An antibacterial peptide composition, preparation method and application

Abstract

The application provides an antibacterial peptide composition, a preparation method and application, and comprises a composite probiotic core layer, an antibacterial lipopeptide loading layer and a CaCO3 nanocrystal buffered alginate / pectin composite microcapsule coating; the composite probiotic layer comprises a bacteria slurry composed of Ruminococcus lactaris CY12 and S. salivarius 7247 at a ratio of 1:1 in terms of viable bacterial number, and the total viable bacterial number is greater than or equal to 5.0x10 9 CFU / g; and the antibacterial lipopeptide layer comprises SEPs candidate peptides mined from human intestinal metagenome, and the total addition amount is 0.1%-0.5%. The antibacterial peptide composition, the preparation method and the application are used, gastric acid tolerance and colon targeting accuracy better than traditional pH-dependent coating are realized, natural synergy of maintenance and defense is realized, and excellent efficacy can be achieved without genetic engineering modification.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide compositions, and in particular to an antimicrobial peptide composition, its preparation method, and its application. Background Technology

[0002] The gut microbiota plays a crucial role in maintaining host health, participating in various physiological processes such as nutrient metabolism, immune regulation, and pathogen defense. Probiotics, as live microorganisms that colonize the gut, demonstrate significant effects in restoring gastrointestinal function and stabilizing the intestinal mucosal barrier through mechanisms such as improving host microbiota balance, regulating the immune system, inhibiting the growth of pathogenic bacteria, and suppressing endotoxin production. In recent years, the combination therapy of probiotics and antimicrobial peptides has become a research hotspot in the field of gut health due to its unique advantages of synergistic effects and complementary functions.

[0003] However, existing combinations of probiotics and antimicrobial peptides still have the following technical shortcomings: Firstly, the strains used in existing compound probiotic preparations are mostly conventional commercial strains (such as Lactobacillus acidophilus and Lactobacillus plantarum). These strains have limited functions in intestinal barrier repair and immune regulation, making it difficult to achieve the synergistic effect of "functional complementarity between strains." Studies have shown that although Lactobacillus rhamnosus and Lactobacillus reuteri are both commonly used probiotics in intestinal flora regulation research, they differ significantly in their anti-colitis mechanisms: Lactobacillus reuteri performs better in enhancing the intestinal barrier, while Lactobacillus rhamnosus is more advantageous in short-chain fatty acid synthesis. However, current technologies have not yet specifically combined the two to leverage their respective biological advantages.

[0004] Secondly, existing antimicrobial peptide products mainly rely on traditional lipopeptides (such as cyclic lipopeptides derived from Bacillus, such as Surfactin, Fengycin, and Iturin) or natural antimicrobial peptides derived from animals / insects (such as cephalosporins and lactoferrin). These antimicrobial peptides generally suffer from drawbacks such as a broad antimicrobial spectrum and poor selectivity for beneficial bacteria, which can easily cause "collateral damage" to intestinal probiotics during use. Furthermore, traditional lipopeptides have limited activity against multidrug-resistant Gram-negative bacteria. At the same time, oral delivery of antimicrobial peptides faces significant obstacles such as enzymatic degradation, instability, and poor permeability through the gastrointestinal epithelium, resulting in low bioavailability. Current technologies lack effective solutions to this problem.

[0005] Third, existing probiotic delivery systems mostly employ single pH-responsive enteric coatings (such as the Eudragit® series) or simple alginate microcapsules, which suffer from drawbacks such as poor gastric acid tolerance and insufficient precision in colon-targeted release. Studies have found that single alginate microcapsules significantly reduce probiotic survival rates in simulated gastric fluid, failing to meet the requirements for intestinal-targeted delivery. In contrast, microcapsule systems based on CaCO3 nanocrystal-buffered alginate / pectin composites release CO3 into gastric fluid. 2- By neutralizing the local acidic environment, it can achieve "super acid-resistant" protection and precise colon-targeted release, demonstrating significantly enhanced probiotic stability and targeted colonization ability in both in vitro and in vivo experiments. However, this delivery system has not yet been organically combined with the precise delivery of antimicrobial lipopeptides, especially lacking a synergistic design for the sequential release of probiotics and antimicrobial peptides.

