Multifunctional probiotic preparation and preparation method thereof

Abstract

The invention relates to the technical field of probiotic preparations, and particularly discloses a multifunctional probiotic preparation and a preparation method thereof. According to the invention, bifidobacterium animalis Bb-12, lactobacillus rhamnosus GG and phytobacterium plantarum LP are compounded, the three strains have a synergistic effect to realize comprehensive intestinal regulation, modified chitosan is adopted as a coating, a compact structure of a covalent skeleton formed by cross-linking genipin, amino acid and casein phosphopeptides are complexed step by step, gastric juice erosion can be resisted, and the intestinal tract can be fully regulated. Thalli are prevented from being damaged by gastric acid; the amino acid in the coating layer improves the intestinal mucosa adhesion ability of the probiotics, and the casein phosphopeptides promote mineral absorption and cooperate with the probiotics to further strengthen the intestinal regulation effect.

Description

Technical Field

[0001] This invention relates to the field of probiotic preparation technology, and in particular to a multifunctional probiotic preparation and its preparation method. Background Technology

[0002] Probiotics are live microorganisms that colonize the human gut and reproductive system, improving the host's microecological balance and producing definite health benefits. They possess three core characteristics: activity, colonization, and beneficialness. Inactive microorganisms cannot exert their physiological functions. Commonly used gut probiotics include Lactobacillus, Bifidobacterium, and other beneficial bacteria such as Saccharomyces boulardii and Bacillus subtilis. Different strains colonize different parts of the intestine, such as the small and large intestines, each undertaking different physiological functions. The balance of gut microbiota is crucial for gut health. In a healthy state, beneficial bacteria dominate while harmful bacteria are suppressed. Once this balance is disrupted, various intestinal discomforts such as diarrhea, constipation, and bloating can occur. The core function of probiotics is to supplement and strengthen the proportion of beneficial bacteria in the gut, helping to restore the balance of the gut microecology.

[0003] Multifunctional probiotic preparations are highly active compound formulations of probiotics and are currently the most commonly used form of probiotic products for daily gut health. They scientifically combine multiple probiotic strains and are produced as powder using vacuum freeze-drying technology. Compared to ordinary probiotic tablets and oral liquids, compound freeze-drying offers many significant advantages. The compounded strains can cover the entire intestinal tract, and the strains can mutually promote proliferation and achieve synergistic effects. Vacuum freeze-drying technology allows probiotics to enter a dormant state, significantly reducing activity loss during production, storage, and consumption. Most products have an active shelf life of 18-24 months at room temperature. After entering the human gut, they can quickly reactivate and colonize upon contact with water and nutrients. Furthermore, the powder form is fine and easy to dissolve, and can be directly mixed with warm water or added to milk or complementary foods.

[0004] CN121101169A discloses a probiotic chitosan microsphere and its preparation method, belonging to the field of probiotic encapsulation technology. The specific steps of the preparation method are as follows: first, trehalose and L-glutamine are dissolved in water; then, lactic acid bacteria are suspended in the above solution, and subsequently, the mixture is thoroughly mixed with the chitosan solution to obtain a mixed solution; this mixed solution is added dropwise to a sodium tripolyphosphate solution, and the resulting insoluble matter is washed; finally, the microspheres are obtained by freeze-drying. Trehalose and L-glutamine possess both probiotic protectant and nutrient supply functions, and their synergistic effect can significantly improve the survival rate of probiotics; when used in combination with chitosan, sodium tripolyphosphate, and other components, it can comprehensively enhance the environmental tolerance and activity stability of probiotics. Meanwhile, by combining specific curing processes with freeze-drying technology, damage to probiotics caused by high temperatures can be effectively avoided. The resulting microspheres have excellent probiotic protection capabilities, good storage stability, and suitable intestinal release performance, which can effectively improve the survival efficiency of probiotics during storage and practical application, and more fully exert their role in regulating the balance of intestinal flora and improving the health level of the host.

