This study aimed to fabricate and characterize a novel colorimetric indicator designed to detect ammonia (NH3) and monitor meat freshness. The sensing platform was constructed using electrospun nanofibers made from polylactic acid (PLA), which were then impregnated with anthocyanins as a natural pH-sensitive dye, extracted from red cabbage. This research involved investigating the relationship between the various concentrations of anthocyanins and the colorimetric platform\'s efficiency when exposed to ammonia vapor. Scanning electron microscope (SEM) results were used to examine the morphology and structure of the nanofiber mats before and after the dip-coating process. The study also delved into the selectivity of the indicator when exposed to various volatile organic compounds (VOCs) and their stability under extreme humidity levels. Furthermore, the platform\'s sensitivity was evaluated as it encountered ammonia (NH3) in concentrations ranging from 1 to 100 ppm, with varying dye concentrations. The developed indicator demonstrated an exceptional detection limit of 1 ppm of MH3 within just 30 min, making it highly sensitive to subtle changes in gas concentration. The indicator proved effective in assessing meat freshness by detecting spoilage levels in beef over time. It reliably identified spoilage after 10 h and 7 days, corresponding to bacterial growth thresholds (107 CFU/mL), both at room temperature and in refrigerated environments, respectively. With its simple visual detection mechanism, the platform offered a straightforward and user-friendly solution for consumers and industry professionals alike to monitor packaged beef freshness, enhancing food safety and quality assurance.

This study investigated the impact of Brochothrix (B.) thermosphacta and Pseudomonas (Ps.) fragi on the transcriptomes of Photobacterium (P.) phosphoreum and P. carnosum on chicken meat under modified atmosphere (MA) and air atmosphere (AA). P. phosphoreum TMW2.2103 responded to MA with a reduced transcript number related to cell division and an enhanced number related to oxidative stress. Concomitantly, the analysis revealed upregulation of fermentation and downregulation of respiration. It predicts enhanced substrate competition in presence of co-contaminants/MA. In contrast, the strain upregulated the respiration in AA, supposably due to improved substrate accessibility in this situation. For P. carnosum TMW2.2149 the respiration was downregulated, and the pyruvate metabolism upregulated under MA. MA/co-contaminant resulted in multiple upregulated metabolic routes. Conversely, AA/co-contaminant resulted only in minor regulations, showing inability to cope with fast growing competitors. Observations reveal different strategies of photobacteria to react to co-contaminants on meat.

Modified atmosphere packaging (MAP) is a common strategy to selectively prevent the growth of certain species of meat spoiling bacteria. This study aimed to determine the impact of high oxygen MAP (70% O2, 30% CO2, red and white meats) and oxygen-free MAP (70% N2, 30% CO2, also white meat and seafood) on preventing the growth of spoiling photobacteria on meat. Growth of Photobacterium carnosum and P. phosphoreum was monitored in a meat simulation media under different gas mixtures of nitrogen, oxygen, and carbon dioxide, and samples were taken during exponential growth for a comparative proteomic analysis. Growth under air atmosphere appears optimal, particularly for P. carnosum. Enhanced protein accumulation affected energy metabolism, respiration, oxygen consuming reactions, and lipid usage. However, all the other atmospheres show some degree of growth reduction. An increase in oxygen concentration leads to an increase in enzymes counteracting oxidative stress for both species and enhancement of heme utilization and iron-sulfur cluster assembly proteins for P. phosphoreum. Absence of oxygen appears to switch the metabolism toward fermentative pathways where either ribose (P. phosphoreum) or glycogen (P. carnosum) appear to be the preferred substrates. Additionally, it promotes the use of alternative electron donors/acceptors, mainly formate and nitrate/nitrite. Stress response is manifested as an enhanced accumulation of enzymes that is able to produce ammonia (e.g., carbonic anhydrase, hydroxylamine reductase) and regulate osmotic stress. Our results suggest that photobacteria do not sense the environmental levels of carbon dioxide, but rather adapt to their own anaerobic metabolism. The regulation in presence of carbon dioxide is limited and strain-specific under anaerobic conditions. However, when oxygen at air-like concentration (21%) is present together with carbon dioxide (30%), the oxidative stress appears enhanced compared to air conditions (very low carbon dioxide), as explained if both gases have a synergistic effect. This is further supported by the increase in oxygen concentration in the presence of carbon dioxide. The atmosphere is able to fully inhibit P. carnosum, heavily reduce P. phosphoreum growth in vitro, and trigger diversification of energy production with higher energetic cost, highlighting the importance of concomitant bacteria for their growth on raw meat under said atmosphere.

