A composite probiotic preparation for promoting digestion and absorption and a preparation method thereof

Abstract

The application belongs to the technical field of probiotic preparations, and specifically discloses a compound probiotic preparation for promoting digestion and absorption and a preparation method thereof. The preparation comprises a probiotic compound, galactooligosaccharide, an activating agent and glycine; the probiotic compound is composed of plant lactobacillus and rhamnose lactobacillus; and the preparation method comprises the steps of bacterial body culture, carboxylation of galactooligosaccharide, activation coupling, reaction termination and purification, and freeze-drying. In the application, galactooligosaccharide is modified by carboxylation of succinic anhydride, and then, by using an activating agent composed of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide hydrochloride and N-hydroxysuccinimide, a stable amide bond is formed between the carboxylated galactooligosaccharide and the primary amino group on the surface of the probiotic bacterial body, so that the covalent grafting of galactooligosaccharide on the surface of the bacterial body is realized, and the problems of different action of prebiotics and probiotics in the physical mixed preparation and low survival rate of probiotics in gastrointestinal fluid are solved.

Description

Technical Field

[0001] This invention belongs to the field of compound probiotic preparation technology, and relates to a compound probiotic preparation that promotes digestion and absorption and its preparation method. Background Technology

[0002] The gut microbiota is closely related to the human digestive and absorptive functions. Probiotic preparations have been widely used to improve symptoms such as indigestion, bloating, diarrhea, and constipation by supplementing exogenous beneficial bacteria, inhibiting the colonization of pathogenic bacteria, improving the gut microecological environment, and secreting organic acids and digestive enzymes. Currently, most commercially available compound probiotic preparations adopt a physical mixing method, which is to simply mix probiotic powder with prebiotics such as galactooligosaccharides and fructooligosaccharides to make the preparation. This type of physical mixing preparation has the following inherent defects: (1) Probiotics need to be eroded by gastric acid and bile salts during oral administration, and the survival rate is generally low; (2) Free galactooligosaccharides are easily decomposed by gastric acid or prematurely degraded by host digestive enzymes, and may also be competitively consumed by intestinal transit bacteria, resulting in a significant reduction in the effective concentration of the target probiotics; (3) Probiotics and prebiotics migrate at different rates in the gastrointestinal tract, and the two often cannot reach the colon synchronously. Prebiotics cannot be efficiently utilized by the target bacteria, and synergistic effects are difficult to achieve.

[0003] Therefore, there is an urgent need to develop a novel compound probiotic preparation based on molecular-level chemical bonding technology, which can firmly link prebiotics and probiotics at the microscopic scale through covalent bonds, thereby solving the problems of ineffective prebiotic loss and low probiotic survival rate from the source. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a compound probiotic preparation that promotes digestion and absorption, and its preparation method. The core innovation of the present invention lies in the following: using chemical coupling technology, succinic anhydride is used to carboxylate galactooligosaccharides, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are used as activators to convert the carboxylated galactooligosaccharides into N-hydroxysuccinimide esters (NHS esters). These NHS esters undergo nucleophilic substitution reactions with the primary amino groups on the surface proteins and peptidoglycans of probiotic cells, forming stable amide bonds. This covalently grafts galactooligosaccharide molecules onto the surface of the probiotics, constructing a "probiotic-galactooligosaccharide" covalently coupled complex. This design enables the final compound probiotic preparation to solve the technical problems of low prebiotic utilization efficiency and poor probiotic survival rate in existing physically mixed probiotic preparations from the source.

[0005] To achieve the above and other objectives, the technical solution adopted by the present invention is as follows:

[0006] On one hand, the present invention provides a compound probiotic preparation that promotes digestion and absorption. The compound probiotic preparation is prepared from the following raw materials in parts by weight: 25-35 parts of probiotic complex, 10-20 parts of galactooligosaccharide, 0.5-2 parts of activator, and 0.1-0.5 parts of glycine. The probiotic complex is composed of Lactiplantibacillus plantarum and Lactobacillus rhamnosus, and the ratio of the number of live bacteria of the two is 1:1.5-2.5.

[0007] Furthermore, the ratio of viable Lactobacillus plantarum to Lactobacillus rhamnosus in the probiotic complex is preferably 1:2.

[0008] Furthermore, the activator is prepared by mixing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 4:1.

