Strains, culture media, and cultivation

In this research, three different strains of L. monocytogenes were examined. The strain FFH (= DSM 112142; serovar group 4b, 4d, or 4e) was isolated from minced meat in 2011 and the strain FFL 1 (= DSM 112143; serovar group 1/2a or 3a) from Irish smoked organic salmon in 2012. The type strain DSM 20600^T (serovar group 1/2a) was included as the reference strain in this study. The type strain was obtained from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures. The two strains FFH and FFL 1 were deposited at the DSMZ open collection. These strains were assigned to serovar groups by multiplex polymerase chain reaction (PCR) according to the method of Doumith et al. (2004). The adaptive response of strain FFL 1 to low temperature is primarily a fatty acid-dependent mechanism while strains DSM 20600^T and FFH expressed, in addition, MK-mediated response (Seel et al. 2018).

All strains were aerobically cultured in 100 ml tryptic soy broth-yeast extract (TSB-YE) medium composed of tryptic soy broth containing 17.0 g peptone from casein l⁻¹, 3.0 g peptone from soy l⁻¹, 2.5 g d-glucose l⁻¹, 5.0 g sodium chloride l⁻¹, and 2.5 g dipotassium hydrogen phosphate l⁻¹ supplemented with 6.0 g yeast extract l⁻¹ or in 100 ml ultra-high temperature processed milk (UHT-milk, 3.5% fat) using 300 ml Erlenmeyer flasks. The TSB-YE medium or the UHT-milk, respectively, was supplemented with a mixture of the AAA l-phenylalanine, l-tryptophan and l-tyrosine (each of 200, 400, 600, or 800 mg l⁻¹). Non-AAA l-alanine, l-cysteine, and l-serine were used as controls. Water activity (a_w) of the medium was measured with a LabMaster-aw instrument (Novasina, Switzerland). Growth in TSB-YE medium was documented by optical density (OD) at 625 nm with a GENESYS 30 visible spectrophotometer (Thermo Fisher Scientific, USA) and fitted by the Gompertz growth model as described by López et al. (2004). Cultures were prepared in two to six independent replicates, inoculated with 1% (vol/vol) of an overnight culture for growth in TSB-YE medium or 0.02% (vol/vol) for cultivation in milk, and incubated on an orbital shaker at 6 °C and 150 rpm until late exponential phase (OD_625nm = 0.8–1.0). Cultures were harvested by centrifugation (10,000×g for 10 min) at growth temperature and washed thrice with sterile phosphate-buffered saline (PBS) which was adjusted to growth temperature, pH 7.4. Subsequently, this biomass was used for fatty acid analysis, determination of MK-7 content, and temperature stress test. Colonies were cultivated on tryptic soy agar-yeast extract (TSA-YE) medium at 30 °C.

To determine colony forming units (CFU) for the temperature stress test and the growth inhibition test in milk, 50 µl of serial dilutions were plated on TSA-YE medium (90 mm Petri dish) using the exponential mode (ISO 4833-2, ISO 7218 and AOAC 977.27) of the easySpiral automatic plater (Interscience, France). After a 1-day incubation at 37 °C, the CFU were counted for the corresponding dilution steps and the weighted average of enumerated L. monocytogenes was given in CFU ml⁻¹. The results for the temperature stress test and the growth inhibition test in milk were presented as decadic logarithm (log₁₀) decrease or increase relative to the initial CFU ml⁻¹, respectively.