Developments in the fermentation process and quality improvement strategies for mead production.

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 to improve meads' quality and make the production process easier and more efficient, by clarifying the source of unexpected events, describing the implementation of different fermentative microorganisms and using new methodologies.

Transglutaminases: recent achievements and new sources.

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.

Biological activities of commercial bee pollens: antimicrobial, antimutagenic, antioxidant and anti-inflammatory.

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.

Food authentication of commercially-relevant shrimp and prawn species: from classical methods to Foodomics.

Although seafood species identification has traditionally relied on morphological analysis, sometimes this is difficult to apply for the differentiation among penaeid shrimps owing to their phenotypic similarities and to the frequent removal of external carapace during processing. The objective of this review is to provide an updated and extensive overview on the molecular methods for shrimp and prawn species authentication, in which several omics approaches based on protein and DNA analysis are described. DNA-based methods include the amplification by PCR of different genes, commonly the mitochondrial 16S ribosomal RNA and cytochrome oxidase I genes. A recently described method based on RFLP coupled to PCR turned out to be particularly interesting for species differentiation and origin identification. Protein analysis methods for the characterization and detection of species-specific peptides are also summarized, emphasizing some novel proteomics-based approaches, such as phyloproteomics, peptide fragmentation, and species-specific peptide detection by HPLC coupled to multiple reaction monitoring (MRM) MS, the latter representing the fastest method described to date for species authentication in food.

Species identification of the Northern shrimp (Pandalus borealis) by polymerase chain reaction-restriction fragment length polymorphism and proteomic analysis.

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.

Evaluation of a novel 16S rRNA/tRNAVal mitochondrial marker for the identification and phylogenetic analysis of shrimp species belonging to the superfamily Penaeoidea.

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.

Identification of shrimp species in raw and processed food products by means of a polymerase chain reaction-restriction fragment length polymorphism method targeted to cytochrome b mitochondrial sequences.

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.

A polymerase chain reaction-restriction fragment length polymorphism method based on the analysis of a 16S rRNA/tRNA(Val) mitochondrial region for species identification of commercial penaeid shrimps (Crustacea: Decapoda: Penaeoidea) of food interest.

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 the identification of penaeid shrimps in complex processed precooked foods, where this type of shellfish is used as an added-value food ingredient. Sequencing analysis provided new information about the genetic relationships among shrimps not only at the levels of species and genus, but also among different populations at intraspecific level. The 16S rRNA/tRNA(Val) fragment considered in this study seems to be accurate for shrimp species identification in raw and processed foodstuffs and for phylogenetic analysis among penaeid shrimp species.