Stingless Bee Foraging Activity Related to Environmental Aspects.

The environment where bee colonies are inserted must provide the necessary resources for their survival. Given this, any biotic and abiotic changes in the environment can affect the development and survival of the colonies. We evaluated the foraging activity of Plebeia droryana (Friese), Scaptotrigona bipunctata (Lepetelier), and Melipona quadrifasciata (Lepetelier) in areas with different land uses and land cover. These areas were classified as predominantly (i) urbanized/forest (CDA-Cidade das Abelhas), (ii) agricultural (FER-Fazenda Experimental da Ressacada), and (iii) with dense vegetation (SFB-Sitio Florbela). We correlated the morphometric characteristics of the bees with the pollen load transported. Four colonies from each species were installed in the three areas. We recorded light, wind speed, humidity, and temperature and counted the foragers returning with nectar, pollen, and resin. Plebeia droryana and S. bipunctata collected more resin and nectar in with dense vegetation area compared to agricultural area. Scaptotrigona bipunctata collected more pollen in urbanized/forest area and with dense vegetation area, and M. quadrifasciata did not show differences in foraging activity between areas. Plebeia droryana and M. quadrifasciata showed moderate and strong correlations between morphometric characteristics and pollen load. SFB had higher luminosity and wind speed. CDA had higher average temperature. FER had higher humidity. The three species showed positive and negative correlations between temperature and light and foraging in the different areas. Smaller species showed a higher gathering of resources in the area predominantly covered by dense vegetation. The reduction of vegetation cover can affect the resource collection activity of stingless bees.

Antioxidant and antibacterial properties of ethanolic pot-pollen extract of Melipona beecheii and determination of the major components by GC-MS.

Melipona beecheii pot-pollen is a natural product that has barely been studied, unlike other hive products such as honey and propolis. Its application has been reported since ancient times in traditional Mayan medicine, and it is also a functional food with high nutritional value. In the present study, samples of ethanolic pot-pollen extracts from five locations in the Yucatán Peninsula were analyzed to determine their antibacterial and antioxidant properties. All the extracts showed activity against five medically important bacteria; Pseudomonas aeruginosa and Listeria monocytogenes were the most susceptible bacteria in all samples. The evaluated antioxidant activity was higher than that reported by other studies. Palmitic, linoleic, and linolenic fatty acids and their respective ethyl ethers were detected by Gas Chromatography-Mass Spectrometry (GC-MS) in all samples in different concentrations. Based on these results, pot-pollen extract from Mama, Yucatán exhibited the best biological activities (Minimum Inhibitory Concentrations (MICs) between 6 and 40 mg/mL, EC50 DPPH 28 µg/mL, EC50 RP 30 µg/mL), which could be related to a higher content of unsaturated fatty acids and their ethyl esters. The present study demonstrates that M. beecheii pot-pollen has therapeutic potential in addition to its benefits as a nutritional supplement.

Chemical characterisation, thermal analysis, and antibacterial activity of honeys from Caatinga stingless bees of Melipona spp.

In the present study, chemical characterisation, thermal analysis and antibacterial activity of honeys from Melipona spp. with occurrence in Caatinga biome of Brazil. The honeys presented pH from 4.07 to 4.14, density of 1.41 g/cm3 and °Brix value of 79.90. The thermogravimetry (TG) analysis presented six-seven events and differential thermal analysis (DTA) presented three-four endothermic peaks. HPLC fingerprint revealed a predominant presence of gallic acid and vanillin. Antioxidant activity evaluated using in vitro 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) radical scavenging with IC50 values in the range of 14.5404 to 15.2454 mg/mL. The honeys also showed antimicrobial activities against Staphylococcus aureus and Pseudomonas aeruginosa using a modified agar diffusion and microdilution method. The results of the present study demonstrate that the honey from stingless bees by Caatinga biome indicate polyphenol compounds, antioxidant activity and in vitro antimicrobial potential. The analytical methos permitted of fingerprint of samples.

Effect of conA-unbound proteins from Melipona beecheii honey on the formation of Pseudomonas aeruginosa ATCC 27853 biofilm.

Pseudomonas aeruginosa is an opportunistic bacterium that can form a biofilm with the ability to colonize different surfaces and for increasing resistance to antibiotics. An alternative to solve this problem may be the use of non-glucose/mannose glycosylated proteins from Melipona beecheii honey, which are capable of inhibiting the growth of this pathogen. In this work, the antibiofilm activity of the conA-unbound protein fraction (F1) from M. beecheii was evaluated. The crude protein extract (CPE) and the F1 fraction inhibited the P. aeruginosa biofilm growth above 80% at 4 and 1.3 µg/mL, respectively. These proteins affected the structure of the biofilm, as well as fleQ and fleR gene expressions involved in the formation and regulation of the P. aeruginosa biofilm. The results demonstrated that the F1 fraction proteins of M. beecheii honey inhibit and affect the formation of the P. aeruginosa biofilm.

