Leticia M. Estevinho

Antioxidant and antimicrobial effects of phenolic compounds extracts of Northeast Portugal honey

Phenolic compounds of dark and clear honeys from Trás-os-Montes of Portugal were extracted with Amberlite XAD-2 and evaluated for their antioxidant and antimicrobial activities. The antioxidant effect was studied using the in vitro test capacity of scavenge the 2,2-diphenyl-1-picryhydrazyl (DPPH) free radical and of reducing power of iron (III)/ferricyanide complex. The antimicrobial activity was screened using three Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Staphylococcus lentus) and three Gram-negative bacteria (Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli). The results obtained from the partial identification of honey phenolic compounds by high-performance liquid chromatography with a diode array detector showed that p-hydroxibenzoic acid, cinnamic acid, naringenin, pinocembrin and chrysin are the phenolic compounds present in most of the samples analyzed. Antioxidant potential was dependent of honey extract concentration and the results showed that dark honey phenolic compounds had higher activity than the obtained from clear honey. In the biological assays, results showed that S. aureus were the most sensitive microrganisms and B. subtilis, S. lentus, K. pneumoniae and E. coli were each moderately sensitive to the antimicrobial activity of honey extracts. Nevertheless, no antimicrobial activity was observed in the test with P. aeruginosa.

Wild and commercial mushrooms as source of nutrients and nutraceuticals

In order to promote the use of mushrooms as source of nutrients and nutraceuticals, several experiments were performed in wild and commercial species. The analysis of nutrients included determination of proteins, fats, ash, and carbohydrates, particularly sugars by HPLC-RI. The analysis of nutraceuticals included determination of fatty acids by GC-FID, and other phytochemicals such as tocopherols, by HPLC-fluorescence, and phenolics, flavonoids, carotenoids and ascorbic acid, by spectrophotometer techniques. The antimicrobial properties of the mushrooms were also screened against fungi, Gram positive and Gram negative bacteria. The wild mushroom species proved to be less energetic than the commercial sp., containing higher contents of protein and lower fat concentrations. In general, commercial species seem to have higher concentrations of sugars, while wild sp. contained lower values of MUFA but also higher contents of PUFA. alpha-Tocopherol was detected in higher amounts in the wild species, while gamma-tocopherol was not found in these species. Wild mushrooms revealed a higher content of phenols but a lower content of ascorbic acid, than commercial mushrooms. There were no differences between the antimicrobial properties of wild and commercial species. The ongoing research will lead to a new generation of foods, and will certainly promote their nutritional and medicinal use.

Total phenols, antioxidant potential and antimicrobial activity of walnut (Juglans regia L.) green husks

The total phenols content and antioxidant and antimicrobial activities were studied in walnut (Juglans regia L.) green husks aqueous extracts of five different cultivars (Franquette, Mayette, Marbot, Mellanaise and Parisienne). Total phenols content was determined by colorimetric assay and their amount ranged from 32.61 mg/g of GAE (cv. Mellanaise) to 74.08 mg/g of GAE t (cv. Franquette). The antioxidant capacity of aqueous extracts was assessed through reducing power assay, scavenging effects on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and beta-carotene linoleate model system. A concentration-dependent antioxidative capacity was verified in reducing power and DPPH assays, with EC50 values lower than 1 mg/mL for all the tested extracts. The antimicrobial capacity was screened against Gram positive and Gram negative bacteria, and fungi. All the extracts inhibited the growth of Gram positive bacteria, being Staphylococcus aureus the most susceptible one with MIC of 0.1 mg/mL for all the extracts. The results obtained indicate that walnut green husks may become important in the obtainment of a noticeable source of compounds with health protective potential and antimicrobial activity.

Bioactive properties and chemical composition of six walnut (Juglans regia L.) cultivars

The chemical composition, antioxidant potential and antimicrobial activity were studied in six walnuts (Juglans regia L.) cultivars (cv. Franquette, Lara, Marbot, Mayette, Mellanaise and Parisienne) produced in Portugal. Concerning their chemical composition the main constituent of fruits was fat ranging from 78.83% to 82.14%, being the nutritional value around 720kcal per 100g of fruits. Linoleic acid was the major fatty acid reaching the maximum value of 60.30% (cv. Lara) followed by oleic, linolenic and palmitic acids. The aqueous extracts of walnut cultivars were investigated by the reducing power assay, the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and beta-carotene linoleate model system. All the walnut extracts exhibited antioxidant capacity in a concentration-dependent manner being the lowest EC(50) values obtained with extracts of cv. Parisienne. Their antimicrobial capacity was also checked against gram positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus) and gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae) and fungi (Candida albicans, Cryptococcus neoformans), revealing activity against the different tested microorganisms.

