30%_CO 2 /70%_N 2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101. Furthermore, species-specific metabolic pathways enabling different and preferential carbon source utilization were identified, which enable non-competitive coexistence of respective bacteria on meat, resulting in synergistic spoilage. In conclusion, this study gives mechanistically explanations of their acknowledged status as typical spoilage organisms on MAP meats.

High oxygen modified atmosphere packaging is a commonly applied method to prolong the minimum shelf life of fresh (red) meats. Upon spoilage, changes of the initial oxygen concentration and microbiome composition can be observed. Thus, we classified the typical representative meat spoiling bacteria Brochothrix (B.) thermosphacta TMW2.2101 and the four lactic acid bacteria (LAB) Carnobacterium (C.) divergens TMW2.1577, C. maltaromaticum TMW2.1581, Leuconostoc (L.) gelidum subsp. gelidum TMW2.1618, and L. gelidum subsp. gasicomitatum TMW2.1619 along their oxygen consuming capacity, which can indicate the timeline of microbiome and sensorial changes. All bacteria were grown in a model system employing gas tight glass bottles containing meat simulation media and under modified atmosphere (70% O2 and 30% CO2). Oxygen concentrations of media and headspaces were monitored over time and the oxygen uptake rate (OUR) was calculated for all species. All bacteria were able to consume dissolved oxygen, with B. thermosphacta TMW2.2101 exhibiting a 31-times higher OUR per single cell in 60 h. Furthermore, all strains showed significant growth enhancement in the presence of heme indicating respiratory activity. Comparative genomic and physiological analyses predict the activity of a respiratory chain for all species upon high oxygen atmosphere. An additional cytochrome aa3 oxidase is suggested to be responsible for the increased OUR of B. thermosphacta TMW2.2101. Furthermore, B. thermosphacta TMW2.2101 revealed highest oxidative stress tolerance compared to the other bacteria, facilitating a higher respiratory activity. Coupling of respiration and fermentation via regeneration of NADH can be a competitive advantage for meat spoiling bacteria resulting in a higher cell count and possibly accelerated spoilage. The exhibited highest capacity for oxygen consumption of B. thermosphacta compared to LAB in vitro also suggests a higher contribution of this bacterium to the change in the atmosphere upon spoilage of MAP meats in situ.

Significance and Impact of the Study: This study demonstrates applicability of an incorporated oxygen sensor spot to non-invasively determine microbial headspace oxygen consumption in high oxygen modified atmosphere packages (MAP). Furthermore, exploitation of this technology for the prediction of the onset of meat spoilage prior to human perception is demonstrated. Thus, this study provides proof of concept for a novel rapid and easily implementable method, which can be used to reduce meat disposal as waste in retail by individual assessment of single high oxygen MAP.

The ability of certain Pseudomonas (P.) species to grow or persist in anoxic habitats by either denitrification, acetate fermentation, or arginine fermentation has been described in several studies as a special property. Previously, we had isolated strains belonging to the species P. lundensis, P. weihenstephanensis, and P. fragi from anoxic modified atmosphere packaged (MAP) minced beef and further proved their anaerobic growth in vitro on agar plates. This follow-up study investigated the anaerobic growth of two strains per respective species in situ on inoculated chicken breast filet under 100% N2 modified atmosphere. We were able to prove anaerobic growth of all six strains on chicken breast filet with cell division rates of 0.2–0.8/day. Furthermore, we characterized the anaerobic metabolic lifestyle of these Pseudomonas strains by comparative proteomics, upon their cultivation in meat simulation media, which were constantly gassed with either air or 100% N2 atmospheres. From these proteomic predictions, and respective complementation by physiological experiments, we conclude that the Pseudomonas strains P. fragi, P. weihenstephanensis, P. lundensis exhibit a similar anaerobic lifestyle and employ arginine fermentation via the arginine deiminase (ADI) pathway to grow anaerobically also on MAP meats. Furthermore, glucose fermentation to ethanol via the ED-pathway is predicted to enable long term survival but no true growth, while respiratory growth with nitrate as alternative electron acceptor or glucose fermentation to acetate could be excluded due to absence of essential genes. The citric acid cycle is partially bypassed by the glyoxylate shunt, functioning as the gluconeogenetic route without production of NADH2 under carbon limiting conditions as e.g., in packaged meats. Triggered by an altered redox balance, we also detected upregulation of enzymes involved in protein folding as well as disulfide bonds isomerization under anoxic conditions as a counteracting mechanism to reduce protein misfolding. Hence, this study reveals the mechanisms enabling anaerobic grow and persistence of common meat-spoiling Pseudomonas species, and further complements the hitherto limited knowledge of the anaerobic lifestyle of Pseudomonas species in general.

Aims Coagulase‐negative Staphylococcus xylosus strains are used as starter organisms for sausage fermentation. As those strains have to cope with low pH‐values during fermentation, the aim of this study was to identify the acid adaptation mechanisms of S. xylosus TMW 2.1523 previously isolated from salami. Methods and Results A comparative proteomic study between two different acid tolerant mutants was performed. Therefore, both S. xylosus mutants were grown pH‐static under acid stress (pH 5·1) and reference conditions (pH 7·0). Proteomic data were supported by metabolite and cell membrane lipid analysis. Staphylococcus xylosus acid stress adaptation is mainly characterized by a metabolic change towards neutral metabolites, enhanced urease activity, reduced ATP consumption, an increase in membrane fluidity and changes in the membrane thickness. Conclusion This study corroborates mechanisms as previously described for other Gram‐positive bacteria. Additionally, the adjustment of membrane structure and composition in S. xylosus TMW 2.1523 play a prominent role in its acid adaptation. Significance and Impact of the Study This study demonstrates for the first time changes in the membrane lipid composition due to acid stress adaptation in staphylococci.