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ABSTRACT: A specific real-time PCR quantification method combined with a propidium monoazide sample treatment step was developed to determine quantitatively the viable population of the Photobacterium phosphoreum species group in raw modified atmosphere packed salmon. Primers were designed to amplify a 350 bp fragment of the gyrase subunit B gene (gyrB) of P. phosphoreum. The specificity of the two primers was demonstrated by using purified DNA from 81 strains of 52 different bacterial species. When these primers were used for real-time PCR in pure culture, a good correlation (R(2) of 0.99) was obtained between this method and conventional enumeration on marine agar (MA). Quantification was linear over 5 log units as confirmed by using inoculated salmon samples. On naturally contaminated fresh salmon, the new real-time PCR method performed successfully with a quantification limit of 3 log (CFU/g). A correlation coefficient (R(2)) of 0.963 was obtained between the PCR method and classic enumeration on MA followed by identification of colonies (290 isolates identified by real-time PCR or by 16S rDNA sequencing). A good correlation with an R(2) of 0.940 was found between the new PCR method and an available specific conductance method for P. phosphoreum. This study presents a rapid tool for producing reliable quantitative data on viable P. phosphoreum in fresh salmon in 6 hours. This new culture-independent method will be valuable for future fish inspection, the assessment of raw material quality in fish processing plants and for studies on the ecology of this important specific spoilage microorganism.
Applied and environmental microbiology 02/2013; · 3.69 Impact Factor
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ABSTRACT: The spoilage potential of eight bacterial groups/species (Serratia spp., Hafnia alvei, Brochothrix thermosphacta, Carnobacterium maltaromaticum, Shewanella baltica, Lactococcus piscium, Photobacterium phosphoreum, "other Enterobacteriaceae" [containing one strain of Moellerella sp., Morganella sp. and Pectobacterium sp.]) isolated from spoiled raw salmon fillets stored under modified atmosphere packaging (MAP) was evaluated by inoculation into sterile raw salmon cubes followed by storage for 12days at 8°C. Microbial growth and sensory changes were monitored during the storage period. The dominant spoilage bacteria were C. maltaromaticum, H. alvei and P. phosphoreum. In order to further characterize their spoilage potential and to study the effect of their interactions, each of these 3 specific spoilage organisms (SSO) and two mixed-cultures, C. maltaromaticum/H. alvei and C. maltaromaticum/P. phosphoreum were tested in the sterile salmon model system using a combination of complementary methods: molecular (PCR-TTGE), sensory, chemical and conventional microbiological analyses. It was concluded that, in the mixed-culture inoculated samples, the dominant species determined the spoilage characteristics. The volatile fraction of P. phosphoreum inoculated samples was analyzed by solid-phase microextraction (SPME) followed by gas chromatography coupled to mass spectrometry (GC-MS). Among the specific volatile compounds present on P. phosphoreum spoiled inoculated samples, acetic acid was correlated with sensory analysis and can be proposed as a raw salmon spoilage marker.
International journal of food microbiology 01/2013; 160(3):227-38. · 3.01 Impact Factor
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ABSTRACT: Brochothrix thermosphacta, a Gram-positive bacterium, is considered as the predominant spoilage microbiota of modified atmosphere packing (MAP) shrimp and fish. Traditional methods currently used to detect B. thermosphacta in foods are time-consuming and labour-intensive. The aim of this study was to develop a real-time PCR quantification method combined with a propidium monoazide (PMA) sample treatment step to monitor the population of B. thermosphacta in cooked shrimp and salmon. The specificity of the two primers MO405 and MO404 used to amplify a 70 bp fragment of the 16S rRNA gene was demonstrated by using purified DNA from 30 strains, among 21 bacterial species including 22 reference strains. Using these primers for real-time PCR and in pure culture, a good correlation was obtained between real-time PCR and the conventional plating method. Quantification was linear over 7-log units using artificially inoculated samples. The method performed successfully when tested on naturally contaminated cooked shrimp and fresh salmon, with a minimum threshold of 1.9×10² CFU/g for accurate quantification of B. thermosphacta. The correlation between the B. thermosphacta counts obtained by real-time PCR and plate counts on naturally contaminated shrimp and salmon was high (R²=0.895). Thus, this study presents a rapid tool for producing reliable quantitative data on B. thermosphacta in cooked shrimp and fresh salmon.
Food Microbiology 05/2012; 30(1):173-9. · 3.28 Impact Factor
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ABSTRACT: In order to characterise the spoilage related to microbiota of raw salmon, a combination of culture-dependent and -independent methods, including PCR-TTGE, was used to analyse 3 raw salmon batches stored for 3 days at chilled temperature in modified atmosphere packaging (MAP) (50% CO₂/50% N₂) or under vacuum. Sensory evaluation, microbiological enumeration and chemical analysis were performed after 3, 7 and 10 days of storage. At the onset of spoilage, 65 bacterial isolates were picked from the plates. Thus, 13 different genera or species were identified by phenotypic and molecular tests: Serratia spp., Photobacterium phosphoreum, Yersinia intermedia, Hafnia alvei, Buttiauxella gaviniae, Pseudomonas sp., Carnobacterium maltaromaticum, Carnobacterium divergens, Lactococcus piscium, Lactobacillus fuchuensis, Vagococcus carniphilus, Leuconostoc gasicomitatum and Brochothrix thermosphacta. The PCR-TTGE profiles and band identification enabled a shift of the dominant populations during the storage to be visualised for all the batches, probably due to the temperature change and the packaging. At the beginning of storage, Pseudomonas sp. dominated the raw salmon microbiota while in the following days (7 and 10), P. phosphoreum and L. piscium were identified as the main bacterial groups. This study enhances the knowledge of MAP and vacuum-packed raw salmon spoilage microbiota.
Food Microbiology 05/2012; 30(1):164-72. · 3.28 Impact Factor
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ABSTRACT: The spoilage potential of six bacterial species isolated from cooked and peeled tropical shrimps (Brochothrix thermosphacta, Serratia liquefaciens-like, Carnobacterium maltaromaticum, Carnobacterium divergens, Carnobacterium alterfunditum-like and Vagococcus penaei sp. nov.) was evaluated. The bacteria were inoculated into shrimps, packaged in a modified atmosphere and stored for 27 days at 8 °C. Twice a week, microbial growth, as well as chemical and sensory changes, were monitored during the storage period. The bacteria mainly involved in shrimp spoilage were B. thermosphacta, S. liquefaciens-like and C. maltaromaticum whose main characteristic odours were cheese-sour, cabbage-amine and cheese-sour-butter, respectively. The volatile fraction of the inoculated shrimp samples was analysed by solid-phase microextraction (SPME) and gas chromatography coupled to mass spectrometry (GC-MS). This method showed that the characteristic odours were most likely induced by the production of volatile compounds such as 3-methyl-1-butanal, 2,3-butanedione, 2-methyl-1-butanal, 2,3-heptanedione and trimethylamine.
International journal of food microbiology 06/2011; 147(3):195-202. · 3.01 Impact Factor
