Peeking into the Stingers: A Comprehensive SWATH-MS Study of the European Hornet Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae) Venom Sac Extracts.

This study aimed to investigate the venom sac extracts (VSEs) of the European hornet (EH) Vespa crabro (Linnaeus, 1758) (Hymenoptera: Vespidae), focusing on the differences between stinging females, gynes (G), and workers (W), at the protein level. Using a quantitative "Sequential Window Acquisition of all Theoretical Fragment Ion Mass Spectra" (SWATH-MS) analysis, we identified and quantified a total of 240 proteins. Notably, within the group, 45.8% (n = 110) showed significant differential expression between VSE-G and VSE-W. In this set, 57.3% (n = 63) were upregulated and 42.7% (n = 47) downregulated in the G. Additionally, the two-hundred quantified proteins from the class Insecta belong to sixteen different species, six of them to the Hymenoptera/Apidae lineage, comprising seven proteins with known potential allergenicity. Thus, phospholipase A1 (Vesp v 1), phospholipase A1 verutoxin 2b (VT-2b), hyaluronidase A (Vesp v 2A), hyaluronidase B (Vesp v 2B), and venom allergen 5 (Vesp v 5) were significantly downregulated in the G, and vitellogenin (Vesp v 6) was upregulated. Overall, 46% of the VSE proteins showed differential expression, with a majority being upregulated in G. Data are available via ProteomeXchange with identifier PXD047955. These findings shed light on the proteomic differences in VSE between EH castes, potentially contributing to our understanding of their behavior and offering insights for allergy research.

Proteomics of Vespa velutina nigrithorax Venom Sac Queens and Workers: A Quantitative SWATH-MS Analysis.

Health risks caused by stings from Vespa velutina nigrithorax (VV), also known as the yellow-legged Asian hornet, have become a public concern, but little is known about its venom composition. This study presents the proteome profile of the VV's venom sac (VS) based on Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). The study also performed proteomic quantitative analysis and examined the biological pathways and molecular functions of the proteins in the VS of VV gynes (i.e., future queens [SQ]) and workers [SW]. The total protein content per VS was significantly higher in the SW than in the SQ (274 ± 54 µg/sac vs. 175 ± 22 µg/sac; p = 0.02). We quantified a total of 228 proteins in the VS, belonging to 7 different classes: Insecta (n = 191); Amphibia and Reptilia (n = 20); Bacilli, γ-Proteobacteria and Pisoniviricetes (n = 12); and Arachnida (n = 5). Among the 228 identified proteins, 66 showed significant differential expression between SQ and SW. The potential allergens hyaluronidase A, venom antigen 5 and phospholipase A1 were significantly downregulated in the SQ venom.

What We Know about Sting-Related Deaths? Human Fatalities Caused by Hornet, Wasp and Bee Stings in Europe (1994-2016).

Epidemiology of Hymenopteran-related deaths in Europe, based on official registers from WHO Mortality Database (Cause Code of Death: X23), are presented. Over a 23-year period (1994-2016), a total of 1691 fatalities were recorded, mostly occurring in Western (42.8%) and Eastern (31.9%) Europe. The victims tended to concentrate in: Germany (n = 327; 1998-2015), France (n = 211; 2000-2014) and Romania (n = 149; 1999-2016). The majority of deaths occurred in males (78.1%) between 25-64 years (66.7%), and in an "unspecified place" (44.2%). The highest X23MR (mortality rate) were recorded in countries from Eastern Europe (0.35) followed by Western (0.28), Northern (0.23) and Southern Europe (0.2). The countries with the highest and lowest mean X23MR were Estonia (0.61), Austria (0.6) and Slovenia (0.55); and Ireland (0.05), United Kingdom (0.06) and the Netherlands (0.06), respectively. The X23 gender ratio (X23GR; male/female) of mortality varied from a minimum of 1.4 for Norway to a maximum of 20 for Slovenia. Country-by-country data show that the incidence of insect-sting mortality is low and more epidemiological data at the regional level is needed to improve our understanding of this incidence. With the expansion of non-native Hymenopteran species across Europe, allergists should be aware that their community's exposures are continually changing.