[0009] On the other hand, the present invention also provides a method for preparing a compound probiotic preparation that promotes digestion and absorption, comprising the following steps:

[0010] S1. Cultivation and Collection of Probiotic Complexes: *Lactobacillus plantarum* and *Lactobacillus rhamnosus* were inoculated into MRS liquid medium and anaerobically cultured at 37°C for 20-22 hours. After cultivation, the bacterial cells were collected by centrifugation at 6000 rpm for 10 minutes at 4°C. The cells were washed twice with sterile phosphate buffer (pH 7.2), then once with MES buffer, and resuspended in MES buffer. The bacterial suspension concentration was adjusted to 1×10⁻⁶. 10 -5×10 10 CFU / mL was used to obtain bacterial suspensions of each strain. The bacterial suspensions of the two strains were mixed according to the ratio of live bacteria to obtain probiotic suspensions for later use.

[0011] S2. Carboxyl activation of galactooligosaccharides: Galactooligosaccharides were dissolved in anhydrous dimethyl sulfoxide, and then succinic anhydride and 4-dimethylaminopyridine were added. The mixture was stirred at 40°C for 24 hours. After the reaction was completed, the reaction solution was slowly poured into anhydrous ethanol, and a precipitate was formed. The precipitate was collected by centrifugation, washed twice with anhydrous ethanol, dried under vacuum at room temperature, and then dissolved in deionized water. The product was dialyzed and freeze-dried to obtain purified carboxylated galactooligosaccharides.

[0012] S3. Probiotic Coupling: The carboxylated galactooligosaccharides obtained in step S2 are dissolved in MES buffer to prepare a solution. Then, an activator is added to the solution and activated at room temperature for 15 minutes to obtain an activated mixture. During this process, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in the activator activates the carboxyl groups on the carboxylated galactooligosaccharide molecules to generate an unstable O-acylisourea intermediate. This intermediate reacts with N-hydroxysuccinimide to form N-hydroxysuccinimide ester (NHS ester). Then, the probiotic suspension from step S1 is mixed with the activated mixture at a volume ratio of 1:1 and gently stirred at 4°C for 2-4 hours. During the reaction, the NHS ester undergoes a nucleophilic substitution reaction with the primary amino groups (-NH2) on the surface proteins / peptidoglycans of the probiotic suspension to form stable amide bonds (-CO-NH-), thereby covalently grafting the galactooligosaccharide molecules onto the surface of the bacteria.

[0013] S4. Reaction Termination and Purification: Add glycine to the above reaction system and stir for 15 minutes to quench any remaining activated esters. Then, collect the bacterial cells after reaction by centrifugation at 4°C and 6000 rpm for 10 minutes. Wash three times with sterile phosphate buffer (pH 7.2) to completely remove unreacted substances. Finally, resuspend the bacterial cells in a lyophilization protectant solution and adjust the viable count to 2 × 10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension;

[0014] S5. Freeze-drying: The bacterial suspension from step S4 is dispensed into freeze-drying pans and subjected to vacuum freeze-drying according to the following procedure: pre-freeze at -40°C for 3 hours, first dry at -20°C and 0.2 mbar for 24 hours, second dry at 25°C and 0.05 mbar for 6 hours, pulverize after freeze-drying, and pass through a 60-mesh sieve to obtain the compound probiotic preparation that promotes digestion and absorption.

[0015] Furthermore, the freeze-drying protectant solution in step S4 is prepared by mixing skim milk powder, trehalose, glycerin and deionized water in a mass ratio of 5:4:2:89.

[0016] This invention also provides an application of a compound probiotic preparation that promotes digestion and absorption. The application involves using the prepared compound probiotic preparation to prepare capsules or tablets. The capsules are prepared by mixing the dried compound probiotic preparation with microcrystalline cellulose as a flow aid, and filling the mixture into hydroxypropyl methylcellulose enteric-coated capsule shells. The tablets are prepared by using a dry direct compression process to compress the compound probiotic preparation, adding crospovidone as a disintegrant, and then controlling the compression pressure within the range of 8-12 kN.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] The core innovation of the compound probiotic preparation obtained by this invention lies in the use of chemical bonding technology to upgrade the traditional "loose physical mixing" of galactooligosaccharides and probiotics to "tight molecular-level bonding". Specifically, in the preparation process, this invention utilizes an activator system composed of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to initiate a precise catalytic reaction under a mild, weakly acidic environment. This reaction allows the succinic anhydride-modified galactooligosaccharides to undergo a nucleophilic substitution reaction with the proteins and peptidoglycans on the surface of the probiotic cells, ultimately locking the two together firmly through "amide bonds". This surface modification network with covalent bonds as the absolute connection points solves the problems of asynchronous action of galactooligosaccharides and probiotics in the gastrointestinal tract and low prebiotic utilization efficiency in traditional physically mixed preparations, ensuring that galactooligosaccharides are always anchored on the surface of the cells, achieving in-situ nutrient supply and protection.

