Characterization and interactions of spoilage of Pseudomonas fragi C6 and Brochothrix thermosphacta S5 in chilled pork based on LC-MS/MS and screening of potential spoilage biomarkers.

Pseudomonas and Brochothrix are the main spoilage organisms in pork, and each of these plays an essential role in the spoilage process. However, the effect of co-contamination of these two organisms in pork has not been elucidated. The changing bacterial communities during spontaneous spoilage of pork at 4 °C were evaluated using high-throughput sequencing. The dominant spoilage bacteria were isolated and these were identified as Pseudomonas fragi C6 and Brochothrix thermosphacta S5. Chilled pork was then experimentally contaminated with these strains, individually and in combination, and the progression of spoilage was assessed by analyzing various physicochemical indicators. These included total viable counts (TVC), pH, color, total volatile basic nitrogen (TVB-N), and detection of microbial metabolites. After 7 days of chilled storage, co-contaminated pork produced higher TVC and TVB-N values than mono-contaminated samples. Metabolomic analysis identified a total of 8,084 metabolites in all three groups combined. Differential metabolites were identified, which were involved in 38 metabolic pathways. Among these pathways, the biosynthesis of alkaloids derived from purine and histidine was identified as an important pathway related to spoilage. Specifically, histidine, histamine, AMP, IMP, GMP, succinic acid, and oxoglutaric acid were identified as potential spoilage biomarkers. The study showed that the combined presence of P. fragi C6 and B. thermosphacta S5 bacteria makes chilled pork more prone to spoilage, compared to their individual presence. This study provides insights that can assist in applying appropriate techniques to maintain quality and safety changes in meat during storage and further the assessment of freshness.

Functionally coherent transcriptional responses of Jatropha curcas and Pseudomonas fragi for rhizosphere mediated degradation of pyrene.

Pyrene is an extremely hazardous, carcinogenic polycyclic aromatic hydrocarbon (PAH). The plant-microbe interaction between Pseudomonas fragi DBC and Jatropha curcas was employed for biodegradation of pyrene and their transcriptional responses were compared. The genome of P. fragi DBC had genes for PAH degrading enzymes i.e. dioxygenases and dehydrogenases, along with root colonization (trpD, trpG, trpE and trpF), chemotaxis (flhF and flgD), stress adaptation (gshA, nuoHBEKNMG), and detoxification (algU and yfc). The transcriptional expression of catA and yfc that respectively code for catabolic enzyme (catechol-1, 2-dioxygnase) and glutathione-s-transferase for detoxification functions were quantitatively measured by qPCR. The catA was expressed in presence of artificial root exudate with or without pyrene, and glucose confirming the non-selective approach of bacteria, as desired. Pyrene induced 100-fold increase of yfc expression than catA, while there was no expression of yfc in absence of pyrene. The transcriptome of plant roots, in presence of pyrene, with or without P. fragi DBC inoculation was analysed. The P. fragi DBC could upregulate the genes for plant growth, induced the systemic acquired resistance and also ameliorated the stress response in Jatropha roots.

Biosurfactant from Pseudomonas fragi enhances the competitive advantage of Pseudomonas but reduces the overall spoilage ability of the microbial community in chilled meat.

Biosurfactants from Pseudomonas spp. have been reported to exhibit antibacterial and anti-adhesive properties, but their role during meat spoilage remains unclear. In this study, the biosurfactant was isolated from an isolate of Pseudomonas fragi with strong spoilage potential, and its surface tension and emulsification ability were determined. The chemical and microbial characteristics of the biosurfactant-treated meat samples were periodically analyzed. The results demonstrated that the biosurfactant produced by P. fragi could reduce surface tension and showed good emulsification properties. For the in situ spoilage trials, biosurfactant from P. fragi changed the microbial diversity on meat, helping Pseudomonas establish a dominant position in the population. However, biosurfactant treatment caused chicken meat to exhibit a weaker spoilage state, as indicated by the growth of psychrophilic microorganisms, total volatile basic nitrogen (TVBN) and meat color. These results provide practical information for understanding the role of P. fragi biosurfactant during chilled meat storage.

Effects of deletion of siderophore biosynthesis gene in Pseudomonas fragi on quorum sensing and spoilage ability.