Chemical Characterization and Biological Activities of Jandaíra Stingless Bee Products (Melipona subnitida, Ducke, 1911): A Brief Review.

Melipona subnitida (Ducke, 1911), a species of stingless bee, popularly known as Jandaíra, has a wide distribution in the Brazilian Northeast region, being an important pollinator of the Caatinga biome. This bee produces products such as honey, geopropolis, pollen (saburá) and wax that are traditionally used for therapeutic purposes and some studies report the biological properties, as well as its chemical composition. This review aimed to select, analyze and gather data published in the literature focusing on the chemical profile and bioactivities described for M. subnitida products. Data collection was carried out through the Capes Journal Portal platform, using the following databases: Web of Science, Scopus, and PubMed. Original articles published in English and Portuguese were included, with no time limitation. The chemical composition of M. subnitida products has been investigated through chromatographic analysis, demonstrating the presence of a variety of phenolic compounds, such as flavonoids and phenylpropanoids, among other classes of secondary metabolites. These products also have several biological activities, including antioxidant, healing, antinociceptive, anti-inflammatory, antidepressant, antidyslipidemic, antiobesity, antifungal, antibacterial and prebiotic. Among the biological activities reported, the antioxidant activity was the most investigated. These data show that products derived from the stingless bee M. subnitida have promising bioactive compounds. This review provides useful information about the bioactivities and chemical profile of Melipona subnitida bee products, and a direction for future research, which should focus on understanding the mechanisms of action associated with the already elucidated pharmacological activities, as well as the bioactive properties of the main isolate's constituents identified in the chemical composition of these products.

First Records of Heartbeats via ECG in a Stingless Bee, Melipona flavolineata (Apidae, Meliponini), during Contention Stress Using Isoflurane as an Anesthetic.

The hemodynamic activity of Melipona flavolineata workers was evaluated during restraint stress for a period of 30 min. The observed parameters were power variation in the elapsed time, and subsequently, six periods of one second were divided and called A, B, C, D, E and F; in each period, the electrocardiographic parameters were evaluated: spike frequency, amplitude, spike intervals and spike duration. The experiment was carried out with eight worker bees of M. flavolineata, for which electrodes of a nickel-chromium alloy were made. The bees were previously anesthetized with isoflurane and properly contained and fixed in a base for stereotaxis in which the electrode was implanted. All these procedures were performed inside a Faraday cage. The results showed power oscillations during the recording, with the highest energy level being between 300 and 600 s. Spike frequency, spike amplitude, interval between spikes and spike duration parameters underwent changes during the restraint stress period. Thus, the cardiac activity of M. flavolineata can be used as a biomarker and can be used to clarify physiological issues or alterations caused by toxic agents and indicate risk factors for these animals.

Impact of genus (Geotrigona, Melipona, Scaptotrigona) in the targeted 1H-NMR organic profile, and authenticity test by interphase emulsion of honey processed in cerumen pots by stingless bees in Ecuador.

The biodiversity of Ecuadorian stingless bees is almost 200 species. Traditional pot-honey harvest in Ecuador is mostly done from nests of the three genera selected here Geotrigona Moure, 1943, Melipona Illiger, 1806, and Scaptotrigona Moure, 1942. The 20 pot-honey samples collected from cerumen pots and three ethnic honeys "abeja de tierra", "bermejo", and "cushillomishki" were analyzed for qualitative and quantitative targeted 1H-NMR honey profiling, and for the Honey Authenticity Test by Interphase Emulsion (HATIE). Extensive data of targeted organic compounds (41 parameters) were identified, quantified, and described. The three honey types were compared by ANOVA. Amino acids, ethanol, hydroxymethylfurfural, aliphatic organic acids, sugars, and markers of botanical origin. The number of phases observed with the HATIE were one in Scaptotrigona and three in Geotrigona and Melipona honeys. Acetic acid (19.60 ± 1.45 g/kg) and lactic acid (24.30 ± 1.65 g/kg) were particularly high in Geotrigona honey (in contrast to 1.3 g/kg acetic acid and 1.6 g/kg lactic acid in Melipona and Scaptotrigona), and with the lowest fructose + glucose (18.39 ± 1.68) g/100g honey compared to Melipona (52.87 ± 1.75) and Scaptotrigona (52.17 ± 0.60). Three local honeys were tested using PCA (Principal Component Analysis), two were assigned with a correct declared bee origin, but "bermejo" was not a Melipona and grouped with the Scaptotrigona cluster. However after HCA (Hierarchical Cluster Analysis) the three honeys were positioned in the Melipona-Scaptotrigona cluster. This research supports targeted 1H-NMR-based profiling of pot-honey metabolomics approach for multi-parameter visualization of organic compounds, as well as descriptive and pertained multivariate statistics (HCA and PCA) to discriminate the stingless bee genus in a set of Geotrigona, Melipona and Scaptotrigona honey types. The NMR characterization of Ecuadorian honey produced by stingless bees emphasizes the need for regulatory norms. A final note on stingless bee markers in pot-honey metabolites which should be screened for those that may extract phylogenetic signals from nutritional traits of honey. Scaptotrigona vitorum honey revealed biosurfactant activity in the HATIE, originating a fingerprint Honey Biosurfactant Test (HBT) for the genus in this set of pot-honeys.

