Bifidobacterium animalis subsp. Lactis composition for regulating intestines and stomach and preparation method thereof

Abstract

The invention discloses a bifidobacterium animalis subsp. Lactis composition for regulating intestines and stomach and a preparation method of the bifidobacterium animalis subsp. Lactis composition, and relates to the technical field of biology. A rigid hydrophobic group is introduced into sodium alginate for grafting modification, the rigid hydrophobic group of 4-aminoethyl biphenyl is grafted to a sodium alginate molecular chain through 1, 4-butanediol diglycidyl ether, and meanwhile, the sodium alginate molecular chain and cystamine-chitosan into which a disulfide bond is introduced are subjected to electrostatic compounding modification design; the bifidobacterium animalis subsp. Lactis is embedded, so that the survival rate of the bifidobacterium animalis subsp. Lactis in intestines and stomach is effectively improved, and the effective gastrointestinal regulation function of the bifidobacterium animalis subsp. Lactis is realized.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a composition of Bifidobacterium lactis subsp. animalis for regulating the gastrointestinal tract and its preparation method. Background Technology

[0002] Probiotics, as a class of live microorganisms that can produce beneficial effects on the health of the host when ingested in appropriate amounts, have become a research hotspot in the field of functional foods and dietary supplements. Among them, Bifidobacterium animalis subsp. lactis has excellent acid and bile salt resistance and intestinal epithelial cell adhesion ability, and has excellent effects in maintaining the physiological intestinal microecological balance and inhibiting pathogenic bacteria. Therefore, its high survival rate in the gastrointestinal tract is the primary prerequisite for exerting its probiotic effects.

[0003] In the existing technology, microencapsulation technology is regarded as an effective means to improve the survival rate of probiotics in the gastrointestinal tract. However, existing encapsulation materials have certain limitations: on the one hand, some materials disintegrate too quickly in gastric acid and cannot provide effective protection for the strains; on the other hand, some materials overprotect the strains, making it difficult for them to disintegrate quickly and completely in the intestinal environment, resulting in the probiotics not being released in a timely or complete manner.

[0004] In summary, providing a composition of Bifidobacterium lactis subsp. animalis for regulating the gastrointestinal tract and its preparation method is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a composition of Bifidobacterium lactis subsp. animalis for regulating the gastrointestinal tract and its preparation method, so as to solve the problems raised in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing a composition of Bifidobacterium lactis subsp. animalis for regulating the gastrointestinal tract includes the following steps: Step 1: Add modified sodium alginate and cystamine-chitosan to deionized water, adjust the pH to 3.5-4.5, set the temperature to 40-50℃, stir, and obtain a wall material solution; Step 2: Add the bacterial suspension to the wall material solution and stir. Add calcium chloride solution, let stand for 1-2 hours, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

[0007] In a more optimized manner, the raw materials of the wall material solution, by weight, have a modified sodium alginate, cystamine-chitosan, and deionized water in a mass ratio of (0.2~0.8):(0.1~0.4):100.

[0008] A more optimized method for preparing the modified sodium alginate is as follows: sodium alginate is added to deionized water, the pH is adjusted to 8.5-9, the temperature is set to 40-50℃, 1,4-butanediol diglycidyl ether is slowly added dropwise, and the mixture is kept at this temperature for 6-7 hours. Then, 4-aminoethyl biphenyl is added, the temperature is raised to 70-80℃, and the temperature is kept at this temperature for 3-4 hours. The mixture is then cooled to room temperature, the pH is adjusted to 4-4.5, acetone is added and the mixture is soaked for 12-15 hours, filtered, washed, and dried to obtain the modified sodium alginate. The modified sodium alginate raw materials, by weight, consist of: 1-3 parts sodium alginate, 0.3-0.8 parts 1,4-butanediol diglycidyl ether, and 0.5-2 parts 4-aminoethyl biphenyl.

[0009] In a more optimized manner, the preparation method of the cystamine-chitosan is as follows: carboxymethyl chitosan is added to phosphate buffer and stirred evenly. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially. The mixture is activated at room temperature for 20-40 minutes. Then, cystamine dihydrochloride is added and stirred evenly. The mixture is stirred at room temperature for 6-8 hours, dialyzed, and dried to obtain cystamine-chitosan. The raw materials for cystamine-chitosan, by weight, include 0.2-0.6 parts carboxymethyl chitosan, 15-25 parts phosphate buffer, 0.4-1 parts 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.2-0.5 parts N-hydroxysuccinimide, and 0.2-0.6 parts cystamine dihydrochloride.