[0005] CN113892652A discloses an oil-soluble probiotic microcapsule composite freeze-dried powder and its preparation method. The core of this invention lies in employing microcapsule technology to encapsulate active probiotics within microcapsules with a specific capsule wall structure, achieving a dual technological breakthrough: Firstly, the constructed microcapsule wall possesses excellent mechanical strength, stably encapsulating the active probiotics within the capsule cavity, effectively solving industry pain points such as easy inactivation of live bacteria, large fluctuations in bacterial count, short shelf life, and unstable quality in traditional probiotic products; secondly, after entering the human stomach, the capsule wall structure resists the acidic environment of gastric juice and does not decompose. Upon reaching the intestines, it rapidly dissolves and releases probiotics in the intestinal fluid environment, ensuring that the active probiotics efficiently reach the site of action and are absorbed by the intestines, fully exerting their physiological effects.

[0006] It is important to note that the gut-regulating effects of probiotics depend entirely on their activity. Even if multifunctional probiotic formulations overcome the challenges of preserving activity, improper consumption and storage can still lead to a loss of activity. Since the pH of gastric juice is approximately 1-3 when the stomach is empty, making it highly acidic, probiotic strains that have not undergone any protective treatment will experience a significant loss of activity when passing through the stomach. Therefore, addressing this issue is crucial to understanding how probiotics regulate gut function. Summary of the Invention

[0007] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a multifunctional probiotic preparation and a method for preparing the same.

[0008] This invention employs a combination of Bifidobacterium animalis Bb-12, Lactobacillus rhamnosus GG, and Lactobacillus plantarum LP, with the three strains working synergistically to achieve comprehensive intestinal regulation. Bb-12 can regulate the balance of intestinal flora, inhibit the proliferation of harmful bacteria, and enhance the intestinal mucosal barrier function, making it particularly suitable for people with sensitive intestines; the GG strain has outstanding acid and bile salt tolerance, can stably colonize in the intestine, promote the repair of intestinal epithelial cells, and reduce intestinal inflammatory responses; LP can metabolize to produce organic acids, lower the intestinal pH, create a suitable environment for the growth of beneficial bacteria, and at the same time promote intestinal peristalsis, improving both constipation and diarrhea.

[0009] To address the issue of probiotics being destroyed by stomach acid in the stomach, thus hindering their release into the intestines, this invention employs modified chitosan as a coating. The dense structure of the covalent backbone formed by genipin cross-linking, which is stepwise complexed with amino acids and casein phosphopeptides, resists gastric acid erosion and prevents bacterial cell destruction. The low-acyl gellan gum in the coating layer responds to intestinal calcium... 2+ Upon contact with the pH environment, the substance swells and ruptures, enabling targeted release into the intestines. The amino acids in the coating layer enhance the adhesion of probiotics to the intestinal mucosa, while casein phosphopeptides promote mineral absorption, synergistically strengthening the intestinal regulatory effect in conjunction with probiotics.

[0010] To achieve the above objectives, the present invention provides a method for preparing a multifunctional probiotic preparation, comprising the following steps: S1. Add Bifidobacterium animalis Bb-12, Lactobacillus rhamnosus GG, and Lactobacillus plantarum LP to PBS buffer, stir well to obtain a bacterial suspension, and refrigerate for later use. S2. After dissolving xylooligosaccharide, skim milk powder, mannitol and water at 70-80℃, add PEG-400, stir evenly, add bacterial suspension dropwise, mix evenly and freeze dry to obtain core material freeze-dried powder. S3. Add chitosan to water, adjust the pH with acid, and stir to dissolve to obtain a chitosan solution; add whey protein to water and stir evenly at 30-40℃ to obtain a whey protein solution; add the whey protein solution dropwise to the chitosan solution, adjust the pH, add 30wt% genipin aqueous solution, stir at 35-50℃ for 3-5 hours, add 20wt% amino acid aqueous solution, stir for 30-60 minutes, add 20wt% casein phosphopeptide aqueous solution, continue stirring for 1-2 hours, concentrate under reduced pressure to a solid content of 20-30%, and freeze-dry to obtain modified chitosan; S4. Add modified chitosan to water and stir evenly to obtain modified chitosan substrate solution; add low acyl gellan gum and potassium citrate to water and stir evenly at 40-50℃ to obtain crosslinking solution; add crosslinking solution to substrate solution, add propylene glycol and adjust pH to weakly acidic; sieve to obtain coating solution and then coat the core material freeze-dried powder; freeze-dry to obtain multifunctional probiotic preparation.