Human Fatalities Caused by Hornet, Wasp and Bee Stings in Spain: Epidemiology at State and Sub-State Level from 1999 to 2018.

Epidemiology of fatalities in Spain due to hornet, wasp, and bee stings (Cause Code of Death: X23) is described. Over a 20-year period (1999-2018), a total of 78 fatalities were recorded, mostly occurring in males (85.9%), of 65 years and older (52.6%), at "unspecified places" (67.9%), and in the months of July and August (50%). The X23 mortality rates (X23MR) expressed in terms of annual rates and per million inhabitants, varied from 0.02 to 0.19 (mean value ± standard deviation = 0.09 ± 0.05), placing Spain at low levels in comparison with other countries. A more detailed and specific breakdown of the distribution of the yearly deaths at the sub-state level and across communities reveals some striking features. They were more concentrated in the communities of Galicia (35.8%), Andalucía (21.7%), and Castilla y León (12.8%). X23MR were estimated in Galicia at 1.82, 1.10, and 2.22 in 2014, 2016, and 2018, respectively; and in Asturias at 1.88 and 0.97, in 2014 and 2017, respectively. The role of the invasive species Vespa velutina (VV) is examined. Due to its habits, abundance, and broader distribution, the risk that VV represents to human health is unmatched by other Hymenoptera native species.

Asian Hornet, Vespa velutina Lepeletier 1836 (Hym.: Vespidae), Venom Obtention Based on an Electric Stimulation Protocol.

The yellow-legged Asian hornet (Vespa velutina Lepeletier 1836 (Hymenoptera: Vespidae)) is naturally distributed in China, Southeast Asia, and India; however, recently it has been detected outside of its native area, confirmed as being established in South Korea, Europe, and Japan. Health risks and deaths caused by the invasive Vespa velutina stings have become a public health concern, being the most common cause of anaphylaxis due to hymenopterans in some European regions. This in turn has led to increased demand from medical practitioners and researchers for Vespa velutina venom for diagnostic and therapeutic purposes. In this study, a straightforward, quick, and inexpensive method for obtaining Vespa velutina venom by electric stimulation is described. The venom extracts were analyzed by nuclear magnetic resonance spectroscopy (1H-NMR). The availability of Vespa velutina venom will lead to improved diagnostic and therapeutic methods, mainly by venom immunotherapy (VIT), in patients allergic to this invasive species.

Extraction and Physicochemical Characterization of Chitin Derived from the Asian Hornet, Vespa velutina Lepeletier 1836 (Hym.: Vespidae).

Fifteen years ago, at least one multimated female yellow-legged Asian hornet (Vespa velutina Lepeletier 1836) arrived in France, which gave rise to a pan-European invasion. In this study, the isolation and characterization of chitin (CHI) that was obtained from Vespa velutina (CHIVV) is described. In addition, an easy procedure is carried out to capture the raw insect, selectively and with high rates of success. The chitin contents of dry VV was observed to be 11.7%. Fourier transform infrared spectroscopy (FTIR), solid-state NMR (ssNMR), elemental analysis (EA), scanning electron microscopy (SEM), and thermogravimetric analysis (TG) characterized the physicochemical properties of CHIVV. The obtained CHIVV is close to pure (43.47% C, 6.94% H, and 6.85% N), and full acetylated with a value of 95.44%. Additionally, lifetime and kinetic parameters such as activation E and the frequency factor A using model-free and model-fitting methods, were determined. For CHIVV the solid state mechanism that follows the thermodegradation is of type F2 (random nucleation around two nuclei). The invasive Asian hornet is a promising alternative source of CHI, based on certain factors, such as the current and probable continued abundance of the quantity and quality of the product obtained.

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.

Use of propolis in the sanitization of lettuce.