[0019] Thanks to the unique covalently bonded structure and surface micro-remodeling mechanism described above, the compound probiotic formulation of this invention exhibits superior performance compared to physically mixed formulations. First, the covalently grafted galactooligosaccharides form a hydrophilic molecular brush protective layer on the bacterial surface, significantly improving the probiotics' tolerance to gastric acid and bile salts. Second, this covalent binding endows prebiotics with absolute "targeted delivery" characteristics, solving the problem of free galactooligosaccharides being prematurely degraded by host digestive enzymes or competitively consumed by transiting intestinal bacteria after oral administration. Third, the surface-anchored galactooligosaccharides can act as molecular baits, competitively binding to pathogens and reducing harmful bacterial colonization. Simultaneously, the short-chain fatty acids produced by probiotic metabolism repair the intestinal barrier, achieving a dual synergistic effect of pathogen resistance and barrier protection. Furthermore, based on this chemical coupling technology, the compound probiotic formulation prepared by this invention can achieve better digestion and absorption effects, fundamentally eliminating gastrointestinal adverse reactions such as bloating and borborygmus that are easily caused by traditional physical addition of high doses of free prebiotics. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0021] Figure 1 The figures show the test results of the artificial gastrointestinal fluid tolerance of the probiotic preparations prepared in various embodiments and comparative examples of the present invention;

[0022] Figure 2 The in vitro intestinal adhesion rate of the probiotic preparations prepared in the various embodiments and comparative examples of the present invention;

[0023] Figure 3 The figures show the in vitro digestion-promoting activity test results of the probiotic preparations prepared in various embodiments and comparative examples of the present invention. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0026] The materials used in the following implementations include an activator prepared from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 4:1, and a lyophilization protectant solution prepared from skim milk powder, trehalose, glycerol, and deionized water in a mass ratio of 5:4:2:89. Unless otherwise specified, all other materials are new materials purchased from the market, including a sterile phosphate buffer with pH 7.2 and a MES buffer with pH 5.5 at 0.1M.

[0027] Example 1: This example provides a compound probiotic preparation that promotes digestion and absorption. The compound probiotic preparation is prepared from the following raw materials in parts by weight: 30 parts of probiotic complex, 15 parts of galactooligosaccharide, 1 part of activator, and 0.3 parts of glycine; wherein the ratio of viable bacteria of Lactobacillus plantarum to Lactobacillus rhamnosus in the probiotic complex is 1:2.

[0028] This embodiment also provides a method for preparing a compound probiotic preparation that promotes digestion and absorption, including the following steps:

[0029] S1. Cultivation and Collection of Probiotic Complexes: *Lactobacillus plantarum* and *Lactobacillus rhamnosus* were inoculated separately into MRS liquid medium at a 5% inoculum size and anaerobically cultured at 37°C for 20 hours. After cultivation, the bacterial cells were collected by centrifugation at 6000 rpm for 10 minutes at 4°C. The cells were washed twice with 20 mL of sterile phosphate buffer, then once with MES buffer, and resuspended in MES buffer. The concentration of *Lactobacillus plantarum* was adjusted to 1 × 10⁻⁶ using a hemocytometer combined with plate counting. 10 CFU / mL, Lactobacillus rhamnosus concentration up to 2×10 10 CFU / mL, take 5mL of Lactobacillus plantarum suspension and mix with 5mL of Lactobacillus rhamnosus suspension to obtain 10mL of probiotic suspension;

