Jorge Barros-Velázquez

Changes in Biogenic Amines and Microbiological Analysis in Albacore (Thunnus alalunga) Muscle during Frozen Storage

Albacore specimens of extra quality were analyzed for their biogenic amine contents after 1, 3, 6, and 9 months of frozen storage at -18 degrees C or -25 degrees C. A high-performance liquid chromatography method involving a linear elution gradient was optimized for the identification and determination of putrescine, cadaverine, histamine, spermidine, and spermine in albacore tuna. Putrescine was the biogenic amine that showed the highest increase, reaching concentrations of 59.04 ppm (815% of the initial level) and 68.26 ppm (942% of the initial level) in the white muscle of albacore after 9 months of frozen storage at -18 and -25 degrees C, respectively. Cadaverine, histamine, and spermidine concentrations were below 3, 5, and 11 ppm, respectively, after 9 months of frozen storage, while spermidine underwent a significant decrease at both storage temperatures. Microbiological analysis confirmed the absence of species of Enterobacteriaceae in 75% of the albacore specimens after 9 months of frozen storage; coliforms were always below 3 CFU/g. The survival rate of the psychrotrophic microorganisms after 9 months of frozen storage at -25 degrees C was 4.6%, while 38.9 and 92.1% of the aerobic mesophiles present in the white muscle of albacore before freezing survived 9 months of storage at -18 and -25 degrees C, respectively.

Species Differentiation of Seafood Spoilage and Pathogenic Gram-Negative Bacteria by MALDI-TOF Mass Fingerprinting

Species differentiation is important for the early detection and identification of pathogenic and food-spoilage microorganisms that may be present in fish and seafood products. The main 26 species of seafood spoilage and pathogenic Gram-negative bacteria, including Aeromonas hydrophila, Acinetobacter baumanii, Pseudomonas spp., and Enterobacter spp. among others, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) of low molecular weight proteins extracted from intact bacterial cells by a fast procedure. From the acquired spectra, a library of specific mass spectral fingerprints was constructed. To analyze spectral fingerprints, peaks in the mass range of 2000-10 000 Da were considered and representative mass lists of 10-35 peak masses were compiled. At least one unique biomarker peak was observed for each species, and various genus-specific peaks were detected for genera Proteus, Providencia, Pseudomonas, Serratia, Shewanella, and Vibrio. Phyloproteomic relationships based on these data were compared to phylogenetic analysis based on the 16S rRNA gene, and a similar clustering was found. The method was also successfully applied for the identification of three bacterial strains isolated from seafood by comparing the spectral fingerprints with the created library of reference fingerprints. Thus, the proteomic approach demonstrated to be a competent tool for species identification.

Histamine and cadaverine production by bacteria isolated from fresh and frozen albacore (Thunnus alalunga).

Two hundred twenty-seven bacterial strains were isolated from fresh and frozen albacore stored either at -18 or -25 degrees C and investigated for their abilities to produce biogenic amines. As a preliminary screening, all 227 strains were tested in either Niven or Niven modified medium, which allowed the selection of 25 presumptive histamine-producing strains. High-pressure liquid chromatography revealed that only 10 of the 25 strains selected were able to produce low histamine concentrations (<25 ppm) in tryptic soy broth medium supplemented with 2% histidine. None of the 25 strains tested produced putrescine or spermine, whereas 6 strains produced spermidine. Histamine production by Stenotrophomonas maltophilia strain 25MC6 was not prevented at 4 degrees C, and the levels of this amine reached concentrations of 25.8 ppm after 6 days. Three S. maltophilia strains showed strong lysine-decarboxylating activity. Their cadaverine formation capacity was determined by high-pressure liquid chromatography in tryptic soy broth supplemented with 1% lysine; this revealed that the three S. maltophilia strains tested produced more than 700 ppm of cadaverine during the first 24 h of incubation at 37 degrees C. S. maltophilia strain 15MF, initially obtained from fresh albacore tuna, produced up to 2,399 ppm and 4,820 ppm of cadaverine after 24 and 48 h of incubation at 37 degrees C, respectively. To our knowledge, this is the first report on histamine and cadaverine production by strains of the species S. maltophilia, previously known as Pseudomonas and Xanthomonas maltophilia, isolated from fresh and frozen albacore tuna.

Development of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis reference method for the analysis and identification of fish species in raw and heat-processed samples: a collaborative study.