The present study aimed to determine the effectiveness of propolis in reducing the microbial load in ready-to-eat (RTE) and fresh whole head (FWH) lettuces (Lactuca sativa L.) type Batavia. Two sanitizing solutions were employed: sodium hypochlorite (SH) and propolis (PS), during 15 and 30 min. Tap water (TW) was used as a control. Regarding the mean reduction on aerobic mesophiles, psychrotrophic and fecal coliforms, the SH and PS treatments showed the same pattern of variation. In all cases, PS was slightly more effective in the microbiological reduction in comparison with commercial SH. Reductions between two and three log cycles were obtained with PS on aerobic mesophiles and psychrotrophic counts. The information obtained in the present study can be used to evaluate the potential use of propolis as product for sanitizing other vegetables and for developing other food preservation technologies, with impact on human health.

Novel microwave-assisted synthesis of the immunomodulator organotellurium compound ammonium trichloro(dioxoethylene-O,O')tellurate (AS101).

Ammonium trichloro[1,2-ethanediolato-O,O']-tellurate (AS101) is the most important synthetic Te compound from the standpoint of its biological activity. It is a potent immunomodulator with a variety of potential therapeutic applications and antitumoral action in several preclinical and clinical studies. An experimental design has been used to develop and optimize a novel microwave-assisted synthesis (MAOS) of the AS101. In comparison to the results observed in the literature, refluxing Te(IV) chloride and ethylene glycol in acetonitrile (Method A), or by refluxing Te(IV) chloride and ammonium chloride in ethylene glycol (Method B), it was found that the developed methods in the present work are an effective alternative, because although performance slightly decreases compared to conventional procedures (75% vs. 79% by Method A, and 45% vs. 51% by Method B), reaction times decreased from 4 h to 30 min and from 4 h to 10 min, by Methods A and B respectively. MAOS is proving to be of value in the rapid synthesis of compounds with new and improved biological activities, specially based on the benefit of its shorter reaction times.

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.

Effect of delayed icing on biogenic amines formation and bacterial contribution of iced common carp (Cyprinus carpio).

The variation of six biogenic amines (BAs) and total viable count (TVC) in common carp (Cyprinus carpio) stored in ice with 0, 4 and 8 h delay before icing was evaluated in a period of 4 days. Delayed icing led to significant (p < 0.05) increases in TVC throughout the period of storage and showed a good correlation with BAs content. The obtained data showed that putrescine and cadaverine were predominant in all samples and it was indicated that they could be proper indicators to determine the carp quality. Spermidine and spermine increased slightly toward the end of storage and the levels of dangerous BAs (histamine and tyramine) were under the limit over the period. As a result, it is indicated that delaying time affects on formation of BAs and the effect in samples with 8 h delay was significantly (p < 0.05) more than those with 0 and 4 h delay.

Triacylglyceride, antioxidant and antimicrobial features of virgin Camellia oleifera, C. reticulata and C. sasanqua Oils.

Virgin oils obtained from seeds of Camellia oleifera (CO), Camellia reticulata (CR) and Camellia sasanqua (CS) were studied for their triacylglyceride composition, antioxidant and antimicrobial activities. Levels of fatty acids determined by ¹H-nuclear magnetic resonance analysis were similar to those reported for olive oils (82.30%-84.47%; 5.69%-7.78%; 0.26%-0.41% and 8.04%-11.2%, for oleic, linoleic, linolenic and saturated acids, respectively). The CR oil showed the best antioxidant potential in the three in vitro models tested. With regard to EC50 values (µg/mL), the order in DPPH radical-scavenging was CR (33.48) < CO (35.20) < CS (54.87). Effectiveness in reducing power was CR (2.81) < CO (3.09) < CS (5.32). IC50 for LPO inhibition were 0.37, 0.52 and 0.75 µg/mL for CR, CO and CS, respectively. All the oils showed antimicrobial activity, and exhibited different selectivity and MICs for each microorganism tested (E. coli, B. cereus and C. albicans). B. cereus was the less sensitive species (MIC: 52.083 ± 18.042 for CO; 41.667 ± 18.042 for CR; 104.167 ± 36.084 for CS mg/mL) and the E. coli was the most sensitive to camellia oil's effect. The standard gentamicin presented higher MIC for E. coli (4.2) than the CR (MIC= 2.6) and CO (MIC = 3.9) oils.

Effect of Erica sp. honey against microorganisms of clinical importance: study of the factors underlying this biological activity.

