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- Standard methods for pollen researchPublication . Campos, Maria G.; Anjos, Ofélia; Chica, Manuel; Campoy, Pascual; Nozkova, Janka; Almaraz-Abarca, Norma; Barreto, Lidia M.R.C.; Nordi, João Carlos; Estevinho, Leticia M.; Pascoal, Ananias; Paula, Vanessa B.; Choupina, Altino; Dias, L.G.; Tešić, Živoslav L. j.; Mosić, Mirjana D.; Kostić, Aleksandar Ž.; Pešić, Mirjana B.; Milojković-Opsenica, Dušanka M.; Sickel, Wiebke; Ankenbrand, Markus J.; Grimmer, Gudrun; Steffan-Dewenter, Ingolf; Keller, Alexander; Förster, Frank; Tananaki, Chrysoula H.; Liolios, Vasilios; Kanelis, Dimitrios; Rodopoulou, Maria-Anna; Thrasyvoulou, Andreas; Paulo, Luísa; Kast, Christina; Lucchetti, Matteo A.; Glauser, Gaëtan; Lokutova, Olena; Almeida-Muradian, Ligia Bicudo; Szczęsna, Teresa; Carreck, Norman L.“Bee pollen” is pollen collected from flowers by honey bees. It is used by the bees to nourish themselves, mainly by providing royal jelly and brood food, but it is also used for human nutrition. For the latter purpose, it is collected at the hive entrance as pellets that the bees bring to the hive. Bee pollen has diverse bioactivities, and thus has been used as a health food, and even as medication in some countries. In this paper, we provide standard methods for carrying out research on bee pollen. First, we introduce a method for the production and storage of bee pollen which assures quality of the product. Routine methods are then provided for the identification of the pollen’s floral sources, and determination of the more important quality criteria such as water content and content of proteins, carbohydrates, fatty acids, vitamins, alkaloids, phenolic and polyphenolic compounds. Finally, methods are described for the determination of some important bioactivities of bee pollen such as its antioxidant, anti-inflammatory, antimicrobial and antimutagenic properties. Métodos estándar Para la investigación del polen El "polen de abeja" es el polen recogido de las flores por las abejas melíferas. El polen de abeja es utilizado para nutrir a las propias abejas, principalmente para proporcionar jalea real y alimento para las crías, pero también se utiliza para la nutrición humana. Para este último fin, se recoge en la entrada de la colmena en forma de gránulos que las abejas llevan a la colmena. El polen de abeja tiene diversas bioactividades, por lo que se hautilizado como alimento para la salud, e incluso como medicamento en algunos países. En este artículo, proporcionamos métodos estándar para llevar a cabo investigaciones sobre el polen de abeja. En primer lugar, presentamos un método de producción y almacenamiento de polen de abeja que garantiza la calidad del producto. A continuación, se ofrecen métodos de rutina para la identificación de las fuentes florales del polen y la determinación de los criterios de calidad más importantes, como el contenido de agua y de proteínas, carbohidratos, ácidos grasos, vitaminas, alcaloides y compuestos fenólicos y polifenólicos. Por último, se describen métodos para la determinación de algunas bioactividades importantes del polen de abeja, como sus propiedades antioxidantes, antiinflamatorias, antimicrobianas y antimutagénicas.
- Effect of storage conditions on the quality of the bee pollenPublication . Estevinho, Leticia M.; Rocha, Joana; Pascoal, Ananias; Pereira, Ana Paula; Choupina, AltinoBee pollen (BP), is the result of the agglutination of flower pollens, made by worker honey bees, with nectar and salivary substances and collected at the hive entrance. The collection of AP is a relatively recent development, dependent primarily on the basic concept of scraping pollen of the bees' legs as they enter the hive. The objective of this work was to evaluate the effect of storage conditions in the physical-chemical and microbiological parameters of organic bee pollen.
- Effect of temperature on the bioactive properties of bee pollenPublication . Pascoal, Ananias; Rocha, Joana; Choupina, Altino; Pereira, Ana Paula; Santos, Luís; Estevinho, Leticia M.Bee pollen is flower pollen collected by the honey bee, Apis mellifera, for the purpose of feeding its larvae in the early stages of development. lt is recognized to be a valuable apitherapeutic product with potential for medical, health and nutritional applications. The objective of this work was to compares the effect of different storage conditions in the bioactive compounds and biological properties of bee pollen.
- Developments in the fermentation process and quality improvement strategies for mead productionPublication . Iglesias, Antonio; Pascoal, Ananias; Choupina, Altino; Carvalho, Carlos Alfredo Lopes de; Feás, Xesús; Estevinho, Leticia M.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.
- Review: Novel sources and functions of microbial lipases and their role in the infection mechanismsPublication . Pascoal, Ananias; Estevinho, Leticia M.; Martins, Ivone; Choupina, AltinoLipases belong to the family of serine hydrolases, which in turn include various esterase enzymes (E.C.3.1.1.1). They are involved in the cleavage of triacylglycerols to free fatty acids and glycerol in many important biological processes, as for instance routine metabolism of dietary triglycerides to cell signalling and inflammation. Lipases constitute a ubiquitous group of enzymes able to catalyse a number of different reactions, many of them of industrial interest. Particularly, microbial lipases exhibit a wide range of industrial applications, namely in pharmaceutical, food and detergents industry. The aim of this review is to summarize the recent achievements illustrating the importance and the versatility of microbial lipases, including their involvement in infection mechanisms.
