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 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, qPCR using the EF2 primer set was validated for high amplification efficiency ( E = 96.2%, R 2 = 0.997) and no cross-reactivity with 7 feed-relevant bacterial species. Determined by flow cytometry and qPCR, 40 μM PMA could label over 99% of dead cells. The effects of the physiological saline, sodium citrate and artificial intestinal fluid on releasing free cells from coated E. faecium were investigated by the culture method and PMA-qPCR, and the trypsin-containing artificial intestinal fluid could release 94.2% viable cells from coated E. faecium ( P < 0.05). The hexadecyl trimethyl ammonium bromide ( CTAB )-assisted method was developed, which increased the DNA extraction efficiency by 54.3% ( P < 0.05). Collectively, this established PMA-qPCR method had high sensitivity ( E = 101.1%; R 2 = 0.991) and could detect live and dead cells with high accuracy (observed vs. set, R 2 = 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.