Seafood Matrices Research Articles

An Optimized Method for the Simultaneous Analysis of 16 Lipophilic Marine Toxins Using LC–MS/MS with Solid-Phase Extraction Cleanup in Seafood Matrices

An Optimized Method for the Simultaneous Analysis of 16 Lipophilic Marine Toxins Using LC–MS/MS with Solid-Phase Extraction Cleanup in Seafood Matrices

A Critical Analysis of the Opportunities and Challenges of Phage Application in Seafood Quality Control.

Seafood is an important source of food and protein for humans. However, it is highly susceptible to microbial contamination, which has become a major challenge for the seafood processing industry. Bacteriophages are widely distributed in the environment and have been successfully used as biocontrol agents against pathogenic microorganisms in certain food processing applications. However, due to the influence of environmental factors and seafood matrices, using bacteriophages for commercial-scale biocontrol strategies still faces some challenges. This article briefly introduces the current processes used for the production and purification of bacteriophages, lists the latest findings on the application of phage-based biocontrol in seafood, summarizes the challenges faced at the current stage, and provides corresponding strategies for solving these issues.

Green Chemistry to Valorize Seafood Side Streams: An Ecofriendly Roadmap toward Sustainability.

A major challenge facing sustainable seafood production is the voluminous amounts of nutrient-rich seafood side streams consisting of by-catch, processing discards, and process effluents. There is a lack of a comprehensive model for optimal valorization of the side streams. Upcoming green chemistry-based processing has the potential to recover diverse valuable compounds from seafood side streams in an ecofriendly manner. Microbial and enzymatic bioconversions form major green processes capable of releasing biomolecules from seafood matrices under mild conditions. Novel green solvents, because of their low toxicity and recyclable nature, can extract bioactive compounds. Nonthermal technologies such as ultrasound, supercritical fluid, and membrane filtration can complement green extractions. The extracted proteins, peptides, polyunsaturated fatty acids, chitin, chitosan, and others function as nutraceuticals, food supplements, additives, etc. Green processing can address environmental, economic, and technological challenges of valorization of seafood side streams, thereby supporting sustainable seafood production. Green processing can also encourage bioenergy production. Multiple green processes, integrated in a marine biorefinery, can optimize valorization on a zero-waste trade-off, for a circular blue economy. A green chemistry-based valorization framework has the potential to meet the Sustainable Development Goals (SDGs) of the United Nations.

Determination of Domoic Acid in Seafood Matrices using HPLC-UV with Solid Phase Extraction Cleanup

Determination of Domoic Acid in Seafood Matrices using HPLC-UV with Solid Phase Extraction Cleanup

Optimization of a method designed to extract and characterize microplastics in different packaged fish products

The study of the presence of microplastics (MPs) in seafood products is usually carried out by analyzing the gastrointestinal tract. However, this part is mostly removed during food processing. Moreover, few studies have produced results from processed fishery products. The primary objective of this study was to optimize methods for evaluating the presence of MPs in the edible portions of several products available in supermarkets. Seven seafood products, including smoked, canned, marinated, and those stored in polystyrene trays, were selected for analysis, with saithe fillets (Pollachius virens) and canned tuna (Thunnus alalunga) among them. To achieve optimal digestion of the samples, a 10% KOH solution at 40 °C was used. For the fattiest samples, such as marinated anchovies, hydrophobic filters were employed. Additionally, bleach was utilized to lighten dark filters obtained, ensuring readability under the microscope. These parameters were defined to obtain suitable methods for each product. The average concentrations in different seafood matrices ranged from 0.05 ± 0.04 to 0.33 ± 0.08 MP/g for saithe fillets and canned tuna, respectively. After extrapolation of these values to all the particles observed on each filter, the concentrations increased from 0.06 ± 0.05 MP/g to 2.17 ± 0.33 MP/g. Particles identification across all samples, using a microscope coupled with Fournier transform infrared spectroscopy (μ-FTIR), confirmed the presence of MPs, including PET, PP, and EVA.