[0030] S2. Carboxyl activation of galactooligosaccharides: Accurately weigh 1.5 g of galactooligosaccharides and dissolve them in 15 mL of anhydrous dimethyl sulfoxide. Then add 0.9 g of succinic anhydride and 0.05 g of 4-dimethylaminopyridine to the solution. Stir the mixture at 40 °C for 24 hours. After the reaction is complete, slowly pour the reaction solution into 150 mL of anhydrous ethanol to precipitate the product. Centrifuge at 6000 rpm for 5 minutes to collect the precipitate. Resuspend the precipitate in 50 mL of anhydrous ethanol and wash it twice. After vacuum drying at room temperature, dissolve the product in 50 mL of deionized water and dialyze it at 4 °C for 24 hours using a dialysis bag with a molecular weight cutoff of 500 Da. Freeze-dry the dialysate to obtain purified carboxylated galactooligosaccharides.

[0031] S3. Probiotic conjugation: Dissolve the carboxylated galactooligosaccharide obtained in step S2 in 25.6 mL of MES buffer to prepare a 5% (w / v) solution. Then add an activator to the solution and activate at room temperature for 15 minutes to obtain an activated mixture. Then, mix the probiotic suspension from step S1 with the activated mixture at a volume ratio of 1:1 and gently stir at 4°C for 3 hours.

[0032] S4. Reaction Termination and Purification: Add glycine to the above reaction system and stir for 15 minutes to quench any remaining activated esters. Then, collect the bacterial cells by centrifugation at 4°C and 6000 rpm for 10 minutes. Wash three times with 30 mL of sterile phosphate buffer to completely remove unreacted substances. Finally, resuspend the bacterial cells in a lyophilization protectant solution and adjust the viable count to 2 × 10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension;

[0033] S5. Freeze-drying: The bacterial suspension from step S4 is dispensed into freeze-drying pans and subjected to vacuum freeze-drying according to the following procedure: pre-freeze at -40°C for 3 hours, first dry at -20°C and 0.2 mbar for 24 hours, second dry at 25°C and 0.05 mbar for 6 hours, pulverize after freeze-drying, and pass through a 60-mesh sieve to obtain the compound probiotic preparation that promotes digestion and absorption.

[0034] Example 2: This example provides a compound probiotic preparation that promotes digestion and absorption. The compound probiotic preparation is prepared from the following raw materials in parts by weight: 25 parts of probiotic complex, 10 parts of galactooligosaccharide, 0.5 parts of activator, and 0.1 parts of glycine; wherein the ratio of viable bacteria of Lactobacillus plantarum to Lactobacillus rhamnosus in the probiotic complex is 1:1.5.

[0035] This embodiment also provides a method for preparing a compound probiotic preparation that promotes digestion and absorption, including the following steps:

[0036] S1. Cultivation and Collection of Probiotic Complexes: *Lactobacillus plantarum* and *Lactobacillus rhamnosus* were inoculated separately into MRS liquid medium at a 5% inoculum size and anaerobically cultured at 37°C for 20 hours. After cultivation, the bacterial cells were collected by centrifugation at 6000 rpm for 10 minutes at 4°C. The cells were washed twice with 20 mL of sterile phosphate buffer, then once with MES buffer, and resuspended in MES buffer. The concentration of *Lactobacillus plantarum* was adjusted to 1 × 10⁻⁶ using a hemocytometer combined with plate counting. 10 CFU / mL, Lactobacillus rhamnosus concentration up to 1.5 × 10⁻⁶ 10 CFU / mL, take 5mL of Lactobacillus plantarum suspension and mix with 5mL of Lactobacillus rhamnosus suspension to obtain 10mL of probiotic suspension;

[0037] S2. Carboxyl activation of galactooligosaccharides: Accurately weigh 1.0 g of galactooligosaccharides and dissolve them in 10 mL of anhydrous dimethyl sulfoxide. Then add 0.6 g of succinic anhydride and 0.03 g of 4-dimethylaminopyridine to the solution. Stir the mixture at 40 °C for 24 hours. After the reaction is complete, slowly pour the reaction solution into 100 mL of anhydrous ethanol to precipitate the product. Collect the precipitate by centrifugation at 6000 rpm for 5 minutes. Resuspend the precipitate in 50 mL of anhydrous ethanol and wash it twice. After each wash, centrifuge at 6000 rpm for 5 minutes to collect the precipitate. After vacuum drying at room temperature, dissolve the product in 40 mL of deionized water and dialyze it at 4 °C for 24 hours using a dialysis bag with a molecular weight cutoff of 500 Da. Change the deionized water every 8 hours. After dialysis, remove the dialysate and freeze-dry it to obtain purified carboxylated galactooligosaccharides.