Siderophores are important factors in the spoilage process of Pseudomonas fragi, considered to be one of the main spoilage bacterium of tuna, and the secretion of siderophores is regulated by quorum sensing (QS). This study aimed to construct a mutant with the deletion of the siderophore synthetase gene of P. fragi (MS-10), and to explore its effects on the growth, QS, and spoilage potential of P. fragi. The results showed that the deletion of the siderophore biosynthesis gene slowed down the growth rate of the strain. The apoptosis rate increased by 27.7 % compared with that of the wild-type strain at 4 °C for 48 h. Biofilm formation, extracellular protease expression, and signal molecule production were all significantly lower in the mutant strain compared with the wild-type strain. The total viable count and the histamine content showed that the tuna sterile fish block inoculated with the wild-type strain exceeded the acceptable standards by 5 days and was completely spoiled by 7 days, whereas the mutant strain exceeded the acceptable standards by 6 days and was completely spoiled by 9 days. The pH, texture, and other indicators showed that the variation range of the mutant strain was lower than that of the wild-type strain. The deletion of the siderophore biosynthesis gene reduced the spoilage ability of P. fragi. Based on the results, the development of novel preservation agents targeting the control of the siderophore biosynthesis gene could be a new idea for the preservation of aquatic products.

Influences of flavonoids from Sedum aizoon L. on biofilm formation of Pseudomonas fragi.

Pseudomonas fragi (P. fragi) is one of the main categories of bacteria responsible for the spoilage of chilled meat. In the processing and preservation of chilled meat, it is easy to form biofilms on the meat, leading to the development of slime on the meat, which becomes a major quality defect. Flavonoids, as one of the critical components of secondary plant metabolites, are receiving increasing attention for their antibacterial activity. Flavonoids in Sedum aizoon L. (FSAL), relying on its prominent antibacterial activity, are of research importance in food preservation and other applications. This article aims to investigate the effect of FSAL on the biofilm formation of P. fragi, to better apply FSAL to the processing and preservation of meat products. The disruption of cellular structure and aggregation properties by FSAL was demonstrated by the observation of the cellular state within the biofilm. The amount of biofilm formation was determined by crystal violet staining, and the content of polysaccharides and proteins in the extracellular wrapped material was determined. It was shown that the experimental concentrations of FSAL (1.0 MIC) was able to inhibit biofilm formation and reduce the main components in the extracellular secretion. The swimming motility assay and the downregulation of flagellin-related genes confirmed that FSAL reduced cell motility and adhesion. The downregulation of cell division genes and the lowering of bacterial metabolic activity suggested that FSAL could hinder bacterial growth and reproduction within P. fragi biofilms. KEY POINTS: • FSAL inhibited the activity of Pseudomonas fragi in the dominant meat strain • The absence of EPS components affected the formation of P. fragi biofilms • P. fragi has reduced adhesion capacity due to impaired flagellin function.

Pseudomonas fragi biofilm on stainless steel (at low temperatures) affects the survival of Campylobacter jejuni and Listeria monocytogenes and their control by a polymer molybdenum oxide nanocomposite coating.

Pseudomonas spp. are widely distributed bacteria on surfaces in the food production and processing environment, where they form extracellular polymeric substance rich biofilms that interact with other bacteria. In this study, the influence of biofilm of Pseudomonas fragi ATCC 4973 on Listeria monocytogenes ATCC 19115 and Campylobacter jejuni NCTC 11168 was investigated at 5 °C and 15 °C on stainless steel in broth and food homogenates (fish or chicken meat). Stainless steel was then coated with PVDF-HFP/PVP/MoO3 nanocomposite and examined for surface changes (scanning electron microscope, static contact angle, Vickers hardness and elastic modulus). The effect of the prepared nanocomposite coating on P. fragi and on L. monocytogenes and C. jejuni was evaluated in mono- and co-culture. P. fragi produced more biofilm at 15 °C than at 5 °C, especially when food homogenates were used as growth media. Co-cultivation with pathogens did not affect biofilm production by P. fragi, but significant changes were observed in L. monocytogenes and C. jejuni, resulting in a decrease and increase, respectively, in the determined number of culturable biofilm cells. The first change was probably due to competition for the surface, and the second to the oxygen gradient. Stainless steel was then coated with a PVDF-HFP/PVP/MoO3 nanocomposite, which was characterised by lower roughness and higher wettability, but lower hardness compared to uncoated stainless steel. The prepared nanocoating showed bactericidal activity when tested in phosphate buffered saline. When used in food homogenates, a reduction of over 95 % in bacterial counts was observed. An abundant biofilm of P. fragi proved protective to L. monocytogenes and C. jejuni against the functionalised nanocomposite surface when tested in food homogenates. The control of spoilage Pseudomonas spp., which are common in the food production and processing environment, is important for reducing the contamination of food with spoilage bacteria and with pathogens such as L. monocytogenes and C. jejuni, which may be present in the same environment. The PVDF-HFP/PVP/MoO3 nanocomposite showed good potential for use as a coating for food contact surfaces, but possible migration of nanoparticles from the nanocomposite coating to food should be evaluated before its commercial use.

