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.

The complex multicellular morphology of the food spoilage bacteria Brochothrix thermosphacta strains isolated from ground chicken.

Herein we describe a highly structured, filamentous growth phenotype displayed by an isolate of the food spoilage microorganism Brochothrix thermosphacta. The growth morphology of this B. thermosphacta strain (strain BII) was dependent on environmental factors such as the growth media, incubation temperatures, and the inoculum concentration. Inoculation of cultures in highly dilute suspensions resulted in the formation of isolated, tight aggregates resembling fungal growth in liquid media. This same strain also formed stable, mesh-like structures in 6-well tissue culture plates under specific growth conditions. The complex growth phenotype does not appear to be unique to strain BII but was common among B. thermosphacta strains isolated from chicken. Light and electron micrographs showed that the filaments of multiple BII cells can organize into complex, tertiary structures resembling multistranded cables. Time-lapse microscopy was employed to monitor the development of such aggregates over 18 h and revealed growth originating from short filaments into compact ball-like clusters that appeared fuzzy due to protruding filaments or cables. This report is the first to document this complex filamentous growth phenotype in a wild-type bacterial isolate of B. thermosphacta.

Analysis of microbiome in raw chicken meat from butcher shops and packaged products in South Korea to detect the potential risk of foodborne illness.

Chicken meat is one of the most widely consumed meats worldwide. The microbiota on the whole body of chicken is a potential source of foodborne pathogens that can be transmitted to humans during the preparation of raw meat. However, to date, there have been no studies comparing the microbiota of packaged chicken products and those of raw chicken carcasses from butcher shops, although such information could be useful for identifying sources of contamination in cases of food poisoning. We addressed this in the present study by analyzing the microbiota of 80 chicken meat samples collected from various butcher shops and processing plants in South Korea with the Illumina MiSeq system based on the 16S rRNA gene sequence. The bacterial amounts in chicken samples were estimated by quantitative real-time PCR. Although different microbial members were present in unpackaged meat from butcher shops as compared to those in packaged products from commercial sources, seasonal differences (sample obtained in January vs. July) in microbiota were more significant even in the packaged products from the same company. We also investigated the influence of contaminated foodborne pathogen on the indigenous microbiota (64 chicken samples) by artificially inoculated with Salmonella enterica serotype Virchow on chicken carcasses under various conditions, and carrying out 16S rRNA gene and whole metagenome sequencing. The amount of contaminated Salmonella in chicken meat samples was the highest and lowest in samples stored at 27 °C and 4 °C after washing, respectively. Additionally, the relative abundance of virulence genes was detected lower in samples stored at 4 °C after washing in both butcher shop and commercial samples. These results could be useful for reducing the risk of foodborne illness caused by cross-contamination during the preparation of chicken meat.

Genotypic and phenotypic characterization of the food spoilage bacterium Brochothrix thermosphacta.

Microbial food spoilage is responsible for significant economic losses. Brochothrix thermosphacta is one of the major bacteria involved in the spoilage of meat and seafood. Its growth and metabolic activities during food storage result in the production of metabolites associated with off-odors. In this study, we evaluated the genotypic and phenotypic diversity of this species. A collection of 161 B. thermosphacta strains isolated from different foods, spoiled or not, and from a slaughterhouse environment was constituted from various laboratory collections and completed with new isolates. A PCR test based on the rpoB gene was developed for a fast screening of B. thermosphacta isolates. Strains were typed by MALDI-TOF MS, rep-PCR, and PFGE. Each typing method separated strains into distinct groups, revealing significant intra-species diversity. These classifications did not correlate with the ecological origin of strains. The ability to produce acetoin and diacetyl, two molecules associated with B. thermosphacta spoilage, was evaluated in meat and shrimp juices. The production level was variable between strains and the spoilage ability on meat or shrimp juice did not correlate with the substrate origin of strains. Although the B. thermosphacta species encompasses ubiquitous strains, spoiling ability is both strain- and environment-dependent.

