Papers by Ananias Pascoal
Analytical Biochemistry, Jun 1, 2009
In this study, we infer the phylogenetic relationships within commercial shrimp using sequence da... more In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA) Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.
Food Chemistry, 2008
A novel PCR-RFLP method was evaluated as a tool to assess the incidence of incorrect labelling of... more A novel PCR-RFLP method was evaluated as a tool to assess the incidence of incorrect labelling of prawns and shrimps in commercial food products. The whole method can be performed in less than 8 h in only one day of work. PCR amplification with primers 16Scru4/16Scru3, targeted to the amplification of a ca. 530 bp region of 16S rRNA and tRNA Val mitochondrial genes, was coupled to restriction analysis with AluI, TaqI or HinfI. Forty-one commercial food products were considered. The molecular method considered allowed the identification of up to 17 different prawn and shrimp species in all the processed products considered. Seven (28%) of the 25 food products declaring one or more species in their labels were incorrectly labelled. Authentication was successfully assessed in commercial peeled products subjected to industrial processing, in which none of the products displayed labelling at species level. Overall, incorrect labelling was detected in 10 (24.4%) of the 41 commercial products tested, while another 16 samples (39%) exhibited incomplete labelling. The molecular method evaluated in this study proved to be a rapid and easy-to-perform two-step analytical approach to achieve species identification of commercial whole specimens of frozen prawns and shrimps and in peeled processed products where such raw materials are included as added-value ingredients.
Animal Production Science, 2016
Electrophoresis, 2008
A simple PCR-RFLP method has been developed for the identification of 19 penaeid shrimp species o... more A simple PCR-RFLP method has been developed for the identification of 19 penaeid shrimp species of food interest belonging to the superfamily Penaeoidea. Preliminary amplification, sequencing and alignment of a 960 bp fragment of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial region allowed the design of 16Scru4/16Scru4 primers, constructed on well-conserved mitochondrial sequences of the penaeid shrimp species considered. Such primers afforded the amplification of an internal 515-535 bp region of the 16S rRNA/tRNA(Val) genes that, when subjected to cleavage with AluI, TaqI and HinfI, provided species-specific restriction patterns. Moreover, the proposed method also allowed the definition of different intraspecific restriction types between different populations of Litopenaeus vannamei, Farfantepenaeus notialis, Fenneropenaeus merguiensis, Metapenaeus sp., Melicertus latisulcatus and Pleoticus muelleri of different origins. The method described here was also successfully applied for ...
Microbiology for Surgical Infections, 2014
Food and Bioprocess Technology, 2008
This review emphasizes the importance of novel biopreservation strategies and their application t... more This review emphasizes the importance of novel biopreservation strategies and their application to ensure seafood quality and safety especially within the context of increasing demand for minimally processed aquatic food products. The paper addresses the major hazards linked to spoilage and pathogenic bacteria found in fresh and processed aquatic foods, mainly ready-to-eat seafood subjected to short-term storage, and the biological strategies that can be used to minimize their growth. This is followed by an overview of current knowledge about the inhibiting bacteriocin-producing lactic acid bacteria isolated from aquatic food products or that is being evaluated for ensuring safety on seafood and seafood products as well as the characteristics of their bacteriocins. The different strategies for the biopreservation of aquatic food products, such as protective cultures or spray drying, and their current and future applications for the preservation of seafood products are also explored. Finally, novel antimicrobial active and intelligent packaging strategies based on antimicrobials film allowing controlled release of bacteriocins to refrigerated aquatic food products are also discussed.
Molecules (Basel, Switzerland), 2014
Mead is a traditional alcoholic drink derived from the fermentation of diluted honey in the prese... more Mead is a traditional alcoholic drink derived from the fermentation of diluted honey in the presence of appropriate yeast. Its modern production, in general terms, involves the addition of nutrients to initial diluted honey, pasteurization, yeast inoculation, fermentation and removal of impurities. Undesirable events along the process have been reported; among them, we highlight: delayed or arrested fermentations, modified and unpleasant sensory and quality parameters of the final product. These problems have been linked to the inability of yeasts to accomplish their role in extreme growth conditions. Emphasis has also been placed on the long fermentation times required, ranging from weeks to months, particularly when traditional procedures are applied and when the honey concentration is low. A series of alterations to the must and technological changes have been proposed in order to optimize the mead production process. In this context, this review examines the evidence that aims t...