Temporal Changes in Gut Microbiota Composition and Pollen Diet Associated with Colony Weakness of a Stingless Bee.

Compared to honeybees and bumblebees, the effect of diet on the gut microbiome of Neotropical corbiculate bees such as Melipona spp. is largely unknown. These bees have been managed for centuries, but recently an annual disease is affecting M. quadrifasciata, an endangered species kept exclusively by management in Southern Brazil. Here we report the results of a longitudinal metabarcoding study involving the period of M. quadrifasciata colony weakness, designed to monitor the gut microbiota and diet changes preceding an outbreak. We found increasing amounts of bacteria associated to the gut of forager bees 2 months before the first symptoms have been recorded. Simultaneously, forager bees showed decreasing body weight. The accelerated growth of gut-associated bacteria was uneven among taxa, with Bifidobacteriaceae dominating, and Lactobacillaceae decreasing in relative abundance within the bacterial community. Dominant fungi such as Candida and Starmerella also decreased in numbers, and the stingless bee obligate symbiont Zygosaccharomyces showed the lowest relative abundance during the outbreak period. Such changes were associated with pronounced diet shifts, i.e., the rise of Eucalyptus spp. pollen amount in forager bees' guts. Furthermore, there was a negative correlation between the amount of Eucalyptus pollen in diets and the abundance of some bacterial taxa in the gut-associated microbiota. We conclude that diet and subsequent interactions with the gut microbiome are key environmental components of the annual disease and propose the use of diet supplementation as means to sustain the activity of stingless bee keeping as well as native bee pollination services.

Cinnamoyloxy-mammeisin, a coumarin from propolis of stingless bees, attenuates Th17 cell differentiation and autoimmune inflammation via STAT3 inhibition.

T helper 17 (Th17) lymphocytes play a critical role in the pathogenesis of autoimmune diseases, mainly by producing the pro-inflammatory cytokine interleukin-17 (IL-17). Therefore, Th17 lymphocytes have been considered a strategic target for drug discovery and development. In this study, we investigated the activity and possible mechanisms of action of a 4-phenyl coumarin isolated from propolis, named cinnamoyloxy-mammeisin (CNM), in Th17 cell differentiation and the development of experimental Th17-dependent autoimmune encephalomyelitis (EAE). Our data showed that in vitro Th17 cell differentiation was attenuated by CNM treatment in a concentration-dependent manner (1, 3, and 10 µM). This was associated with a reduction in the release of IL-17 (35% inhibition) and interleukin-22 (IL-22, 51% inhibition). Th17-differentiated cells exposed to CNM also downregulated the expression of Th17 hallmarked cell genes, such as RAR-related orphan receptor c (Rorc, 51% inhibition), and interleukin-23 receptor (Il23r, 64% inhibition), indicating possible upstream molecular mechanisms. Mechanistically, CNM significantly reduced the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) during in vitro Th17 cell differentiation. In vivo treatment with CNM (100 µg/kg) reduced the clinical signs of EAE, which was associated with a reduction in Central Nervous System demyelination, neuroinflammation, and Th17 response in the spinal cord and inguinal lymph nodes. Consistent with this, CNM also effectively attenuated human Th17 differentiation in vitro. Collectively, our results highlight the potential of CNM as a new molecule that can modulate Th17 cells via inhibition of STAT3 signaling and, as a result, reduce autoimmune inflammation.

Antimicrobial and antioxidant activity of proteins isolated from Melipona beecheii honey.

