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.

Quality assessment of ice-stored tropical yellowfin tuna (Thunnus albacares) and influence of vacuum and modified atmosphere packaging.

Metagenomic, microbial, chemical and sensory analyses of Thunnus albacares from Martinique stored in ice (AIR - 0 °C), vacuum (VP - 4/8 °C) and modified atmosphere packaging (MAP - 4/8 °C) (70% CO2 - 30% O2) were carried out. The organoleptic rejection of AIR tuna was observed at day 13 when total bacterial counts equaled 10(6)-10(7) CFU g(-1). No extension of shelf-life was provided by VP and MAP. According to 16S rRNA gene sequence analyzed by Illumina MiSeq and PCR-TTGE, Rhodanobacter terrae was the main species of the freshly caught tuna. At the sensory rejection time, Brochothrix thermosphacta and Pseudomonas dominated the AIR products while B. thermosphacta alone or a mix of B. thermosphacta, Enterobacteriaceae and lactic acid bacteria (LAB) dominated the microbiota of MAP and VP products, respectively. The pH value remained stable in all trials, ranging from 5.77 to 5.97. Total volatile basic nitrogen (TVBN) and trimethylamine (TMA-N) concentrations were weak and not significantly different between batches. Lipid oxidation increased in the samples containing O2 (MAP > AIR). The initial concentration of histamine was high (75-78 mg kg(-1)) and stable up to 8 days but then significantly decreased in all trials to reach 25-30 mg kg(-1), probably due to the presence of histamine-decomposing bacteria.

Genetic diversity analysis of isolates belonging to the Photobacterium phosphoreum species group collected from salmon products using AFLP fingerprinting.

An accurate amplified fragment length polymorphism (AFLP) method, including three primer sets for the selective amplification step, was developed to display the phylogenetic position of Photobacterium isolates collected from salmon products. This method was efficient for discriminating the three species Photobacterium phosphoreum, Photobacterium iliopiscarium and Photobacterium kishitanii, until now indistinctly gathered in the P. phosphoreum species group known to be strongly responsible for seafood spoilage. The AFLP fingerprints enabled the isolates to be separated into two main clusters that, according to the type strains, were assigned to the two species P. phosphoreum and P. iliopiscarium. P. kishitanii was not found in the collection. The accuracy of the method was validated by using gyrB-gene sequencing and luxA-gene PCR amplification, which confirmed the species delineation. Most of the isolates of each species were clonally distinct and even those that were isolated from the same source showed some diversity. Moreover, this AFLP method may be an excellent tool for genotyping isolates in bacterial communities and for clarifying our knowledge of the role of the different members of the Photobacterium species group in seafood spoilage.

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.

Origin and ecological selection of core and food-specific bacterial communities associated with meat and seafood spoilage.

The microbial spoilage of meat and seafood products with short shelf lives is responsible for a significant amount of food waste. Food spoilage is a very heterogeneous process, involving the growth of various, poorly characterized bacterial communities. In this study, we conducted 16S ribosomal RNA gene pyrosequencing on 160 samples of fresh and spoiled foods to comparatively explore the bacterial communities associated with four meat products and four seafood products that are among the most consumed food items in Europe. We show that fresh products are contaminated in part by a microbiota similar to that found on the skin and in the gut of animals. However, this animal-derived microbiota was less prevalent and less abundant than a core microbiota, psychrotrophic in nature, mainly originated from the environment (water reservoirs). We clearly show that this core community found on meat and seafood products is the main reservoir of spoilage bacteria. We also show that storage conditions exert strong selective pressure on the initial microbiota: alpha diversity in fresh samples was 189±58 operational taxonomic units (OTUs) but dropped to 27±12 OTUs in spoiled samples. The OTU assemblage associated with spoilage was shaped by low storage temperatures, packaging and the nutritional value of the food matrix itself. These factors presumably act in tandem without any hierarchical pattern. Most notably, we were also able to identify putative new clades of dominant, previously undescribed bacteria occurring on spoiled seafood, a finding that emphasizes the importance of using culture-independent methods when studying food microbiota.