This study aimed to determine the factors (phenolic compounds, flavonoids, sugars or H2O2) that contribute the most to the antimicrobial activity of heather honey samples against four yeasts and four bacteria with medical importance. To discard the effect of H2O2 in the antimicrobial activity, catalase was added. To evaluate the osmotic pressure's effect, artificial honey was also used. Phenolic compounds and flavonoids were determined and Pearson's correlation analysis was performed to assess whether these correlated with antimicrobial activity. The amount of phenolic compounds ranged from 630.89 ± 5.21 GAE kg-1 to 718.92 ± 4.41 GAE kg-1, while the flavonoids varied between 450.72 ± 5.67 CAE kg-1 and 673.98 ± 4.33 CAE kg-1. For the bacteria, the minimum inhibitory concentration (MIC) of the honey without catalase ranged from 1.01 ± 0.50% to 10.00 ± 4.72% and was between 2.00 ± 0.94% and 13.27 ± 5.23% for honey with catalase. Concerning the yeasts, the MICs was between 13.16 ± 4.08% and 20.00 ± 5.09% for honey without catalase and between 14.95 ± 4.16% and 25.67 ± 5.50% for honey with catalase. The elucidation of the antimicrobial factors and action mechanisms is essential for the correct use of honey in therapeutic applications.

Commercial bee pollen with different geographical origins: a comprehensive approach.

Since the primordial of humanity, pollen has been considered a good source of nutrients and energy. Its promising healing properties have also been referred to. The present study aimed to characterize, for the first time, eight commercial pollens from Portugal and Spain available on the market studying the legislation on labeling, pollinic origin, physicochemical and microbiological analyses and identification of yeasts. Eleven botanical families were found amongst the samples. The most abundant family and the most dominant pollen was Cistaceae. The moisture content, ash, a(w), pH, reducing sugars, carbohydrates, proteins, lipids and energy were analyzed and the specific parameters were within the specifications required by some countries with legislation regarding these parameters. Microbiologically commercial pollen showed acceptable safety for the commercial quality and hygiene. All samples showed negative results for toxigenic species. The microorganisms studied were aerobic mesophiles, yeasts and moulds, coliforms, Escherichia coli, Staphylococcus aureus, Salmonella and sulfite-reducing Clostridium. During the work, six yeasts species were isolated from pollen, with Rhodotorula mucilaginosa being the most abundant, as it was present in four samples.

Comprehensive study of honey with protected denomination of origin and contribution to the enhancement of legal specifications.

In this study the characterization of a total of 60 honey samples with Protected Denomination of Origin (PDO) collected over three harvests (2009-2011, inclusive), from the Northeast of Portugal was carried out based on the presence of pollen, physicochemical and microbiological characteristics. All samples were found to meet the European Legislation, but some didn't meet the requirements of the PDO specifications. Concerning the floral origin of honey, our results showed the prevalence of rosemary (Lavandula pedunculata) pollen. The microbiological quality of all the analyzed samples was satisfactory, since fecal coliforms, sulfite-reducing clostridia and Salmonella were absent, and molds and yeasts were detected in low counts. Significant differences between the results were studied using one-way analysis of variance (ANOVA), followed by Tukey's HSD test. The samples were submitted to discriminant function analysis, in order to determine which variables differentiate between two or more naturally occurring groups (Forward Stepwise Analysis). The variables selected were in this order: diastase activity, pH, reducing sugars, free acidity and HMF. The pollen spectrum has perfect discriminatory power. This is the first study in which a honey with PDO was tested, in order to assess its compliance with the PDO book of specifications.

Organic bee pollen: botanical origin, nutritional value, bioactive compounds, antioxidant activity and microbiological quality.