Quantification of insecticides in commercial seafood sold in East Asian markets: risk assessment for consumers

The extraction of 21 insecticides and 5 metabolites was performed using an optimized and validated QuEChERS protocol that was further used for the quantification (GC–MS/MS) in several seafood matrices (crustaceans, bivalves, and fish-mudskippers). Seven species, acquired from Hong Kong and Macao wet markets (a region so far poorly monitored), were selected based on their commercial importance in the Indo-Pacific region, market abundance, and affordable price. Among them, mussels from Hong Kong, together with mudskippers from Macao, presented the highest insecticide concentrations (median values of 30.33 and 23.90 ng/g WW, respectively). Residual levels of fenobucarb, DDTs, HCHs, and heptachlors were above the established threshold (10 ng/g WW) for human consumption according to the European and Chinese legislations: for example, in fish-mudskippers, DDTs, fenobucarb, and heptachlors (5-, 20- and tenfold, respectively), and in bivalves, HCHs (fourfold) had higher levels than the threshold. Risk assessment revealed potential human health effects (e.g., neurotoxicity), especially through fish and bivalve consumption (non-carcinogenic risk; ΣHQLT > 1), and a potential concern of lifetime cancer risk development through the consumption of fish, bivalves, and crustaceans collected from these markets (carcinogenic risk; ΣTCR > 10–4). Since these results indicate polluted regions, where the seafood is collected/produced, a strict monitoring framework should be implemented in those areas to improve food quality and safety of seafood products.Graphical

Functional proteins through green refining of seafood side streams.

Scarcity of nutritive protein is a major global problem, the severity of which is bound to increase with the rising population. The situation demands finding additional sources of proteins that can be both safe as well as acceptable to the consumer. Food waste, particularly from seafood is a plausible feedstock of proteins in this respect. Fishing operations result in appreciable amounts of bycatch having poor food value. In addition, commercial processing results in 50 to 60% of seafood as discards, which consist of shell, head, fileting frames, bones, viscera, fin, skin, roe, and others. Furthermore, voluminous amounts of protein-rich effluents are released during commercial seafood processing. While meat from the bycatch can be raw material for proteinous edible products, proteins from the process discards and effluents can be recovered through biorefining employing upcoming, environmental-friendly, low-cost green processes. Microbial or enzyme treatments release proteins bound to the seafood matrices. Physico-chemical processes such as ultrasound, pulse electric field, high hydrostatic pressure, green solvent extractions and others are available to recover proteins from the by-products. Cultivation of photosynthetic microalgae in nutrient media consisting of seafood side streams generates algal cell mass, a rich source of functional proteins. A zero-waste marine bio-refinery approach can help almost total recovery of proteins and other ingredients from the seafood side streams. The recovered proteins can have high nutritive value and valuable applications as nutraceuticals and food additives.

On-chip immunomagnetic separation of allergens from myofibrillar proteins of seafoods for rapid allergy tests.

The main objective of the current study is to establish an on-chip strategy to analyze the allergens existing in the myofibrillar proteins of seafood matrices. On-chip immunomagnetic separation (IMS) of allergens derived from large yellow croaker (Larimichthys crocea), swimming crab (Portunus trituberculatus) and black tiger shrimp (Penaeus monodon), as model seafoods, was studied. The analysis of the tryptic digests of on-chip captured proteins, combined with protein database searches, was performed for the identification of allergens by MALDI-TOF MS with a 0% false-positive rate. By using the sera of ten seafood-allergic patients, six allergens were identified from the myofibrillar proteins of the large yellow croaker. The present protocol was successfully validated by ELISA and can be applied in blood tests for food allergies with a single drop of blood. The current protocol is presumed to be applied in the identification of potential allergens in other organisms.

Combination of Direct Viable Count and Fluorescent In Situ Hybridization (DVC-FISH) as a Potential Method for Identifying Viable Vibrioparahaemolyticus in Oysters and Mussels.

