Propolis phenolic compounds extraction using response surface methodology

Propolis is a complex resinous substance prepared by bees (as, Apis mellifera) using compounds collected from plants, buds and exudates, which are then mixed with wax, pollen and enzymes ofbees' saliva [1, 2]. It is used to seal openings and cracks in the hive walls, to isolate animal calcasses inside the beehive, and in the protection against parasites. This product hás attained much interest due to its chemical composition and fünctional properties. Recent publications emphasize its biological and health enhancing properties as, for instance, immunomodulatory, antitumoral, antimicrobial, anti-inflammatory. antioxidant, antiviral and antifungal [3, 4].Fmancial support received fi-om PRODER, "PA 54811 Appreciation ofapiculture by-products - Mead and vinegar production of honey", is gratefully acknowledged.info:eu-repo/semantics/publishedVersio

Caraterização química e biológica do própolis da “serra de Bornes” por TLC

O própolis é uma substância resinosa, obtida pelas abelhas Apis mellifera, considerado como um produto “antibiótico natural” que desempenha um papel importante na defesa da colmeia, protegendo-a de microrganismos, fungos, bactérias e vírus. Este produto possui na sua composição uma grande variedade de compostos, salientando-se os compostos fenólicos, aos quais se atribuem forte poder antioxidante e atividade antimicrobiana. Neste trabalho usaram-se duas amostras de própolis de origem geográfica diferente: Bornes e Lousã. Estudou-se a composição polínica, os teores totais de compostos fenólicos e flavonoides, bem como, as propriedades antioxidantes na amostra de Bornes. Da análise do extrato de própolis por TLC (cromatografia em camada fina), efetuaram-se estudos da atividade antimicrobiana usando-se extratos de manchas removidas da placa de TLC, selecionadas por terem grande concentração de compostos (manchas mais escuras). Os extratos das manchas separadas na placa de TLC foram analisados por HPLC e MS. A amostra de Lousã foi analisada no seu conteúdo em teores de compostos fenólicos totais e flavonoides totais. A análise polínica da amostra de própolis de Bornes mostrou que nesta predominavam os pólens Erica sp. e Castanea sp. com valores de abundância de 41% e 32% , respetivamente. A análise do perfil químico de amostras de própolis de Bornes e Lousã baseou-se nos teores de compostos fenólicos totais e flavonoides totais. Os teores de compostos fenólicos totais foram determinados pelo método de Folin-Ciocalteu, utilizando como padrão o ácido gálico. Os resultados obtidos foram de 91,5 ± 0,7 mg/L e de 117 ± 2 mg/L para as amostras de Bornes e Lousã, respetivamente. O doseamento de flavonoides totais baseou-se no método espetrofotométrico usando o reagente cloreto de alumínio e como padrão, a quercetina. Os resultados obtidos foram de 23,4 ± 0,8 mg/L e de 32,9 ± 0,1 mg/L para as amostras de Bornes e Lousã, respetivamente. A atividade antioxidante foi avaliada pelo método do poder redutor e pelo efeito bloqueador de radicais livres de DPPH (2,2-difenil-1-picrilhidrazil) para a amostra de própolis de Bornes. Para o método do poder redutor os resultados evidenciaram valores de EC50 de 0,33 ± 0,02 mg/mL, e para o método de DPPH demonstraram valores mais baixos de EC50, 0,072 ± 0,003 mg/mL. O extrato de própolis de Bornes foi analisado por TLC (sílica gel 60 com fluorescência a 254 nm) usando a técnica bidimensional com o objetivo de obter uma primeira separação de compostos para posteriormente serem identificados por HPLC-DAD e MS. A separação dos compostos fenólicos na placa de TLC foi visualizada usando reagentes ou radiação UV 254 nm. Os reagentes usados na visualização das manchas referentes aos compostos fenólicos separados na placa de TLC, foram: cloreto de alumínio, cloreto de ferro, Folin-Ciocalteu, DPPH e vanilina. Para analisar a atividade antimicrobiana selecionaram-se manchas de TLC com grande concentração de compostos (manchas mais escuras) provenientes do própolis de Bornes. A extração dos compostos da sílica gel da mancha selecionada foi efetuada usando-se o solvente dimetilsulfóxido (DMSO). Os compostos presentes nas manchas de TLC isoladas e testadas inibiram os microrganismos em estudo (Salmonella spp., Escherichia coli, Bacillus cereus, Staphylococcus aureus e Candida albicans). O método de HPLC com detetor DAD foi usado com o objetivo de analisar os extratos da sílica gel de todas as manchas identificadas na placa de TLC, de forma a identificar a que família de compostos fenólicos pertencem os picos obtidos em cada cromatograma. Nos cromatogramas de cada extrato verificou-se sempre a presença de vários picos mostrando que a separação por TLC não permite um isolamento total dos compostos. A identificação da família do composto fenólico de cada pico presente nos cromatogramas foi feita por comparação com padrões puros, e só nos casos de se ter uma “semelhança elevada” nos espetros UV é que