[0010] Ideally, the volume ratio of the bacterial suspension to the wall material solution is (1~2):(4~6); and the concentration of the calcium chloride solution is 1~2wt%.

[0011] In a more optimized manner, the preparation method of the bacterial suspension is as follows: the probiotics are activated, cultured in an expanded manner, centrifuged, concentrated, washed, and centrifuged again in sequence, and then placed in physiological saline containing a protectant to prepare the bacterial suspension.

[0012] More preferably, the probiotic is Bifidobacterium animalis subsp. lactis CP-9, which was deposited at the China Center for Type Culture Collection on November 24, 2014, with accession number CCTCC No: M2014588.

[0013] Ideally, the concentration of the bacterial suspension is 5 × 10⁻⁶. 9 ~5×10 10 CFU / mL.

[0014] More optimally, the mass ratio of the protective agent to physiological saline is (1~5):20; the protective agent is skim milk powder and trehalose in a mass ratio of (1~4):1.

[0015] A method for preparing a Bifidobacterium lactis composition for regulating the gastrointestinal tract.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This invention achieves effective gastrointestinal regulation of Bifidobacterium animalis subsp. lactis by grafting sodium alginate with rigid hydrophobic groups and electrostatically combining it with cystamine-chitosan with disulfide bonds.

[0017] First, by grafting the rigid hydrophobic group of 4-aminoethyl biphenyl onto the sodium alginate molecular chain through 1,4-butanediol diglycidyl ether, the 4-aminoethyl biphenyl has a hydrophobic benzene ring structure, which can play a good supporting role and improve the mechanical properties of the gel. At the same time, it can effectively block the penetration and diffusion of hydrogen ions in gastric acid. Through the hydrophobic effect, it can inhibit the excessive swelling of the composition, so that it can still maintain a high degree of integrity in the strong gastric acid environment, significantly reduce the risk of exposure and death of probiotics due to the rapid swelling or disintegration of the wall material, and greatly improve the gastric survival rate of probiotics. Secondly, a disulfide bond is introduced through a grafting reaction between the carboxyl groups of carboxymethyl chitosan and the amino groups of cystamine. Then, a wall material solution is formed by electrostatic composite coagulation and modified sodium alginate. The wall material solution encapsulates probiotics to obtain a composition. The disulfide bonds in the composition are specifically cleaved by glutathione, which destroys the electrostatic interaction network that maintains the stability of the composition structure. At the same time, the enzymes of the gut microbiota further degrade the wall material fragments. In addition, the competitive action of phosphate ions and calcium ions in the gut weakens the gel network structure of the encapsulated wall material and calcium ions, achieving synergistic disintegration inside and outside, allowing the encapsulated Bifidobacterium lactis subsp. animalis to disintegrate rapidly and completely, and achieving efficient and precise release of Bifidobacterium lactis subsp. animalis. Detailed Implementation

[0018] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0019] In the following embodiments, the parts are by weight. It should be noted that there are no special restrictions on the purchase manufacturers of all raw materials involved in this invention. Exemplary examples include: sodium alginate (CAS: 9005-38-3, molecular weight 35000), 1,4-butanediol diglycidyl ether (CAS: 2425-79-8, purity 99.5%), 4-aminoethylbiphenyl (CAS: 712-76-5, purity 98%), carboxymethyl chitosan (CAS: 83512-85-0, molecular weight 500000), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (CAS: 7084-11-9), N-hydroxysuccinimide (CAS: 6066-82-6), and cystamine dihydrochloride (CAS: 56-17-7, purity 97%).

[0020] The probiotic is Bifidobacterium animalis subsp. lactis CP-9, which was deposited at the China Center for Type Culture Collection on November 24, 2014, with accession number CCTCC No: M2014588.

[0021] Example 1: A method for preparing a composition of Bifidobacterium lactis subsp. animalis for regulating the gastrointestinal tract, specifically including the following steps: I. Preparation of modified sodium alginate: Add 2 parts of sodium alginate to 50 parts of deionized water, adjust the pH to 8.5, set the temperature to 45℃, slowly add 0.5 parts of 1,4-butanediol diglycidyl ether, maintain for 6.5 hours, then add 1 part of 4-aminoethyl biphenyl, raise the temperature to 75℃, maintain for 3.5 hours, cool to room temperature, adjust the pH to 4.5, add acetone and soak for 14 hours, filter, wash and dry to obtain modified sodium alginate.