The spoilage of vacuum-packed meat by Clostridium estertheticum complex (CEC), which is accompanied by or without production of copious amounts of gas, has been linked to the acetone-butyrate-ethanol fermentation, but the mechanism behind the variable gas production has not been fully elucidated. The reconstruction and comparison of intra- and interspecies metabolic pathways linked to meat spoilage at the genomic level can unravel the genetic basis for the variable phenotype. However, this is hindered by unavailability of CEC genomes, which in addition, has hampered the determination of genetic diversity and its drivers within CEC. Therefore, the current study aimed at determining the diversity of CEC through comprehensive comparative genomics. Fifty CEC genomes from 11 CEC species were compared. Recombination and gene gain/loss events were identified as important sources of natural variation within CEC, with the latter being pronounced in genomospecies2 that has lost genes related to flagellar assembly and signaling. Pan-genome analysis revealed variations in carbohydrate metabolic and hydrogenases genes within the complex. Variable inter- and intraspecies gas production in meat by C. estertheticum and Clostridium tagluense were associated with the distribution of the [NiFe]-hydrogenase hyp gene cluster whose absence or presence was associated with occurrence or lack of pack distention, respectively. Through comparative genomics, we have shown CEC species exhibit high genetic diversity that can be partly attributed to recombination and gene gain/loss events. We have also shown genetic basis for variable gas production in meat can be attributed to the presence/absence of the hyp gene cluster.

Photobacterium (P.) is a genus widely studied in regards to its association with and ubiquitous presence in marine environments. However, certain species (P. phosphoreum, P. carnosum, P. iliopiscarium) have been recently described to colonize and spoil raw meats without a marine link. We have studied 27 strains from meat as well as 26 strains from marine environments in order to probe for intraspecies marine/terrestrial subpopulations and identify distinct genomic features acquired by environmental adaptation. We have conducted phylogenetic analysis (MLSA, ANI, fur, codon usage), search of plasmids (plasmidSPADES), phages (PHASTER), CRISPR-cas operons (CRISPR-finder) and secondary metabolites gene clusters (antiSMASH, BAGEL), in addition to a targeted gene search for specific pathways (e.g. TCA cycle, pentose phosphate, respiratory chain) and elements relevant for growth, adaptation and competition (substrate utilization, motility, bioluminescence, sodium and iron transport). P. carnosum appears as a conserved single clade, with one isolate from MAP fish clustering apart that doesn\'t, however, show distinct features that could indicate different adaptation. The species harbors genes for a wide carbon source utilization (glycogen/starch, maltose, pullulan, fucose) for colonization of diverse niches in its genome. P. phosphoreum is represented by two different clades on the phylogenetic analyses not correlating to their origin or distribution of other features analyzed that can be divided into two novel subspecies based on genome-wide values. A more diverse antimicrobial activity (sactipeptides, microcins), production of secondary metabolites (siderophores and arylpolyenes), stress response and adaptation (bioluminescence, sodium transporters, catalase, high affinity for oxygen cytochrome cbb3 oxidase, DMSO reductase and proton translocating NADH dehydrogenase) is predicted compared to the other species. P. iliopiscarium was divided into two clades based on source of isolation correlating with phylogeny and distribution of several traits. The species shows traits common to the other two species, similar carbon utilization/transport gene conservation as P. carnosum for the meat-isolated strains, and predicted utilization of marine-common DMSO and flagellar cluster for the sea-isolated strains. Results additionally suggest that photobacteria are highly prone to horizontal acquisition/loss of genetic material and genetic transduction, and that it might be a strategy for increasing the frequency of strain- or species-specific features that offers a growth/competition advantage.

The high perishability of fresh meat results in short sales and consumption periods, which can lead to high amounts of food waste, especially when a fixed best-before date is stated. Thus, the aim of this study was the development of a real-time dynamic shelf-life criterion (DSLC) for fresh pork filets based on a multi-model approach combining predictive microbiology and sensory modeling. Therefore, 647 samples of ma-packed pork loin were investigated in isothermal and non-isothermal storage trials. For the identification of the most suitable spoilage predictors, typical meat quality parameters (pH-value, color, texture, and sensory characteristics) as well as microbial contamination (total viable count, Pseudomonas spp., lactic acid bacteria, Brochothrix thermosphacta, Enterobacteriaceae) were analyzed at specific investigation points. Dynamic modeling was conducted using a combination of the modified Gompertz model (microbial data) or a linear approach (sensory data) and the Arrhenius model. Based on these models, a four-point scale grading system for the DSLC was developed to predict the product status and shelf-life as a function of temperature data in the supply chain. The applicability of the DSLC was validated in a pilot study under real chain conditions and showed an accurate real-time prediction of the product status.

Photobacterium spp. occur frequently in marine environments but have been recently also found as common spoilers on chilled meats. The environmental conditions in these ecological niches differ especially regarding salinity and ambient pressure. Linking the occurrence of photobacteria in different niches may elucidate its ecology and bring insights for the food industry. We investigated tolerance of Photobacterium (P.) phosphoreum and P. carnosum strains to high hydrostatic pressure and salinity and aligned our observations with presence of relevant genes. The strains were isolated from packaged meats and salmon (or the sea) to identify adaptations to marine and terrestrial habitats. Growth of all P. carnosum strains was reduced by 40 MPa hydrostatic pressure and >3% sodium chloride, suggesting loss of traits associated with marine habitats. In contrast, P. phosphoreum strains were only slightly affected, suggesting general adaptation to marine habitats. In accordance, these strains had gene clusters associated with marine niches, e.g. flagellar and lux-operons, being incomplete in P. carnosum. Occurrence of P. carnosum strains on packaged salmon and P. phosphoreum strains on meats therefore likely results from cross-contamination in meat and fish processing. Still, these strains showed intermediate traits regarding pressure- and halotolerance, suggesting developing adaptation to their respective environment.