Proteins from Melipona beecheii honey were purified by concanavalin A (conA) affinity chromatography and eluted with a stepwise glucose gradient into fractions named F2-F5. The conA-unbound fraction (F1) was further separated by molecular exclusion into fractions named MbF1-1,2 and MbF1-3. All fractions were evaluated for antibacterial activity against foodborne pathogens and antioxidant capacity. F1 fraction possessed highest antimicrobial activity against S. aureus, L. monocytogenes, S. Typhimurium, E. coli and P. aeruginosa with MIC's 1.4 ± 0.2, 15 ± 1, 39 ± 2, 1 ± 0.1, and 75 ± 2 µg/mL, respectively. F1, MbF1-1,2 and MbF1-3 had bactericidal effect except against P. aeruginosa. When the antioxidant capacity of the fractions was determined, F2 had the highest antioxidant activity measured by DPPH radical scavenging activity (IC50 = 2.4 ± 0.4 µg/µL) and reducing power of Fe(III) (IC50 = 1.8 ± 0.2 µg/µL). We provide evidence that M. beecheii honey proteins possess broad spectrum antibacterial and antioxidant activity, the latter probably through their reducing agent and free radical scavenger properties.

Propolis from native Stingless Bees: ultrasound-assisted extraction / Propóleos de abejas nativas sin aguijón: extracción asistida por ultrasonido

BACKGROUND: Propolis has been considered a highly valuable material due to its therapeutic properties. However, in Colombia, the commercialization of propolis is limited not only by low production but also by the little knowledge about its efficient extraction. Therefore, finding an optimal and economical extraction method to obtain propolis is a necessity for beekeepers that would open new possibilities for industrial use and, therefore, for the market. OBJECTIVES:The objective of this study was to evaluate a conventional and ultrasound-assisted extraction method, seeking to obtain the highest yield and a high amount of content of bioactive compounds in propolis extracts. METHODS: The extraction was carried out for three crude propolis from different types of bees: Tetragoniscaangustula or Angelita (ANG), Meliponaeburnea or Melipona(MEL), and Scaptotrigonaspp (SCT). The extracts were characterized by color, pH, visual appearance, solid content, antioxidant capacity, total polyphenol content, and bacterial inhibition capacity. RESULTS: The highest extraction performance was obtained when the ultrasound-assisted method was used, especially for the ANG extract, which in addition to presenting inhibition for gram-negative (E. coli) and gram-positive (S. Aureus) bacteria, had the best antioxidant activity with a value of 545 mg GAE / 100 g of sample and total polyphenol content of 1,884 mg GAE / 100 g of sample. CONCLUSIONS: Ultrasound-assisted extraction can be considered a low-cost alternative to increase the extraction performance of crude propolis, together with its total polyphenol content and antioxidant capacity, without altering its physical properties

ANTECEDENTES: El propóleos ha sido considerado un material de alto valor por sus propiedades terapéuticas. Sin embargo, en Colombia la comercialización de propóleos está limitada no solo por la baja producción sino también por el incipiente conocimiento sobre la extracción eficiente de este. Por ello, encontrar un método de extracción óptimo y económico para la obtención de propóleos es una necesidad para los apicultores que abriría nuevas posibilidades para el uso industrial y por tanto para el mercado. OBJETIVOS: El objetivo de este estudio fue evaluar un método de extracción convencional y asistido por ultrasonido (US) buscando el mayor rendimiento y alto contenido de compuestos bioactivos en extractos de propóleos. MÉTODOS: La extracción se realizó para tres propóleos crudos de diferentes tipos de abejas Tetragonisca angustula o Angelita(ANG), Melipona eburnea o Melipona (MEL) y Scaptotrigona spp (SCT). Todos los extractos se caracterizaron por su color, pH, apariencia visual, contenido de sólidos, capacidad antioxidante, contenido total de polifenoles y capacidad de inhibición bacteriana. RESULTADOS: El mayor rendimiento de extracción se obtuvo cuando se usó el método asistido por ultrasonido y específicamente para el extracto ANG, que además de presentar inhibición para bacterias gram negativas (E. coli) y gram positivas (S. Aureus), tuvo la mejor actividad antioxidante con un valor de 545 mg GAE / 100 g de muestra y contenido total de polifenoles de 1884 mg GAE / 100 g de muestra. CONCLUSIONES: La extracción asistida por ultrasonido puede considerarse una alternativa de bajo costo para aumentar el rendimiento de extracción del propóleos crudo, así como su contenido total de polifenoles y capacidad antioxidante sin alterar sus propiedades físicas

Modulation of gut microbiota by Mantequilla and Melipona honeys decrease low-grade inflammation caused by high fructose corn syrup or sucrose in rats.

