# 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

## TL;DR

This study characterizes a direct-fed microbial from calf rumen and shows it improves gut barrier function in rats.

## Contribution

The study introduces a low-cost method for characterizing DFMs and demonstrates their protective effect on gut health.

## Key findings

- The DFM has a protein/carbohydrate ratio of 1.3:1 and a surface charge of -32.6 mV, indicating colloidal stability.
- Streptococcus was the dominant genus in the DFM, identified via 16S rRNA sequencing.
- DFM supplementation reduced intestinal permeability and increased gut villi and Goblet cells in rats.

## 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.

The beneficial effects of a direct-fed microbial (DFM) isolated from calf rumen fluid were evaluated and found to enhance the intestinal barrier by reducing the absorption of the marker fluorescein isothiocyanate dextran. Additionally, physicochemical techniques, including Fourier-transform infrared spectroscopy, as well as size distribution and zeta-potential analysis were employed as low-cost and rapid alternatives to standard methods, providing preliminary support for effectively characterizing the DFM.

Graphical Abstract