Improving simultaneously the quality and safety of cooked and peeled shrimp using a cocktail of bioprotective lactic acid bacteria.

Tropical shrimp is of considerable economic importance in the world but is highly perishable due to microbial and chemical degradation. Biopreservation is a food preservation technology based on the addition of "positive" bacteria able to kill or prevent the growth of undesirable microorganisms. Two strains of lactic acid bacteria (LAB) have previously been selected for a biopreservation strategy: Lactococcus piscium CNCM I-4031, for its ability to prevent the sensory deterioration of seafood and Carnobacterium divergens V41, which inhibits growth of Listeria monocytogenes. The objective was to test the association of the two strains to improve both the quality and safety of shrimp. In a first trial, the two LAB were inoculated alone or in a cocktail in cooked and peeled shrimp (CPS) Penaeus vannamei at 5×105CFU/g. Chemical, sensory and microbiological analyses by culture-dependent and -independent methods were performed during storage under modified atmosphere packaging (MAP) at 8°C. The results were compared to a non-inoculated batch. In a second trial, the same experiments were repeated in the presence of 102CFU/g of L. monocytogenes RF191. The microbiota of CPS was composed of LAB, Shewanella spp. and Enterobacteriaceae. Brochothrix thermosphacta was not detected. L. piscium and C. divergens reached 108 and 109CFU/g, respectively, in 7days and did not inhibit each other when co-inoculated. L. piscium reduced L. monocytogenes by 1Log (CFU/g) for 28days. C. divergens had an immediate listericidal effect lasting 7days. A regrowth of L. monocytogenes was then observed but the count was always 2 to 5Log (CFU/g) lower than in the control. No additional or synergic effect between protective strains was observed and the cocktail had the same inhibitory effect as C. divergens alone. C. divergens was very effective at preventing the sensory deterioration of CPS. This may be related to the inhibition of Shewanella and Enterobacteriaceae. However, the panelists could detect the presence of C. divergens during the first 10days of storage, with slight unpleasant odors and flavors. L. piscium improved the sensory quality of CPS for 14days only. In co-culture, L. piscium eliminated the off-odors and flavors released by C. divergens in the early stage of storage and the co-culture allowed maintaining a good quality of CPS throughout the storage. Therefore, the use of a cocktail of L. piscium CNCM I-4031 and C. divergens V41 is recommended in a strategy of biopreservation of shrimp.

Identification and Quantification of Volatile Chemical Spoilage Indexes Associated with Bacterial Growth Dynamics in Aerobically Stored Chicken.

Volatile organic compounds (VOCs) as chemical spoilage indexes (CSIs) of raw chicken breast stored aerobically at 4, 10, and 21 °C were identified and quantified using solid phase microextraction (SPME) combined with gas chromatography-mass spectrometry (GC-MS). The growth dynamics of total viable count (TVC), psychrotrophs, Pseudomonas spp., lactic acid bacteria (LAB), Brochothrix thermosphacta and H2 S producing bacteria were characterized based on maximum growth rates (µmax ), maximal microbial concentration (Nmax ) and at the moment of microbial shelf life (Svalues ), calculated from Gompertz-fitted growth curves. Pseudomonas spp. was predominant species, while B. thermosphacta was characterized by the highest µmax . The microbiological and sensory shelf lives were estimated based on TVC, Pseudomonas spp., and B. thermosphacta counts and sensory evaluation, respectively. Among 27 VOCs identified by GC-MS in spoiled chicken samples, ethanol (EtOH), 1-butanol-3-methyl (1But-3M), and acetic acid (C2 ) achieved the highest Pearson's correlation coefficients of 0.66, 0.61, and 0.59, respectively, with TVC, regardless of storage temperature. Partial least squares (PLS) regression revealed that the synthesis of 1But-3M and C2 was most likely induced by the metabolic activity of B. thermosphacta and LAB, while EtOH was attributed to Pseudomonas spp. The increase in concentration of selected volatile spoilage markers (EtOH, 1But-3M, and C2 ) in the headspace over spoiled chicken breast was found to be statistically significant (P < 0.05) with TVC growth. These findings highlight the possibility of analyzing the combination of 3 selected spoilage markers: EtOH, 1But-3M, and C2 as rapid evaluation for poultry quality testing using SPME-GC-MS.