se avançou com a identificação do grupo a que pertence o composto fenólico. Dos identificados, a família de compostos fenólicos mais abundante foi a dos ácidos hidroxicinâmicos. Neste trabalho faz-se uma breve apresentação de resultados preliminares obtidos com o método MS, com o objetivo de dar continuidade a este trabalho no futuro. Este método ainda está a ser estudado nas suas condições experimentais de forma a identificar todos os compostos fenólicos presentes nos extratos das manchas separadas na placa de TLC. Os primeiros resultados mostram que os compostos presentemente identificados são derivados do ácido cafeico e da pinobanksina. Propolis is a resinous substance obtained by honey bees Apis mellifera, a product considered "natural antibiotic" which plays an important role in defending the hive, protecting it from microorganisms, fungi, bacteria and viruses. This product has a large variety of compounds in its composition, giving greater emphasis to the phenolic compounds, which are attributed strong antioxidant and antimicrobial activities. This work used two propolis samples of different geographical origin: Bornes and Lousã. It was studied the pollen composition, the total content of flavonoids and phenolic compounds, as well as the antioxidant properties of the Bornes sample. Analysis with propolis extracts separated by TLC (Thin-Layer Chromatography), were made in studies of antimicrobial activity by using extracts of stains removed from the TLC plate, selected for having large concentration of compounds (darker stains). The extracts of stains separated on TLC plate were analyzed by HPLC and MS. The Lousã sample was analyzed in their content in phenolic compounds and total flavonoids. Pollen analysis of the Bornes propolis sample showed that the predominant pollens Erica sp. and Castanea sp. had abundance values of 41% and 32%, respectively. The analysis of the chemical profile of Bornes and Lousã propolis samples was based on the content of total phenolic compounds and flavonoids. The concentrations of total phenolics were determined by Folin-Ciocalteu, using gallic acid as standard. The results were 91,5 ± 0,7 mg/L and 117 ± 2 mg/L for the samples of Bornes and Lousã, respectively. The assay of total flavonoids was based on the spectrophotometric method using the reagent aluminum chloride and as standard compound, quercetin. The results were 23,4 ± 0,8 mg/L and 32,9 ± 0,1 mg/L for samples of Bornes and Lousã, respectively. The Bornes sample antioxidant activity was evaluated by the method of reducing power and the blocking effect of free radicals of DPPH (2,2-diphenyl-1-picrylhydrazyl) for the sample. The assay of reducing power showed EC50 values of 0,33 ± 0,02 mg/mL, and the DPPH assay showed lower values of EC50, 0,072 ± 0,003 mg/mL. The Bornes propolis extract was analyzed by TLC (silica gel 60 with fluorescence at 254 nm) using the two-dimensional technique in order to obtain an initial separation of compounds for later identification by HPLC-DAD and MS. The separation of the phenolic compounds in the TLC plate was visualized using UV irradiation at 254 nm or chemical reagents. The reagents used in the visualization of stains pertaining to phenolic compounds separated on TLC plate, were: aluminum chloride, iron chloride, Folin-Ciocalteu, DPPH and vanillin. To analyze the antimicrobial activity were selected TLC stains with high concentrations of compounds (darker stains) from the Bornes propolis two-dimensional TLC analysis. The compounds extraction from the selected silica gel stain was carried out using the solvent dimethylsulfoxide (DMSO). The extracted compounds of the TLC spots isolated and tested inhibited the growth of the microorganisms under study (Salmonella spp., Escherichia coli, Bacillus cereus, Staphylococcus aureus e Candida albicans). The HPLC method with DAD detector was used to analyze the extracts of silica gel of all stains identified on TLC plate in order to identify the family of phenolic compounds belonging to the peaks in each chromatogram obtained. In the chromatograms of each extract, it was verified the presence of several peaks showing that separation by TLC does not permit a total isolation of each compound. The identification of the phenolic compounds family in each peak in the chromatograms was done by comparison with pure standards. Only in cases of having a "high similarity" in the UV spectrum, it was possible to advance with the identification of the family to which compound phenolic belongs. Of the identified family of phenolic compounds, the most abundant was hydroxycinnamic acid. This work makes a brief presentation of preliminary results obtained with the MS method, being a component of this work that remains to be completed, in order to identify all phenolic compounds present in the extracts of stains separated on TLC plate. Initial results show that the compounds identified are presently caffeic acid derivatives and pinobanksina