Vibrio parahaemolyticus is a human food-borne pathogen with the ability to enter the food chain. It is able to acquire a viable, non-cultivable state (VBNC), which is not detected by traditional methods. The combination of the direct viable count method and a fluorescent in situ hybridization technique (DVC-FISH) makes it possible to detect microorganisms that can present VBNC forms in complex samples The optimization of the in vitro DVC-FISH technique for V. parahaemolyticus was carried out. The selected antibiotic was ciprofloxacin at a concentration of 0.75 μg/mL with an incubation time in DVC broth of 5 h. The DVC-FISH technique and the traditional plate culture were applied to detect and quantify the viable cells of the affected pathogen in artificially contaminated food matrices at different temperatures. The results obtained showed that low temperatures produced an important logarithmic decrease of V. parahaemolyticus, while at 22 °C, it proliferated rapidly. The DVC-FISH technique proved to be a useful tool for the detection and quantification of V. parahaemolyticus in the two seafood matrices of oysters and mussels. This is the first study in which this technique has been developed to detect viable cells for this microorganism.

Enhanced Cytotoxicity of Cadmium by a Sulfated Polysaccharide from Abalone.

Consumption of seafood is a common route of cadmium ion (Cd2+) exposure to consumers. The seafood matrices may alter the toxicity profile of Cd2+ due to the interaction between Cd2+ and biomacromolecules in seafood. In this study, enhanced cytotoxicity of Cd2+ was found in the presence of an abalone gonad sulfated polysaccharide (AGSP) and the mechanism was investigated at a metabolic level. The formation of the AGSP-Cd2+ complex was demonstrated by isothermal titration calorimetry. The level of reactive oxygen species (ROS) increased and mitochondrial membrane potential reduced upon exposure to the AGSP-Cd2+ complex as compared with those of Cd2+ exposure. The decreased cell viability after incubation with the AGSP-Cd2+ complex also suggested enhanced Cd2+ toxicity induced by AGSP. The metabolomics and lipidomics analysis revealed that, compared with the Cd2+ group, the AGSP-Cd2+ downregulated the phospholipid metabolism and resulted in more serious damage in the cellular membrane. The lipid metabolism disorder, in turn, amplified the generation of ROS, leading to a decrease in cell viability. These results provided new evidence of the enhanced Cd2+ toxicity upon interaction with seafood polysaccharides, and much attention should be paid to the effect of food ingredients on heavy metal ion toxicity.

A novel method based on infrared spectroscopic inception-resnet networks for the detection of the major fish allergen parvalbumin

We have developed a novel approach that involves inception-resnet network (IRN) modeling based on infrared spectroscopy (IR) for rapid and specific detection of the fish allergen parvalbumin. SDS-PAGE and ELISA were used to validate the new method. Through training and learning with parvalbumin IR spectra from 16 fish species, IRN, support vector machine (SVM), and random forest (RF) models were successfully established and compared. The IRN model extracted highly representative features from the IR spectra, leading to high accuracy in recognizing parvalbumin (up to 97.3%) in a variety of seafood matrices. The proposed infrared spectroscopic IRN (IR-IRN) method was rapid (~20 min, cf. ELISA ~4 h) and required minimal expert knowledge for application. Thus, it could be extended for large-scale field screening and identification of parvalbumin or other potential allergens in complex food matrices.