[0038] S3. Probiotic conjugation: Dissolve the carboxylated galactooligosaccharide obtained in step S2 in 24.6 mL of MES buffer to prepare a 5% (w / v) solution. Then add an activator and activate at room temperature for 15 minutes to obtain an activated mixture. Then, mix the probiotic suspension from step S1 with the activated mixture at a volume ratio of 1:1 and gently stir at 4°C for 2 hours.

[0039] S4. Reaction Termination and Purification: Add glycine to the above reaction system and stir for 15 minutes to quench any remaining activated esters. Then, collect the bacterial cells by centrifugation at 4°C and 6000 rpm for 10 minutes. Wash three times with 30 mL of sterile phosphate buffer to completely remove unreacted substances. Finally, resuspend the bacterial cells in a lyophilization protectant solution and adjust the viable count to 2 × 10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension;

[0040] S5. Freeze-drying: The bacterial suspension from step S4 is dispensed into freeze-drying pans and subjected to vacuum freeze-drying according to the following procedure: pre-freeze at -40°C for 3 hours, first dry at -20°C and 0.2 mbar for 24 hours, second dry at 25°C and 0.05 mbar for 6 hours, pulverize after freeze-drying, and pass through a 60-mesh sieve to obtain the compound probiotic preparation that promotes digestion and absorption.

[0041] Example 3: This example provides a compound probiotic preparation that promotes digestion and absorption. The compound probiotic preparation is prepared from the following raw materials in parts by weight: 35 parts of probiotic complex, 20 parts of galactooligosaccharide, 2 parts of activator, and 0.5 parts of glycine; wherein the ratio of viable bacteria of Lactobacillus plantarum to Lactobacillus rhamnosus in the probiotic complex is 1:2.5.

[0042] This embodiment also provides a method for preparing a compound probiotic preparation that promotes digestion and absorption, including the following steps:

[0043] S1. Cultivation and Collection of Probiotic Complexes: *Lactobacillus plantarum* and *Lactobacillus rhamnosus* were inoculated separately into MRS liquid medium at a 5% inoculum size and anaerobically cultured at 37°C for 21 hours. After cultivation, the bacterial cells were collected by centrifugation at 6000 rpm for 10 minutes at 4°C. The cells were washed twice with 20 mL of sterile phosphate buffer, then once with MES buffer, and resuspended in MES buffer. The concentration of *Lactobacillus plantarum* was adjusted to 2 × 10⁻⁶ using a hemocytometer combined with plate counting. 10 CFU / mL, Lactobacillus rhamnosus concentration up to 5×10 10 CFU / mL, take 5mL of Lactobacillus plantarum suspension and mix with 5mL of Lactobacillus rhamnosus suspension to obtain 10mL of probiotic suspension;

[0044] S2. Carboxyl activation of galactooligosaccharides: Accurately weigh 2.0 g of galactooligosaccharides and dissolve them in 20 mL of anhydrous dimethyl sulfoxide. Then, add 1.2 g of succinic anhydride and 0.067 g of 4-dimethylaminopyridine to the solution. Stir the mixture at 40 °C for 24 hours. After the reaction is complete, slowly pour the reaction solution into 200 mL of anhydrous ethanol to precipitate the product. Centrifuge at 6000 rpm for 5 minutes to collect the precipitate. Resuspend the precipitate in 60 mL of anhydrous ethanol and wash it twice. After vacuum drying at room temperature, dissolve the product in 50 mL of deionized water and dialyze it at 4 °C for 24 hours using a dialysis bag with a molecular weight cutoff of 500 Da. Freeze-dry the dialysate to obtain purified carboxylated galactooligosaccharides.

[0045] S3. Probiotic conjugation: Dissolve the carboxylated galactooligosaccharide obtained in step S2 in 40 mL of MES buffer to prepare a 5% (w / v) solution. Take 10 mL of this solution and add an activator to it. Activate at room temperature for 15 minutes to obtain an activated mixture. Then, mix the probiotic suspension from step S1 with the activated mixture at a volume ratio of 1:1 and gently stir at 4°C for 4 hours.

