Fe-TA-GN包被猪源屎肠球菌PL84的制备及其体外环境耐受性评价

Preparation and evaluation of Enterococcus faecium PL84 encapsulated with Fe-TA-GN for enhanced tolerance in simulated gastrointestinal conditions

摘要:

饲用益生菌对维持畜禽健康、促进畜禽生产具有重要作用。然而，多数益生菌的应用受限于其对动物肠道环境胁迫(酸、胆盐、温度)的敏感性，微囊化包被技术是提升其稳定性的关键手段。目的 以自主分离的具有益生特性的屎肠球菌(Enterococcus faecium) PL84为材料，构建金属-多酚-益生元(Fe-TA-GN)复合包被体系，并验证该体系对PL84的保护效果，为其产业化应用提供技术支撑。方法 通过优化包被参数(Fe³⁺与TA的比例、GN的浓度)，结合电镜观察，同时采用CCK-8法检测包被材料对小鼠肠道上皮细胞(IEC-6)活性的影响。通过酸性、胆盐、温度及模拟胃肠液(SGF/SIF)环境的体外耐受性试验评价包被体系的保护效果。结果 PL84在对数生长期时菌体活性最佳，适用于包被。当Fe³⁺与TA的化学计量比为1:3时PL84-Fe-TA复合颗粒的纳米粒度最小，形成致密的金属-多酚网络；当GN浓度为0.4 mg/mL时Fe-TA-GN保护层的zeta电位最高，结构最稳定。扫描电镜(SEM)观察显示，PL84-Fe-TA-GN表面的保护层均匀连续。体外耐受性实验表明，PL84-Fe-TA-GN在pH 3.0、胆盐浓度0.6%的条件下存活率极显著高于未包被的PL84 (P<0.01)；在60 ℃处理后，其存活率较未包被菌株提升了16.29%，且在人工模拟胃液(simulated gastric fluid, SGF)、模拟肠液(simulated intestinal fluid, SIF)中的存活率分别提升了20.80%、13.53%。包被材料(Fe-TA、GN、Fe-TA-GN)对PL84的活性无显著影响(P>0.05)。结论 当Fe³⁺与TA的化学计量比为1:3、GN浓度为0.4 mg/mL时，金属-多酚-益生元复合包被体系稳定，可显著提升屎肠球菌PL84的环境耐受性，且包被材料具有良好的生物安全性，为菌株PL84的产业化应用奠定了技术基础。

Abstract:

Probiotic additives for feed play a crucial role in maintaining the health and improving the production performance of livestock and poultry. However, the application of most probiotics is limited by their sensitivity to environmental stresses (e.g., acid, bile salt, and temperature) in the animal intestinal tract, and microencapsulation serves as a key approach to enhance their stability.Objective This study constructed a metal-polyphenol-prebiotic (Fe-TA-GN) composite coating system for the probiotic strain Enterococcus faecium PL84 isolated by us and verified its protective effect on PL84, aiming to provide technical support for the industrial application of the strain.Methods Coating parameters (Fe³⁺-TA molar ratio and GN concentration) were optimized. Scanning electron microscopy (SEM) and the CCK-8 assay were employed to evaluate the effects of coating materials on the viability of mouse intestinal epithelial cells (IEC-6). The protective effect of the coating system was assessed through in vitro tolerance tests under acidic, bile salt, thermal conditions, as well as in simulated gastrointestinal fluids.Results E. faecium PL84 exhibited the highest cell viability during the logarithmic growth phase, being suitable for microencapsulation. When the molar ratio of Fe³⁺ to TA was 1:3, the PL84-Fe-TA composite particles showed the smallest nanoscale particle size and formed a dense metal-polyphenol network. At a GN concentration of 0.4 mg/mL, the Fe-TA-GN coating layer achieved the highest zeta potential and optimal structural stability. SEM revealed a uniform and continuous surface coating layer of PL84-Fe-TA-GN. In vitro tolerance assays demonstrated that the survival rate of PL84-Fe-TA-GN was higher than that of uncoated PL84 under conditions of pH 3.0 and 0.6% bile salt (P<0.01). After treatment at 60 ℃, the survival rate of the coated strain increased by 16.29% compared with that of uncoated PL84. Additionally, the survival rates of PL84-Fe-TA-GN in simulated gastric fluid and simulated intestinal fluid improved by 20.8% and 13.53%, respectively. The coating materials (Fe-TA, GN, and Fe-TA-GN) had no significant effect on the viability of PL84 (P>0.05).Conclusion When the molar ratio of Fe³⁺ to TA is 1:3 and the GN concentration is 0.4 mg/mL, the metal-polyphenol-prebiotic composite coating system is stable and can significantly enhance the environmental tolerance of E. faecium PL84. Moreover, the coating materials possess good biocompatibility, laying a solid technical foundation for the industrial application of E. faecium PL84.