[0006] Fourth, the discovery of existing antimicrobial lipopeptides largely relies on traditional microbial fermentation screening or chemical synthesis of known sequences, resulting in low efficiency and long development cycles for the discovery of novel antimicrobial lipopeptides. In recent years, metagenomic mining technology based on artificial intelligence has provided a new pathway for the original innovation of antimicrobial peptides. Researchers developed the AMP-SEMiner framework, integrating protein language models, structural clustering, and evolutionary analysis, systematically mining over 1.6 million novel antimicrobial peptide candidates from the human gut metagenomics. Experimental verification showed that five of these candidates exhibited antimicrobial efficacy surpassing that of traditional antibiotics. However, these newly discovered antimicrobial peptides derived from human gut symbiotic bacteria have not yet been applied to gut health-related composite products. Summary of the Invention

[0007] The purpose of this invention is to provide an antimicrobial peptide composition, preparation method and application, which achieves better gastric acid tolerance and colon targeting accuracy than traditional pH-dependent coating, realizes natural synergy of maintenance and defense, and can exert excellent efficacy without genetic engineering modification.

[0008] To achieve the above objectives, the present invention provides an antimicrobial peptide composition comprising a composite probiotic core layer, an antimicrobial lipopeptide loading layer, and a CaCO3 nanocrystal buffered alginate / pectin composite microcapsule coating. The compound probiotic layer comprises a bacterial slurry composed of *Lactobacillus rhamnosus* CY12 and *Lactobacillus salivarius* 7247 in a 1:1 live bacteria ratio, with a total live bacteria count ≥ 5.0 × 10⁻⁶. 9 CFU / g; The antimicrobial lipopeptide layer contains SEP candidate peptides mined from the human gut metagenomics, with a total addition amount of 0.1%-0.5%.

[0009] Preferably, the SEP candidate peptides are selected from one or more of Prevotellin-2, bacteroidin-1, fusobacticin-1, or their derivatives.

[0010] Preferably, the antibacterial lipopeptide layer is pre-encapsulated with nanoliposomes, wherein the molar ratio of soybean phospholipids:cholesterols:SEPs in the nanoliposome composition is 8:1:0.5, and the particle size is 80-150 nm.

[0011] Preferably, the core layer of the compound probiotics also includes three layers of freeze-drying protectants, the mass composition of which is 10wt% skim milk, 5wt% trehalose, and 0.2wt% gelatin.

[0012] Preferably, the mass ratio of the three-layer freeze-drying protectant to the bacterial sludge is 1:(1~3). The mass ratio of the compound probiotic core layer to the antimicrobial lipopeptide loading layer is (5~10):1.

[0013] A method for preparing an antimicrobial peptide composition includes the following steps: Preparation of the core layer of compound probiotics: CY12 and LS7247 were fermented at a live bacteria ratio of 1:1, and the bacterial sludge was collected by centrifugation. The bacterial sludge was mixed with a three-layer freeze-drying protectant, pre-frozen, and then vacuum freeze-dried to obtain freeze-dried probiotic powder. Preparation of antibacterial lipopeptide loading layer: SEPs were mixed with soybean phospholipids and cholesterol in a molar ratio of 8:1:0.5, and nanoliposomes were prepared by thin film hydration method; Preparation of CaCO3 nanocrystal-buffered alginate / pectin composite microcapsules: 0.1M CaCl2 and 0.1M Na2CO3 solutions were mixed in equimolar ratio and stirred at room temperature to generate CaCO3 nanocrystals. Sodium alginate and pectin were dissolved in deionized water at a mass ratio of (9~1):1, with a final concentration of 2% (w / v) for sodium alginate and pectin. Then, CaCO3 nanocrystals were added to obtain a CaCO3 nanocrystal suspension. The freeze-dried probiotic powder was mixed with nanoliposomes, and then CaCO3 nanocrystal suspension was added and stirred to disperse the mixture. The mixture was dripped into a cross-linking bath containing 1% CaCl2 to form alginate / pectin / CaCO3 composite microcapsules; The antimicrobial peptide composition was obtained by curing in a crosslinking bath, washing, and freeze-drying.

