A novel method to quantify viable Enterococcus faecium during feed manufacturing

Enterococcus faecium (E. faecium) is vulnerable to feed manufacturing, and viability loss in pellet feed has led to compromised probiotic efficacy. This study aimed to develop a propidium monoazide (PMA)-qPCR method to quantify viable E. faecium in feed and to investigate the protective effect of different coating technologies during feed manufacturing. First, High amplication efficiency (E = 96.2%, R² = 0.997) and no cross-reactivity with 7 feed-related bacteria was validated by EF2 primers and no cross-reactivity with 7 feed-relevant bacterial species. An optimal concentration of PMA at 40 μM was confirmed to bind to over 99% of deal cells by flow cytometry. Artificial intestinal fluid with trysin could release 94.2% cells from coated E. faecium, which was superior than physiological saline, sodium citrate, artificial intestinal fluid (P < 0.05). With the addition of hexadecyl trimethyl ammonium bromide (CTAB), the DNA extraction efficiency for coating E. faecium was increased by 54.3%. Collectively, this established PMA-qPCR method had high sensitivity (E = 101.1%; R² = 0.991) and could detect live and dead cells with high accuracy (observed vs. set, R² = 0.998). Next, using this method, the survival of different coated E. faecium during feed manufacturing was investigated through a split-split-plot design. As the conditioning temperature rose from 65 °C to 85 °C, overall survival decreased from 72.4% to 12.2% (P < 0.05), in which uncoated and post-encapsulated E. faecium significantly dropped from 85.7% to 8.1% and 13.8% to 3.2% from conditioning to pelleting at 65 °C (P < 0.05), with no further change after cooling. In contrast, pre-encapsulated E. faecium exhibited significantly higher survival than both uncoated and post-encapsulated products at all processing steps and temperatures (P < 0.05). In conclusion, the PMA-qPCR method is valuable to quantify viable E. faecium in feed. The pre‑encapsulated E. faecium tested in this study could improve resistance to heat and mechanical force compared to uncoated and post-encapsulated formulations.