Spoilage potential of Vagococcus salmoninarum in preservative-free, MAP-stored brown shrimp and differentiation from Brochothrix thermosphacta on streptomycin thallous acetate actidione agar.

AIMS: During a previous study concerning brown shrimp (Crangon crangon), selective streptomycin thallous acetate actidione (STAA) agar was used to determine the growth of Brochothrix thermosphacta. However, the growth of Vagococcus salmoninarum on this medium was also noticed. This study explores the spoilage potential of this organism when inoculated on sterile shrimp. METHODS AND RESULTS: Isolates growing on STAA were identified using (GTG)5 clustering followed by partial 16S rRNA gene sequence analysis. Their biochemical spoilage potential was analysed for H2 S production and enzymatic activities were tested using an APIZYM test. Headspace solid phase micro-extraction (SPME) and gas chromatography-mass spectrometry (GC-MS) were used to analyse the volatile organic compounds (VOCs) produced during storage of inoculated shrimp. CONCLUSION: Fifty-five per cent of isolates taken from STAA could be identified as V. salmoninarum, while no apparent morphological difference with B. thermosphacta isolates was identified upon the prescribed incubation conditions. For isolates identified as V. salmoninarum, production of 2-heptanone, 2-nonanone, 2-undecanone was found, as was the possibility to form H2 S. SIGNIFICANCE AND IMPACT OF THE STUDY: When using the STAA medium for detecting B. thermosphacta, one should consider the possible abundant presence of V. salmoninarum as well. Based on this study, V. salmoninarum does not exhibit great spoilage potential, although it can produce H2 S and formed VOCs which are also found in other spoiled seafood products.

Selection of bioprotective cultures for preventing cold-smoked salmon spoilage.

Biopreservation is a natural technology of food preservation, which consists of inoculating food with microorganisms selected for their antibacterial properties. The objective of this study was to select lactic acid bacteria (LAB) to improve the quality of cold-smoked salmon (CSS). In this work, different strains representative of the 4 dominant species, identified in a previous study by pyrosequencing the 16S rRNA gene, were isolated and their spoiling potential in CSS blocks, sterilized by ionization, was assessed by twelve trained panelists along the vacuum storage at 8°C. Photobacterium phosphoreum, Brochothrix thermosphacta and Serratia proteamaculans released strong off-odors whereas the spoiling potential of Carnobacterium divergens was weaker. The spoiling capacity of Lactococcus piscium EU2241, Leuconostoc gelidum EU2247, Lactobacillus sakei EU2885, Staphylococcus equorum S030674 and 4 commercial starters was tested by the same method and 2 strains were eliminated due to off-odor production. The effect of the 6 selected LAB against the 4 specific spoiling organisms (SSOs) selected was tested by challenge tests in sterile CSS blocks. The protective effect of the LAB differed from one SSO to another and no correlation could be established between the sensory improvement, SSO inhibition, and the implantation or acidification of protective cultures (PCs). All the PCs except L. piscium reduced the off-odors released by P. phosphoreum although some of them had no effect on its growth. S. equorum, which did not grow in CSS, favored the implantation of P. phosphoreum but prevented its off-odor formation. L. piscium was the only strain that prevented the spoilage of B. thermosphacta and S. proteamaculans although it did not grow very well and did not acidify the product. L. gelidum EU2247 inhibited the growth of these 2 SSOs and lowered the pH but had no effect on the sensory quality. Finally, L. piscium was tested in 2 naturally contaminated products, with a positive effect on 1 batch. This effect was not correlated with the microbial ecosystem as determined by acultural and cultural techniques. Based on these results, the selection strategy is discussed.