[0022] II. Preparation of cystamine-chitosan: 0.4 parts of carboxymethyl chitosan were added to 20 parts of phosphate buffer and stirred until homogeneous. Then, 0.7 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.4 parts of N-hydroxysuccinimide were added sequentially. The mixture was activated at room temperature for 30 minutes. Then, 0.4 parts of cystamine dihydrochloride were added and stirred until homogeneous. The mixture was stirred at room temperature for 7 hours, dialyzed, and dried to obtain cystamine-chitosan.

[0023] III. Preparation of Bifidobacterium lactis subsp. animalis composition: Step 1: Add 0.5 parts of modified sodium alginate and 0.3 parts of cystamine-chitosan to 100 parts of deionized water, adjust the pH to 4, set the temperature to 45℃, and stir for 45 minutes to obtain the wall material solution; Step 2: Activate, expand, centrifuge, concentrate, wash, and centrifuge again of Bifidobacterium animalis subsp. lactis CP-9 in sequence for later use; add 2.5 parts skim milk powder and 1 part trehalose to 20 parts physiological saline, add the prepared Bifidobacterium animalis subsp. lactis CP-9, stir, and prepare a solution with a concentration of 5 × 10⁻⁶. 10 CFU / mL bacterial suspension; Step 3: Mix and stir the bacterial suspension and wall material solution, add 2wt% calcium chloride solution, let stand for 1.5 hours, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

[0024] In this embodiment, the volume ratio of the bacterial suspension, wall material solution, and calcium chloride solution is 0.5:5:14.

[0025] Example 2: A method for preparing a composition of Bifidobacterium lactis subsp. animalis for regulating the gastrointestinal tract, specifically including the following steps: Preparation of modified sodium alginate: Add 1 part sodium alginate to 50 parts deionized water, adjust the pH to 8.5, set the temperature to 40℃, slowly add 0.3 parts 1,4-butanediol diglycidyl ether, maintain for 6 hours, then continue to add 0.5 parts 4-aminoethyl biphenyl, raise the temperature to 70℃, keep at that temperature for 3 hours, cool to room temperature, adjust the pH to 4.5, add acetone and soak for 12 hours, filter, wash and dry to obtain modified sodium alginate.

[0026] Preparation of cystamine-chitosan: 0.2 parts of carboxymethyl chitosan were added to 20 parts of phosphate buffer and stirred until homogeneous. Then, 0.4 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.2 parts of N-hydroxysuccinimide were added sequentially. The mixture was activated at room temperature for 20 minutes. Then, 0.2 parts of cystamine dihydrochloride were added and stirred until homogeneous. The mixture was stirred at room temperature for 6 hours, dialyzed, and dried to obtain cystamine-chitosan.

[0027] III. Preparation of Bifidobacterium lactis subsp. animalis composition: Step 1: Add 0.2 parts of modified sodium alginate and 0.1 parts of cystamine-chitosan to 100 parts of deionized water, adjust the pH to 3.5, set the temperature to 40℃, and stir for 30 minutes to obtain the wall material solution; Step 2: Activate, expand, centrifuge, concentrate, wash, and centrifuge again of Bifidobacterium animalis subsp. lactis CP-9 in sequence for later use; add 1 part skim milk powder and 1 part trehalose to 20 parts physiological saline, add the prepared Bifidobacterium animalis subsp. lactis CP-9, stir, and prepare a solution with a concentration of 5 × 10⁻⁶. 10 CFU / mL bacterial suspension; Step 3: Mix the bacterial suspension and wall material solution according to the volume ratio, add 2wt% calcium chloride solution, let stand for 1 hour, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

[0028] In this embodiment, the volume ratio of the bacterial suspension, wall material solution, and calcium chloride solution is 1:4:10.

[0029] Example 3: A method for preparing a composition of Bifidobacterium lactis subsp. animalis for regulating the gastrointestinal tract, specifically including the following steps: I. Preparation of modified sodium alginate: Add 3 parts of sodium alginate to 50 parts of deionized water, adjust the pH to 8.5, set the temperature to 50℃, slowly add 0.8 parts of 1,4-butanediol diglycidyl ether, maintain for 7 hours, then continue to add 2 parts of 4-aminoethyl biphenyl, raise the temperature to 80℃, maintain for 4 hours, cool to room temperature, adjust the pH to 4.5, add acetone and soak for 15 hours, filter, wash and dry to obtain modified sodium alginate.