Microbiological and Physicochemical Evaluation of Hydroxypropyl Methylcellulose (HPMC) and Propolis Film Coatings for Cheese Preservation

Dairy products are highly susceptible to contamination from microorganisms. This study aimed to evaluate the efficacy of hydroxypropyl methylcellulose (HPMC) and propolis film as protective coatings for cheese. For this, microbiological analyses were carried out over the cheese' ripening period, focusing on total mesophilic bacteria, yeasts and moulds, lactic acid bacteria, total coliforms, Escherichia coli, and Enterobacteriaceae. Physicochemical parameters (pH, water activity, colour, phenolic compounds content) were also evaluated. The statistical analysis (conducted using ANOVA and PERMANOVA) showed a significant interaction term between the HPMC film and propolis (factor 1) and storage days (factor 2) with regard to the dependent variables: microbiological and physicochemical parameters. A high level of microbial contamination was identified at the baseline. However, the propolis films were able to reduce the microbial count. Physicochemical parameters also varied with storage time, with no significant differences found for propolis-containing films. Overall, the addition of propolis to the film influenced the cheeses' colour and the quantification of phenolic compounds. Regarding phenolic compounds, their loss was verified during storage, and was more pronounced in films with a higher percentage of propolis. The study also showed that, of the three groups of phenolic compounds (hydroxybenzoic acids, hydroxycinnamic acids, and flavonoids), hydroxycinnamic acids showed the most significant losses. Overall, this study reveals the potential of using HPMC/propolis films as a coating for cheese in terms of microbiological control and the preservation of physicochemical properties.This research was funded by the Project-PHARMAPITOX-Desenvolvimento de um coletor inovador e protocolo para purificação da apitoxina para uso nas indústrias farmacêutica e cosmética (Development of an innovative collector and protocol for the purification of apitoxin for use in the pharmaceutical and cosmetic industries). This project was co-financed by the European Regional Development Fund (FEDER), NORTE-01-0247-FEDER-113540, through the Competitiveness and Internationalisation Operational Programme—PORTUGAl2020. The authors are grateful to Nelson De Moura of the company Ecoapis Unipessoal Lda (http://www.ecoapis.pt (accessed on 15 March 2023)) for supplying the propolis samples; Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2020).info:eu-repo/semantics/publishedVersio