[0046] S4. Reaction Termination and Purification: Add glycine to the above reaction system and stir for 15 minutes to quench any remaining activated esters. Then, collect the bacterial cells by centrifugation at 4°C and 6000 rpm for 10 minutes. Wash three times with 30 mL of sterile phosphate buffer to completely remove unreacted substances. Finally, resuspend the bacterial cells in a lyophilization protectant solution and adjust the viable count to 2 × 10⁻⁶. 10 CFU / mL was used to obtain a bacterial suspension;

[0047] S5. Freeze-drying: The bacterial suspension from step S4 is dispensed into freeze-drying pans and subjected to vacuum freeze-drying according to the following procedure: pre-freeze at -40°C for 3 hours, first dry at -20°C and 0.2 mbar for 24 hours, second dry at 25°C and 0.05 mbar for 6 hours, pulverize after freeze-drying, and pass through a 60-mesh sieve to obtain the compound probiotic preparation that promotes digestion and absorption.

[0048] Example 4: The difference between this example and Example 1 is that step S5 in the preparation method of a compound probiotic preparation that promotes digestion and absorption is modified to: freeze drying: The bacterial suspension from step S4 is dispensed into a freeze-drying tray and subjected to vacuum freeze drying according to the following procedure: pre-freezing at -40°C for 3 hours, first drying at -20°C and 0.2 mbar for 24 hours, second drying at 25°C and 0.05 mbar for 6 hours, after freeze drying, it is pulverized and passed through a 60-mesh sieve, and then mixed with microcrystalline cellulose as a flow aid, and filled into hydroxypropyl methylcellulose enteric-coated capsule shells to obtain the compound probiotic preparation that promotes digestion and absorption. The remaining steps and raw materials are the same as in Example 1.

[0049] Example 5: The difference between this example and Example 1 is that step S5 in the preparation method of a compound probiotic preparation that promotes digestion and absorption is modified to: freeze drying: The bacterial suspension from step S4 is dispensed into freeze-drying pans and subjected to vacuum freeze drying according to the following procedure: pre-freezing at -40°C for 3 hours, first drying at -20°C and 0.2 mbar for 24 hours, second drying at 25°C and 0.05 mbar for 6 hours, after freeze drying, it is pulverized and passed through a 60-mesh sieve, and then a dry direct tableting process is adopted, with cross-linked povidone added as a disintegrant, and the tableting pressure is controlled at 10 kN to obtain the compound probiotic preparation that promotes digestion and absorption. The remaining steps and raw materials are the same as in Example 1.

[0050] Comparative Example 1: This comparative example provides a compound probiotic preparation, which differs from Example 1 in that the raw materials do not include an activator. The corresponding preparation method is modified in step S3 as follows: the carboxylated galactooligosaccharide obtained in step S2 is dissolved in 25.6 mL of MES buffer to prepare a 5% (w / v) solution, and allowed to stand at room temperature for 15 minutes without adding any activator. Then, the probiotic suspension from step S1 is mixed with the above solution at a volume ratio of 1:1, and the mixture is gently stirred at 4°C for 3 hours. The remaining steps are the same as in Example 1.

[0051] Comparative Example 2: This comparative example provides a compound probiotic preparation, which differs from Example 1 in that the carboxylation of galactooligosaccharides and the probiotic coupling step are not performed in the preparation steps. That is, step S2 in the preparation method is modified to: physical dispersion of galactooligosaccharides: accurately weigh 1.5g of galactooligosaccharides and dissolve them in 30mL of MES buffer, and stir magnetically until completely dissolved to obtain an galactooligosaccharide solution; step S3 is modified to: physical mixing and contact: mix the galactooligosaccharide solution obtained in step S2 with the probiotic suspension obtained in step S1 at a volume ratio of 1:1, and gently stir the mixture at 4°C for 3 hours. This step is only a physical contact between galactooligosaccharide molecules and probiotic cells, and no covalent coupling reaction occurs; the remaining steps are the same as in Example 1.