[0030] II. Preparation of cystamine-chitosan: 0.6 parts of carboxymethyl chitosan were added to 20 parts of phosphate buffer and stirred until homogeneous. Then, 1 part of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.5 parts of N-hydroxysuccinimide were added sequentially. The mixture was activated at room temperature for 40 minutes. Then, 0.6 parts of cystamine dihydrochloride were added and stirred until homogeneous. The mixture was stirred at room temperature for 8 hours, dialyzed, and dried to obtain cystamine-chitosan.

[0031] III. Preparation of Bifidobacterium lactis subsp. animalis composition: Step 1: Add 0.8 parts of modified sodium alginate and 0.4 parts of cystamine-chitosan to 100 parts of deionized water, adjust the pH to 4.5, set the temperature to 50℃, and stir for 60 minutes to obtain the wall material solution; Step 2: Activate, expand, centrifuge, concentrate, wash, and centrifuge again of Bifidobacterium animalis subsp. lactis CP-9 in sequence for later use; add 4 parts skim milk powder and 1 part trehalose to 20 parts physiological saline, add the prepared Bifidobacterium animalis subsp. lactis CP-9, stir, and prepare a solution with a concentration of 5 × 10⁻⁶. 10 CFU / mL bacterial suspension; Step 3: Mix and stir the bacterial suspension and wall material solution, add 2wt% calcium chloride solution, let stand for 2 hours, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

[0032] In this embodiment, the volume ratio of the bacterial suspension, wall material solution, and calcium chloride solution is 2:6:18.

[0033] Comparative Example 1: Based on Example 1, the composition of the wall material solution was adjusted, wherein sodium alginate was not modified and was used to prepare a composition of Bifidobacterium lactis subsp. animalis. The rest were the same as in Example 1. The specific steps are as follows: I. Preparation of cystamine-chitosan: 0.4 parts of carboxymethyl chitosan were added to 20 parts of phosphate buffer and stirred until homogeneous. Then, 0.7 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.4 parts of N-hydroxysuccinimide were added sequentially. The mixture was activated at room temperature for 30 minutes. Then, 0.4 parts of cystamine dihydrochloride were added and stirred until homogeneous. The mixture was stirred at room temperature for 7 hours, dialyzed, and dried to obtain cystamine-chitosan. II. Preparation of Bifidobacterium lactis subsp. animalis composition: Step 1: Add 0.5 parts sodium alginate and 0.3 parts cystamine-chitosan to 100 parts deionized water, adjust the pH to 4, set the temperature to 45℃, stir for 45 minutes to obtain the wall material solution; Step 2: Activate, expand, centrifuge, concentrate, wash, and centrifuge again of Bifidobacterium animalis subsp. lactis CP-9 in sequence for later use; add 2.5 parts skim milk powder and 1 part trehalose to 20 parts physiological saline, add the prepared Bifidobacterium animalis subsp. lactis CP-9, stir, and prepare a solution with a concentration of 5 × 10⁻⁶. 10 CFU / mL bacterial suspension; Step 3: Mix and stir the bacterial suspension and wall material solution, add 2wt% calcium chloride solution, let stand for 1.5 hours, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

[0034] In Comparative Example 1, the volume ratio of the bacterial suspension, wall material solution, and calcium chloride solution is 0.5:5:14.