[0011] Furthermore, the mass ratio of Bifidobacterium animalis Bb-12, Lactobacillus rhamnosus GG, Lactobacillus plantarum LP, and PBS buffer is 1-3:1-3:1-2:5-10; the viable count of Bifidobacterium animalis Bb-12 is >1.0×10⁻⁶. 11 CFU / g; viable count of Lactobacillus rhamnosus GG > 1.5 × 10⁻⁶ 11 CFU / g; viable count of *Lactobacillus plantarum* LP > 2.0 × 10⁻⁶ 11 CFU / g.

[0012] Furthermore, the mass ratio of the xylooligosaccharide, skim milk powder, mannitol, PEG-400, water, and bacterial suspension is 1-2:1.4-1.5:1-2:0.01-1:5-15:0.5-2.

[0013] Furthermore, the mass ratio of chitosan to water is 1-1.5:15-25.

[0014] Furthermore, the mass ratio of whey protein to water is 1-2:5-10.

[0015] Furthermore, the mass ratio of chitosan to whey protein, genipin, amino acids, and casein phosphopeptide is 1-2:0.1-1:0.1-0.5:0.2-0.5:0.1-0.3.

[0016] Furthermore, the mass ratio of the modified chitosan to water is 1-2:100-150.

[0017] Furthermore, the mass ratio of the low-acyl gellan gum, potassium citrate, and water is 1-2:0.5-0.8:30-40.

[0018] Furthermore, the amino acid polymer is one of polyglutamic acid, polythreonine, or polyserine.

[0019] Preferably, the preparation method of the multifunctional probiotic preparation includes the following steps, in parts by weight: S1. Add 1-3 parts of Bifidobacterium animalis Bb-12, 1-3 parts of Lactobacillus rhamnosus GG, and 1-2 parts of Lactobacillus plantarum LP to 5-10 parts of PBS buffer, stir well to obtain a bacterial suspension, and refrigerate for later use. S2. Dissolve 1-2 parts xylooligosaccharide, 1.4-1.5 parts skim milk powder, 1-2 parts mannitol and 5-15 parts water at 70-80℃, then add 0.01-1 parts PEG-400, stir evenly, cool to room temperature and add 0.5-2 parts bacterial suspension, mix evenly and freeze dry to obtain core material freeze-dried powder. S3. Add 10-15 parts of chitosan to 150-250 parts of water, adjust the pH to 5-5.5 with acid, and stir to dissolve to obtain a chitosan solution; add 5-10 parts of whey protein to 25-50 parts of water and stir evenly at 30-40℃ to obtain a whey protein solution; add the whey protein solution dropwise to the chitosan solution, adjust the pH, add 10-20 parts of 30wt% genipin aqueous solution, stir at 35-50℃ for 3-5 hours, then add 15-25 parts of 20wt% amino acid aqueous solution, stir for 30-60 minutes, then add 5-15 parts of 20wt% casein phosphopeptide aqueous solution, continue stirring for 1-2 hours, concentrate under reduced pressure to a solid content of 20-30%, and freeze-dry to obtain modified chitosan; S4. Add 1-2 parts of modified chitosan to 100-150 parts of water and stir evenly to obtain modified chitosan substrate solution; add 1-2 parts of low acyl gellan gum and 0.5-0.8 parts of potassium citrate to 30-40 parts of water and stir evenly at 40-50℃ to obtain crosslinking solution; add crosslinking solution to substrate solution, add propylene glycol and then adjust pH to 5.5-6 with sodium citrate buffer; after sieving to obtain coating solution, coat the core material freeze-dried powder; freeze-dry to obtain multifunctional probiotic preparation.