[0052] Comparative Example 3: This comparative example provides a single-strain probiotic preparation. The difference between this and the other examples in Example 1 is that the probiotic complex in the raw materials was replaced with an equal weight of *Lactobacillus plantarum*. The corresponding step S1 was modified as follows: *Lactobacillus plantarum* was inoculated into MRS liquid medium at a 5% inoculum and cultured anaerobically at 37°C for 20 hours. After the culture, the cells were collected by centrifugation at 6000 rpm for 10 minutes at 4°C. The cells were washed twice with 20 mL of sterile phosphate buffer, then washed once with MES buffer, and resuspended in MES buffer. The concentration of *Lactobacillus plantarum* was adjusted to 3 × 10⁻⁶ using a hemocytometer combined with plate counting. 10 CFU / mL, take 10mL of Bacillus plantarum suspension to obtain probiotic suspension; the remaining steps are the same as in Example 1.

[0053] Experimental methods:

[0054] Artificial gastrointestinal fluid tolerance test: The probiotic preparations prepared in Examples 1-5 and Comparative Examples 1-3 were respectively placed in simulated gastric fluid (pH 2.0, containing 3 g / L pepsin) for 2 hours, and then transferred to simulated intestinal fluid (pH 6.8, containing 1 g / L trypsin) for 2 hours. Samples were taken at 0 and 2 hours, and the viable bacteria count was determined by plate counting method. The survival rate (%) was calculated. Each sample was measured in triplicate. The results are as follows: Figure 1 As shown.

[0055] In vitro intestinal adhesion test: Caco-2 cells were seeded in 24-well plates and cultured until a dense monolayer formed. The probiotic preparations prepared in Examples 1-5 and Comparative Examples 1-3 were resuspended in PBS buffer and added to the wells. The plates were incubated at 37°C and 5% CO2 for 2 hours. After incubation, the cells were washed three times with PBS buffer to remove unadhered bacteria. Cells were then lysed by digestion with 0.25% trypsin. After dilution, the number of viable bacteria adhering to the cells was counted using a plate count method, and the adhesion rate (%) was calculated. The results are shown below. Figure 2 As shown.

[0056] In vitro digestion-promoting activity test: A lipase inhibition / promotion assay was used. 0.05 g of each of the preparations from the examples and comparative examples was dissolved in 5 mL of PBS buffer, and 1 mL of 4-nitrophenyl octanoate solution was added. After preheating at 37°C for 5 minutes, 1 mL of porcine pancreatic lipase solution was added, and the mixture was quickly mixed. The absorbance change over time was immediately measured at 405 nm, and the enzyme reaction rate was calculated. The digestion promotion rate = The result is as follows Figure 3 As shown.

[0057] Animal in vivo experiments: Forty SPF-grade male Kunming mice were acclimatized for 3 days. Except for the blank control group, the remaining groups were administered rhubarb decoction daily by gavage to establish a spleen-deficiency indigestion model. After successful model establishment, the mice were randomly divided into: blank control group, model control group, Example 1 group, Comparative Example 1 group, Comparative Example 2 group, and Comparative Example 3 group. The Example 1 and Comparative Example 2 groups were administered 0.20g of the Example or Comparative Example preparation daily by gavage, while the blank control group and model control group were administered an equal volume of physiological saline daily by gavage for 7 consecutive days. On day 8, the mice were sacrificed, and the contents of the cecum were aseptically collected to determine the viable bacterial count. Blood samples were also collected to determine serum D-xylose (reflecting small intestinal absorption function) and gastrin levels (reflecting digestive function).

[0058] Table 1 Results of in vivo animal experiments

[0059]