[0035] Comparative Example 2: Based on Example 1, the composition of the wall material solution was adjusted. In step 2 of the wall material solution preparation process, only carboxymethyl chitosan was used to prepare the Bifidobacterium animalis subsp. lactis composition. The rest was the same as in Example 1. The specific steps are as follows: I. Preparation of modified sodium alginate: Add 2 parts of sodium alginate to 50 parts of deionized water, adjust the pH to 8.5, set the temperature to 45℃, slowly add 0.5 parts of 1,4-butanediol diglycidyl ether, maintain for 6.5 hours, then add 1 part of 4-aminoethyl biphenyl, raise the temperature to 75℃, maintain for 3.5 hours, cool to room temperature, adjust the pH to 4.5, add acetone and soak for 14 hours, filter, wash and dry to obtain modified sodium alginate; II. Preparation of Bifidobacterium lactis subsp. animalis composition: Step 1: Add 0.5 parts of modified sodium alginate and 0.3 parts of carboxymethyl chitosan to 100 parts of deionized water, adjust the pH to 4, set the temperature to 45℃, stir for 45 minutes to obtain the wall material solution; Step 2: Activate, expand, centrifuge, concentrate, wash, and centrifuge again of Bifidobacterium animalis subsp. lactis CP-9 in sequence for later use; add 2.5 parts skim milk powder and 1 part trehalose to 20 parts physiological saline, add the prepared Bifidobacterium animalis subsp. lactis CP-9, stir, and prepare a solution with a concentration of 5 × 10⁻⁶. 10 CFU / mL bacterial suspension; Step 3: Mix and stir the bacterial suspension and wall material solution, add 2wt% calcium chloride solution, let stand for 1.5 hours, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

[0036] In Comparative Example 2, the volume ratio of the bacterial suspension, wall material solution, and calcium chloride solution is 0.5:5:14.

[0037] Comparative Example 3: Based on Example 1, the composition of the wall material solution was adjusted, and 4-aminoethylbiphenyl was not introduced to prepare a composition of Bifidobacterium lactis subsp. animalis. All other steps remained the same as in Example 1. The specific steps are as follows: I. Preparation of modified sodium alginate: Add 2 parts of sodium alginate to 50 parts of deionized water, adjust the pH to 8.5, set the temperature to 45℃, slowly add 0.5 parts of 1,4-butanediol diglycidyl ether, maintain for 6.5 hours, cool to room temperature, adjust the pH to 4.5, add acetone and soak for 14 hours, filter, wash and dry to obtain modified sodium alginate.

[0038] II. Preparation of cystamine-chitosan: 0.4 parts of carboxymethyl chitosan were added to 20 parts of phosphate buffer and stirred until homogeneous. Then, 0.7 parts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 0.4 parts of N-hydroxysuccinimide were added sequentially. The mixture was activated at room temperature for 30 minutes. Then, 0.4 parts of cystamine dihydrochloride were added and stirred until homogeneous. The mixture was stirred at room temperature for 7 hours, dialyzed, and dried to obtain cystamine-chitosan.

[0039] III. Preparation of Bifidobacterium lactis subsp. animalis composition: Step 1: Add 0.5 parts of modified sodium alginate and 0.3 parts of cystamine-chitosan to 100 parts of deionized water, adjust the pH to 4, set the temperature to 45℃, and stir for 45 minutes to obtain the wall material solution; Step 2: Activate, expand, centrifuge, concentrate, wash, and centrifuge again of Bifidobacterium animalis subsp. lactis CP-9 in sequence for later use; add 2.5 parts skim milk powder and 1 part trehalose to 20 parts physiological saline, add the prepared Bifidobacterium animalis subsp. lactis CP-9, stir, and prepare a solution with a concentration of 5 × 10⁻⁶. 10 CFU / mL bacterial suspension; Step 3: Mix and stir the bacterial suspension and wall material solution, add 2wt% calcium chloride solution, let stand for 1.5 hours, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

[0040] In this embodiment, the volume ratio of the bacterial suspension, wall material solution, and calcium chloride solution is 0.5:5:14.

[0041] Comparative Example 4: Based on Example 1, the composition of the wall material solution was adjusted. A wall material solution was prepared using conventional sodium alginate and chitosan to prepare a Bifidobacterium lactis composition. All other steps remained the same as in Example 1. The specific steps are as follows: Preparation of Bifidobacterium lactis subsp. animalis composition: Step 1: Add 0.5 parts sodium alginate and 0.3 parts chitosan to 100 parts deionized water, adjust the pH to 4, set the temperature to 45℃, and stir for 45 minutes to obtain the wall material solution.

[0042] Step 2: Activate, expand, centrifuge, concentrate, wash, and centrifuge again of Bifidobacterium animalis subsp. lactis CP-9 in sequence for later use; add 2.5 parts skim milk powder and 1 part trehalose to 20 parts physiological saline, add the prepared Bifidobacterium animalis subsp. lactis CP-9, stir, and prepare a solution with a concentration of 5 × 10⁻⁶. 10 CFU / mL bacterial suspension; Step 3: Mix and stir the bacterial suspension and wall material solution, add 2wt% calcium chloride solution, let stand for 1.5 hours, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

[0043] In this embodiment, the volume ratio of the bacterial suspension, wall material solution, and calcium chloride solution is 0.5:5:14.