[0014] Preferably, the mass ratio of freeze-dried probiotic powder, nanoliposomes and CaCO3 nanocrystals is 1:(0.1~0.2):(0.03~0.09).

[0015] Preferably, the mass ratio of the mixed solution of CaCO3 nanocrystals, sodium alginate, pectin, and deionized water is 1:6.

[0016] Preferably, the freeze-dried probiotic powder has a freeze-drying survival rate of ≥90% and a moisture content of ≤5%.

[0017] The use of an antimicrobial peptide composition in the preparation of a medicament or functional food for the prevention of antibiotic-associated diarrhea, Clostridium difficile infection or treatment of intestinal infections caused by multidrug-resistant Gram-negative bacteria.

[0018] Therefore, the present invention employs the above-mentioned antimicrobial peptide composition, preparation method, and application, and the technical effects are as follows: Advanced colon-targeted delivery: Utilizing CaCO3 nanocrystal buffered alginate / pectin composite microcapsule technology, it achieves superior gastric acid tolerance and colon-targeting accuracy compared to traditional pH-dependent coatings.

[0019] The complementary combination of two strains: CY12 (barrier repair) and LS7247 (bacteriocin secretion) complement each other, achieving a natural synergy of "maintenance and defense", and can exert excellent efficacy without genetic engineering.

[0020] Wide applicability: It can be prepared as lyophilized powder, enteric-coated capsules or oral suspensions for the prevention and treatment of antibiotic-associated diarrhea, Clostridium difficile infection and intestinal infection caused by multidrug-resistant Gram-negative bacteria, with broad application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic cross-sectional view of the three-layer structure of the microcapsule of the present invention; Figure 2 This is a schematic diagram of time-sequenced release closed-loop management. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] Lipopeptide screening process: This invention utilizes AI tools (such as AMPidentifier) ​​to identify candidate AMPs-6 from metagenomics in a high-throughput manner. This method has been validated in top journals such as *Cell* and *Microbiome* in 2024-2025, and is characterized by high efficiency, accuracy, and original innovation, significantly different from existing technologies that rely on natural product extraction or chemical synthesis.

[0025] Example 1 A method for preparing an antimicrobial peptide composition includes the following steps: S1. Preparation of the compound probiotic layer Lactobacillus rhamnosus CY12 and Lactobacillus salivarius LS7247 were inoculated into MRS medium and anaerobically cultured at 37°C for 24 hours. The bacterial sludge was collected by centrifugation (4°C, 6000 rpm, 15 minutes). The sludge was mixed with a three-layer freeze-drying protectant at a ratio of 1:2 (w / w). The protectant formulation consisted of 10% skim milk, 5% trehalose, and 0.2% gelatin. After pre-freezing, the mixture was vacuum freeze-dried, pulverized, and passed through an 80-mesh sieve to obtain freeze-dried probiotic powder with a freeze-drying survival rate of 92.4%.

[0026] S2. Preparation of antibacterial lipopeptide layer Candidate SEPs were mined from the human gut metagenomic database using the AI ​​tool AMPidentifier, and Prevotellin-2 and its derivatives were identified as the main candidate peptides. Prevotellin-2 (encoded by Prevotellacopri) with a purity >95% was synthesized using solid-phase chemical synthesis. SEPs were mixed with soybean phospholipids and cholesterol in a molar ratio of 8:1:0.5, and nanoliposomes were prepared using a thin-film hydration method, with particle sizes controlled at 80-150 nm.

[0027] Preparation of S3.CaCO3 nanocrystal suspension Prepare 0.1 M CaCl2 and 0.1 M Na2CO3 solutions, and adjust the pH to 10.5. At room temperature, add the CaCl2 solution dropwise to the Na2CO3 solution while stirring at 6000 rpm. Collect the precipitate, wash and dry it to obtain CaCO3 nanocrystals with a particle size of 50-100 nm, which are then suspended in deionized water.