Cuantificacio´n de tres clases feno´ licas y contenido feno´ lico total de extractos de propo´ leos utilizando un u´ nico espectro UV-Vis

This work presents a methodology for simultaneously measuring the total content of three classes of phenolic compounds (hydroxybenzoic acids, hydroxycinamic acids and flavonoids), as well the total phenolic content using a single UVvis spectrum. To test the methodology, samples of propolis were used. Firstly, an experimental design based in the Response Surface Methodology established that the best hydroethanolic solution for propolis phenolic compounds extraction had 80% of ethanol. Secondly, calibration models were developed with multiple linear regression, coupled with the leaps algorithm for variable selection, using UV-vis spectra of mixed standard solutions (gallic acid, ferulic acid and quercetin mixtures), all with orthogonal concentrations established by a multilevel fractional factorial design. The model’s estimation and prediction performance had linearity between predicted and expected concentration values, with slope ranging from 0.996–1.04 and determination coefficients higher than 0.995. A quality control solution presented acceptable repeatability and accuracy (relative standard deviation percentage and percentage relative error were lower than 4.8%). Analysis of propolis phenolic compounds extracts showed acceptable precision (relative standard deviation percentages lower than 1.6%) and acceptable accuracy (recoveries assays ranged between 98 and 112%). Overall, the present multi-parametric analytical technique can be a first approach for chemical characterization of phenolic compounds extracts, such as that obtained from the propolis samples, considering its advantages of simplicity, rapidity, precision and accuracy. Moreover, this methodology can be adapted to other sample matrices, provided that the typical UV-vis absorption spectra of their classes of phenolic compounds shows partial overlapping, allowing multivariate calibration.Este trabajo presenta una metodologı´a para medir simulta´neamente el contenido total de tres clases de compuestos feno´ licos (a´cidos hidroxibenzoicos, a´cidos hidroxicina´micos y flavonoides), ası´ como el contenido feno´ lico total usando un solo espectro UV-Vis. Para probar la metodologı´a, se utilizaron muestras de propo´ leos. En primer lugar, un disen˜o experimental basado en la metodologı´a de la respuesta en superficie establecio´ que la mejor solucio´n hidroetano´ lica para la extraccio´n de compuestos feno´ licos de propo´ leos tenı´a un 80% de etanol. En segundo lugar, se desarrollaron modelos de calibracio´n con regresio´n lineal mu´ ltiple, junto con el algoritmo de saltos para la seleccio´n variable, utilizando espectros UV-Vis de soluciones esta´ndar mixtas (a´cido ga´lico, a´cido feru´lico y mezclas de quercetina), todas con concentraciones ortogonales establecidas por un disen˜o factorial fraccional multinivel. La estimacio´n del modelo y el rendimiento de la prediccio´n tenı´an linealidad entre los valores de concentracio´n previstos y esperados, con una pendiente que variaba de 0,996 a 1,04 y coeficientes de determinacio´n superiores a 0,995. Una solucio´n de control de calidad presento´ aceptable repetibilidad y precisio´n (porcentaje de desviacio´n esta´ndar relativa y porcentaje de error relativo inferiores al 4,8%). El ana´lisis de extractos de compuestos feno´ licos de propo´ leos mostro´ una precisio´n aceptable (porcentajes de desviacio´n esta´ndar relativa inferior al 1,6%) y una precisio´n aceptable (los ensayos de recuperacio ´n variaron entre 98 y 112%). En general, la presente te´cnica analı´tica multiparame´trica puede ser un primer enfoque para la caracterizacio´n quı´mica de extractos de compuestos feno´ licos, como el obtenido a partir de las muestras de propo´ leos, considerando sus ventajas de simplicidad, rapidez, precisio´n y precisio´ n. Adema´s, esta metodologı´a puede adaptarse a otras matrices de muestra, siempre que los espectros de absorcio´n UV-Vis tı´picos de sus clases de compuestos feno´ licos muestren solapamiento parcial, permitiendo la calibracio´n multivariante.This work was financially supported by CQ-VR, Centro de Química – Vila Real, University of Trás-os-Montes e Alto Douro [Project UID/QUI/00616/2013]; Centre of Molecular and Environmental Biology, University of Minho [Project UID/ BIA/04050/2013]; funded by FEDER (Fundo Europeu de Desenvolvimento Regional) through COMPETE2020 (Programa Operacional Competitividade e Internacionalização, POCI); by national funds through FCT (Fundação para a Ciência e a Tecnologia, Portugal); by Project 56343–“New products derived from sausage”, Measure 4.1 – Cooperation for Innovation, PRODER program, Portugal.info:eu-repo/semantics/publishedVersio