[0044] Performance testing: The animal Bifidobacterium lactis subsp. compositions prepared in Examples 1-3 and Comparative Examples 1-4 were tested for encapsulation efficiency, survival rate in simulated gastric and intestinal fluids, and stability. Specific data are shown in the table below. Encapsulation efficiency test: A sodium bicarbonate solution with a pH of 8 and a concentration of 0.2 mol / L and a trisodium citrate solution with a concentration of 0.06 mol / L were mixed to obtain an encapsulation solution. 1 g of Bifidobacterium animalis subsp. lactis composition was mixed with 9 mL of the encapsulation solution and washed. The viable bacteria count was determined by plate counting method. The calculation formula is: Encapsulation efficiency (%) = viable bacteria count after encapsulation / viable bacteria count before encapsulation × 100%.

[0045] Survival rate test in simulated gastric juice: A sodium chloride solution with a pH of 2 and a concentration of 0.09 mg / mL was mixed with a 0.3% (w / v) pepsin solution to obtain simulated gastric juice. 1 g of Bifidobacterium animalis subsp. lactis composition was mixed with 9 mL of simulated gastric juice and cultured in a shaker at 37°C and 200 r / min for 5 hours. After decapsulation and washing, the number of viable bacteria was determined by plate count method. The calculation formula is: Survival rate (%) = Number of viable bacteria after treatment with simulated gastric juice / Number of viable bacteria before treatment with simulated gastric juice × 100%.

[0046] Survival rate test in simulated intestinal fluid: PBS solution with pH 8 was mixed with 0.1% (w / v) trypsin solution and 0.3% (w / v) sodium cholate, filtered to remove bacteria to obtain simulated intestinal fluid. 1g of Bifidobacterium animalis subsp. lactis composition was mixed with 9mL of simulated intestinal fluid and incubated in a water bath at 37℃ for 3 hours. After decapsulation and washing, the number of viable bacteria was determined by plate count method. The calculation formula is: Survival rate (%) = number of viable bacteria after treatment with simulated intestinal fluid / number of viable bacteria before treatment with simulated intestinal fluid × 100%.

[0047]

[0048] Conclusion: As can be seen from the data in the table, the *Bifidobacterium lactis* compositions prepared in Examples 1-3 all exhibited excellent encapsulation performance and resistance to gastrointestinal stress. This is because the crosslinking modification of sodium alginate by 1,4-butanediol diglycidyl ether and the hydrophobic modification by 4-aminoethyl biphenyl enhanced the structural stability of sodium alginate. After activation, the carboxymethyl chitosan underwent a grafting reaction with cysteine ​​dihydrochloride, successfully introducing a disulfide and amide crosslinking structure, which improved the acid resistance and intestinal responsiveness of the wall material. Subsequently, the composite wall material formed a uniform and stable encapsulation network through electrostatic interaction under acidic conditions, effectively improving the resistance of the *Bifidobacterium lactis* compositions to gastrointestinal stress.

[0049] Comparing Example 1 with Comparative Examples 1-4, it can be seen that Comparative Example 1, due to the lack of modification of sodium alginate, has a loose molecular chain structure, resulting in decreased compatibility with cystamine-chitosan and weakened encapsulation efficiency and acid resistance. Comparative Example 2, lacking cystamine-chitosan, lacks the structural stability and intestinal targeting provided by disulfide crosslinking, making the wall material prone to disintegration in the gastrointestinal environment and insufficient viable bacterial protection. Comparative Example 3, without the introduction of 4-aminoethylbiphenyl, lacks hydrophobic modification of sodium alginate, leading to a reduced survival rate in simulated intestinal fluid. Comparative Example 4, using conventional sodium alginate and chitosan, lacks specific crosslinking and functional modification, resulting in a loose wall material structure with poor acid and enzyme resistance, unable to resist the erosion of gastric acid and digestive enzymes in the gastrointestinal environment, thus exhibiting the lowest encapsulation efficiency and survival rate. In summary, the cross-linking-hydrophobic composite modification of sodium alginate, the cysteine ​​grafting modification of carboxymethyl chitosan, and the composite wall material system formed by the two modified polymers can effectively improve the encapsulation efficiency and gastrointestinal tolerance of Bifidobacterium lactis, achieving efficient protection and targeted delivery of probiotics.