[0020] The present invention also provides a multifunctional probiotic preparation, which is prepared by the above method.

[0021] The beneficial effects of this invention are: 1. Compared with existing technologies, this invention uses a combination of Bifidobacterium animalis Bb-12, Lactobacillus rhamnosus GG, and Lactobacillus plantarum LP. Bb-12 can regulate the balance of intestinal flora, inhibit the proliferation of harmful bacteria, and enhance the intestinal mucosal barrier function; the GG strain has outstanding acid and bile salt resistance, can stably colonize in the intestine, promote the repair of intestinal epithelial cells, and reduce intestinal inflammatory response; LP can metabolize to produce organic acids, lower the pH value of the intestine, create a suitable environment for the growth of beneficial bacteria, and at the same time promote intestinal peristalsis, improving both constipation and diarrhea in the intestine.

[0022] 2. This invention uses modified chitosan as a coating. The dense structure formed by the covalent backbone of genipin crosslinking and the stepwise complexation with amino acids and casein phosphopeptides can resist gastric acid erosion and prevent the bacteria from being destroyed by gastric acid. The low-acyl gellan gum in the coating layer can respond to intestinal calcium... 2+ In response to pH conditions, the bacteria swell and rupture to achieve targeted release into the intestines. The amino acids in the coating layer enhance the adhesion of probiotics to the intestinal mucosa, while casein phosphopeptides promote mineral absorption, synergistically strengthening the intestinal regulatory effect in conjunction with probiotics. Detailed Implementation

[0023] Bifidobacterium animalis Bb-12, catalog number: WN-BZ16207, is sourced from Wuhan Huanna Biotechnology Co., Ltd.

[0024] Lactobacillus rhamnosus GG, ATCC53103, is derived from the American Type Culture Collection.

[0025] Lactobacillus plantarum LP, CGMCC 1.2469, was derived from the China General Microbiological Culture Collection Center.

[0026] Bifidobacterium animalis Bb-12, Lactobacillus rhamnosus GG, and Lactobacillus plantarum LP were activated and cultured separately. Each strain was first inoculated into the culture medium and activated for 14 hours at 37℃, under a micro-anaerobic environment and pH 6.5 until the viable bacterial concentration was ≥5×10⁻⁶. 9 Seed culture was prepared at CFU / mL; then, the seed culture of each strain was transferred to the same food-grade compound culture medium at a 5% inoculation rate for scale-up culture, maintained at 37℃ under micro-anaerobic conditions, with the pH of the system finely adjusted to 5.5 during the culture process, and cultured for 18 hours until the viable cell concentration of each fermentation broth was ≥1.5×10⁻⁶. 11 CFU / mL, and bacterial cells of each strain were collected separately by low-temperature centrifugation.

[0027] Whey protein, model number: WPC80, sourced from MSG, USA.

[0028] Low-acyl gellan gum, batch number: RK20210423, sourced from Muque.

[0029] Polyglutamic acid, Mw=80000-100000Da.

[0030] Polythreonine, Mw=70000-90000Da.

[0031] Polyserine, Mw=40000-70000Da.

[0032] PBS buffer, pH 6.5, 0.1 mol / L.

[0033] Sodium citrate buffer, pH=6.0, 0.1mol / L.