Several studies have shown that consumption of honey is associated with various health benefits. However, there is scarce evidence on whether honeys modify the intestinal microbiota by preventing the inflammatory response in the host. Therefore, the aim of the present work was to study the effect of Melipona (Mel) and Mantequilla (Mtq) honeys, which contain different bioactive compounds and antioxidant capacity on gut microbiota and metabolic consequences in comparison with other sweeteners, in particular sucrose (S) and high fructose corn syrup (HFCS) in rats. The results of the present work showed that both honeys have polyphenols, flavonoids, antioxidant and bactericidal activities. Rats fed with both honeys gained less weight and body fat by increasing energy expenditure compared to S or HFCS and increased gene expression of antioxidant enzymes mediated by the transcription factor Nrf2. Analysis of the gut microbiota showed that consumption of both honeys modified the beta-diversity compared to those fed S or HFCS resulting in increased abundance of a specific cluster of bacteria of the Clostridium genus particularly Coprococcus eutactus, Defluviitalea saccharophila, Ruminicoccus gnavus and Ruminicoccus flavefaciens. As a result of the changes in the gut microbiota, there was a decrease in LPS- and TLR4-mediated low-grade inflammation and an increase in sIgA. Consumption of both honeys prevented glucose intolerance and increased adipocyte size compared to S or HFCS. In conclusion, consumption of MtqH or MelH can reduce metabolic endotoxemia by modifying the gut microbiota to prevent glucose intolerance.

Tragedy of the commons in Melipona bees revisited.

Melipona stingless bees display a paradoxical overproduction of queens, which are later eliminated by nest-mate workers. Mechanistically, it was suggested that the monoterpenoid geraniol deposited into newly provisioned cells by adult bees would cause larvae to develop into queens in Melipona beecheii. This system could be evolutionarily stable if many of these new queens were to leave the nest and parasitize other genetically unrelated colonies nearby, as was shown to occur in a congeneric species. Here, we use microsatellite markers to test whether queen overproduction could be a strategy by which adult workers control the caste fate of the developing larvae to export copies of their own genes to the rest of the population via queen parasitism in M. beecheii. In addition, we re-examined whether artificially increasing the levels of geraniol indeed caused larvae to develop as queens rather than workers. Contrary to our prediction, we found no evidence for queen parasitism in M. beecheii and observed no effect of geraniol on the rearing of new queens. Together, these results support the original 'tragedy of the commons' hypothesis for queen overproduction in Melipona bees, where individual larvae selfishly bias their development towards the queen pathway according to their best evolutionary interests.

Stingless bee honey: An overview of health benefits and main market challenges.

This review aimed to evaluate the nutraceutical and medicinal effects of stingless bee honey (SBH) by bringing a discussion focused on the main known in vitro/in vivo health-promoting effects. SBH has a high-water content, slight sweetness, acidic flavor, fluid texture, and slow crystallization. The type and concentration of phenolic compounds and consequent antioxidant activity were mainly associated with the floral sources, geographical location, bee species, and processing steps. SBH has anti-inflammatory, antimicrobial (against spoilage and pathogenic microorganisms), anti-diabetic, and skin aging delay activities in in vitro tests. It has also shown antioxidant and hypolipidemic effects, can protect from injuries caused by dyslipidemia, possess anti-inflammatory activity against chronic subclinical systemic inflammation and anti-diabetic properties, and can control and prevent Staphylococcus aureus infection on infected wound healings in in vivo tests (rats). However, clinical trials are crucial for the probation of the medicinal and nutraceutical properties of SBH. Despite this, there are still no general norms and/or quality standards for this type of honey. The information summarized in this review is important to add value to this little-consumed food, providing helpful information to spread knowledge about its benefits, assisting future studies, and raising perspectives for its recognition as a functional food. Furthermore, it may encourage the creation of standard quality for the production and marketing of SBH. PRACTICAL APPLICATIONS: Previous studies have already summarized the chemical profile and physicochemical properties of stingless bee honey (SBH) and its potential health properties. However, no study has performed an overview of the potential nutraceutical and medicinal effects of SBH, presenting results from in vitro and in vivo investigations. Therefore, this review is the first study to overview the potential nutraceutical and medicinal effects of SBH, showing results of in vitro/in vivo health-promoting effects. The bioactivity of SBH is related to bee species and floral sources. The SBH has anti-inflammatory, antimicrobial, anti-diabetic, and antioxidant in vitro activity. It has also shown hypolipidemic effects and protection from injuries caused by dyslipidemia in rats.