Potential inhibitory effect of carbon dioxide on the spoilage behaviors of Pseudomonas fragi in high-oxygen packaged beef during refrigerated storage.

Pseudomonas fragi is a dominant meat spoilage organism under high-oxygen modified atmosphere packaging (HiOx-MAP). This work investigated the effects of CO2 on P. fragi growth and the related spoilage phenomena of HiOx-MAP beef. Minced beef incubated with P. fragi T1, a strain owning the strongest spoilage potential among isolates, was stored under CO2-enriched HiOx-MAP (TMAP; 50% O2/40% CO2/10% N2) or non-CO2 HiOx-MAP (CMAP; 50% O2/50% N2) at 4 °C for 14 days. Compared to CMAP, TMAP maintained sufficient O2 levels to endow beef with higher a* values and meat color stability due to lower P. fragi counts from day 1 (P < 0.05). TMAP samples also showed lower (P < 0.05) lipase activity and protease activity within 14-days and 6-days than CMAP samples respectively. TMAP delayed the significantly increased pH and total volatile basic nitrogen contents occurred in CMAP beef during storage. Despite TMAP markedly promoted the lipid oxidation associated with higher concentrations of hexanal and 2,3-octanedione than CMAP (P < 0.05), TMAP beef retained an acceptable organoleptic odor due to a CO2-inhibition on the microbial-induced 2,3-butanedione and ethyl 2-butenoate formation. This study provided a comprehensive insight into the antibacterial mechanism of CO2 on P. fragi in HiOx-MAP beef.

Meat-derived Escherichia coli and Pseudomonas fragi manage to co-exist in dual-species biofilms by adjusting gene-regulated competitive strength.

Pseudomonas fragi and Escherichia coli are considered as common colonizers of fresh and spoilage meat, where they tend to live in the proximity. In this study, we primarily tested interplay patterns between different isolates of these two species in two-by-two combinations grown on stainless steel surfaces as dual-species biofilms. Results showed that these two species presented competition as major observed interplay patterns as biofilms progressed independent of bacterial strains and growth temperatures (15 °C and 25 °C). One dual-species combination was proposed as a representative to further explore dynamic patterns of interaction strength between these two species, with species colonization order taken into consideration as a biological effector. We firstly reported that prior colonization of one species significantly decreased the initiatively colonized cell counts of counterpart species by one to three orders of magnitude when competing for limited adhesion surface, under which E. coli was observed to be more aggressive in surface colonization as compared to P. fragi. However, the spatial structure and microbial composition of mature dual-species biofilms were not observed to be significantly affected. Our findings also shed new light on the evidence that E. coli and P. fragi, respectively, enhanced their biofilm formation capabilities by upregulating expression level of genes that encoded Type 1 fimbriae and phosphate response regulator as dual-species consortia progressed, which could serve as a crucial factor that improved the difficulty of food biocontrol.

Nitrogen removal characterization and functional enzymes identification of a hypothermia bacterium Pseudomonas fragi EH-H1.