Recent advances in applied microbiology: Editorial

The importance of microbiology has grown exponentially since the development of genomics, transcriptomics, and proteomics, making it possible to clarify microbial biogeochemical processes and their interactions with macroorganisms in both health and disease. Particular attention is being payed to applied microbiology, a discipline that deals with the application of microorganisms to specific endeavors, whose economic value is expected to exceed USD 675.2 billion by 2024. In the Special Issue “Recent Advances in Applied Microbiology”, twenty-four papers were published (four reviews and twenty original research papers), covering a wide range of subjects within applied microbiology, including: microbial pathogenesis, the health-promoting properties of microorganisms and their by-products, food conservation, the production of alcoholic beverages, bioremediation and the application of microbiology to several industrial processes.info:eu-repo/semantics/publishedVersio

Influence of the storage conditions on the quality of bee pollen

Bee pollen is a healthful food product with a good nutritional profile and therapeutic properties. However, the storage conditions may affect its composition and characteristics. This study aimed to evaluate the effect of the storage conditions on chemical composition of different monofloral bee pollen samples, namely acidity (pH), water activity, total acidity and the content of fibre, ash, reducing sugars, protein, lipids, total phenols and total flavonoids. Nine bee pollen samples were harvested in three places in the Northeast of Portugal and divided into two aliquots: one was frozen at −20°C, while the other was dried at 42°C, until reaching moisture of 6–8%. Even though differences in the botanical origin are a significant factor explaining the variation between samples, the storage method was also found to be a highly significant factor for several parameters: reducing sugars, lipids, total phenols and total flavonoids. Higher counts were obtained on the frozen bee pollen samples regarding aerobic mesophiles and moulds and yeasts. Even so, for all samples and conservation methods, the values were below those given by the standards. Our study suggests that it is better to consume bee pollen frozen at −20°C in comparison to that dried in an electric oven.Centro de Estudos Florestais is a research unit funded by Fundação para a Ciência e Tecnologia, (UID/ AGR/00239/2019).info:eu-repo/semantics/publishedVersio

Propolis microencapsulation by double emulsion solvent evaporation approach: Comparison of different polymeric matrices and extract to polymer ratio

Propolis, a heterogeneous matrix produced by honeybees, is an antioxidant amongst other valuable biological properties. Nevertheless, the practical application of propolis, especially for the food industry, is still limited due to its relative high hydrophilicity and strong/unpleasant taste and aroma. Accordingly, microencapsulation arises a potential alternative approach to protect propolis compounds while allowing to mask its unpleasant sensory properties. The purpose of this study was to microencapsulate propolis extracts in lipophilic polymer-carriers using alternative propolis extract to polymer ratios – 1/4, 1/2 and 3/4 – by double emulsion solvent evaporation approach. The product yield was higher than the previously reported in the literature (80.3 ± 3.7% versus 68%). The encapsulation efficiencies (considering the encapsulated antioxidants and phenolic compounds) were independent from the propolis extract to polymer ratio, for the studied lipophilic polymer-carriers. The results of this work provide promising approaches for the manufacture of propolis extracts-loaded microparticles for further incorporation in functional matrices.info:eu-repo/semantics/publishedVersio

