ABSTRACT: Modelling the growth and interaction between Brochothrix thermosphacta, Pseudomonas spp. and Leuconostoc gelidum in minced pork meat samples.
Project description:Oxygen and carbon dioxide are common protective gases used in modified atmosphere packaging (MAP) of meat. Within the package, they selectively suppress members of the spoilage microbiome, reshaping it to adapted species concomitantly growing upon MAP. Thus, this species must exhibit adaptation mechanisms to withstand the inhibitory effect of carbon dioxide and oxygen, and cope with selective nutrition on MAP meat. In order to uncover these mechanisms, the typical representative meat-spoiling bacteria Brochothrix (B.) thermosphacta TMW2.2101 and 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 were grown in a meat simulation medium under a controlled, sterile environment, aerated constantly with either air, 100%_N2, 30%_CO2/70%_O2 or 30%_CO2/70%_N2. Growth dynamics were monitored and a label-free quantitative mass spectrometric approach was employed to determine changes within the bacterial proteomes in response to the different gas atmospheres. Revealed bacterial tolerance to modified atmospheres (MA) comprise two possible scenarios: Either bacteria were intrinsically adapted to MA, exhibiting no proteomic regulation of enzymes (L. gelidum subsp. gelidum and gasicomitatum) or, tolerance was provided by varying specific metabolic adaptation (B. thermosphacta, C. divergens, C. maltaromaticum). In detail, metabolic adaptation mechanisms to oxygen comprised an enhanced oxidative stress reduction response, adjustment of the pyruvate metabolism and catabolic oxygen consuming reactions. Adaptation to carbon dioxide was characterized by an upregulation of proteins involved in intracellular pH homeostasis, maintenance of osmotic balance and alteration of the fatty acid composition of the cell membrane. We furthermore predict species-specific strategies for different and preferential carbon source utilization enabling a non-competitive coexistence on meat and resulting in a synergistic spoilage. We conclude that a gas atmosphere containing 30%_CO2/70%_O2 has no inhibitory effect on the analyzed prominent meat-spoiling bacteria whereas 30%_CO2/70%_N2 predictively inhibits C. divergens TMW21577 and B. thermosphacta TMW2.2101 but not the other three species. This gives a mechanistically explanation of their acknowledged status as typical spoilage organisms on packaged meats.
Project description:To find a promoter upregulated in the presence of rotten meat, we exposed B. subtilis 168 to the volatiles of rotten meat (mixed beef/pork) and performed a microarray comparing it to B. subtilis which was not exposed to the meat. The results where used to build iGEM Groningen 2012s Food Warden, a spoiled meat detector. Find more information at: 2012.igem.org/Team:Groningen
Project description:To find a promoter upregulated in the presence of rotten meat, we exposed B. subtilis 168 to the volatiles of rotten meat (mixed beef/pork) and performed a microarray comparing it to B. subtilis which was not exposed to the meat. The results where used to build iGEM Groningen 2012s Food Warden, a spoiled meat detector. Find more information at: 2012.igem.org/Team:Groningen One condition design; including dye swap, two technical replicates and two experimental replicates
Project description:This study applied peptidomics to investigate potential biomarkers for evaluating pork-meat freshness. Meat samples stored at -2, 4, 10, and 25 °C were collected at specific time points to evaluate meat freshness indicators (color, total viable count, pH, and total volatile basic nitrogen). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profile was analyzed, and substantial protein degradation (myosin heavy chain, paramyosin, troponin) was detected at the end of storage, regardless of the temperature. Peptidomics analysis was performed using a UHPLC-LTQ-Orbitrap mass spectrometer, and the potential peptide marker MVHMASKE was filtered via multivariate analysis and quantified by parallel reaction monitoring combined with external standard quantitation. In addition, the relationship between peptide content and change in meat freshness was verified using real-life samples and the content of MVHMASKE showed an obvious decline during storage, presenting a period of pork meat from fresh to spoilage. This study provides favorable evidences to evaluate pork meat freshness by mass spectrometry-based pep-tidomics.
Project description:Previous studies have evaluated pork quality by omics methods. However, proteomics coupled with metabolomics to investigate pork freshness by using pork exudates has not been reported. This study determined the changes in profiles of peptides and metabolites in exudates from pork stored at different temperatures (25, 10, 4, and -2 ℃). Multivariate statistical analysis revealed similar changes in profiles in exudates collected from pork stored at -2 and 4 ℃, and additional changes following storage at higher temperatures. We identified peptides from 7 proteins and 30 metabolites differing in abundance between fresh and spoiled pork. Significant correlations be-tween pork quality and most of the peptides from these 7 proteins and 30 metabolites were found. The present study provides insight into changes in peptide and metabolite profiles of exudates from pork during storage at different temperatures and our analysis suggest that such changes can be used as markers for pork spoilage.
Project description:Mostly, lactic acid bacteria (LAB), including food-spoilage-associated, grow in communities consisting of several microbial species. The interspecies interactions eventually shape the structure and global activity of a given microbial community. Generally, the knowledge on system level responses of LAB (especially food-spoilage-associated) during such interactions is very limited. To study transcriptome responses during interactions between three MAP meat-spoilage-associated LAB (Leuconostoc gelidum subsp. gasicomitatum LMG 18811T, Lactococcus piscium MKFS47 and Lactobacillus oligofermentans LMG 22743T) we grew them separately in individual cultures and in mixed cultures pairwise (three combinations) and all together (triple culture) in three replicates on a glucose-containing growth medium (MRS) under microaerobic conditions at 25 C, samples were taken at three time points (3, 5 and 11 h) and extracted RNA were sequenced. The experiments were performed in two batches. At first (batch 1), co-cultivation of Le. gelidum and Lc. piscium accompanied with their individual cultures was performed and processed. The raw RNA-seq data for the individual culture of Lc. piscium from the batch 1 were uploaded earlier and are available in the ArrayExpress database under accession number E-MTAB-3245. Later (batch 2), two other pairwise cultures (Le. gelidum + Lb. oligofermentans and Lc. piscium + Lb. oligofermentans) and the triple culture were grown together with the individual cultures of all three LAB. Designations used for the sample names: G: Le. gelidum; P: Lc. piscium; O: Lb. oligofermentans; GO, PO, PG: pairwise cultures of the corresponding species; OPG: triple culture; b1: batch 1; b2: batch 2. Example: 3G2_b1: 3 h, Le. gelidum, 2nd replicate, batch 1; 11PO3_b2: 11 h, pairwise culture of Lc. piscium and Lb. oligofermentans, 3d replicate, batch 2. One sample (5PO3_b2) had very low number of reads ~ 9000, and, therefore, was not uploaded under this project. RNA extraction and library construction were done analogously as in the study (Andreevskaya M et al., 2015. Appl. Environ. Microbiol. 81:38003811, doi: 10.1128/AEM.00320-15). Ribosomal RNA was omitted. Libraries were sequenced in five lanes using SOLiD 5500XL (Life technologies, Foster City, Ca, USA) to produce 75 bp single-end reads. For the data submission, xsq files obtained from SOLiD 5500XL machine, were converted into fastq files. Adapter sequences were removed using cutadapt 1.4.1.