A novel hypothermic strain, Pseudomonas fragi EH-H1, was found to effectively perform heterotrophic nitrification and aerobic denitrification at 15 °C. This strain could consume 100 %, 100 % and 99.95 % of ammonium (54.90 mg∙L-1), nitrate (56.12 mg∙L-1) and nitrite (54.15 mg∙L-1), accompanied by peak removal rates of 5.51, 3.63 and 3.14 mg/L/h, respectively. The ammonium was removed preferentially during simultaneous nitrification and denitrification. Notably, the elimination rate of the toxic nitrite nitrogen remained approximately 3.14 mg/L/h, whether supplemented with ammonium or not. Stepwise inhibition experiments revealed that the key enzymes of ammonia monooxygenase (AMO) and nitrite oxidoreductase (NiR) for nitrification and denitrification coexisted in strain EH-H1. AMO, nitrate reductase and NiR were successfully expressed and detected at 0.637, 0.239 and 0.018 U/mg proteins, respectively. Overall, strain EH-H1 had an outstanding ability to remove nitrogen at low temperatures and could provide guidance for cryogenic wastewater treatment.

Respiratory Depression as Antibacterial Mechanism of Linalool against Pseudomonas fragi Based on Metabolomics.

Linalool showed a broad-spectrum antibacterial effect, but few studies have elucidated the antibacterial mechanism of linalool on Pseudomonas fragi (P. fragi) to date. The present study aimed to uncover the antimicrobial activity and potential mechanism of linalool against P. fragi by determining key enzyme activities and metabolites combined with a high-throughput method and metabolomic pathway analysis. As a result, linalool had excellent inhibitory activity against P. fragi with MIC of 1.5 mL/L. In addition, the presence of linalool significantly altered the intracellular metabolic profile and a total of 346 differential metabolites were identified, of which 201 were up-regulated and 145 were down-regulated. The highlight pathways included beta-alanine metabolism, pantothenic acid and CoA metabolism, alanine, aspartate and glutamate metabolism, nicotinate and nicotinamide metabolism. Overall, linalool could cause metabolic disorders in cells, and the main metabolic pathways involved energy metabolism, amino acid metabolism and nucleic acid metabolism. In particular, the results of intracellular ATP content and related enzymatic activities (ATPase, SDH, and GOT) also highlighted that energy limitation and amino acid disturbance occurred intracellularly. Together, these findings provided new insights into the mechanism by which linalool inhibited P. fragi and theoretical guidance for its development as a natural preservative.

Valorization of cheese whey to lactobionic acid by a novel strain Pseudomonas fragi and identification of enzyme involved in lactose oxidation.

BACKGROUND: Efficient upgrading of inferior agro-industrial resources and production of bio-based chemicals through a simple and environmentally friendly biotechnological approach is interesting Lactobionic acid is a versatile aldonic acid obtained from the oxidation of lactose. Several microorganisms have been used to produce lactobionic acid from lactose and whey. However, the lactobionic acid production titer and productivity should be further improved to compete with other methods. RESULTS: In this study, a new strain, Pseudomonas fragi NL20W, was screened as an outstanding biocatalyst for efficient utilization of waste whey to produce lactobionic acid. After systematic optimization of biocatalytic reactions, the lactobionic acid productivity from lactose increased from 3.01 g/L/h to 6.38 g/L/h in the flask. In batch fermentation using a 3 L bioreactor, the lactobionic acid productivity from whey powder containing 300 g/L lactose reached 3.09 g/L/h with the yield of 100%. Based on whole genome sequencing, a novel glucose dehydrogenase (GDH1) was determined as a lactose-oxidizing enzyme. Heterologous expression the enzyme GDH1 into P. putida KT2440 increased the lactobionic acid yield by 486.1%. CONCLUSION: This study made significant progress both in improving lactobionic acid titer and productivity, and the lactobionic acid productivity from waste whey is superior to the ever reports. This study also revealed a new kind of aldose-oxidizing enzyme for lactose oxidation using P. fragi NL20W for the first time, which laid the foundation for further enhance lactobionic acid production by metabolic engineering.

Unraveling the antibacterial mechanism of 3-carene against Pseudomonas fragi by integrated proteomics and metabolomics analyses and its application in pork.