Organic bee pollen (BP, n = 22) harvested from the Douro International Natural Park (DINP, Portugal) was studied. Nine botanical families were found in the mixture of the samples. The water activity and pH ranged 0.21-0.37 and 4.3-5.2, respectively. The BP analyses averaged 67.7% carbohydrates, 21.8% crude protein, 5.2% crude fat and 2.9% ash. The energy ranged from 396.4 to 411.1 kcal/100 g. The principal fatty acid found was linolenic, followed by linoleic acid, palmitic acid and oleic acid. The phenolic and flavonoid contents varied from 12.9 to 19.8 mg of gallic acid equivalents/g of extract and from 4.5 to 7.1 mg of catechin equivalents/g of extract, respectively. The scavenger activity and ß-carotene bleaching assays values (EC50) were 3.0 ± 0.7 mg/mL and 4.6 mg/mL ± 0.9 mg/mL, respectively. E. coli, sulphite-reducing Clostridia, Salmonella and S. aureus were not found. Since there are studies indicating appreciable differences among BPs from different regions, the full characterization of BP from diverse origins still appears to be a sound research priority in order to obtain reliable data about this beehive product.

¹H-nuclear magnetic resonance analysis of the triacylglyceride composition of cold-pressed oil from Camellia japonica.

Camellia japonica (CJ) has oil-rich seeds, but the study of these oils has received little attention and has mainly focused only on their health properties. In the present work the relative composition of the fatty acid (FA) components of the triglycerides in cold-pressed oil from CJ is studied by ¹H-NMR. The results obtained were: 75.75%, 6.0%, 0.17% and 18.67%, for oleic, linoleic, linolenic and saturated FA respectively. Levels of C18 unsaturated FA found in CJ oil were similar to those reported for olive oils. We also checked the possibility of using ¹³C-NMR spectroscopy; however, the results confirmed the drawback of ¹³C over ¹H-NMR for the study of FA components of CJ triglycerides due to its low gyromagnetic ratio and its very low natural abundance.

Antimicrobial activity, phenolic profile and role in the inflammation of propolis.

Nowadays a great amount of information regarding chemical and biological aspects of bee products is available in the literature, but few data on its therapeutic uses are found. The aim of this study was to evaluate the phenolic profile, the in vitro antimicrobial activity and effect in the hyaluronidase enzyme (widely related with the inflammation process) of propolis harvested in Portugal. The efficacy of three extracts (hydro-alcoholic, methanolic and aqueous) was also compared. It was chosen the hydro-alcoholic extract, because this was the most effective for extracting phenolic compounds. The antimicrobial activity was accessed in Gram-positive and Gram-negative bacteria and yeasts, isolated from different biological fluids and the results were then compared with the obtained for reference microorganisms. The propolis from Bragança was the one that possessed the highest polyphenols' content. The sample from Beja showed the less significant inhibition of the hyaluronidase enzyme. Concerning the antimicrobial activity, Candida albicans was the most resistant and Staphylococcus aureus the most sensitive. The reference microorganisms were more sensitive than the ones isolated from biological fluids.

Organic honey from Trás-Os-Montes region (Portugal): chemical, palynological, microbiological and bioactive compounds characterization.

At the present time, the quality, integrity, sanitation and nutritional value of honeys receive attention on an international level due to the increasing content of chemicals in the aforementioned matrix. This work was conducted to evaluate the quality of 75 organic honey samples from the Trás-Os-Montes region (Portugal). Mean values obtained for physico-chemical parameters were: pH 3.7; 15.6% moisture; 0.26mS/cm electrical conductivity; 0.25% ash; 1.1mg/kg HMF; 15.3 Gothe diastase activity; 40.3meq/kg free acidity; 67.8% invert sugars and 2.7% apparent sucrose. All honey samples can be classified as monofloral Erica sp., as showed by pollen features. The amounts of phenols and flavonoids in the samples were also determined. In respect to sanitary quality (fecal coliforms) and safety (sulfite-reducing clostridia and Salmonella), all organic honey samples were negative. Furthermore, yeast and molds were detected in low counts, with mean values obtained of 5.5cfu/g and the value of total aerobic mesophiles obtained from honeys was established in 1.3×10(2)cfu/g±7.5×10(1)cfu/g. The levels of flavonoids had a stronger impact on both mesophiles (p=0.0004) and molds (p=0.0138) than the sucrose concentration (p=0.001 and 0.0278; respectively). The results reported in this study should be introduced in the organic honey label, and may help beekeepers, the industry, researchers and consumers better understand honey properties.