Evaluation of the spoilage potential of bacteria isolated from spoiled cooked whole tropical shrimp (Penaeus vannamei) stored under modified atmosphere packaging.

The spoilage potential of isolates belonging to five bacterial groups/species (Shewanella baltica, Carnobacterium maltaromaticum, Aeromonas salmonicida, Vibrio sp., "other Gamma-Proteobacteria" [containing one strain of Pseudoalteromonas sp. and one strain of Psychrobacter sp.]) isolated from spoiled cooked and whole tropical shrimp stored under modified atmosphere packaging (MAP) was evaluated by inoculation into ionized cooked and peeled tropical shrimp followed by storage for 32 days at 8 °C. Microbial growth and sensory changes were monitored during the storage period. The major spoilage bacterial isolate groups were C. maltaromaticum and S. baltica. In order to characterize their spoilage potential further and to study the effect of their interactions, each of these two specific spoilage organisms (SSO) and one mixed-culture, C. maltaromaticum/S. baltica, were tested 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, both species groups imposed their spoilage characteristics.

Development of a rapid real-time PCR method as a tool to quantify viable Photobacterium phosphoreum bacteria in salmon (Salmo salar) steaks.

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 rRNA gene 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 bacteria in fresh salmon in 6 h. This new culture-independent method will be valuable for future fish inspection, the assessment of raw material quality in fish processing plants, and studies on the ecology of this important specific spoilage microorganism.

Evaluation of the spoilage potential of bacteria isolated from spoiled raw salmon (Salmo salar) fillets stored under modified atmosphere packaging.

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.

Characterisation of the spoilage microbiota in raw salmon (Salmo salar) steaks stored under vacuum or modified atmosphere packaging combining conventional methods and PCR-TTGE.

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.

Sensory and physicochemical evolution of tropical cooked peeled shrimp inoculated by Brochothrix thermosphacta and Lactococcus piscium CNCM I-4031 during storage at 8°C.

This study investigated the sensory quality and physicochemical evolution (pH, glucose, l-lactic acid, biogenic amine, free amino-acids and volatile compounds) during storage at 8°C of cooked peeled shrimp inoculated with the specific spoilage bacteria Brochothrix thermosphacta alone or mixed with the protective strain Lactococcus piscium CNCM I-4031. Growth of both bacteria was monitored at regular intervals during storage by microbial counts and the thermal temperature gradient gel electrophoresis (TTGE) technique. Bacterial counts showed that L. piscium and B. thermosphacta inoculated at 7 log CFU/g and 3 log CFU/g were well adapted to shrimp, reaching a maximum level of 9 log CFU/g after 4days and 10days respectively. In mixed culture, the growth of B. thermosphacta was reduced by 3.2±0.1 log CFU/g. The TTGE technique allowed monitoring the colonisation of the strains on the shrimp matrix and confirming the dominance of L. piscium in mixed culture throughout the experiment. Sensory analysis confirmed that B. thermosphacta spoiled the product after 11days, when its cell number attained 8 log CFU/g with the emission of strong butter/caramel off-odours. This sensory profile could be linked to the production of 2,3 butanedione, cyclopentanol, 3-methylbutanol, 3-methylbutanal, 2-methylbutanal, 4-methyl-3-chloro-3-pentanol and ethanol, which were produced in more significant quantities in the B. thermosphacta batch than in the batches in which the protective strain was present. On the contrary, TVBN and TMA were not suitable as quality indicators for B. thermosphacta spoilage activity. In the products where the protective L. piscium strain was present, no adverse effect on sensory quality was noted by the sensory panels. Moreover, biogenic amine assessment did not show any histamine or tyramine production by this strain, underlining its safety profile. Both strains produced lactic acid (1850mg/kg in L. piscium and B. thermosphacta batch on days 3 and 10 respectively; 3830mg/kg on day 7 in mixed culture) and the pH decrease from 6.6±0.0 to 5.9±0.1 was similar in all batches. Lactic acid production or competition for free amino-acid was not involved in the inhibition mechanism; however rapid glucose consumption by L. piscium could partially explain the growth limitation of the spoilage micro-organism. This study demonstrated the spoilage characteristic of B. thermosphacta and the usefulness of L. piscium as a bioprotective culture for tropical cooked peeled shrimp without any adverse effect on the sensory quality of the product.

