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- Projeto BEEHAPPY: sanidade da abelha melífera (Apis mellifera L.) dos Açores: comparação dos padrões epidemiológicos num laboratório natural únicoPublication . Pinto, M. Alice; Henriques, Dora; Rodrigues, Pedro João; Miranda, Joachim; Martín-Hernández, Raquel; Amaral, Joana S.O BEEHAPPY é um projeto financiado pela Fundação para a Ciência e a Tecnologia (FCT), é coordenado pelo CIMO e envolve uma equipa de 16 especialistas multidisciplinares provenientes de várias instituições nacionais (CIMO-Centro de Investigação de Montanha, REQUIMTE-Rede de Química e Tecnologia, CEDRI -Centro de Investigação em Digitalização e Robótica Inteligente) e estrangeiras (SLU-Swedish University of Agricultural Sciences, CIAPA-IRIAF- Centro de Investigación Apícola y Agroambiental- Instituto Regional de Investigación y Desarrollo Agralimentario y Forestal, INIA-Peru- Instituto Nacional de Innovación Agraria). O BEEHAPPY centra-se nas populações de abelha dos Açores e tem como principais objectivos (i) o estudo da interação entre a abelha e as suas principais doenças (viroses e Nosemose) e (ii) o desenvolvimento de ferramentas com vista a identificar as populações de abelha (ferramenta informática) e avaliar a autenticidade do mel dos Açores (ferramenta molecular), o qual apresenta um grande potencial comercial. Para se atingirem tais objetivos, irão ser executadas 10 tarefas dedicadas à abelha (Tarefas 1, 4, 5, 6, 7, 8, 9), patogénios (Tarefas 1, 2, 3) e mel (Tarefa 10; Figura 1).
- Mass spectrometry‐based approaches to assess the botanical authenticity of dietary supplementsPublication . Grazina, Liliana; Mafra, Isabel; Monaci, Linda; Amaral, Joana S.Dietary supplements are legally considered foods despite frequently including medicinal plants as ingredients. Currently, the consumption of herbal dietary supplements, also known as plant food supplements (PFS), is increasing worldwide and some raw botanicals, highly demanded due to their popularity, extensive use, and/orwell-established pharmacological effects, have been attaining high prices in the international markets. Therefore, botanical adulteration for profit increase can occur along the whole PFS industry chain, from raw botanicals to plant extracts, until final PFS. Besides the substitution of highvalue species, unintentional mislabeling can happen in morphologically similar species. Both cases represent a health risk for consumers, prompting the development of numerous works to access botanical adulterations in PFS. Among different approaches proposed for this purpose, mass spectrometry (MS)-based techniques have often been reported as the most promising, particularly when hyphenated with chromatographic techniques. Thus, this review aims at describing an overview of the developments in this field, focusing on the applications of MS-based techniques to targeted and untargeted analysis to detect botanical adulterations in plant materials, extracts, and PFS.
- Chemical characterization, antioxidant and antimicrobial activities of winemaking industry by-productsPublication . Duarte, Cristina Nogueira; Dias, Maria Inês; Heleno, Sandrina A.; Santos-Buelga, Celestino; Dias, Rolando; Barros, Lillian; Amaral, Joana S.The exploitation of bioactive compounds trom agri-food by-products has been attracting an increasing interest from different industries within a circular economy context since several of compounds may have health properties or be used as natural colorants or preservatives. So far, several studies have been developed on the characterization of red wine grape pomace and its components (seeds, skins, and stems). However, less attention has been paid to other by-products, such as grape pomace from white wine, the residues after distillation, wine lees, and diatomaceous earth, the latter used in the filtration of wine and corresponding to about 250 tons/year of waste in Portugal. In this work, samples of these by-products were evaluated for their chemical composition by HPLC-DAD-ESI-Msn and bioactivities (antioxidant activity by TBARS, DPPH, and reducing power assays, and antimicrobial activity against eight bacteria and two fungi). Fifteen non-anthocyanin phenolic compounds were found, including 5 phenolic acids, 4 flavan-3-0Is, 2 0- glycosylated flavanols, 3 flavanol aglycones, and one unknown. In the red wine byproducts 12 anthocyanins were detected, most being malvidin derivatives. Wine lees and white grape pomace before distillation presented the highest contents of phenolic compounds. Ali samples showed antibacterial and antifunga! activity against most of the tested microorganisms, with red and white grape pomace and diatomaceous earth having the best bacteriostatic activity, while the lees stood out against fungi. Ali samples showed promising antioxidant capacity, with very good results obtained on TBARS for the white pomace after distillation and diatomaceous earth. Overall, the results show that besides grape pomace other wine industry byproducts are also good sources of bioactive compounds with high potential for exploitation.