- An overview of the bioactive compounds, therapeutic properties and toxic effects of apitoxinPublication . Pascoal, Ananias; Estevinho, Maria Manuela; Choupina, Altino; Sousa-Pimenta, Mário; Estevinho, Leticia M.Apitoxin, also known as bee venom (BV), is produced in two specialized abdominal glands of worker bees and is used as a defence weapon of bees’ colony (Choi et al., 2015; Zhang et al., 2016). In addition, this natural product has been used since the ancient times to relieve pain and to treat chronic inflammatory diseases such as rheumatoid arthritis, tendinitis, bursitis, skin conditions and even neurologic disorders (Moreno and Giralt, 2015). This natural product is a light-yellow liquid, characterized by a bitter taste, pungent smell and a pH ranging from 4.5 to 5.5 (Eze et al., 2016; Hossen et al., 2017). Some studies have described that 88% of BV is water, yet its specific composition varies depending on bee’ species and season (Moreno and Giralt, 2015). Indeed, BV has been reported to contain a complex of biologically active compounds among which enzymes (phospholipase A2 [PLA2], lysophospholipase, hyaluronidase, acid phosphomonoesterase; α-glucosidase, dipeptidyl peptidase IV and vitellogenin), peptides (melittin [MLT], apamin, mast cell degranulating [MCD], mastocytolytic peptide, scapin, adolapin, minimine; apidaecin; tertiapin; melittin F; cadiopep; procamine A, B, pamine, and proteaseinhibitors), biogenic amines (histamine, dopamine, noradrenaline, norepinephrine, neurotransmitters) and other compounds such as amino acids (γ-aminobutyric acid, α-amino acids), carbohydrates (glucose, fructose), pheromones (Iso-pentyl acetate, n-buttyl acetate, iso-pentanol, n-hexyl acetate, n-octyl acetate, 2-nonanol, n-decyl acetate, benzyl acetate, benzyl alcohol and (2)-11 –eicosen-1-ol) and minerals (P, Ca and Mg) (Nguyen et al., 2015; Tusiimire et al., 2015; Rady et al., 2017; Moga et al., 2018; Lee et al., 2018). Among these compounds, MLT is the main therapeutic ingredient of BV, representing between 40 and 60% of the dry weight (Abd-Elhakim et al., 2014).
- Effect of temperature on the bioactive properties of bee pollenPublication . Pascoal, Ananias; Rocha, Joana; Choupina, Altino; Pereira, Ana Paula; Santos, Luís; Estevinho, Leticia M.Bee pollen is flower pollen collected by the honey bee, Apis mellifera, for the purpose of feeding its larvae in the early stages of development. It is recognized to be a valuable apitherapeutic product with potential for medical, health and nutritional applications. The objective of this work was to compares the effect of different storage conditions in the bioactive compounds and biological properties of bee pollen. The amount in phenolics compounds determined by the method of Folin-Ciocalteu varied from 32.64 to 48.40 mg GAE/g, for the dehydrated and refrigerated samples, respectively. Significant differences were verified among the two conservation processes. the amount in total flavonoids determined by the method chloride of aluminum were not verified among significant differences in the samples (6.58 mg CAE/g) and (6.99 mg CAE/g).Concerning the amount of total flavonoids determined by the method of aluminum chloride were not significant differences between the dehydrated (6.58 mg of DEA / g) and refrigerated samples (CAE 6.99 mg / g). The antioxidant capacity of BP extracts was assessed through the scavenging effect on DPPH (2.2-diphenyl-1-picrylhydrazyl) and reducing power. The concentration-dependent antioxidative capacity was verified in DPPH and reducing power assays. Low values of EC50 on DPPH scavenging assay were obtained for fresh and dehydrated samples 0.74 and 1.16 mg/mL, respectively. For reducing power the values obtained for the refrigerated samples and dehydrated were similar. The high activity of refrigerated samples could be related with the conservation process. Regarding to phenolics compounds determined by HPLC method, were identified four (4) families, flavones being the most abundant one in the refrigerated and dehydrated samples. We also verified that the presence of pollen differentially affected the growth of bacterium Gram-positive (Staphylococcus xylosus, Staphylococcus epidermidis), Gram-negative (Shigella spp, Klebsiella pneumoniae) and yeasts (Cândida parapilosis, Cândida menbranifaciens, Cândida gllabrata) under study, depending this on the microorganism and the method of BP conservation.
- Transglutaminases: recent achievements and new sourcesPublication . Martins, Ivone; Matos, Mauro; Costa, Rodrigo Arthur Fonseca; Silva, Fátima H.; Pascoal, Ananias; Estevinho, Leticia M.; Choupina, AltinoTransglutaminases 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.