[0028] S4. Hybridization and Microencapsulation Freeze-dried probiotic powder and nanoliposomes were mixed at a mass ratio of 5:1. A CaCO3 nanocrystal suspension (CaCO3 solids amounting to 6% of the probiotic powder mass) was added, along with sodium alginate and pectin (sodium alginate:pectin = 9:1, final concentration 2% w / v). The mixture was stirred and dispersed. The mixture was then dropped into a cross-linking bath containing 1% CaCl2 at a constant rate and allowed to solidify for 30 minutes to form alginate / pectin / CaCO3 composite microcapsules. These were collected by centrifugation, freeze-dried, and the final product was obtained. A schematic diagram of the finished product is shown below. Figure 1 As shown.

[0029] Example 2 (Probiotic Proportion Increase Plan) The results are basically the same as in Example 1, except that the mass ratio of freeze-dried probiotic powder to nanoliposomes is adjusted to 10:1, the amount of CaCO3 solids is adjusted to 9% (upper limit) of the mass of probiotic powder, and the ratio of sodium alginate to pectin is 3:1 (increasing the pectin ratio to enhance the synergistic effect of the gel network).

[0030] Example 3 (High Antibacterial Lipopeptide Regimen) The formulation is basically the same as Example 1, except that the loading of SEPs in the nanoliposomes is increased to 1.5 times that of the original formulation (i.e., the molar ratio of soybean phospholipids:cholesterol:SEPs = 8:1:0.75), and the mass ratio of probiotic powder to nanoliposomes is adjusted to 5:1 (to maintain a lower ratio to accommodate more liposomes), and the amount of CaCO3 solids is adjusted to 3% of the mass of probiotic powder (lower limit).

[0031] Comparative Example 1 (without antibacterial lipopeptide layer, only probiotic microcapsules) The product is basically the same as in Example 1, except that no antimicrobial lipopeptide layer (SEPs nanoliposomes) is added. Instead, a probiotic microcapsule product is prepared by encapsulating freeze-dried probiotic powder in alginate / pectin / CaCO3 microcapsules.

[0032] Comparative Example 2 (without CaCO3 nanocrystal buffer layer, only alginate / pectin microcapsules) The process is basically the same as in Example 1, except that the outermost layer of the microcapsule does not have a CaCO3 nanocrystal buffer layer, and only alginate / pectin composite microcapsule is used for encapsulation (the amount of CaCO3 solid is 0).

[0033] Table 1 Summary of Test Results

[0034] Comparative Example 2, lacking a CaCO3 nanocrystal buffer layer, showed insufficient protection of the microcapsules in gastric acid, resulting in a gastric juice release rate as high as 15.3%, significantly exceeding the 10% standard. The examples with CaCO3 buffer and Comparative Example 1 both exhibited gastric juice release rates below 8%, meeting the standard. This study demonstrates that adding CaCO3 to the alginate / pectin system can significantly improve the survival rate of probiotics in simulated gastric juice.

[0035] All examples met the quality standard of >90%. Comparative Example 2, due to the lack of CaCO3 buffering, resulted in partial disintegration and release of the microcapsules in gastric juice, leading to reduced remaining contents upon reaching the colon and a colonic release rate slightly below 90%. SEPs target bacterial membranes for sterilization and can regulate intestinal symbiotic bacteria, combating pathogens while reshaping the gut microbiota. All samples containing SEPs showed activation rates exceeding 80%, indicating that the nanoliposome encapsulation combined with the CaCO3 buffer delivery system effectively protects SEPs and achieves colon-targeted activation. Regarding the release mechanism of the antimicrobial peptide composition at different time points, such as... Figure 2 As shown in Table 2.

[0036] Table 2 shows the sequential release of the three-layer structure of the present invention in vivo according to a precise timing sequence.