Fresh meat is commonly packaged in modified atmosphere to decelerate spoilage processes. The applied gas mixture affects the growth of spoilage organisms and selectively shapes the spoilage community. In this study, we investigated the impact of O2 and CO2 on the growth of Photobacterium (P.) phosphoreum and P. carnosum strains in situ on chicken meat by packaging under different modified atmospheres (air, 70% O2/30% CO2, 70% N2/30% CO2, 100% N2). Combination of 70% O2 and 30% CO2 resulted in significant growth reduction of the analyzed strains, suggesting inhibitory effects of both gases in combination. In contrast, 30% CO2 alone had only a minor effect and photobacteria are supposed to have a growth advantage over other meat spoilers in this atmosphere. Additionally, single growth of the strains in the different atmospheres was compared when challenged with the presence of Pseudomonas (Ps.) fragi or Brochothrix (B.) thermosphacta as prominent co-contaminants in different ratios (10:1, 1:1, 1:10). Presence of co-contaminants resulted in increased cell numbers of P. carnosum TMW2.2149 but reduced or unchanged cell numbers of P. phosphoreum TMW2.2103 in most packaging atmospheres. The initial ratio of photobacteria and co-contaminants defined the relative abundance during storage but did not change the type of the interaction. Our results suggest either a commensalistic (P. carnosum) or competitive interaction (P. phosphoreum) of photobacteria and co-contaminants on modified atmosphere packaged chicken, respectively. Furthermore, in a mix comprising seven prominent spoilers, strains of both Photobacterium species prevailed as a constant part of the spoilage microbiome during 7 days of refrigerated storage on chicken meat packaged under O2/CO2 atmosphere.

Biofilms are formed by microorganisms protected by a self-produced matrix, most often attached to a surface. In the food processing environments biofilms endanger the product safety by the transmission of spoilage and pathogenic bacteria. In this study, we characterised the biofilm formation of the following eleven strains isolated from biofilms in a meat-processing environment: Acinetobacter harbinensis BF1, Arthrobacter sp. BF1, Brochothrix thermosphacta BF1, Carnobacterium maltaromaticum BF1, Kocuria salsicia BF1, Lactococcus piscium BF1, Microbacterium sp. BF1, Pseudomonas fragi BF1, Psychrobacter sp. BF1, Rhodococcus erythropolis BF1, Stenotrophomonas sp. BF1. We applied whole- genome sequencing and subsequent genome analysis to elucidate genetic features associated with the biofilm lifestyle. We furthermore determined the motility and studied biofilm formation on stainless steel using a static mono-species biofilm model mimicking the meat processing environment. The biomass and the EPS components carbohydrates, proteins and extracellular DNA (eDNA) of the biofilms were investigated after seven days at 10 °C. Whole-genome analysis of the isolates revealed that all strains except the Kocuria salsicia BF1 isolate, harboured biofilm associated genes, including genes for matrix production and motility. Genes involved in cellulose metabolism (present in 82% of the eleven strains) and twitching motility (present in 45%) were most frequently found. The capacity for twitching was confirmed using plate assays for all strains except Lactococcus piscium BF1, which showed the lowest motility behaviour. Differences in biofilm forming abilities could be demonstrated. The bacterial load ranged from 5.4 log CFU/cm2 (Psychrobacter sp. isolate) to 8.7 log CFU/cm2 (Microbacterium sp. isolate). The amount of the matrix components varied between isolates. In the biofilm of six strains we detected all three matrix components at different levels (carbohydrates, proteins and eDNA), in two only carbohydrates and eDNA, and in three only carbohydrates. Carbohydrates were detected in biofilms of all strains ranging from 0.5 to 4.3 μg glucose equivalents/cm2. Overall, the Microbacterium sp. strain showed the highest biofilm forming ability with high bacterial load (8.7 log CFU/cm2) and high amounts of carbohydrates (2.2 μg glucose equivalents/cm2), proteins (present in all experiments) and eDNA (549 ng/cm2). In contrast, Brochothrix thermosphacta was a weak biofilm former, showing low bacterial load and low levels of carbohydrates in the matrix (6.2 log CFU/cm2 and 0.5 μg glucose equivalents/cm2). This study contributes to our understanding of the biofilm forming ability of bacteria highly abundant in the meat processing environment, which is crucial to develop strategies to prevent and reduce biofilm formation in the food producing environment.

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.