Microbiological spoilage and investigation of volatile profile during storage of sea bream fillets under various conditions.

Volatile organic compound (VOC) profile was determined during storage of sea bream (Sparus aurata) fillets under air and Modified Atmosphere Packaging (MAP - CO2/O2/N2: 60/10/30) at 0, 5 and 15°C. Microbiological, TVB-N (Total Volatile Base Nitrogen) and sensory changes were also monitored. Shelf-life of sea bream fillets stored under air was 14, 5 and 2days (d) at 0, 5 and 15°C respectively, while under MAP was 18, 8, and 2d at 0, 5 and 15°C respectively. At the end of shelf life, the total microbial population ranged from 7.5 to 8.5logcfu/g. Pseudomonas spp. were among the dominant spoilage microorganisms in all cases, however growth of Brochothrix thermosphacta and Lactic Acid Bacteria (LAB) were favoured under MAP compared to air. TVB-N production was favoured at higher temperatures and under air compared to lower temperatures and MAP. TVB-N increased substantially from the middle of storage and its value never reached concentrations higher than 30-35mgN/100g, which is the legislation limit, making it a poor chemical spoilage index (CSI). A lot of alcohols, aldehydes, ketones and ethyl esters that were detected in the present study have been reported as bacterial metabolites, others as products of chemical oxidation while others as aroma constituents. VOCs such as 3-methylbutanal, acetic acid, ethanol, ethyl esters of isovaleric and 2-methylbutyric acids, 1-penten-3-ol, 1-octen-3-ol and cis-4-heptenal appeared from the early or middle stages and increased until the end of storage. From those only 3-methylbutanal, acetic acid, ethanol and the ethyl esters have been reported as microbial origin, making them potential CSI candidates of sea bream fillets.

Genotypic characterization of Brochothrix thermosphacta isolated during storage of minced pork under aerobic or modified atmosphere packaging conditions.

A total of 306 colonies were isolated from the selective medium for Brochothrix spp., during the spoilage of minced pork stored at 0, 5, 10 and 15 °C and packed aerobically and under modified atmosphere packaging conditions (MAP). Brochothrix biodiversity was assessed by Pulsed Field Gel Electrophoresis (PFGE), and representative strains were further analysed by Rep-PCR using primer (GTG)5 and Sau-PCR with primers SAG1 and SAG2. Although, different results were obtained from the different methods, a significant diversity among isolates recovered from aerobic conditions was observed. On the contrary, isolates from MAP showed a lower degree of heterogeneity. The storage conditions affected the Brochothrix diversity, the strains isolated in the initial stage being different from the ones present at the final stage of storage at chill temperatures. A representative number of isolates, based on the results of the clustering by molecular methods, were subjected to 16S rRNA gene sequencing, revealing that all belonged to Brochothrix thermosphacta.

The prevalence and some metabolic traits of Brochothrix thermosphacta in meat and meat products packaged in different ways.

BACKGROUND: The effect of Brochothrix thermosphacta on the quality of meat and meat products is of vital importance in connection with Regulation EC/178/2002 extending the definition of unsafe foodstuffs to encompass all those which are unfit for human consumption. This study aimed to determine the prevalence of B. thermosphacta in meat and meat products packaged under different conditions and to estimate the effect of B. thermosphacta strains on product quality based on their protein and lipid degradation activity. RESULTS: B. thermosphacta was absent in only two of 132 samples. All other samples were contaminated with this bacterium (10(1) to 10(9) cfu g(-1) meat and 10(2) to 10(8) cfu g(-1) meat product). In products stored under high-oxygen atmosphere Brochothrix cells accounted for almost 100% total mesophilic count (TMC) and below 50% TMC in oxygen-free atmosphere. While the tested B. thermosphacta strains did not show any proteolytic activity, most of them displayed lipolytic activity at 25 °C and some even at 4 °C. CONCLUSION: B. thermosphacta is commonly present in meat and meat products packaged in different ways. This bacterium can display lipolytic activity also at refrigeration temperature. Its over-proliferation can be inhibited through vacuum packaging or packaging under a modified atmosphere with reduced oxygen content.