Efficiency of two digestion methods in determining the presence of metals (Cd, Cu, Cr, Pb and Zn) in geopropolis produced by Melipona scutellaris / Eficacia de dos métodos de digestión para determinar metales (Cd, Cu, Cr, Pb y Zn) en geopropóleos producidos por Melipona scutellaris / Eficiência de dois métodos de digestão para determinar metais (Cd, Cu, Cr, Pb e Zn) na geoprópolis produzida por Melipona scutellaris

Abstract This study evaluates the efficiency of acid extraction and total digestion to determine the presence of metals in geopropolis produced by the stingless bee Melipona scutellaris. Geopropolis samples were collected at five meliponaries in the city and in the metropolitan region of Salvador, Bahia State, Brazil. The sample treatment methods comprised acid extraction and total digestion. The Inductively Coupled Plasma Optical Emission Spectrometry (ICP OES) technique was used to quantify Cd, Cu, Cr, Pb, and Zn. Geopropolis samples submitted to both digestion methods showed statistical differences. For both methods, Cr and Zn showed the highest concentrations, while those of Cd were the lowest. The Cr concentration for determination by total digestion was 37.53 mg/kg, while for acid extraction it was 32.90 mg/kg. For Zn, the concentration was 17.65 mg/kg and 8.85 mg/kg for total digestion and acid extraction, respectively. The total digestion method showed the highest concentrations of the metals evaluated; however, acid extraction (USEPA 3050b) is a more straightforward procedure for metal evaluation in geopropolis samples and presented values that support the use of geopropolis as a bioindicator. The acid extraction method USEPA 3050b, in combination with detection using ICP OES, showed efficiency in analyses carried out to determine metals in geopropolis.

Resumen El objetivo de este artículo fue evaluar la eficacia da extracción ácida y de la digestión total para determinación de metales absorbidos en geopropóleos producidos por Melipona scutellaris. Se colectaron muestras de geopropóleos en cinco meliponarios localizadas en la región metropolitana del Salvador, estado de Bahia, Brasil. Se utilizaron como métodos de tratamiento para muestras, la extracción ácida y la digestión total. Para determinar los metales Cd, Cu, Cr, Pb y Zn se utilizó Espectroscopia de Emisión Óptica con Plasma Acoplado Inductivamente (ICP OES). Se encontraron diferencias estadísticas en la concentración de metales hallados en las muestras de geopropóleos sometidas a los diferentes métodos de digestión. Para ambos métodos de digestión, las mayores concentraciones de metal fueron observadas para Cr y Zn. Entre los metales evaluados Cd presentó la menor concentración. La concentración de Cr para la determinación por digestión total fue de 37,53 mg/kg y para la extracción ácida fue de 32,90 mg/ kg. Para el Zn, la concentración fue de 17,65 mg/kg y 8,85 mg/kg para la digestión total y la extracción ácida, respectivamente. El método de digestión total mostró los mayores valores para concentraciones de los metales evaluados. Entretanto, la extracción ácida, USEPA 3050b, es un procedimiento más simple para la evaluación de metales en muestras de geopropóleos y también mostró valores que pueden satisfacer la necesidad de su uso en evaluaciones de colmenas como bio-indicador. El método de extracción ácida USEPA 3050b en combinación con la detección a través de ICP OES se mostró eficiente para el análisis de metales en geopropóleos.

Resumo O objetivo deste estudo foi avaliar a eficiência da extração ácida e da digestão total para determinação de metais adsorvidos na geoprópolis produzida por Melipona scutellaris. Foram coletadas amostras de geoprópolis em cinco meliponários, situados em Salvador, Estado da Bahia e região metropolitana. Foram utilizados como métodos de tratamento das amostras a extração ácida e a digestão total. Para determinação dos metais Cd, Cu, Cr, Pb e Zn utilizou-se a Espectrometria de Emissão Óptica com Plasma Indutivamente Acoplado (ICP OES). Houve diferença estatística na concentração de metais encontrados nas amostras de geoprópolis submetidas aos diferentes métodos de digestão. Para ambos os métodos de digestão as maiores concentrações de metais foi observada para o Cr e Zn. Dentre os metais avaliados o Cd apresentou a menor concentração. A concentração de Cr para determinação por digestão total foi de 37,53 mg/kg e para extração ácida de 32,90 mg/ kg. Para o Zn, a concentração foi de 17,65 mg/kg e 8,85 mg/kg para digestão total e extração ácida, respectivamente. O método de digestão total apresentou os maiores valores para concentrações dos metais avaliados. No entanto, a extração ácida, USEPA 3050b, é um procedimento mais simples para a avaliação dos metais em amostras de geoprópolis e também apresentou valores que podem satisfazer a necessidade de utilização na avaliação deste produto da colmeia como bioindicador. O método de extração ácida USEPA 3050b em combinação com a detecção por ICP OES mostrou eficiência para análise de metais em geoprópolis.