- Chemical composition and biological activity of Commelina erecta: an edible wild plant consumed in BrazilPublication . Cavichi, Lucas V.; Liberal, Ângela; Dias, Maria Inês; Mandim, Filipa; Pinela, José; Kostić, Marina; Soković, Marina; Kalschne, Daneysa Lahis; Fernandes, Ângela; Canan, Cristiane; Barros, Lillian; Amaral, Joana S.In recent years, the interest in products of natural origin has boosted the exploitation and use of plants as food and sources of bioactive compounds, especially wild plants widely used in different cultures for several purposes. Commelina erecta is a wild edible plant (WEP) traditionally used as food and medicine, about which few studies exist. Thus, this study aimed at enhancing the knowledge about its nutritional, chemical and bioactive profile, considering different plant parts and development stages, in order to increase its inclusion in the diet of South American communities. The nutritional profile was found to be similar to other WEP frequently consumed in Brazil. Thirteen phenolic compounds (HPLC-DAD-ESI/MS) were tentatively identified, with apigenin, luteolin and quercetin derivatives being the most abundant. Fructose and oxalic acid were the major sugar and organic acid, respectively, in the aerial parts of C. erecta, and four isoforms of tocopherols were also identified. Regarding the plant’s antioxidant activity, the EC50 values varied between 18.4 and 1060 µg/mL in the inhibition of lipid peroxidation assay (TBARS) and between 53 and 115 µg/mL in the oxidative haemolysis inhibition (OxHLIA) assay. The hydroethanolic extract obtained from stems at the flowering stage also presented anti-inflammatory activity. In general, all the extracts evidenced promising antimicrobial activity. Altogether, these results reinforce the traditional use of this plant species as food and medicine to support the diet of needier populations and also promote food sovereignty and sustainability.
- DNA-based methods as a powerful tool for the entomological authentication of honeyPublication . Honrado, Mónica; Quaresma, Andreia; Lopes, Ana; Pinto, M. Alice; Henriques, Dora; Amaral, Joana S.Honey is a food widely consumed worldwide and much appreciated for its nutritional, organoleptic and health properties. However, it is also considered one of the food products most prone to be adulterated in the EU. Up until now, honey authenticity addressed mainly the issue of sugars addition and botanical origin. Still, increased attention has recently been paid to honey entomological origin as it also relates to its geographical origin since honeybees carrying mitochondrial DNA (mtDNA) of distinct ancestries can be found across Europe. While in Portugal mtDNA of the autochthonous subspecies Apis mellifera iberiensis belongs to the African (A) lineage, in the northeastern part of Iberia African mitotypes are replaced by mitotypes of western European (M-lineage) ancestry. The native distribution of the M-lineage A. m. mellifera expands from the Pyrenees to Scandinavia and from the British Isles to the Ural Mountains while the C-lineage A. m. ligustica and A. m. carnica subspecies are naturally found in the Apennine and Balkan peninsulas, respectively [1]. Also, certain honeys holding the protected designation of origin (PDO) label should be produced by autochthonous A. mellifera subspecies, as mentioned in their EU geographical indications register.
- Extraction of essential oils from the residues of two shrub species aiming for their revalorization: chemical characterization and antioxidant, antimicrobial and cytotoxic activitiesPublication . Xavier, Virginie; Heleno, Sandrina A.; Prieto Lage, Miguel A.; Amaral, Joana S.; Mediavilla Ruiz, Irene; Esteban Pascual, Luis Saul; Ferreira, Isabel C.F.R.; Barros, LillianIn recent years, essential oils have been subject of research for their bioactive properties, such as antimicrobial, fungitoxic, anti-viral, anti-inflammatory and antioxidant activities. Owing to these properties they are potentially interesting for diverse industries including the food industry since one of its main problems concerns microbes and associated toxins that are responsible for food spoilage. Although the application of essential oils in the food industry may have some limitations, such as impact on the organoleptic properties and low solubility, different delivery strategies such as nanoencapsulation, active packaging and coatings are promising technologies that may overcome these issues without compromising nutritional properties in food systems [1]. In this view, increased knowledge on the composition and activity of different essential oils is needed, particularly regarding novel potential sources of essential oils such as agricultural wastes or species grown in marginal lands, on a perspective of circular economy. Therefore, in the scope of the BeonNAT project, biomass from different tree and shrub species are being screened as possible sources of essential oils and respective bioactivity evaluated. In this work, the essential oil extracted by steam distillation from the branches (>20mm) of two shrub species grown in Spain, Juniperus communis L. and Cistus ladanifer L., was characterized for their chemical composition by gas chromatography coupled with mass spectrometry (GC-MS) as well as for their antioxidant, antimicrobial, antiinflammatory and cytotoxic activities. GC-MS analysis allowed the identification of 98.1% of compounds in J. communis EO, corresponding to a total of 63 identified compounds, alfa-pinene being the major compound (35.1%), followed by limonene (15.0%), sabinene (6.7%), cis-tujopsene (8.0%), beta-myrcene (3.2%) and beta-caryophyllene (3.5%). In general, the chemical composition is in agreement with that of juniper berry EO, defined in the European