[0037] Therefore, the present invention, by employing the above-mentioned antimicrobial peptide composition, preparation method and application, achieves better gastric acid tolerance and colon targeting accuracy than traditional pH-dependent coating, realizes natural synergy of maintenance and defense, and can exert excellent efficacy without genetic engineering modification.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An antimicrobial peptide composition, characterized in that, The coating consists of an alginate / pectin composite microcapsule, including a core layer of compound probiotics, an antimicrobial lipopeptide loading layer, and CaCO3 nanocrystal buffer. The compound probiotic layer comprises a bacterial slurry composed of *Lactobacillus rhamnosus* CY12 and *Lactobacillus salivarius* 7247 in a 1:1 live bacteria ratio, with a total live bacteria count ≥ 5.0 × 10⁻⁶. 9 CFU / g; The antimicrobial lipopeptide layer contains SEP candidate peptides mined from the human gut metagenomics, with a total addition amount of 0.1%-0.5%.

2. The antimicrobial peptide composition, preparation method, and application according to claim 1, characterized in that, The SEP candidate peptides are selected from one or more of Prevotellin-2, bacteroidin-1, fusobacticin-1, or their derivatives.

3. The antimicrobial peptide composition, preparation method, and application according to claim 1, characterized in that, The antibacterial lipopeptide layer is pre-encapsulated with nanoliposomes. The molar ratio of soybean phospholipids:cholesterol:SEPs in the nanoliposome composition is 8:1:0.5, and the particle size is 80-150nm.

4. The antimicrobial peptide composition, preparation method, and application according to claim 1, characterized in that, The core layer of the compound probiotics also includes three layers of freeze-drying protectants, which consist of 10 wt% skim milk, 5 wt% trehalose, and 0.2 wt% gelatin.

5. The antimicrobial peptide composition, preparation method, and application according to claim 1, characterized in that, The mass ratio of the three-layer freeze-drying protectant to the bacterial sludge is 1:(1~3). The mass ratio of the compound probiotic core layer to the antimicrobial lipopeptide loading layer is (5~10):

1.

6. A method for preparing an antimicrobial peptide composition, characterized in that, Includes the following steps: Preparation of the core layer of compound probiotics: CY12 and LS7247 were fermented at a live bacteria ratio of 1:1, and the bacterial sludge was collected by centrifugation. The bacterial sludge was mixed with a three-layer freeze-drying protectant, pre-frozen, and then vacuum freeze-dried to obtain freeze-dried probiotic powder. Preparation of antibacterial lipopeptide loading layer: SEPs were mixed with soybean phospholipids and cholesterol in a molar ratio of 8:1:0.5, and nanoliposomes were prepared by thin film hydration method; Preparation of CaCO3 nanocrystal-buffered alginate / pectin composite microcapsules: 0.1M CaCl2 and 0.1M Na2CO3 solutions were mixed in equimolar ratio and stirred at room temperature to generate CaCO3 nanocrystals. Sodium alginate and pectin were dissolved in deionized water at a mass ratio of (9~1):1, with a final concentration of 2% (w / v) for sodium alginate and pectin. Then, CaCO3 nanocrystals were added to obtain a CaCO3 nanocrystal suspension. The freeze-dried probiotic powder was mixed with nanoliposomes, and then CaCO3 nanocrystal suspension was added and stirred to disperse the mixture. The mixture was dropped into a cross-linking bath containing 1% CaCl2 to form alginate / pectin / CaCO3 composite microcapsules; The antimicrobial peptide composition was obtained by curing in a crosslinking bath, washing, and freeze-drying.

7. The method for preparing an antimicrobial peptide composition according to claim 6, characterized in that, The mass ratio of freeze-dried probiotic powder, nanoliposomes and CaCO3 nanocrystals is 1:(0.1~0.2):(0.03~0.09).

8. The method for preparing an antimicrobial peptide composition according to claim 6, characterized in that, The mass ratio of CaCO3 nanocrystals to a mixed solution of sodium alginate, pectin, and deionized water is 1:

6.

9. The method for preparing an antimicrobial peptide composition according to claim 6, characterized in that, The freeze-dried probiotic powder has a freeze-drying survival rate of ≥90% and a moisture content of ≤5%.

10. The use of an antimicrobial peptide composition in the preparation of a medicament or functional food for the prevention of antibiotic-associated diarrhea, Clostridium difficile infection or the treatment of intestinal infections caused by multidrug-resistant Gram-negative bacteria.

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