Food Chemistry, 2011
Polymerase chain reaction-based methodologies have been developed for the identification of three... more Polymerase chain reaction-based methodologies have been developed for the identification of three commercially-relevant penaeid shrimp species, these were: Litopenaeus vannamei, Penaeus monodon and Fenneropenaeus indicus in food products. Such three species represent more than 80% of the whole farmed shrimp production worldwide and may be fraudulently replaced by species exhibiting lower value such as Litopenaeus stylirostris, Penaeus semisulcatus and Fenneropenaeus merguiensis, respectively. For it, preliminary sequencing of a mitochondrial sequence of ca. 530 bp in the 16S rRNA/tRNA Val mitochondrial region was performed in nearly 20 penaeid shrimp species of commercial relevance. Careful analysis of such sequences allowed the design of primers PNVF/PNVR, which allowed the combined identification of P. monodon and L. vannamei, and PNIF/PNIR, which allowed the specific identification of F. indicus. In addition, P. monodon and L. vannamei could be easily differentiated by either restriction with TspE1 or by amplification with novel primers MPNF/MPNR, specific for P. monodon. The proposed specific methods improve current general identification methods of these species based on more general RFLP analyses. In addition, these methods can be easily completed in less than 8 h.
Food Chemistry, 2008
A novel PCR-RFLP method was evaluated as a tool to assess the incidence of incorrect labelling of... more A novel PCR-RFLP method was evaluated as a tool to assess the incidence of incorrect labelling of prawns and shrimps in commercial food products. The whole method can be performed in less than 8 h in only one day of work. PCR amplification with primers 16Scru4/16Scru3, targeted to the amplification of a ca. 530 bp region of 16S rRNA and tRNA Val mitochondrial genes, was coupled to restriction analysis with AluI, TaqI or HinfI. Forty-one commercial food products were considered. The molecular method considered allowed the identification of up to 17 different prawn and shrimp species in all the processed products considered. Seven (28%) of the 25 food products declaring one or more species in their labels were incorrectly labelled. Authentication was successfully assessed in commercial peeled products subjected to industrial processing, in which none of the products displayed labelling at species level. Overall, incorrect labelling was detected in 10 (24.4%) of the 41 commercial products tested, while another 16 samples (39%) exhibited incomplete labelling. The molecular method evaluated in this study proved to be a rapid and easy-to-perform two-step analytical approach to achieve species identification of commercial whole specimens of frozen prawns and shrimps and in peeled processed products where such raw materials are included as added-value ingredients.
Food and Chemical Toxicology, 2014
Bee pollen is considered, since memorable times, a good source of nourishing substances and energ... more Bee pollen is considered, since memorable times, a good source of nourishing substances and energy. The present study aimed to evaluate the biological activities of eight commercial bee pollens purchased from the market. The origin of sample A was not specified in the labeling; samples B, C, D and G were from Portugal and the remaining were from Spain. The sample E presented the highest value of phenolics (32.15 ± 2.12 mg/g) and the H the lowest (18.55 ± 095 mg/g). Sample C had the highest value of flavonoids (10.14 ± 1.57 mg/g) and sample H the lowest (3.92 ± 0.68 mg/g). All the samples exhibited antimicrobial activity, being Staphylococcus aureus the most sensitive and Candida glabrata the most resistant of the microorganisms studied. All the samples exhibited antimutagenic activity, even though some samples were more effective in decreasing the number of gene conversion colonies and mutant colonies. Regarding the antioxidant activity, assessed using two methods, the more effective was sample B. The anti-inflammatory activity, assessed using the hyaluronidase enzyme, was highest in samples B and D. Pearson's correlation coefficients between polyphenols, flavonoids, antioxidant activity and antimicrobial activity were computed. It was also performed a discriminant analysis.