Pharmacopoeia and ISO 8897 standard, except for limonene (15.0%) which was slightly higher than the defined range (Eur. Ph of 2-12% and ISO standard of 2-8%). For C. ladanifer EO, 61 compounds were identified corresponding to 92.8% of the total compounds, with viridiflorol being the main compound (24.0%), followed by alfa-pinene (19.3%), ledol (6.9%), camphene (6.7%) and bornyl acetate (5.0%), which is in good agreement with previous data [2]. Both oils showed potential against the panel of bacteria selected according to their importance in public health and foodborne diseases, highlighting the rock-rose EO that showed interesting activity against Escherichia coli, Morganella morganii, Pseudomonas aeruginosa, Enterococcus faecalis, Listeria monocytogenes and methicillin resistant Staphylococcus aureus in a concentration range of 0.039-2.5%(v/v). Regarding the antioxidant activity, both oils showed promising results, with EC50 values of 1.35 ± 0.19 mg/mL and 1.30 ± 0.07 mg/mL in the reducing power assay and 68% and 83% inhibition of oxidation according to the cellular antioxidant activity assay, for J. communis and C. ladanifer, respectively. The essential oils showed antiinflammatory (IC50 of 24±1 µg/mL and 21±2 µg/mL for juniper and rock-rose-rose, respectively) and cytotoxic activity, with the best results obtained with the rock-rose EO in the inhibition of stomach-AGS, colon-CaCo, breast-MCF-7 and lung-NCI-H460 cancer cell lines (GI50 between 47±5 µg/mL and 58±1 µg/mL). Juniper EO did not evidenced cytotoxicity in non-tumoral Vero cells at the highest tested concentration (400 µg/mL) wich can be an indicator of its safety. Overall, the results demonstrated that shrubs biomass can be a source of EO with similar composition to that reported for respective berries and leaves. The EOs showed interesting antibacterial and antioxidant activity thus being potential candidates for further studies on their safety and potential application in food systems.
- COI Metabarcoding as a Novel Approach for Assessing the Honey Bee Source of European HoneyPublication . Honrado, Mónica; Quaresma, Andreia; Henriques, Dora; Pinto, M. Alice; Amaral, Joana S.Honey is a widely consumed food product frequently subjected to adulteration, with the mislabeling of its botanical or geographical origin being one of the most common practices. Determining the entomological origin of honey is particularly challenging but of high relevance for ensuring its authenticity, especially for products with protected designation of origin (PDO) labels. This study presents a novel DNA metabarcoding approach targeting a highly informative 406 bp fragment of the cytochrome c oxidase I (COI) gene to differentiate among the three major mitochondrial lineages (A, M, and C) of honey bees (Apis mellifera L.) native to Europe. The target region was selected based on the calculated fixation index (FST), which is frequently used in Population Genetics as a measure of differentiation between populations. The approach was validated with 11 honey samples of known entomological origin and applied to 44 commercial honeys from 13 countries. The approach demonstrated high sensitivity, accurately identifying the entomological origin of honey, including samples produced by honey bees of varying ancestries, which could not be resolved by previous methods based on real-time PCR coupled with high-resolution melting (PCR-HRM) analysis. The results demonstrate the effectiveness of COI metabarcoding in verifying honey authenticity and highlight the predominance of C-lineage honey bees in the production of commercial honeys from northwestern Europe. This finding suggests a limited presence of the native M-lineage ancestry, underscoring the need for conservation efforts.
- Evaluation of the phenolic profile of Castanea sativa mill. By-products and their antioxidant and antimicrobial activity against multiresistant bacteriaPublication . Silva, Vanessa; Falco, Virgílio; Dias, Maria Inês; Barros, Lillian; Silva, Adriana; Capita, Rosa; Alonso-Calleja, Carlos; Amaral, Joana S.; Igrejas, Gilberto; Ferreira, Isabel C.F.R.; Poeta, PatríciaThe chestnut industry generates a large amount of by-products. These agro-industrial wastes have been described as potential sources of phenolic compounds with high bioactive potential. Therefore, we aimed to extract the phenolic compounds from chestnut by-products and assess their antioxidant potential and evaluate their antimicrobial activity against multidrug resistant bacteria. The individual phenolic compounds in the ethanolic extracts of chestnut shell, inner shell, bur, and leaves were characterized by HPLC-DAD/electrospray ionization (ESI)-MS. The antioxidant properties were determined by DPPH and ABTS assays. The minimum inhibitory concentration (MIC) and the antimicrobial susceptibility was performed using the Kirby–Bauer disc di usion method against 10 bacterial strains. The major phenolic compounds identified in the extracts were trigalloyl-HHDP-glucose, gallic acid, quercetin, and myricetin glycoside derivatives. All chestnut by-products presented promising antioxidant activity in both assays, with leaf samples the ones presenting the highest antioxidant capacity. The inner shell’s extract was e ective against all Gram-positive and two Gram-negative bacteria; nevertheless, all extracts showed antibacterial activity. Staphylococcus epidermidis showed susceptibility to all extracts while none of the extracts was able to suppress the growth of Escherichia coli and Salmonella enteritidis. Chestnut by-products are a source of phenolic compounds with prominent antioxidant and antimicrobial activities. Nevertheless, further studies should be conducted to assess the correlation between phenolic compounds and the bioactivities obtained.