Evaluation of physic contamiants and contamination with coliforms, molds and yeasts of honey from the Northern Brazil / Avaliação de contaminantes físicos e contaminação com coliformes, bolores e leveduras de mel do Norte do Brasil

The Mercosur Technical Regulation for honey determines microbiological parameters for total coliforms and molds and/ yeasts, and absence of physical contaminants, of any nature. The purpose of this study was to verify the biological and physical contamin of Apis mellifera ligustica and Melipona fasciculata, commercialized in the northeastern region of the State of Pará, Brazil. The fourteen samples from informal trade of the municipalities of Bragança, Capanema, Nova Timboteua, São João de Pirabas, Salinas and Tracauateua were analyzed for the Most Probable Number of colifroms at 35ºC, Fungus and Yeast counts, besides the detection of dirt by Macro and Microscopy techniques. The results obtained from the microbiological analyzes were compared to the standard required by MERCOSUR, for coliforms. 57.14% of the samples were non-standard and ranged from <3.0 NMP/g to 20 NMP/g. The molds and/ yeast counts ranged from 1x10¹ CFU/g to 8.6x10² CFU/g, with atotal of 21.42% of the samples being in disagreement with the legislation. From the macroscopic analysis, it was observed that, although 50% of the samples did not present any apparent dirt, microscopically it was possible to observe dirt in 100% of the samples of honey analyzed, which is out of the standard required by the Brazilian legislation. This shows the low sanitary quality of honey commercialized in this region of the state of Pará.

O Regulamento Técnico do Mercosul para mel, determina parâmetros microbiológicos para coliformes totais e fungos e/leveduras, e ausência de contaminantes físicos, de qualquer natureza. A proposta do presente estudo foi verificar a contaminação biológica e física em méis de abelha das espécies Apis mellifera ligustica e Melipona fasciculata, comercializados na região Nordeste do Estado do Pará, Brasil. As amostras provenientes do comercio informal dos de municípios de, Bragança, Capanema, Nova Timboteua, São João de Pirabas, Salinas e Tracauateua, foram analisadas quanto a determinação do Número Mais Provável de coliformes a 35ºC, Contagem de Fungos e/leveduras, além da detecção de sujidades pelas técnicas de Macro e Microscopia. Os resultados obtidos a partir das análises microbiológicas foram comparadas ao padrão exigido pelo MERCOSUL, para coliformes, 57,14% das amostras encontravam-se fora do padrão variaram de <3,0 NMP/g a 20NMP/g. Já a contagem de fungos e/leveduras variou de 1x10¹ UFC/g a 8,6x10² UFC/g, no total de 21,42 % das amostras apresentaram-se em desacordo com a legislação. A partir da análise macroscópica observou-se que, embora 50% das amostras não apresentassem sujidades aparentes, porém microscopicamente foi possível observar a presença sujidades em 100% das amostras de méis analisadas encontrando-se fora do padrão exigido pela legislação Brasileira. Demostrando a baixa qualidade sanitária do mel comercializado nessa região do estado do Pará.

Antibacterial activity of honeys from Amazonian stingless bees of Melipona spp. and its effects on bacterial cell morphology.

BACKGROUND: Stingless bee honey has great therapeutic potential, especially as an antimicrobial agent. In the present study, we evaluated the in vitro antibacterial potential of honey from Melipona spp. with occurrence in Rio Branco-AC and Xapuri-AC from the Amazonian region. Samples were collected from the species Melipona eburnea, Melipona grandis, Melipona flavolineata and Melipona seminigra. The antibacterial activity of the honey samples was tested against standard Gram-positive and Gram-negative bacteria and two strains isolated from bovine mastitis. RESULTS: In the agar diffusion assay, we observed antibacterial activity for the four honeys against the tested strains. The honey from M. flavolineata showed a minimmum inhibitory concentration (MIC) lower than 3.12% (v/v). The minimum bactericidal concentration values were larger than the MIC for most of the microorganisms tested. Scanning electron microscopy (SEM) showed the damaging effect of the honey of M. flavolineata on Staphylococcus aureus cells, as well as its inhibitory effect on cell division. CONCLUSION: The results of the present study demonstrate that the honey from stingless bees possesses in vitro antimicrobial activity against pathogenic bacteria. The effects observed by SEM show that honey from the Amazonian stingless bee M. flavolineata has promising therapeutic potential as a future antimicrobial agent. © 2020 Society of Chemical Industry.

Mollicute Anti-Adhesive and Growth Inhibition Properties of the Methanolic Extract of Propolis from the Brazilian Native Bee Melipona quadrifasciata.