Survival of Methicillin-Resistant Staphylococcus aureus in Fish and Shrimp under Different Storage Conditions

Foods that are extensively handled during preparation and stored without refrigeration are often associated with staphylococcal food poisoning. This problem is more confounding when contaminating strains belong to the methicillin-resistant Staphylococcus aureus (MRSA) group. In this study, we investigated the survivability of MRSA in two seafood matrices under different storage conditions. MRSA was inoculated at 6 and 3 log CFU/g into all sample groups of peeled shrimp (Parapeneopsis stylifera) stored at -20°C, Bombay duck fish (Harpadon nehereus) stored in ice, and dried Bombay duck fish stored at 30 ± 2°C. The populations of MRSA in frozen peeled shrimp inoculated with MRSA at 6 log CFU/g were reduced by 1.52 log CFU/g, whereas in samples inoculated with 3 log CFU/g levels remained stable after 60 days of storage. In fresh Bombay duck fish inoculated with 6 log CFU/g and stored in ice for 18 days, MRSA levels decreased by 2.75 log CFU/g. In contrast, in fresh fish inoculated with 3 log CFU/g the total viable count increased by 3.02 log CFU/g over 16 days of ice storage. In dried fish stored at 30 ± 2°C, MRSA levels declined by 3.27 log CFU/g in samples inoculated with 6 log CFU/g and by 0.91 log CFU/g in samples inoculated with 3 log CFU/g. These results suggest that the survival of MRSA depends on the temperature of storage and the inoculum level. In our study, MRSA survival was higher when inoculated at 3 log CFU/g regardless of the seafood matrix and storage temperature.

Natural products with preservative properties for enhancing the microbiological safety and extending the shelf-life of seafood: A review.

Seafood is highly perishable, presenting a rapid loss of its quality soon after capture. Temperature is the critical parameter that impacts on seafood shelf-life reduction, allowing the growth of foodborne pathogens and spoilage microorganisms. In recent years, the search by additional methods of preserving seafood has increased, able to ensure quality and safety. Several natural preservatives have highlighted and gained considerable attention from the scientific community, consumers, industry, and health sectors as a method with broad action antimicrobial and generally economical. Natural preservatives, from different sources, have been widely studied, such as chitosan from animal sources, essential oils, and plant extracts from a plant source, lactic acid bacteria, and bacteriocins from microbiological sources and organic acid from different sources, all with great potential for use in seafood systems. This review focuses on the natural preservatives studied in seafood matrices, their forms of application, concentrations usually employed, their mechanisms of action, factors that interfere in their use and the synergistic effect of the interactions among the natural preservatives, with a focus for maintenance of quality and ensure of food safety.

The Fate of Bacteria in Human Digestive Fluids: A New Perspective Into the Pathogenesis of Vibrio parahaemolyticus.

Vibrio parahaemolyticus causes the most seafood-attributed gastroenteritis outbreaks worldwide and studies on its pathogenesis during passage through the human digestive fluids are limited. An in vitro continuous model system mimicking passage through saliva, gastric and intestinal fluid was used to study the survival, morphology and virulence-related gene expression of a total of sixty pathogenic, and non-pathogenic V. parahaemolyticus strains. The changes to these three characteristics for the sixty V. parahaemolyticus strains were minimal on passage through the saliva fluid. No V. parahaemolyticus strains survived passage through gastric fluid with low pH values (2.0 and 3.0) and the cells, examined microscopically, were severely damaged. However, when the pH of gastric fluid increased to 4.0, the bacterial survival rate was 54.70 ± 1.11%, and the survival rate of pathogenic strains was higher when compared to non-pathogenic strains. Even though the bactericidal effect of intestinal fluid was lower than gastric fluid, virulence-related gene expression was enhanced in the intestinal fluid. Seafood matrices can significantly raise the pH level of gastric fluid and thus aid the survival of V. parahaemolyticus through passage from human gastric acid and progression of pathogenesis in the intestinal fluid. We confirmed these phenomena in the in vitro continuous digestion model.