- Development of a loop-mediated isothermal amplification assay for the rapid detection of Styphnolobium japonicum (L.) Schott as an adulterant of Ginkgo biloba (L.)Publication . Rodrigues, Vânia; Honrado, Mónica; Santos, Joana; Pinto, M. Alice; Amaral, Joana S.; Rodrigues, VâniaSpecies adulteration is a concern in herbal products, especially when plant substitutes of lower economic value replace valuable botanicals. Styphnolobium japonicum is well known as a potential adulterant of Ginkgo biloba, which is one of the most demanded medicinal plants due to its wide use in pharmaceuticals, food supplements, and traditional medicine. Despite bearing some resemblance to ginkgo's flavonol composition, S. japonicum lacks many of G. biloba's desired therapeutic properties. To prevent adulteration practices, it is crucial to implement rigorous quality control measures, including fast and simple diagnostic tools that can be used on-field. Purpose: This study aims to develop for the first time a species-specific loop-mediated isothermal amplification (LAMP) method for the fast identification of S. japonicum in ginkgo-containing products. Methods: A set of four specific primers (SjF3, SjB3, SjFIP, and SjBIP) and loop primers (SjLF and SjLB) were designed for a LAMP based assay using the 5.8S partial sequence and the internal transcribed spacer 2 of nuclear ribosomal DNA of S. japonicum. Results: The successful amplification of the LAMP assay was inspected through visual detection, with the highest intensity recorded at the optimal conditions set at 68 °C for 40 min. The primers showed high specificity and were able to accurately discriminate S. japonicum from G. biloba and 49 other species of medicinal plants. Furthermore, the proposed LAMP assay proved to be fast, selective, and highly sensitive, as demonstrated by the absolute and relative limits of detection, which were reached at 0.5 pg for S. japonicum DNA and 0.01 % S. japonicum in G. biloba, respectively. Conclusions: This novel approach allows easy identification and discrimination of S. japonicum as a potential adulterant of G. biloba, thus being a useful tool for quality control. Compared to chromatographic or PCR-based methods, the assay proved to be fast, sensitive and did not require expensive equipment, thus offering the possibly usage in field analysis.
- Chemical characterization and bioactive properties of wine lees and diatomaceous earth towards the valorization of underexploited residues as potential cosmeceuticalsPublication . Duarte, Cristina Nogueira; Taofiq, Oludemi; Dias, Maria Inês; Heleno, Sandrina A.; Santos-Buelga, Celestino; Barros, Lillian; Amaral, Joana S.Annually, wine production is responsible for generating large quantities of residues, which are frequently disposed of and not valorized. So far, different studies have been conducted on grape pomace, yet less attention has been paid to other residues, such as wine lees and diatomaceous earth used in wine filtration. In this context, this study aimed to evaluate and compare the phenolic profile of these underexploited winemaking residues and assess their biological potential based on their antioxidant, antimicrobial, cytotoxic, and anti-aging activities (inhibition of tyrosinase and collagenase). Twenty-nine phenolic compounds, including twelve anthocyanins, were tentatively identified in the residues, with red grape pomace showing the highest diversity of compounds. The diatomaceous earth presented the highest content of non-anthocyanin phenolic compounds, being particularly rich in flavan-3-ols and myricetin-O-hexoside, and also presenting two anthocyanins. This sample also showed a high antioxidant activity, evidencing the best result in the reducing power assay. The red wine lees extract, despite showing a low content of phenolic compounds and less antioxidant activity, presented the highest inhibition capacity of bacteria growth. The extracts did not exhibit cytotoxicity against keratinocyte (up to 400 mu g/mL) and fibroblast (up to 100 mu g/mL) skin cell lines. However, the capacity of inhibiting tyrosinase and collagenase was low for the lees and diatomaceous earth, contrary to the grape pomace, seeds, and skins extracts that showed promising results, evidencing its potential as a cosmeceutical. Overall, this study highlights for the first time the potential of diatomaceous earth, an underexploited winemaking waste, in the obtention of added-value extracts and/or ingredients for cosmetic industry.