Pseudomonas fragi is primarily responsible for the spoilage of various foods, especially meat. The aim of this study was to investigate the antibacterial mechanism of 3-carene against P. fragi. 3-Carene treatment decreased the phospholipid content and the fluidity of the cell membrane, induced reactive oxygen species (ROS) generation and affected respiratory chain dehydrogenase, oxoglutarate dehydrogenase and citrate synthase in P. fragi. Metabolomics and proteomics analyses further showed that in the presence of 3-carene, 519 proteins, 136 metabolites in positive ion mode and 100 metabolites in negative ion mode were differentially expressed. These proteins and metabolites were primarily involved in amino acid metabolism, fatty acid degradation, the tricarboxylic acid cycle (TCA cycle) and other processes. Consequently, the stimulation of 3-carene altered cell membrane properties, disturbed important amino acid and energy metabolism, and even caused oxidative stress. Additionally, the results of total viable counts and the total volatile base nitrogen indicated that 3-carene could significantly improve the preservation of refrigerated pork. This study suggested that 3-carene has promising potential to be developed as a food preservative.

Involvement of AprD in regulating biofilm structure, matrix secretion, and cell metabolism of meat-borne Pseudomonas fragi during chilled storage.

Pseudomonas fragi is by far one of the most threatening species in the spoilage of chilled meat that is stored under aerobic conditions. The membrane protein AprD is a well-established regulator controlling protease secretion in Pseudomonas spp. However, its exact roles in modulating metabolic pathways and spoilage potential of P. fragi at the molecular level remain undefined. Here, an in-frame deletion mutation of aprD was used to explore the impacts on their biofilm structure, matrix secretion, and cell metabolism. The results showed that ΔaprD formed relatively disorganized loose aggregation in biofilm, resulting in a thinner structure and more dead cells. Meanwhile, marked changes in the content of extracellular carbohydrates and proteins were observed. Furthermore, intracellular metabolomic profiling revealed the involvement of aprD in several cellular metabolic pathways, mostly including the carbohydrate pathway, amino acid pathway, and nucleotide pathway, while the characterization of extracellular metabolism clarified the variations in the spoilage-related metabolites (e.g., creatine, IMP, spermine, fatty acids, amino acids, and oligopeptides) could be highly correlated with aprD deletion. In this finding, we indicated that aprD could be responsible for cell reproduction and in situ spoilage potential of P. fragi NMC25 during chilled storage by controlling related metabolism and nutrients utilization. Thus, our results will contribute to an improved understanding of the regulatory mechanism of aprD gene in meat spoilage contaminated with P. fragi, which can be valuable to ensure the quality and safety of meat.

Green synthesis of fluorescent carbon dots with antibacterial activity and their application in Atlantic mackerel (Scomber scombrus) storage.

Antimicrobial materials prepared from natural products could provide new ways to preserve seafood and extend the shelf life. Herein, four kinds of fluorescent carbon dots were prepared using onion, ginger, garlic, and fish through one-step hydrothermal synthesis. The four prepared carbon dots were nearly spherical and nanosized, with amorphous structure, neutral charge and good water dispersibility. The onion and garlic carbon dots contained more sulfur elements than the ginger and fish carbon dots. Interestingly, the onion carbon dots exhibited the best antibacterial activity against Pseudomonas fragi with good stability over a wide pH range. In addition, the onion carbon dots also exhibited antimicrobial activity against representative Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The minimum inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of onion carbon dots against Pseudomonas fragi were 2 mg mL-1 and 4 mg mL-1, respectively. The integrity of the cell wall and the cell membrane were damaged for Pseudomonas fragi, and the extracellular alkaline phosphatase (AKP) and ATP activity also increased after exposure to the onion carbon dots, thus leading to a decrease in the cell viability and alteration of the cellular morphology for Pseudomonas fragi. Furthermore, the preservation effect of onion carbon dots on Atlantic mackerel evaluated by storage at 4 °C revealed that the onion carbon dots significantly reduced drip loss, total volatile basic nitrogen (TVB-N) value and total viable counts (TVC) value, and extended the shelf life of Atlantic mackerel by 2 days. This finding suggests that onion carbon dots have potential to be applied as a bacteriostatic agent for aquatic products.

De novo genome assembly and analysis unveil biosynthetic and metabolic potentials of Pseudomonas fragi A13BB.