[0050] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a composition of Bifidobacterium lactis subsp. animalis for regulating the gastrointestinal tract, characterized in that: The process includes the following steps: Step 1: Add modified sodium alginate and cystamine-chitosan to deionized water, adjust the pH to 3.5~4.5, set the temperature to 40~50℃, stir, and obtain the wall material solution; Step 2: Add the bacterial suspension to the wall material solution and stir. Add calcium chloride solution, let stand for 1-2 hours, filter, wash and dry to obtain the Bifidobacterium animalis subsp. lactis composition.

2. The method for preparing a composition of *Bifidobacterium lactis* subsp. *majorheicum* for regulating the gastrointestinal tract according to claim 1, characterized in that: In the raw materials of the wall material solution, the mass ratio of modified sodium alginate, cystamine-chitosan, and deionized water is (0.2~0.8):(0.1~0.4):100 by weight.

3. The method for preparing a composition of *Bifidobacterium lactis* subsp. *majorheicum* for regulating the gastrointestinal tract according to claim 1, characterized in that: The modified sodium alginate is prepared as follows: sodium alginate is added to deionized water, the pH is adjusted to 8.5-9, the temperature is set to 40-50℃, 1,4-butanediol diglycidyl ether is slowly added dropwise, and the temperature is maintained for 6-7 hours. Then, 4-aminoethyl biphenyl is added, the temperature is raised to 70-80℃, and the temperature is maintained for 3-4 hours. The temperature is then lowered to room temperature, the pH is adjusted to 4-4.5, acetone is added and soaked for 12-15 hours, and the mixture is filtered, washed, and dried to obtain modified sodium alginate. The modified sodium alginate raw materials, by weight, consist of: 1-3 parts sodium alginate, 0.3-0.8 parts 1,4-butanediol diglycidyl ether, and 0.5-2 parts 4-aminoethyl biphenyl.

4. The method for preparing a composition of *Bifidobacterium lactis* subsp. *majorheicum* for regulating the gastrointestinal tract according to claim 1, characterized in that: The preparation method of cystamine-chitosan is as follows: carboxymethyl chitosan is added to phosphate buffer and stirred evenly. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide are added sequentially. The mixture is activated at room temperature for 20-40 minutes. Then, cystamine dihydrochloride is added and stirred evenly. The mixture is stirred at room temperature for 6-8 hours, dialyzed, and dried to obtain cystamine-chitosan. The raw materials for cystamine-chitosan, by weight, include 0.2-0.6 parts carboxymethyl chitosan, 15-25 parts phosphate buffer, 0.4-1 parts 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 0.2-0.5 parts N-hydroxysuccinimide, and 0.2-0.6 parts cystamine dihydrochloride.

5. The method for preparing a composition of *Bifidobacterium lactis* subsp. *animal* for regulating the gastrointestinal tract according to claim 1, characterized in that: The volume ratio of the bacterial suspension to the wall material solution is (1~2):(4~6); the concentration of the calcium chloride solution is 1~2wt%.

6. The method for preparing a composition of *Bifidobacterium lactis* subsp. *majorheicum* for regulating the gastrointestinal tract according to claim 1, characterized in that: The preparation method of the bacterial suspension is as follows: the probiotics are activated, cultured in an expanded manner, centrifuged, concentrated, washed, and centrifuged again in sequence, and then placed in physiological saline containing a protectant to prepare the bacterial suspension.

7. The method for preparing a composition of *Bifidobacterium lactis* subsp. *majorheicum* for regulating the gastrointestinal tract according to claim 1, characterized in that: The concentration of the bacterial suspension is 5 × 10⁻⁶. 9 ~5×10 10 CFU / mL.

8. The method for preparing a composition of *Bifidobacterium lactis* subsp. *animal* for regulating the gastrointestinal tract according to claim 6, characterized in that: The mass ratio of the protective agent to physiological saline is (1~5):20; the protective agent is skim milk powder and trehalose in a mass ratio of (1~4):

1.

9. The animal Bifidobacterium lactis composition prepared by the method for preparing a gastrointestinal regulating animal Bifidobacterium lactis composition according to any one of claims 1 to 8.

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