Changes in the composition of the bacterial flora on tray-packaged pork during chilled storage analyzed by PCR-DGGE and real-time PCR.

In this study, a polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to investigate the changes in the composition of the bacterial population of tray-packaged pork during chilled storage. Relative quantitative real-time PCR was further used to evaluate the predominant spoilage bacteria obtained from DGGE analysis for their relative amount to the total bacteria in meat samples. DGGE analysis of the V3 and V6-V8 regions of the 16S rRNA gene showed that Pseudomonas were the predominant bacterial species at the end of the monitoring period. Real-time PCR expressed as the ΔΔC(T) method showed that the average 2(-ΔΔC)(T) values increased continually during the storage period from less than 0.001 at day 0 to 4.438 at the end of the monitoring, which indicated that the proportions of Pseudomonas within the total bacteria in meat samples increased. Both methods confirmed that Pseudomonas was the predominant spoilage bacteria. Practical Application: This study uses new techniques to identify bacteria in fresh retail pork and to follow changes in the bacterial population during 12 d refrigerated storage. Pseudomonads were found to increase with storage time, becoming the dominant flora after 12 d.

In vitro synthesis of biogenic amines by Brochothrix thermosphacta isolates from meat and meat products and the influence of other microorganisms.

Twenty Brochothrix thermosphacta strains tested for biogenic amines (BAs) production, formed histamine (6.6-16.2 mg/kg) and tyramine (18.7-35.4 mg/kg) but neither putrescine nor cadaverine. Six of the twenty strains were also investigated in respect of their influence on the synthesis of BAs by Escherichia coli, Pseudomonas sp., Proteus mirabilis and Lactobacillus sakei. In pure culture Escherichia coli produced all of the studied amines (histamine, tyramine, putrescine and cadaverine) with a total concentration of 167.7 mg/kg, P. mirabilis produced a total of 56.7 mg/kg histamine, tyramine and cadaverine, while Lactobacillus sakei and Pseudomonas sp. produced histamine and tyramine, totaling 37.9 and 35.2 mg/kg respectively. All B. thermosphacta promoted cadaverine production by Escherichia coli which increased by 12-68%, and some of them contributed to the appearance of this amine among the metabolites of Pseudomonas. The presence of B. thermosphacta decreased the potential ability of P. mirabilis to produce BAs.

Interactions between bacterial isolates from modified-atmosphere-packaged artisan-type cooked ham in view of the development of a bioprotective culture.

Growth and metabolite production of three dominant bacterial isolates (Carnobacterium divergens 3M14, Leuconostoc carnosum 3M42, and Brochothrix thermosphacta RMS6) from modified-atmosphere-packaged (MAP), artisan-type cooked ham were assessed for their interactions in view of the development of a bioprotective culture. During monoculture experiments in cooked ham simulation medium, Leuc. carnosum 3M42 converted the available glucose into lactic acid and ethanol, whereas the two other strains produced additional metabolites such as acetic acid and 3-methyl-1-butanol. When grown in co-culture, Leuc. carnosum 3M42 suppressed the growth and metabolism of B. thermosphacta RMS6. In contrast, a co-culture of the latter bacterium with C. divergens 3M14 led to a variety of spoilage-related metabolic compounds. Subsequently, experiments with a commercial cooked ham product indicated that Leuc. carnosum 3M42 dominated the meat matrix and improved acceptability of the product over time, hence acting as a bioprotective culture for MAP, artisan-type cooked ham.