[0034] Example 1 A method for preparing a multifunctional probiotic preparation includes the following steps, in parts by weight: S1. Add 2 parts of Bifidobacterium animalis Bb-12, 2 parts of Lactobacillus rhamnosus GG, and 1 part of Lactobacillus plantarum LP to 10 parts of PBS buffer, stir well to obtain a bacterial suspension, and refrigerate for later use; the viable count of Bifidobacterium animalis Bb-12 is >1.0×10⁻⁶. 11 CFU / g; viable count of Lactobacillus rhamnosus GG > 1.5 × 10⁻⁶ 11 CFU / g; viable count of *Lactobacillus plantarum* LP > 2.0 × 10⁻⁶11 CFU / g; S2. Dissolve 1 part xylooligosaccharide, 1.44 parts skim milk powder, 1.5 parts mannitol and 10 parts water at 75°C by stirring. Add 0.05 parts PEG-400, cool to room temperature and stir evenly. Add 2 parts bacterial suspension dropwise, mix evenly and freeze dry to obtain freeze-dried core material powder. S3. Add 10 parts of chitosan to 200 parts of water, then add 1 mol / L dilute hydrochloric acid to adjust the pH to 5.5 and stir to dissolve to obtain a chitosan solution; add 5 parts of whey protein to 30 parts of water and stir evenly at 35℃ to obtain a whey protein solution; add the whey protein solution dropwise to the chitosan solution, adjust the pH to 7.5 with sodium citrate buffer, then add 10 parts of 30wt% genipin aqueous solution, stir at 40℃ for 4 hours, then add 20 parts of 20wt% polyserine aqueous solution, stir for 40 minutes, then add 10 parts of 20wt% casein phosphopeptide aqueous solution, continue stirring for 1.5 hours, concentrate under reduced pressure to a solid content of 25%, and freeze-dry to obtain modified chitosan; S4. Add 1 part of modified chitosan to 120 parts of water and stir evenly to obtain modified chitosan substrate solution; add 1 part of low acyl gellan gum and 0.6 parts of potassium citrate to 35 parts of water and stir evenly at 45°C to obtain crosslinking solution; add crosslinking solution to substrate solution, add 2 parts of propylene glycol and then adjust pH to 6 with sodium citrate buffer; pass through 200 mesh sieve to obtain coating solution, then coat the core material freeze-dried powder; freeze-dry to obtain multifunctional probiotic preparation.

[0035] Example 2 It is basically the same as Example 1, except that polyserine is replaced with polythreonine.

[0036] Example 3 It is basically the same as Example 1, except that polyserine is replaced with polyglutamic acid.

[0037] Compare with Example 1 A method for preparing a multifunctional probiotic preparation includes the following steps, in parts by weight: S1. Add 2 parts of Bifidobacterium animalis Bb-12, 2 parts of Lactobacillus rhamnosus GG, and 1 part of Lactobacillus plantarum LP to 10 parts of PBS buffer, stir well to obtain a bacterial suspension, and refrigerate for later use; the viable count of Bifidobacterium animalis Bb-12 is >1.0×10⁻⁶. 11 CFU / g; viable count of Lactobacillus rhamnosus GG > 1.5 × 10⁻⁶ 11 CFU / g; viable count of *Lactobacillus plantarum* LP > 2.0 × 10⁻⁶ 11 CFU / g; S2. Dissolve 1 part xylooligosaccharide, 1.44 parts skim milk powder, 1.5 parts mannitol and 10 parts water at 75°C by stirring. Add 0.05 parts PEG-400, stir evenly, cool to room temperature and add 2 parts bacterial suspension. Mix evenly and freeze dry to obtain a multifunctional probiotic preparation.

[0038] Test Example 1 The survival and release characteristics of the multifunctional probiotic preparation were investigated using in vitro simulated gastric and intestinal environments.

[0039] 1.0 g of multifunctional probiotic preparation was weighed and added to 9 mL of artificial simulated gastric fluid (10 g / L pepsin, dissolved in 0.10 mol / L dilute hydrochloric acid, diluted with water to volume, adjusted to pH=1.5, and filtered for sterilization) and artificial simulated intestinal fluid (10 g / L trypsin, 6.80 g / L potassium dihydrogen phosphate, diluted with water to volume, adjusted to pH=7.4, and filtered for sterilization). After shaking at 37℃ and 180 r / min for 2 h and 4 h respectively, samples were taken to determine the number of viable bacteria and calculate the survival rate and release rate.