OBJECTIVES: The role of rhizosphere microbiome in supporting plant growth under biotic stress is well documented. Rhizobacteria ward off phytopathogens through various mechanisms including antibiosis. We sought to recover novel antibiotic-producing bacterial strains from soil samples collected from the rhizosphere. Pseudomonas fragi A13BB was recovered as part of this effort, and the whole genome was sequenced to facilitate mining for potential antibiotic-encoding biosynthetic gene clusters. DATA DESCRIPTION: Here, we report the complete genome sequence of P. fragi A13BB obtained from de novo assembly of Illumina MiSeq and GridION reads. The 4.94 Mb genome consists of a single chromosome with a GC content of 59.40%. Genomic features include 4410 CDSs, 102 RNAs, 3 CRISPR arrays, 3 prophage regions, and 37 predicted genomic islands. Two ß-lactone biosynthetic gene clusters were identified; besides, metabolic products of these are known to show antibiotic and/or anticancer properties. A siderophore biosynthetic gene cluster was also identified even though P. fragi is considered a non-siderophore producing pseudomonad. Other gene clusters of broad interest identified include those associated with bioremediation, biocontrol, plant growth promotion, or environmental adaptation. This dataset unveils various un-/underexplored metabolic or biosynthetic potential of P. fragi and provides insight into molecular mechanisms underpinning these attributes.

Characterization of the biofilm matrix composition of psychrotrophic, meat spoilage pseudomonads.

Psychrotrophic Pseudomonas species are the key spoilage bacteria of aerobically stored chilled meat. These organisms readily form biofilms on meat under refrigerated conditions leading to consumer rejection and associated economic losses. Limited information is available on the matrix composition of the biofilms formed by these bacteria. We quantified and characterized the main components of the matrix of mono-species biofilms of selected Pseudomonas fragi and Pseudomonas lundensis strains using chemical analysis and Raman spectroscopy. The biofilms were grown at 10 °C and 25 °C on nitro-cellulose membranes placed on surface sterilized beef cuts. Extra-cellular polymeric substances of the matrix were extracted in soluble and bound forms and were chemically assessed for total carbohydrates, proteins and extra-cellular DNA. Both Pseudomonas species showed a significant increase in total carbohydrates and total proteins when grown at 10 °C as compared to 25 °C. Extra-cellular DNA did not show a strong correlation with growth temperature. Raman spectra were obtained from planktonic bacteria and membrane grown biofilms at 10 °C and 25 °C. Higher levels of guanine were detected in planktonic cells as compared to biofilm cells. This study suggests that psychrotrophic Pseudomonas species may respond to cold stress by increasing extra-cellular polymer secretions.

Electrochemical detection of food-spoiling bacteria using interdigitated platinum microelectrodes.

The fast and non-destructive detection of bacterial attachment on food contact surfaces is important for the prevention of the unwanted formation of biofilms. Biofilms constitute a protected growth mode that allows bacteria to survive even in hostile environments. Therefore, the fast detection of bacterial attachment may be an effective strategy for biofilm control. In this study cyclic voltammetry (CV) was used to detect Bacillus subtilis ssp. subtilis, Paenibacillus polymyxa, Pseudomonas fragi attachment on interdigitated microelectrodes. The differences in current between the uncolonized sterile microelectrodes and the microelectrodes after bacterial attachment were determined. In addition, the surface coverage of microelectrodes was visualized using microscopy techniques. The results showed that the cyclic voltammetry in combination with interdigitated platinum microelectrodes can be used to detect bacterial biofilms.

Lactose oxidase: A novel activator of the lactoperoxidase system in milk for improved shelf life.