A collaborative study was carried out in seven European labs with the aim of achieving a sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) standard operation procedure to identify fish species in raw and cooked samples. Urea and SDS‐containing solutions were evaluated as extractants. Several preelectrophoretic operations — such as treatment with RNase/DNase, ultrafiltration and desalting — and up to ten types of gels and three SDS‐PAGE systems were considered. The SDS‐containing solution allowed a higher protein extractability than urea. Unlike urea extraction, SDS extraction seemed not to be influenced so much by the state of the sample (raw, cooked at 60oC, cooked at 85oC). Desalting, ultrafiltration or treatment with RNase/DNase did not improve the discriminatory power of the protein patterns. Commercial homogeneous 15% ExcelGels, especially when they were silver stained, yielded good results and afforded higher reproducibility, thus allowing a better matching of results among the laboratories participating in this collaborative study. Under the optimized technical conditions described above, all the fish species tested, either raw and cooked, yielded reproducible and discriminant species‐specific protein patterns.

Characterization and partial sequencing of species-specific sarcoplasmic polypeptides from commercial hake species by mass spectrometry following two-dimensional electrophoresis

The Merluccidae family comprises marine species, some of them of high commercial value and others less appreciated, whose commercialization in Europe under the generic name of “hake” is highly remarkable. The potential of proteomics was employed in this study with the aim of achieving the differential characterization of five different hake species: Merluccius merluccius (European hake), M. australis (Southern hake), M. hubbsi (Argentinian hake), M. gayi (Chilean hake), and M. capensis (Cape hake), some of them very closely related. Species‐specific polypeptides were observed for the five hake species studied in isoelectric focusing (IEF) and/or two‐dimensional electrophoresis (2‐DE) high‐resolution gels. The peptide mass maps of two polypeptide groups, previously selected by 2‐DE analysis as potentially species‐specific, were obtained by “in‐gel” tryptic digestion, followed by matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). Analysis of group A polypeptides (with pI in the range of 5.0–5.5 and molecular mass of 17 kDa), allowed the differential classification of the hake species into two groups: the East Atlantic coast group and the West Atlantic coast group. Moreover, the peptide mass‐maps from the heat‐resistant parvalbumin fraction (pI below 4.5; molecular mass <12 kDa) allowed the detection of a peptide characteristic of M. australis not present in the other four hake species tested. A specific 17 kDa protein from M. merluccius was also partially sequenced by nanospray‐ion trap‐tandem MS, revealing a high homology with rat nucleoside diphosphate kinase A (NDKA). This work opens the way to the application of proteomics to the differential characterization of commercial hake species at the molecular level.

Characterization of biogenic amine-producing Stenotrophomonas maltophilia strains isolated from white muscle of fresh and frozen albacore tuna

Abstract Three strains — one of them psychrotrophic and two mesophilic — of Stenotrophomonas maltophilia, an emerging pathogen involved in an increasing number of clinical syndromes, were isolated from fresh and frozen-stored (6 months at −25°C) albacore tuna in a routine screening of histamine-forming bacteria. All the three strains showed histidine decarboxylase activity when assayed in a 2% histidine–0.0005% pyridoxal-HCl containing trypticase soy broth and further analyzed by HPLC. Although all three strains produced less than 25 ppm of histamine, they showed strong lysine-decarboxylating activity, cadaverine being produced at concentrations ranging from 1736 to 4821 ppm after 48 h in 0.0005% pyridoxal-HCl–trypticase soy broth supplemented with 1% lysine. Strong secretion of extracellular lipase and protease was also observed in all three strains. The psychrotrophic strain S. maltophilia 5PC6 was able to produce both extracellular lipase and protease even at 4 and 7°C, respectively. Phenotyping assays — including the investigation of up to 40 biochemical tests and 15 susceptibility assays — revealed slight differences among the three strains. Genotyping experiments — based on RFLP analysis and Southern blot hybridizations — indicated that strains 15MF and 25MC6 were closely related but confirmed that all three strains recovered were different.