Chemical composition and biological properties of portuguese wild mushrooms: a comprehensive study

The chemical composition and biological properties of Portuguese wild mushrooms ( Cantharellus cibarius, Hypholoma fasciculare, Lepista nuda, Lycoperdon molle, Lycoperdon perlatum, Ramaria botrytis, Tricholoma acerbum) were evaluated in order to assess these products as sources of nutrients and nutraceuticals. The analyzed mushrooms contain very useful phytochemicals such as phenolics, tocopherols, ascorbic acid, and carotenoids. All of the species proved to have antioxidant activity (measured by four different methods), being more significant for R. botrytis (EC 50 values < 1 mg/mL). Lycoperdon species were resistant to all of the tested microorganisms, and the other samples revealed antimicrobial activity selectively against Gram-positive bacteria, with very low minimal inhibitory concentration, in some cases, even lower than the standard. The combination of bioactive compounds and rich nutritional composition (high contents in protein and carbohydrates, low content in fat with the precious contribution of unsaturated fatty acids and the absence of trans fatty acids) in the mushroom makes it a very special food.

Antioxidant properties, total phenols and pollen analysis of propolis samples from Portugal

Pollen analysis, total phenols content and antioxidant activity were studied for the first time in Portuguese propolis samples from Bornes and Fundão regions. Total phenols content was determined by colorimetric assay and their amount was of 329 mg/g of GAE in Bornes sample and 151 mg/g of GAE in Fundão propolis. The antioxidant capacity of propolis extracts was assessed through the scavenging effects on DPPH (2,2-diphenyl-1-picrylhydrazyl) and reducing power of iron (III)/ ferricyanide complex assays. A concentration-dependent antioxidative capacity was verified in DPPH and reducing power assays. Low values of EC50 on DPPH scavenging assay were obtained for Bornes and Fundão propolis (of 6.22 microg/mL and 52.00 microg/mL, respectively). For reducing power the values were 9.00 microg/mL, for Bornes propolis, and 55.00 microg/mL, for Fundão propolis. The high activity of propolis from Bornes could be related with their different pollen composition. The results obtained indicate that Portuguese propolis is an important source of total phenols showing antioxidant properties that could be beneficial for human health.

Physicochemical, microbiological and antimicrobial properties of commercial honeys from Portugal.

The present study aimed to characterize five commercial honeys available in the Portuguese market in respect to their floral origins, physicochemical parameters and microbial safety and commercial quality assessment. Pollen profile, colour, moisture content, ash, acidity, electrical conductivity, pH, reducing sugars, apparent sucrose and HMF were the parameters analysed in each honey sample. Aerobic mesophiles, moulds and yeasts, fecal coliforms and sulphite-reducing clostridia were the microbial contaminants of interest studied. The antimicrobial effect against four fermentative yeasts was determined. Concerning the physicochemical parameters, all honey samples were found to meet European Legislation (EC Directive 2001/110) for all parameters, except for HMF and apparent sucrose. Microbiologically, the commercial quality was considered good and all samples showed to be negative in respect to safety parameters. We also verified that the presence of honey differentially affected the growth of fermentative yeasts under study, depending on the type of yeast, but this growth was not significantly influenced by the type of honey used.

Honeybee-collected pollen from five Portuguese Natural Parks: Palynological origin, phenolic content, antioxidant properties and antimicrobial activity

The aim of this study was to determine the palynological origin, phenolic content, antioxidant and antimicrobial properties of pollen from five Portuguese Natural-Parks [Parque Nacional Peneda Gerês (PNPG); Parque Natural do Montesinho (PNM); Parque Natural do Alvão (PNA); Parque Natural da Serra da Estrela (PNSE) and Parque Natural do Douro Internacional (PNDI)]. Eight families were found in the mixture of bee pollen: Rosaceae, Cistaceae, Boraginaceae, Asteraceae, Fagaceae, Ericaeae, Myrtaceae and Fabaceae. The phenolic compounds content, determined by the Folin-Ciocalteu method, varied between 10.5 and 16.8 mg of gallic acid equivalents/g of extract (mg GAE/g) found in bee pollen from PNM and PNDI, respectively. The free radical scavenging measured showed the highest effective extract - PNM with EC(50) 2.16, followed by PND with 2.24 mg/mL. In the β-carotene bleaching assay the same behaviour as in the DPPH method was verified. We also verified that the presence of pollen differentially affected the growth of bacteria Gram-positive, Gram-negative and yeasts under study, depending this on the microorganism and the pollen used. This is an important study since, as far we know, it is the first time that Portuguese bee pollen from Natural Parks was studied, and their characterization can increase their economic value.

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. Pearsons correlation coefficients between polyphenols, flavonoids, antioxidant activity and antimicrobial activity were computed. It was also performed a discriminant analysis.

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 β-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.