Fast and environmental-friendly methods for the determination of polybrominated diphenyl ethers and their metabolites in fish tissues and feed

Environmental-friendly, cost-effective and fast methods were developed and validated for the analysis of seven PolyBrominated Diphenyl Ethers (PBDEs) and eight methoxylated PBDEs (MeO-PBDEs) in three distinct seafood matrices (muscle, liver and plasma) and feed using a Quick, Easy, Cheap, Efficient, Rugged and Safe (QuEChERS) extraction approach for solid samples and a Dispersive Liquid-Liquid Microextraction method (DLLME) for plasma. Instrumental analyses were performed with gas chromatography coupled to triple quadrupole mass spectrometry using electron impact source (GC-EI-MS/MS) and negative ion chemical ionization (GC-NICI-MS) to assess BDE-209. Statistical validation showed recoveries for all target substances near 100% with average Relative Standard Deviation (RSD) lower than 9% and recovery standards higher than 65% (average RSD below 20%). Average calculated Method Detection Limits (MDLs) were lower than 65 pg·g−1 wet weight (WW) for muscle, 5.35 ng·g−1 WW for liver, 4.50 ng·g−1 WW for feed, and 0.60 ng·mL−1 for plasma samples. Quality assurance and quality control practices were comprehensively described. Methods scored high in an analytical Eco-scale, thus being classified as “an excellent green analysis”. Finally, real seafood samples collected in local markets and local fishermen were analyzed. Positive samples presented both PBDEs and MeO-PBDEs in safe amounts (0.28–125.80 ng·g−1 WW) for human consumption.

Determination of a major allergen in fish samples by simple and effective label-free capillary electrophoretic analysis after background suppression in ion-exchange chromatography

A simple analytical method using ion-exchange chromatography (IEC) coupled with capillary zone electrophoresis (CZE) was newly developed for detection and quantification of parvalbumin (PV), a major allergen, in fish. The procedure of IEC for simple enrichment of PV crude extract was optimized. By using 25 mmol/L borate-borax buffer with 15 kV separation voltage at pH 9.2 for CZE separation, the migration time, separation efficiency and electrophoretic resolution greatly improved. Under the optimal conditions, PV was determined in 2.8 min, with the limit of detection (LOD) at 0.71 μg/mL (S/N = 3) and the recoveries at 89.6%–104.7%. We also found that only when the concentration of PV was above LOD reported here was the protein capable of stimulating human mast cell degranulation, indicating the biological significance of the LOD. Finally, the use of this method to analyze fish samples with simple sample preparation highlights the applicability for detection of allergens in seafood matrices.

Development of a colony hybridization method for the enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in shellfish

Vibrio parahaemolyticus is a marine microorganism, recognized as cause of gastroenteritis outbreaks associated with seafood consumption.In this study the development and the in-house validation of a colony hybridization method for the enumeration of total and potentially pathogenic V. parahaemolyticus is reported. The method included a set of three controls (process, hybridization and detection control) for the full monitoring of the analytical procedure. Four digoxigenin-labeled probes were designed for pathogenic strains enumeration (tdh1, tdh2, trh1 and trh2 probes) and one for total V. parahaemolyticus count (toxR probe). Probes were tested on a panel of 70 reference strains and 356 environmental, food and clinical isolates, determining the inclusivity (tdh: 96.7%, trh: 97.8%, toxR: 99.4%) and the exclusivity (100% for all probes). Accuracy and linearity of the enumeration were evaluated on pure and mixed cultures: slopes of the regression lines ranged from 0.957 to 1.058 depending on the target gene and R2 was greater than or equal to 0.989 for all reactions. Evaluation was also carried on using four experimentally contaminated seafood matrices (shellfish, finfish, crustaceans and cephalopods) and the slopes of the curves varied from 0.895 (finfish) to 0.987 (cephalopods) for the counts of potentially pathogenic V. parahaemolyticus (R2≥0.965) and from 0.965 to 1.073 for total V. parahaemolyticus enumeration (R2≥0.981). Validation was performed on 104 naturally contaminated shellfish samples, analyzed in parallel by colony hybridization, ISO/TS 21872-1 and MPN enumeration. Colony hybridization and ISO method showed a relative accuracy of 86.7%, and a statistically significant correlation was present between colony hybridization enumeration and MPN results (r=0.744, p<0.001).The proposed colony hybridization can be a suitable alternative method for the enumeration of total and potentially pathogenic V. parahaemolyticus in seafood.