[0040] Table 1

[0041] As can be seen from Table 1, when probiotic freeze-dried powder is not coated for protection, its survival rate is very low in the highly acidic gastric juice environment, and almost no probiotics can survive after 4 hours.

[0042] In this embodiment, modified chitosan is used as a coating. The dense structure formed by the covalent backbone of genipin crosslinking and the stepwise complexation with amino acids and casein phosphopeptides can resist gastric acid erosion and prevent the bacteria from being destroyed by gastric acid. 2+ Upon contact with the pH environment, the substance swells and ruptures, enabling targeted release into the intestines. The amino acids in the coating layer enhance the adhesion of probiotics to the intestinal mucosa, while casein phosphopeptides promote mineral absorption, synergistically strengthening the intestinal regulatory effect in conjunction with probiotics.

[0043] The probiotics in the examples can achieve a relatively long release in the intestine, thereby achieving a regulatory effect on the intestine. Example 3 still showed a lower release rate than other examples after 4 hours. This may be because polyglutamic acid has a dicarboxyl structure, and the γ-position polymerization method allows the side chain carboxyl groups to be fully exposed, which makes the electrostatic complexation efficiency with chitosan higher, resulting in stronger coating tightness and achieving a slower release effect, which is beneficial for long-term regulation. Test Example 2 Healthy, 4-week-old SPF-grade mice of similar size (18-22g) were selected and randomly divided into 5 groups of 25 mice each. All mice were placed in the same standard rearing environment and allowed free access to food for 3 days to acclimatize. All mice were artificially infected with Enterocynoid Escherichia coli (EAEC) to establish a diarrhea model: EAEC cultured to the logarithmic growth phase was adjusted to a concentration of 1×10⁻⁶ with sterile saline. 9 The mice were infected by gavage with CFU / mL at a dose of 0.2mL / 10g mouse body weight. After infection, the mice were observed for 24 hours. If all mice showed typical diarrhea symptoms (unformed, watery or pasty feces, accompanied by rapid breathing, ruffled and messy fur, significantly reduced activity, and food intake decreased by ≥50% compared with before infection), the diarrhea model was successfully established.

[0044] After successful model construction, the corresponding multifunctional probiotic preparations for each group were dissolved in water to prepare a concentration of 0.01 g / mL, and administered via gavage at a dose of 0.1 mL / 10 g mouse body weight. Group 5 served as a blank control group, receiving only an equal volume of water via gavage. All groups were administered gavage twice daily, with a 12-hour interval between doses. After the two gavages, mice were observed continuously for 8 hours, with symptom changes recorded every 2 hours. Observational indicators included respiratory status, body surface condition, fecal characteristics, activity level, and dietary intake. The criteria for diarrhea cure were defined as: stable breathing without rapid or abnormal breathing; smooth, non-fluffy fur; fully formed feces without loose or watery stools; activity level returned to normal without huddling or lethargy; and dietary intake returned to pre-infection levels. After the experiment, the number of cured mice in each group was counted, and the diarrhea cure rate was calculated as (number of cured mice in each group / total number of mice in each group) × 100%.

[0045] Table 2

[0046] As shown in Table 2, the multifunctional probiotic preparation prepared in this invention has a good effect on intestinal regulation. This invention uses a compound of Bifidobacterium animalis Bb-12, Lactobacillus rhamnosus GG, and Lactobacillus plantarum LP. Bb-12 can regulate the balance of intestinal flora, inhibit the proliferation of harmful bacteria, and enhance the intestinal mucosal barrier function. The GG strain has outstanding acid and bile salt resistance, can stably colonize in the intestine, promote the repair of intestinal epithelial cells, and reduce intestinal inflammatory response. LP can metabolize to produce organic acids, lower the pH value of the intestine, create a suitable environment for the growth of beneficial bacteria, and at the same time promote intestinal peristalsis, improving both constipation and diarrhea in the intestine.

