredients include the following parts by weight: 35-45 parts targeted controlled-release live bacteria microspheres, 5-10 parts antioxidant sugar complex, 10-20 parts active peptide complex powder, 3-5 parts complex mineral elements, 0.3-0.5 parts complex vitamins, 5-10 parts moisture-proof coating excipients, and 5-10 parts maltodextrin.

2. The compound probiotic preparation according to claim 1, characterized in that, The preparation method of the targeted controlled-release live bacteria microspheres is as follows: Hydroxypropyl methylcellulose and inulin were added to deionized water and stirred until homogeneous. Then, magnesium carbonate and sodium citrate were added, and the mixture was heated and stirred to obtain a sol matrix. After cooling, Lactobacillus plantarum cells were added to the sol matrix. After shearing emulsification, the mixture was spray-granulated to obtain targeted controlled-release live bacteria microspheres.

3. The compound probiotic preparation according to claim 2, characterized in that, The viable count of *Lactobacillus plantarum* cells is ≥1.0 × 10⁻⁶. 11 CFU / g.

4. The compound probiotic preparation according to claim 1, characterized in that, The method for preparing the antioxidant glycosaminoglycan complex is as follows: β-Cyclodextrin and D-aloxose were added to deionized water, heated and stirred, cooled to 35-45℃, vitamin C palmitate and d-α-tocopherol acetate were added, and the mixture was sheared and emulsified under nitrogen protection. Stachyose was then added, stirred evenly, recrystallized, filtered, the precipitate was collected, washed, and freeze-dried to obtain the antioxidant sugar complex.

5. The compound probiotic preparation according to claim 1, characterized in that, The active peptide compound powder is composed of fish collagen peptides, fructooligosaccharides and L-arabinose in a mass ratio of 10-15:3-5:2-4.

6. The compound probiotic preparation according to claim 1, characterized in that, The complex mineral elements are selected from one or more of calcium citrate, zinc gluconate, ferric pyrophosphate, sodium selenite, and tricalcium phosphate; the complex vitamins are selected from one or more of vitamin B1, vitamin B2, vitamin B6, vitamin B12, and vitamin D3.

7. The compound probiotic preparation according to claim 1, characterized in that, The moisture-proof coating material is composed of sodium octenyl succinate starch and gum arabic in a mass ratio of 2-3:

1.

8. A method for preparing a compound probiotic preparation according to any one of claims 1-7, characterized in that, Includes the following steps: S1. Mix the targeted controlled-release live bacteria microspheres, antioxidant sugar complex and maltodextrin, stir evenly to obtain a premixed composite powder; S2. Add the moisture-proof coating material to deionized water and stir evenly to obtain the moisture-proof coating solution; S3. Atomize and spray the moisture-proof coating liquid onto the surface of the premixed composite powder to obtain the coating intermediate; S4. The coating intermediate is dried by fluidized bed drying and cooled by fluidized bed cooling. Then it is mixed with active peptide compound powder, compound mineral elements and compound vitamins and stirred evenly to obtain a compound probiotic preparation.

9. The method for preparing the compound probiotic preparation according to claim 8, characterized in that, The atomization spraying parameters in step S3 include: fluidized bed inlet air temperature of 35-38℃, outlet air temperature of 25-28℃, atomization pressure of 0.15-0.25MPa, and spraying rate of 15-25mL / min.

10. The use of the compound probiotic preparation according to any one of claims 1-7 in the preparation of health foods or pharmaceuticals for assisting in enhancing the intestinal colonization ability of probiotics.