Mead production using immobilized cells of saccharomyces cerevisiae: Reuse of sodium alginate beads

This work studied the production of mead using second category honey and the immobilized cells of Saccharomyces cerevisiae in sodium alginate, with concentrations of 2% and 4%, and their reuse in five successive fermentations. The immobilized cells with 4% alginate beads were mechanically more stable and able to allow a greater number of reuses, making the process more economical. The fermentation’s consumption of sugars with free cells (control) and immobilized cells showed a similar profile, being completed close to 72 h, while the first use of immobilized cells finished at 96 h. The immobilized cells did not significantly influence some oenological parameters, such as the yield of the consumed sugars/ethanol, the alcohol content, the pH and the total acidity. There was a slight increase in the volatile acidity and a decrease in the production of SO2. The alginate concentrations did not significantly influence either the parameters used to monitor the fermentation process or the characteristics of the mead. Mead fermentations with immobilized cells showed the release of cells into the wort due to the disintegration of the beads, indicating that the matrix used for the yeast’s immobilization should be optimized, considering the mead production medium.The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2019).info:eu-repo/semantics/publishedVersio

Enzyme inhibitory potential of Ligustrum lucidum Aiton berries

Ligustrum lucidum Aiton and its berries have been used in Chinese traditional medicine for around two thousand years. In the present study, L. lucidium berries harvested in two regions of Portugal were studied. Haemolytic activity and inhibition of oxidative haemolysis as well as the enzyme inhibitory activities ( -amylase enzyme and acetylcholinesterase) were assessed. Results suggest that the different biological activities varied according to the region where samples were collected. Results demonstrated that the sample obtained from region R1 was the most efficient extract for all parameters evaluated, presenting the lowest values of IC50, 10.67 0.46 g/mL for the inhibition of erythrocyte oxidative haemolysis, 58.28 3.77 g/mL for the -amylase enzyme and 67.67 _+ 2.10 ug/mL for the acetylcholinesterase inhibition. L. Lucidum berries may be an interesting source of compounds for use in the development of the therapeutic armamentarium for diseases where enzymatic disruption is believed to play a roleinfo:eu-repo/semantics/publishedVersio

Conventional and emergent technologies for honey processing: A perspective on microbiological safety, bioactivity, and quality

Honey is a natural food of worldwide economic importance. Over the last decades, its potential for food, medical, cosmetical, and biotechnological applications has been widely explored. One of the major safety issues regarding such applications is its susceptibility to being contaminated with bacterial and fungi spores, including pathogenic ones, which may impose a hurdle to its consumption in a raw state. Another factor that makes this product particularly challenging relies on its high sugar content, which will lead to the formation of hydroxymethylfurfural (HMF) when heated (due to Maillard reactions). Moreover, honey’s bioactivity is known to be affected when it goes through thermal processing due to its unstable and thermolabile components. Therefore, proper food processing methodologies are of utmost importance not only to ensure honey safety but also to provide a high-quality product with low content of HMF and preserved biological properties. As so, emerging food processing technologies have been employed to improve the safety and quality of raw honey, allowing, for example, to reduce/avoid the exposure time to high processing temperatures, with consequent impact on the formation of HMF. This review aims to gather the literature available regarding the use of conventional and emergent food processing technologies (both thermal and nonthermal food processing technologies) for honey decontamination, preservation/enhancement of honey biological activity, as well as the sensorial attributes.Thanks are due to the University of Aveiro and FCT/MCT for the financial support for the LAQV/REQUIMTE and CIMO research Units (FCT UID/QUI/50006/2020 and UIDB/00690/2020, respectively) through national funds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement, and to the Portuguese NMR Network. The authors Hana Scepankova and Carlos A. Pinto would like to thank also FCT/MCT for the Ph.D. grants (SFRH/BD/88133/2012 and SFRH/BD/137036/2018).info:eu-repo/semantics/publishedVersio