ENHANCED RECOVERY OF SEVERELY HEAT-INJURED, THERMOTOLERANT LISTERIA-MONOCYTOGENES FROM USDA AND FDA PRIMARY ENRICHMENT MEDIA USING A NOVEL, SIMPLE, STRICTLY ANAEROBIC METHOD

A novel, simple, strictly anaerobic method was developed, which dramatically enhanced the recovery of severely heat-injured Listeria monocytogenes from pasteurized milk, compared to current aerobic methods. Cells of L. monocytogenes were grown in trypticase soy broth supplemented with 0.6% yeast extract (TSB-YE) at 43 degrees C for 18 h and then severely heat-injured at 62.8 degrees C for 5 min in raw milk in sealed, totally submerged thermal-death-time tubes. Recovery in primary enrichment media was enhanced in all cases by purging the headspace with N-2 gas. The following treatments (in order of effectiveness) significantly improved recovery compared to conventional aerobic controls: addition of filter-sterilized cysteine + N-2 purging > pre-reduced Hungate media + N-2 purging = Oxyrase(R) + lactate + N-2 purging > filter-sterilized cysteine - N-2 purging > Oxyrase(R) - lactate + N-2 purging. Addition of filter-sterilized cysteine to a final concentration of 0.5 g/l with subsequent: N-2 purging increased recovery from 0% to 60% with the U.S. Department of Agriculture (USDA) UVM broth and from 11 to 100% with the Food and Drug Administration (FDA) LEE. Addition of either 1% pyruvate or a IO-fold higher concentration of cysteine (5.0 gn) completely inhibited recovery. Faster and more efficient recovery of L monocytogenes using cysteine and N, purging was attributed to the absence of oxidative stress during primary enrichment. Potential difficulties in recovering very severely injured cells are discussed.