Rapid species identification of seafood spoilage and pathogenic Gram-positive bacteria by MALDI-TOF mass fingerprinting

The rapid identification of food pathogenic and spoilage bacteria is important to ensure food quality and safety. Seafood contaminated with pathogenic bacteria is one of the major causes of food intoxications, and the rapid spoilage of seafood products results in high economic losses. In this study, a collection of the main seafood pathogenic and spoilage Gram‐positive bacteria was compiled, including Bacillus spp., Listeria spp., Clostridium spp., Staphylococcus spp. and Carnobacterium spp. The strains, belonging to 20 different species, were obtained from the culture collections and studied by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). A reference library was created, including the spectral fingerprints of 32 reference strains and the extracted peak lists with 10–30 peak masses. Genus‐specific as well as species‐specific peak masses were assigned and could serve as biomarkers for the rapid bacterial identification. Furthermore, the peak mass lists were clustered with the web‐application SPECLUST to show the phyloproteomic relationships among the studied strains. Afterwards, the method was successfully applied to identify six strains isolated from seafood by comparison with the reference library. Additionally, phylogenetic analysis based on the 16S rRNA gene was carried out and contrasted with the proteomic approach. This is the first time MALDI‐TOF MS fingerprinting is applied to Gram‐positive bacterial identification in seafood, being a fast and accurate technique to ensure seafood quality and safety.

Industrial Applications of Hyperthermophilic Enzymes: A Review

Over the past two decades, research scientists have been involved in the investigation of thermophilic and hyperthermophilic microorganisms owing to the unique features of their enzymic systems. Such in-depth investigations are now on their way to mastering the cloning and industrial exploitation of a broad variety of genes encoding enzymes involved in starch hydrolysis, amino acid biosynthesis, protein hydrolysis, etc. In this work, we review the state of the art and future perspectives of industrial applications of enzymes from hyperthermophilic and extreme thermophilic microorganisms, special attention being paid to the biotechnological methods involved in their industrial exploitation.

Histamine and Biogenic Amine Production by Morganella morganii Isolated from Temperature-Abused Albacore

Histamine-producing bacteria were isolated from albacore stored at 0, 25, 30, and 37 degrees C. They were screened using Nivens differential medium, and their histamine production was confirmed by high-pressure liquid chromatography analysis. The optimum temperature for growth of histamine-producing bacteria was 25 degrees C. The bacterium producing the highest level of histamine was isolated from fish abused at 25 degrees C. It was identified as Morganella morganii by morphological, cultural, biochemical, and antimicrobial characteristics and by the Vitek microbial identification system. The M. morganii isolate was inoculated into tuna fish infusion broth medium, and the effect of temperature was determined for microbial growth and formation of histamine and other biogenic amines. The isolate produced the highest level of histamine, 5,253 ppm, at 25 degrees C in the stationary phase. At 15 degrees C, histamine production was reduced to 2,769 ppm. Neither microbial growth nor histamine formation was detected at 4 degrees C. To determine whether the isolate can also produce other biogenic amines that can potentiate histamine toxicity, production of cadaverine, putrescine, serotonin, tryptamine, tyramine, phenylethylamine, spermidine, and spermine by the isolate was also monitored. Cadaverine, putrescine, and phenylethylamine were detected with microbial growth in the tuna fish infusion broth medium. The optimum temperature for cadaverine, putrescine, and phenylethylamine formation was found to be 25 degrees C, as it was for histamine.

Detection and quantification of spoilage and pathogenic Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR

A new primer-probe set for the detection and quantification of Bacillus cereus, Bacillus licheniformis and Bacillus subtilis by real-time PCR (Rti-PCR) was developed. For it, forty-eight strains belonging to these species were considered. The DNA of these strains was isolated and a fragment of the 16S rRNA gene amplified. The amplicons were sequenced and the obtained sequences were aligned with reference sequences from the GenBank. For the development of the Real-Time PCR (RTi-PCR) methodology based on TaqMan probes, a primer pair and probe, specific for the studied Bacillus spp., were designed. To establish the quantification method, two RTi-PCR standard curves were constructed; one with DNA extracted from a serially-diluted B. cereus culture and a second curve with DNA extracted from a sterilised food product inoculated with serial dilutions of B. cereus. The curves exhibited R(2) values of 0.9969 and 0.9958 respectively. Linear correlations between the log(10) input DNA concentration and the threshold cycle (Ct) values were observed with a magnitude of linearity in the range of 1.65 × 10(1) CFU/mL to 1.65 × 10(6) CFU/mL for both standard curves. The specificity of the designed primers and probe was tested with DNA extracted from B. cereus, B. licheniformis and B. subtilis strains, which gave Ct values between 14 and 15, whereas non-specific amplifications of the DNA from other microbial species of food interest exhibited a Ct value above 28.5. To our knowledge, this method represents the first study about the quantification of spoilage and/or pathogenic B. cereus, B. licheniformis and B. subtilis in food products, with the aim to prevent the presence of these undesirable species in the food chain.