Sensory characteristics of spoilage and volatile compounds associated with bacteria isolated from cooked and peeled tropical shrimps using SPME-GC-MS analysis.

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.

Vagococcus penaei sp. nov., isolated from spoilage microbiota of cooked shrimp (Penaeus vannamei).

A polyphasic taxonomic study, using phenotypic, phylogenetic and genotypic characterization, was performed on five Gram-stain-positive, catalase-negative, coccus-shaped Vagococcus-like bacteria isolated from the spoilage microbiota of cooked shrimp. Comparative 16S rRNA gene sequence analysis indicated that the isolates belonged to the genus Vagococcus. The five isolates shared 100% 16S rRNA gene sequence similarity, and representative strain CD276(T) formed a branch that was distinct from the type strains of the six recognized species of the genus Vagococcus (Vagococcus fluvialis CCUG 32704(T), V. salmoninarum NCFB 2777(T), V. lutrae CCUG 39187(T), V. fessus M2661/98/1(T), V. carniphilus ATCC BAA-340(T) and V. elongatus PPC9(T)). The taxonomic position of strain CD276(T) was clarified using DNA-DNA hybridization, pulsed-field gel electrophoresis of whole-genome DNA, G+C content determination, cell-wall peptidoglycan typing, fatty acid analysis and biochemical characterization. On the basis of this evidence, a novel species, Vagococcus penaei sp. nov., is proposed. The type strain is CD276(T) (=LMG 24833(T) =CIP 109914(T)).

Effect of bacterial interactions on the spoilage of cold-smoked salmon.

Cold-smoked salmon is a lightly preserved fish product in which a mixed microbial flora develops during storage and where the interactive behaviour of micro-organisms may contribute to their growth and spoilage activity. The aim of this study was to assess the effect of the bacterial interactions between the main species contaminating the cold-smoked salmon on bacterial growth, chemical and sensory changes, and spoilage. First, Carnobacterium piscicola, Photobacterium phosphoreum, Lactobacillus sakei, Vibrio sp., Brochothrix thermosphacta and Serratia liquefaciens-like were inoculated as pure cultures on sterile cold-smoked salmon. All bacterial species grew well; Vibrio sp. was the fastest and L. sakei strains developed very rapidly as well with a high maximum cell density on cold-smoked salmon blocks (up to 10(9) cfu g(-1) after 10 days at 8 degrees C). Based on sensory analysis, Vibrio sp. was identified as non-spoilage bacteria, C. piscicola as very lightly and B. thermosphacta as lightly spoiling. L. sakei and S. liquefaciens-like were found to be the most spoiling bacteria. Secondly, C. piscicola and L. sakei, two species frequently occurring in the lactic flora of the product, were inoculated together and each of them in mixed cultures with respectively P. phosphoreum, Vibrio sp., B. thermosphacta, and S. liquefaciens-like. The growth of L. sakei was shown to strongly inhibit most of the co-inoculated strains i.e. P. phosphoreum, B. thermosphacta, S. liquefaciens-like and, to a lesser extent, Vibrio sp. The growth of C. piscicola seemed to be enhanced with B. thermosphacta and to develop earlier with P. phosphoreum and Vibrio sp. Conversely, S. liquefaciens-like and P. phosphoreum were weakly inhibited by C. piscicola. The main observation resulting from the sensory evaluation was the delay in the appearance of the spoilage characteristics in the mixed cultures with L. sakei, in particular L. sakei/ S. liquefaciens-like. On the other hand, the spoilage activity of the non-spoiler strains Vibrio sp. or the moderate spoilage strains B. thermosphacta and C. piscicola was increased when they were associated together. It is concluded that the spoilage behaviour of micro-organisms in mixed culture is significantly different from pure culture and explain the difficulty to find robust quality indices for this product.