[0047] Compared to Control Example 1, the improvement in diarrhea in the Example 1 was faster and more significant. This may be because the probiotics in the Example 1 had less loss of activity, thus enabling them to better regulate the gut.

[0048] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A method for preparing a multifunctional probiotic preparation, characterized in that, Includes the following steps: S1. Add Bifidobacterium animalis Bb-12, Lactobacillus rhamnosus GG, and Lactobacillus plantarum LP to PBS buffer, stir well to obtain a bacterial suspension, and refrigerate for later use. S2. After dissolving xylooligosaccharide, skim milk powder, mannitol and water at 70-80℃, add PEG-400, stir evenly, cool to room temperature and add bacterial suspension dropwise. After mixing evenly, freeze dry to obtain core material freeze-dried powder. S3. Add chitosan to water, adjust the pH with acid, and stir to dissolve to obtain a chitosan solution; add whey protein to water and stir evenly at 30-40℃ to obtain a whey protein solution; add the whey protein solution dropwise to the chitosan solution, adjust the pH, add 30wt% genipin aqueous solution, stir at 35-50℃ for 3-5 hours, add 20wt% amino acid aqueous solution, stir for 30-60 minutes, add 20wt% casein phosphopeptide aqueous solution, continue stirring for 1-2 hours, concentrate under reduced pressure to a solid content of 20-30%, and freeze-dry to obtain modified chitosan; S4. Add modified chitosan to water and stir evenly to obtain modified chitosan substrate solution; add low acyl gellan gum and potassium citrate to water and stir evenly at 40-50℃ to obtain crosslinking solution; add crosslinking solution to substrate solution, add propylene glycol and adjust pH to weakly acidic; sieve to obtain coating solution and then coat the core material freeze-dried powder; freeze-dry to obtain multifunctional probiotic preparation.

2. The method for preparing the multifunctional probiotic preparation as described in claim 1, characterized in that, The mass ratio of Bifidobacterium animalis Bb-12, Lactobacillus rhamnosus GG, Lactobacillus plantarum LP, and PBS buffer is 1-3:1-3:1-2:5-10; the viable count of Bifidobacterium animalis Bb-12 is >1.0×10⁻⁶. 11 CFU / g; viable count of Lactobacillus rhamnosus GG > 1.5 × 10⁻⁶ 11 CFU / g; viable count of *Lactobacillus plantarum* LP > 2.0 × 10⁻⁶ 11 CFU / g.

3. The method for preparing the multifunctional probiotic preparation as described in claim 1, characterized in that, The mass ratio of xylooligosaccharides, skim milk powder, mannitol, PEG-400, water, and bacterial suspension is 1-2:1.4-1.5:1-2:0.01-1:5-15:0.5-2.

4. The method for preparing the multifunctional probiotic preparation as described in claim 1, characterized in that, The mass ratio of chitosan to water is 1-1.5:15-25.

5. The method for preparing the multifunctional probiotic preparation as described in claim 1, characterized in that, The mass ratio of whey protein to water is 1-2:5-10.

6. The method for preparing the multifunctional probiotic preparation as described in claim 1, characterized in that, The mass ratio of chitosan to whey protein, genipin, amino acids, and casein phosphopeptide is 1-2:0.1-1:0.1-0.5:0.2-0.5:0.1-0.

3.

7. The method for preparing the multifunctional probiotic preparation as described in claim 1, characterized in that, The mass ratio of the modified chitosan to water is 1-2:100-150.

8. The method for preparing the multifunctional probiotic preparation as described in claim 1, characterized in that, The mass ratio of the low-acyl gellan gum, potassium citrate, and water is 1-2:0.5-0.8:30-40.

9. The method for preparing the multifunctional probiotic preparation as described in claim 1, characterized in that, The amino acid polymer is one of polyglutamic acid, polythreonine, or polyserine.

10. A multifunctional probiotic preparation, characterized in that, Prepared by the method described in any one of claims 1-9.

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