[0060] observe Figure 1-3As shown in Table 1, Examples 1-5 significantly outperformed Comparative Examples 1-3 and the blank control group in all key indicators. Regarding tolerability and adhesion, the gastric fluid survival rate and cumulative intestinal fluid survival rate of Examples 1-5 were significantly higher than those of Comparative Examples 1-3. This demonstrates that the present invention modifies galactooligosaccharides by carboxylation with succinic anhydride and covalently couples them to the surface of probiotic cells using an activator, forming a stable hydrated protective layer of galactooligosaccharides around the cells. This protective layer effectively neutralizes hydrogen ions in gastric acid, resists the enzymatic action of pepsin, and maintains structural integrity in intestinal fluid, thereby significantly improving the survival rate of probiotics in the gastrointestinal environment. The cell adhesion rate of Examples 1-5 was also significantly higher than that of Comparative Examples 1-3. This indicates that after the present invention stably grafts galactooligosaccharides onto the cell surface via amide bonds, galactooligosaccharides, acting as a "molecular anchor," can specifically recognize and bind to galactolectin receptors on the brush border membrane of Caco-2 cells, achieving active targeted adhesion of probiotics. Figure 3 The results further demonstrate that the formulation of this invention, through covalent coupling technology to anchor galacto-oligosaccharides to the bacterial surface, not only plays a protective role but also acts as a highly efficient biocatalyst carrier, enhancing the rate of enzymatic reactions. Serum D-xylose and gastrin are highly representative clinical indicators in gastroenterology. Observation of the data in Table 1 shows that Example 1 performed excellently in terms of in vivo digestive and absorption indicators. This indicates that the formulation of this invention significantly improves the survival and colonization of probiotics in the intestine through chemical coupling technology, thereby substantially improving the small intestinal absorption function and gastric digestive and secretory function of mice by regulating the intestinal microecology, providing solid pharmacodynamic support for the clinical treatment of malabsorption.

[0061] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0062] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A compound probiotic preparation that promotes digestion and absorption, characterized in that, The compound probiotic preparation is prepared from the following raw materials in parts by weight: 25-35 parts of probiotic complex, 10-20 parts of galactooligosaccharide, 0.5-2 parts of activator, and 0.1-0.5 parts of glycine; the probiotic complex is composed of *Lactobacillus plantarum* and *Lactobacillus rhamnosus*, with a live bacteria ratio of 1:1.5-2.5; the activator is prepared from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a mass ratio of 4:

1.

2. The compound probiotic preparation for promoting digestion and absorption according to claim 1, characterized in that, The ratio of viable Lactobacillus plantarum to Lactobacillus rhamnosus in the probiotic complex is 1:

2.

3. A method for preparing a compound probiotic preparation for promoting digestion and absorption according to any one of claims 1-2, characterized in that, Includes the following steps: S1. Cultivation and collection of probiotic complex: Lactobacillus plantarum and Lactobacillus rhamnosus were inoculated into MRS liquid medium, respectively. After anaerobic culture, the bacterial cells were collected by centrifugation, washed and resuspended in MES buffer to obtain bacterial suspensions of each strain. The bacterial suspensions of the two strains were mixed according to the ratio of viable cells to obtain probiotic suspension. S2. Carboxyl activation of galactooligosaccharides: Galactooligosaccharides were dissolved in anhydrous dimethyl sulfoxide, and then succinic anhydride and 4-dimethylaminopyridine were added to the solution and stirred. The reaction solution was then slowly poured into anhydrous ethanol to precipitate the precipitate. The precipitate was centrifuged, washed, dialyzed, and freeze-dried to obtain carboxylated galactooligosaccharides. S3. Probiotic conjugation: Carboxylated galactooligosaccharides are dissolved in MES buffer to prepare a solution. Then, an activator is added to the solution and activated at room temperature to obtain an activated mixture. The probiotic suspension is then mixed with the activated mixture and stirred to react. S4. Reaction termination and purification: Add glycine to the above reaction system, stir and mix, centrifuge to collect the bacterial cells after the reaction, wash with sterile phosphate buffer at pH 7.2, and then resuspend the bacterial cells in a lyophilization protectant solution to obtain a bacterial suspension. S5. Freeze-drying: The bacterial suspension is dispensed into freeze-drying trays and subjected to vacuum freeze-drying. After freeze-drying, it is pulverized and sieved to obtain the compound probiotic preparation that promotes digestion and absorption.

4. The method for preparing a compound probiotic preparation for promoting digestion and absorption according to claim 3, characterized in that, The specific washing steps for resuspending in MES buffer after washing in step S1 are as follows: first wash with sterile phosphate buffer at pH 7.2, and then wash with MES buffer.

5. The method for preparing a compound probiotic preparation for promoting digestion and absorption according to claim 3, characterized in that, The volume ratio of the probiotic suspension to the activated mixture in step S2 is 1:

1.

6. The method for preparing a compound probiotic preparation for promoting digestion and absorption according to claim 3, characterized in that, The freeze-drying protectant solution in step S4 is composed of skim milk powder, trehalose, glycerin and deionized water in a mass ratio of 5:4.

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