In this study, we show that calcium pectinate beads (CPB) allow the formation of 20 µm spherical microcolonies of the probiotic bacteria Lacticaseibacillus paracasei (formerly designated as Lactobacillus paracasei) ATCC334 with a high cell density, reaching more than 10 log (CFU/g). The bacteria within these microcolonies are well structured and adhere to a three-dimensional network made of calcium-pectinate through the synthesis of extracellular polymeric substances (EPS) and thus display a biofilm-like phenotype, an attractive property for their use as probiotics. During bacterial development in the CPB, a coalescence phenomenon arises between neighboring microcolonies accompanied by their peripheral spatialization within the bead. Moreover, the cells of L. paracasei ATCC334 encased in these pectinate beads exhibit increased resistance to acidic stress (pH 1.5), osmotic stress (4.5 M NaCl), the freeze-drying process and combined stresses, simulating the harsh conditions encountered in the gastrointestinal (GI) tract. In vivo, the oral administration of CPB-formulated L. paracasei ATCC334 in mice demonstrated that biofilm-like microcolonies are successfully released from the CPB matrix in the colonic environment. In addition, these CPB-formulated probiotic bacteria display the ability to reduce the severity of a DSS-induced colitis mouse model, with a decrease in colonic mucosal injuries, less inflammation, and reduced weight loss compared to DSS control mice. To conclude, this work paves the way for a new form of probiotic administration in the form of biofilm-like microcolonies with enhanced functionalities.

在这项研究中，我们表明果胶酸钙珠（CPB）可以形成20 µm益生菌副干酪乳杆菌（原命名为副干酪乳杆菌）ATCC334球形微菌落，细胞密度高，达到10 log（CFU / g）以上）。这些微菌落中的细菌结构良好，并通过胞外聚合物质（EPS）的合成粘附到由果胶酸钙制成的三维网络上，因此表现出生物膜样表型，这对于将其用作益生菌具有吸引力。在CPB中的细菌发育过程中，相邻的微菌落之间会发生聚结现象，并伴随其在微珠内的周边空间化。此外，副干酪乳杆菌的细胞装在这些果胶状微珠中的ATCC334表现出对酸性压力（pH 1.5），渗透压（4.5 M NaCl），冷冻干燥过程和复合应力的增强抵抗力，模拟了胃肠道（GI）遇到的恶劣条件。在体内，CPB配制的副干酪乳杆菌的口服给药小鼠中的ATCC334证明在结肠环境中已成功从CPB基质中释放出了类似生物膜的微菌落。此外，与DSS对照小鼠相比，这些由CPB配制的益生菌具有降低DSS诱导的结肠炎小鼠模型严重程度的能力，同时结肠粘膜损伤减少，炎症减少，体重减轻。总而言之，这项工作为以功能增强的生物膜样微菌落形式的新型益生菌施用铺平了道路。