European Food Research and Technology, 2005
The identification of beef in animal foods is a major concern not only for the prevention of comm... more The identification of beef in animal foods is a major concern not only for the prevention of commercial fraud, but also to avoid safety risks deriving from the presence of prohibited bovine material that might be harmful to both human and animal health. Here we report a novel set of bovine-specific primers, CYTbos1 (forward) and CYTbos2 (reverse), which allow the specific amplification of a 115 base pair fragment of the bovine cytochrome b gene (cytb) between nt 844 (mitochondrial site 15,590) and nt 958 (mitochondrial site 15,704), no cross-reaction being observed with DNA from another 12 frequent commercial meat species. The polymerase chain reaction product obtained is cleaved specifically by endonucleases ScaI and TspE1 to achieve further confirmation evidence. The sensitivity of the proposed method was 0.025%. The CYTbos primers successfully detected bovine DNA in meat samples processed for 20 min at 133 C/300 kPa or for 2 h at 121 C. CYTbos primers also detected bovine DNA in heat-processed commercial meat products exhibiting a complex nature, as well as in bovine specific risk materials. The proposed polymerase chain reaction method, aimed at detecting a small and specific fragment of the bovine mitochondrial DNA, may be especially useful for the direct identification of bovine DNA in foodstuffs subjected to severe heating under overpressure conditions.
European Food Research and Technology, 2004
Polymerase chain reaction (PCR) coupled to restriction-fragment length polymorphism analysis (RFL... more Polymerase chain reaction (PCR) coupled to restriction-fragment length polymorphism analysis (RFLP) was considered for exploring the incidence of incorrect labelling in food products containing one or more meat species. Universal primers CYT b1/CYT b2, which amplify a variable region of the mitochondrial cytochrome b of vertebrates, and endonucleases PalI, MboI, HinfI and AluI were used for this purpose. Fifty food products, nine of them raw or cured and the other 41 subjected to a variety of technological processes such as pre-cooking and freezing, cooking and smoking, dehydration or sterilisation, were investigated. Twenty of the 50 products declared mixtures of meat species on their labels. Fifteen (30%) of the 50 food samples investigated displayed an incorrect qualitative labelling. While this affected only one (11.1%) of the nine raw/cured products, 14 (34.2%) of the 41 products subjected to some type of heat-processing were not correctly labelled. The undeclared presence of turkey was the most frequent concern, since it was detected in seven food products. The complete absence of a declared species of high commercial value-such as beef or roe-deer-was observed in another four cases. The PCR-RFLP method used here proved to be a rapid and easy-to-perform two-step analytical approach to achieve qualitative meat species identification in raw and cooked food products containing one or more different species.
ELECTROPHORESIS, 2008
A novel PCR-RFLP method has been developed for the identification of six commercially relevant pe... more A novel PCR-RFLP method has been developed for the identification of six commercially relevant penaeid shrimp species in raw and processed food products. The method can be completed within 8 h. To implement the method, PCR amplification with the crustF/crustR primers, targeted to the amplification of a ca. 181 bp region of the cytochrome b (cytb) mitochondrial gene in penaeid shrimps, was coupled to restriction analysis with CviJI, DdeI and NlaIV. The method was also applied successfully to the identification of shrimp species in complex processed foods, including this type of shellfish as an added-value food ingredient. The small size of this molecular target facilitates amplification from fresh, frozen, or precooked samples, where DNA fragmentation may be relevant and fragment size critical. We also report the first cytb mitochondrial sequences described to date for the species Farfantepenaeus notialis, Parapenaeus longirostris and Pleoticus muelleri, and these nearly triplicate current knowledge of reference nucleotide sequences in this mitochondrial region for this group of species. The cytb mitochondrial gene may also be considered as a molecular marker for identification and phylogenetic purposes in penaeid shrimp species.