The lactoperoxidase system (LS), an antimicrobial system naturally present in milk that is activated by H2O2, has been used to inhibit microbial outgrowth in raw milk in areas where refrigeration is not viable. This study evaluated lactose oxidase (LO) as a novel activator of the LS. Lactose oxidase oxidizes lactose and produces H2O2 needed for the activation of the LS. The antimicrobial effect of different concentrations of LO with and without components of the LS, thiocyanate (TCN) and lactoperoxidase (LP), was evaluated in model systems and then applied in pasteurized milk and raw milk. In general, an increase in LO caused greater reductions of Pseudomonas fragi in the model systems and treatments were more effective at 6°C than at 21°C. At 6°C, the LO solution at 0.12 and 1.2 g/L showed significantly higher microbial reduction than the control when both added alone and combined with LS components. At 21°C, treatments with 1.2 g/L of LO solution achieved a reduction of >2.93 log cfu/mL in 24 h, but at lower levels there was not a significant reduction from the control. Higher concentrations of TCN led to a greater P. fragi reduction at both temperatures when LO was added alone but not when combined with LP. In pasteurized milk, the LO solution at 0.12 g/L caused a reduction of approximately 1.4 log of P. fragi within 24 h when added alone and a reduction of approximately 2.7 log when combined with LP and TCN. Bacterial counts remained at significantly lower levels than the control during storage, and the TCN-supplemented milk exhibited an approximately 6-log difference from the control by d 7. In raw milk, the total bacterial growth curve showed a longer lag phase when the LS was activated by LO (11.3 ± 1.4 h) compared with the control (4.0 ± 1.0 h), but it was not different from the recommended method (9.4 ± 1.0 h). However, the total bacterial count after 24 h for the sample treated with LO and TCN (5.3 log cfu/mL) was significantly lower compared with the control (7.2 log cfu/mL) and the recommended method (6.1 log cfu/mL). Results from this study suggest that LO is an alternative source of H2O2 that enhances the microbial inhibition achieved by the LS. Lactose oxidase could be used to develop enzyme-based preservation technologies for applications where cold chain access is limited. This enzymatic approach to improving the shelf life of dairy products also represents a novel option for clean label spoilage control.

Strain-Level Diversity Analysis of Pseudomonas fragi after In Situ Pangenome Reconstruction Shows Distinctive Spoilage-Associated Metabolic Traits Clearly Selected by Different Storage Conditions.

Microbial spoilage of raw meat causes huge economic losses every year. An understanding of the microbial ecology associated with the spoilage and its dynamics during the refrigerated storage of meat can help in preventing and delaying the spoilage-related activities. The raw meat microbiota is usually complex, but only a few members will develop during storage and cause spoilage upon the pressure from several external factors, such as temperature and oxygen availability. We characterized the metagenome of beef packed aerobically or under vacuum during refrigerated storage to explore how different packaging conditions may influence the microbial composition and potential spoilage-associated activities. Different population dynamics and spoilage-associated genomic repertoires occurred in beef stored aerobically or in vacuum packaging. Moreover, the pangenomes of Pseudomonas fragi strains were extracted from metagenomes. We demonstrated the presence of specific, storage-driven strain-level profiles of Pseudomonas fragi, characterized by different gene repertoires and thus potentially able to act differently during meat spoilage. The results provide new knowledge on strain-level microbial ecology associated with meat spoilage and may be of value for future strategies of spoilage prevention and food waste reduction.IMPORTANCE This work provides insights on the mechanisms involved in raw beef spoilage during refrigerated storage and on the selective pressure exerted by the packaging conditions. We highlighted the presence of different microbial metagenomes during the spoilage of beef packaged aerobically or under vacuum. The packaging condition was able to select specific Pseudomonas fragi strains with distinctive genomic repertoires. This study may help in deciphering the behavior of different biomes directly in situ in food and in understanding the specific contribution of different strains to food spoilage.

Modified atmosphere packaging decreased Pseudomonas fragi cell metabolism and extracellular proteolytic activities on meat.

Modified atmosphere packaging (MAP) is considered an effective method for extending the shelf life of meat. The use of optimal mixture of gases (CO2 and N2) in food packaging containers has been proved to effectively inhibit the growth of microorganisms in poultry meat. In general, a minimum CO2 concentration range of 20%-30% is required for the inhibitory effect. The aim of this study was to investigate the mechanism by which MAP (CO2/N2 30%/70%) inhibits Pseudomonas fragi, a dominant spoilage microorganism in aerobically stored chilled meat. The cell physiological changes were determined by measuring membrane integrity, membrane potential, ATP level, and extracellular proteolytic activity. The results showed that samples stored under MA retained cell membrane integrity, but lost significant membrane potential and ATP synthesis activity. Furthermore, the peptides issued from 2 structural proteins (myosin and actin) were mainly identified in air samples, indicating that these fragments result from bacterial proteolytic activity while MAP inhibited this activity. Overall, the study found that cell metabolism and extracellular protease activity decreased under MAP conditions. This study showed that MAP is an effective food preservation strategy and revealed mechanisms by which MAP inhibits spoilage.