Physicochemical characterization and 16S rRNA analysis of a direct-fed microbial from calf ruminal fluid and its protective effect on Sprague-Dawley rat gut barrier function.

Abstract

This study aimed to characterize the physicochemical properties and microbiota composition of a direct-fed microbial (DFM) and evaluate its protective effect on intestinal permeability in Sprague-Dawley rats using fluorescein isothiocyanate dextran (FITC-d) as a biomarker. The DFM was further characterized using Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), environmental scanning electron microscopy (ESEM), energy-dispersive X-ray spectroscopy (EDS), and cell surface hydrophobicity (microbial adhesion to hexadecane [MATH] assay). The 16S rRNA gene was sequenced using short-read sequencing. In general, the DFM exhibited the characteristic FTIR bands associated with probiotic cells with a protein/carbohydrate ratio of 1.3:1. It was also found from the DLS analysis that the average particle size and surface electrical potential of the probiotic cells were 1,062 ± 77 nm and -32.6 ± 3.7 mV, respectively. ESEM studies confirmed the size of the cells (1,010 to 1,060 nm), showing a quasi-spherical cocci-type morphology, whereas EDS spectroscopy revealed a higher Nitrogen/Carbone ratio on the cell surface. Moreover, the MATH assay showed the hydrophobic character of the DFM (92% adhesion). Furthermore, based on the 16S rRNA gene analysis, the predominant genus in the DFM was Streptococcus (99%). Regarding the protective effect on the gut barrier, animals supplemented with 10¹¹ CFU/mL exhibited a significantly reduced intestinal permeability compared with the control group. DFM supplementation also increased villi and crypt dimensions and Goblet cells (P < 0.05) in the ileum and cecum. These results demonstrate that the DFM presented adequate surface and colloidal properties that help maintain the functionality of the gut barrier.