ELECTROPHORESIS, 2008
Mercury resistant (Hgr) bacteria were isolated from four terrestrial sites: three containing high... more Mercury resistant (Hgr) bacteria were isolated from four terrestrial sites: three containing high levels of mercury (sites T12, SE, and SO) and one uncontaminated site (SB). The frequencies of Hg' bacteria in the total cultivable populations were 0.05% (SB), 0.69%o (SO), 4.8% (SE), and 25% (T2). Between 35 and 100o of the isolates from the four sites contained DNA sequences homologous to a DNA probe from the mercury resistance (mer) operon of the TnSO1 Hg' determinant. The mer sequences of 10 TnSOl-homologous Hg' determinants from each site were amplified by the polymerase chain reaction, with primers designed to consensus sequences of the mer determinants of TnSOl, Tn2l, and pMJ100, and were classified on the basis of the size of the amplified product and the restriction fragment length polymorphism pattern. Two main groups of amplification product were identified. The first, represented by the T2 and SB isolates and one SE isolate, gave an amplification product indistinguishable in size from that amplified from TnSOl (-1,010 bp). The second group, represented by the SO isolates and the majority of the SE isolates, produced larger amplification products of 1,040 or 1,060 bp. Restriction fragment length polymorphism analysis revealed that each amplification product size group could be further subdivided into five subgroups. APPL. ENvIRON. MICROBIOL. on June 3, 2013 by guest http://aem.asm.org/ Downloaded from
Analytical Biochemistry, 2009
In this study, we infer the phylogenetic relationships within commercial shrimp using sequence da... more In this study, we infer the phylogenetic relationships within commercial shrimp using sequence data from a novel mitochondrial marker consisting of an approximately 530-bp region of the 16S ribosomal RNA (rRNA)/transfer RNA (tRNA) Val genes compared with two other mitochondrial genes: 16S rRNA and cytochrome c oxidase I (COI). All three mitochondrial markers were considerably AT rich, exhibiting values up to 78.2% for the species Penaeus monodon in the 16S rRNA/tRNA Val genes, notably higher than the average among other Malacostracan mitochondrial genomes. Unlike the 16S rRNA and COI genes, the 16S rRNA/tRNA Val marker evidenced that Parapenaeus is more closely related to Metapenaeus than to Solenocera, a result that seems to be more in agreement with the taxonomic status of these genera. To our knowledge, our study using the 16S rRNA/tRNA Val gene as a marker for phylogenetic analysis offers the first genetic evidence to confirm that Pleoticus muelleri and Solenocera agassizi constitute a separate group and that they are more related to each other than to genera belonging to the family Penaeidae. The 16S rRNA/tRNA Val region was also found to contain more variable sites (56%) than the other two regions studied (33.4% for the 16S rRNA region and 42.7% for the COI region). The presence of more variable sites in the 16S rRNA/tRNA Val marker allowed the interspecific differentiation of all 19 species examined. This is especially useful at the commercial level for the identification of a large number of shrimp species, particularly when the lack of morphological characteristics prevents their differentiation.
Analytical Biochemistry, 2012
Genomic and proteomic techniques for species identification of meat and seafood products are bein... more Genomic and proteomic techniques for species identification of meat and seafood products are being widely used. In this study, a genomic approach was used to differentiate Pandalus borealis (the Northern shrimp), which belongs to the superfamily Pandaloidea, from 30 crustaceans consisting of 19 commercially relevant prawns/shrimps species that belong to the superfamily Penaeoidea, which include the families Penaeidae and Solenoceridae, and 11 other crustacean species, including prawns, shrimps, lobsters, and crabs. For this purpose, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was designed based on the amplification of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial regions using the primers 16S-CruF and 16S-CruR. The 966-bp PCR products were produced and cleaved with the restriction enzymes AluI, TaqI, and HinfI, which provided species-specific restriction patterns. In addition, a proteomic approach, based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization-ion trap (ESI-IT) mass spectrometry, was used to identify and characterize new P. borealis-specific peptides that could be useful as potential markers of this species in protein-based detection methods. To our knowledge, this is the first time a molecular method has been successfully applied to identify a wide range of prawn and shrimp species, including P. borealis, for either whole individuals or processed products. However, validation of the methods proposed here is required by applying them to a larger sample of individuals from different populations and geographic origins in order to avoid mainly false-negative results.
Applied Microbiology and Biotechnology, 2014
Transglutaminases are a family of enzymes (EC 2.3.2.13), widely distributed in various organs, ti... more Transglutaminases are a family of enzymes (EC 2.3.2.13), widely distributed in various organs, tissues, and body fluids, that catalyze the formation of a covalent bond between a free amine group and the γ-carboxamide group of protein or peptide-bound glutamine. Besides forming these bonds, that exhibit high resistance to proteolytic degradation, transglutaminases also form extensively cross-linked, generally insoluble, protein biopolymers that are indispensable for the organism to create barriers and stable structures. The extremely high cost of transglutaminase of animal origin has hampered its wider application and has initiated efforts to find an enzyme of microbial origin. Since the early 1990s, many microbial transglutaminase-producing strains have been found, and production processes have been optimized. This has resulted in a rapidly increasing number of applications of transglutaminase in the food sector. However, applications of microbial transglutaminase in other sectors have also been explored, but in a much lesser extent. Our group has identified a transglutaminase in the oomycete Phytophthora cinnamomi, which is able to induct defense responses and disease-like symptoms. In this mini-review, we report the achievements in this area in order to illustrate the importance and the versatility of transglutaminases.
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Papers by Ananias Pascoal