Determination of Polycyclic Aromatic Hydrocarbons in Seafood

The aim of this project is to determine polycyclic aromatic hydrocarbons (PAHs) in several seafood matrices, i.e., fish, and mussel, by the use of comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry (GCXGC-MS). The procedure involves a simple extraction of PAHs, a filtration and concentration step followed by GCXGC-MS analysis. PAHs were determined in fish tissue samples by GCXGC-MS and by a standard high-pressure liquid chromatography fluorescence detector method. A Bland–Altman analysis of the two methods was performed. It indicated equivalence between the two methods. Analysis of mussel tissue (National Institute of Standards and Technology (NIST)-SRM 2974a) was performed. The results were in line with the official results of NIST.

Determination of Polycyclic Aromatic Hydrocarbons in Edible Seafood by QuEChERS‐Based Extraction and Gas Chromatography‐Tandem Mass Spectrometry

A high-throughput analysis of polycyclic aromatic hydrocarbons (PAHs) in edible seafood using QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) based extraction and gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed and validated in 4 seafood matrices--crab, finfish, oyster, and shrimp. The extraction employs QuEChERS with dispersive solid phase extraction (dSPE) using cleanup sorbent that includes C18. The acetonitrile extract is partitioned into hexane and analyzed by GC-MS/MS. The procedure was validated by spike recovery experiments of 15 parent and 5 substituted PAHs at 5ppb and 25 ppb levels in the 4 matrices. Recoveries were 71%-130% with RSDs less than 14%. The limit of quantitation (LOQ) for benzo[a]pyrene are 0.4-2.5 ppb depending on matrix. For all 15 parent PAHs the LOQs are less than 10% of the Levels of Concern established by the U.S. Food and Drug Administration. Good recoveries were also achieved in aged spikes at 5 ppb level in oyster and shrimp. Method accuracy was further evaluated by analyzing NIST Standard Reference Material 1974b. Ten of 14 incurred PAHs corresponding to the parent compounds in the present study fell within the control limits established by the National Oceanic and Atmospheric Administration. Using the present method approximately 20 samples every 24 h can be analyzed with confirmation. The presented analytical method could be used for seafood safety assessments to examine petroleum contaminant levels in edible seafood after an oil spill. The method is fast and sensitive.

Palytoxin in seafood by liquid chromatography tandem mass spectrometry: investigation of extraction efficiency and matrix effect

Blooms of Ostreopsis spp. have been recently reported along the Mediterranean coasts of Spain, France, Italy, and Greece posing serious risks to human health. Occurrence of Ostreopsis spp. may result in palytoxin contamination of seafood and, in order to prevent sanitary risks, the need exists to develop efficient extraction procedures to be coupled to rapid and sensitive monitoring methods of palytoxin-like compounds in seafood. In the present study, the best conditions for both extraction of palytoxin from seafood and palytoxin quantification by using liquid chromatography tandem mass spectrometry (LC-MS/MS) were investigated. Three seafood matrices (mussels, sea-urchins, and anchovies) were selected and five different extraction systems were tested, namely: the official protocol for extraction of lipophilic toxins and various aqueous methanol or acetonitrile solutions (MeOH/H(2)O 1:1, MeOH/H(2)O 8:2, MeCN/H(2)O 8:2 and MeOH 100%). Extraction with MeOH/H(2)O 8:2 provided the best results in terms of accuracy and matrix interference on LC-MS/MS detection of palytoxin. Accuracy and intra-day reproducibility (n = 3) were evaluated for all the selected matrices but only for mussels at three spiking concentration levels, including the provisional limit proposed by the Community Reference Laboratory for marine biotoxins (250 μg kg(-1)). Limits of quantitation of palytoxin in mussels, sea-urchins and anchovies tissues were calculated using matrix-matched standards; taking into account extraction efficiency of MeOH/H(2)O 8:2, they resulted to be 228, 343, and 500 μg kg(-1), respectively.