Hydroalcoholic propolis extracts from the bee species Melipona quadrifasciata have been shown to possess antimicrobial activity against different mollicute strains, but a methanolic extract (ME) could contain an increased diversity of nonpolar bioactive components with a potentially higher antimicrobial activity. The ME obtained by maceration of the propolis sample was fractionated with solvents of different polarities and then, purified by silica gel column chromatography through biomonitoring of its antimicrobial activity against mollicute strains. Analysis by gas chromatography-mass spectrometry (GC/MS) enabled the identification of compounds using the NIST library. Minimum inhibitory concentrations (MICs) of the samples were determined by broth microdilution. Anti-adhesive assays were performed with Mycoplasma pneumoniae cells. The hexane (MIC=62.5 mg/L) and dichloromethane (MIC=125 mg/L) fractions presented the most promising results against M. pneumoniae. They were fractionated into 74 subfractions, and even the best ones did not show better results (MIC>250 mg/L) than their original fractions, likely due to the loss of terpene compounds that seem to act in synergy. The dichloromethane subfraction FD4 was highlighted in the anti-adhesive assay with an inhibitory activity of 21.6 %. A synergistic effect of the nonpolar compounds in M. quadrifasciata propolis may be responsible for its antibacterial activity, but several purified components can improve its anti-adhesive properties.

Characterization of a non-glycosylated fraction from honey proteins of Melipona beecheii with antimicrobial activity against Escherichia coli O157:H7.

AIMS: To analyse the non-glycosylated protein fraction from Melipona beecheii honey for antimicrobial activity against Escherichia coli O157:H7. METHODS AND RESULTS: The proteins from M. beecheii honey were separated according to their degree of glycosylation using Concanavalin A-affinity chromatography. The total protein extract and its fractions were analysed by 1D and 2D electrophoresis. We also determined the antimicrobial and antihaemolytic activities of the total protein extract and the non-glycosylated fraction. Furthermore, we evaluated the effect of this non-glycosylated fraction for the expression of the Stx1, Stx2, EAE and HlyA pathogen genes. Melipona beecheii honey contained at least 24 proteins with molecular weights ranging between 7·6 and 95 kDa and isoelectric points between 3 and 10, three proteins from the 24 are non-glycosylated. The non-glycosylated fraction had an MIC90 of 1·128 µg ml-1 , and this fraction inhibited the haemolytic activity of the pathogen, as well as reduced the expression of Stx1, Stx2 and HlyA. The MbF1-2 protein from the non-glycosylated fraction was sequenced and identified as a homologue of the royal jelly-like protein of Melipona quadrifasciata. CONCLUSIONS: The non-glycosylated protein fraction from M. beecheii honey greatly contributes to antibacterial activity and it is composed of at least three proteins, of which MbF1-2 provided over 50% of the antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study showed significant antimicrobial activity from several proteins present in the honey of M. beecheii. Interestingly, the non-glycosylated protein fraction demonstrated antihaemolytic activity and adversely affected the expression of virulence genes in Escherichia coli O157:H7; these proteins have the potential to be used in developing therapeutic agents against this bacterium.

Determination and uptake of abamectin and difenoconazole in the stingless bee Melipona scutellaris Latreille, 1811 via oral and topic acute exposure.

Bees are considered as important providers of ecosystem services, acting via pollination process in crops and native plants, and contributing significantly to the maintenance of biodiversity. However, the decrease of bee's population has been observed worldwide and besides other factors, this collapse is also related to the extensive use of pesticides. In this sense, studies involving the assessment of adverse effects and the uptake of pesticides by bees are of great concern. This work presents an analytical method for the determination of the insecticide abamectin and the fungicide difenoconazole in the stingless bee Melipona scutellaris exposed via oral and topic to endpoints concentrations of active ingredients (a.i.) alone and in commercial formulations and the discussion about its mortality and uptake. For this purpose, QuEChERS (Quick, Easy, Cheap, Efficient, Rugged and Safe) acetate modified method was used for extraction and pesticides were determined by LC-MS/MS. The validation parameters have included: a linear range between 0.01 and 1.00 µg mL-1; and LOD and LOQ of 0.038 and 0.076 µg g-1 for abamectin and difenoconazole, respectively. The uptake of tested pesticides via oral and topic was verified by the accumulation in adult forager bees, mainly when the commercial product was tested. Mortality was observed to be higher in oral exposure than in topic tests for both pesticides. For abamectin in a commercial formulation (a.i.) no differences were observed for oral or topic exposure. On the other hand, for difenoconazole, topic exposure had demonstrated higher accumulation in bees, according to the increase of received dose. Through the results, uptake and the possible consequences of bioaccumulated pesticides are also discussed and can contribute to the knowledge about the risks involving the exposure of bees to these compounds.