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- Tools to develop dairy ingredients: bioactive and preservative purposesPublication . Khouja, Dora; Fernandes, Filipa Alexandra; Carocho, Márcio; Calhelha, Ricardo C.; Rodrigues, Paula; Zaghdoudi, Khalil; Barros, Lillian; Heleno, Sandrina A.; Ferreira, Isabel C.F.R.Phytosterols can be ingested and consequently absorbed by the human body, leading to a direct absorption competition with cholesterol, thus reducing its absorption [1]. Mushrooms are described as having several bioactive components, such as mycosterols (ergosterol), which, similarly to phytosterols, exhibit a strong hypocholesterolemic potential [2]. Thus, the objective of this work was to obtain mycosterol enriched extracts from A. bisporus L. bio-waste for further incorporation in sheep cottage cheese, in order to develop a functional food with hypocholesterolemic effects. Sheep cottage cheeses with pure ergosterol, and a control cheese with no incorporation were prepared. The extracts were obtained by ultrasound assisted extraction (UAE) and the identification and quantification of their compounds was achieved through HPLC-UV. The toxicity and hypocholesterolemic activity were sought through a CaCo2 cell line. In terms of analysis, the nutritional value was analyzed according to AOAC procedures, the physical parameters included texture, external colour and water activity, and the microbial load (total aerobic mesophiles, enterobacteria, Psychrotrophic bacteria, yeasts, molds and Staphylococcus aureus) was also analyzed along a shelf life of 9 days according to the International Organization for Standardization (ISO) 6887-1:2003 [3,4]. No significant differences were verified for the nutritional parameters. The color of the cheeses incorporated with the extract hinted towards a browner tone when compared to the other two samples (cottage cheese with ergosterol and control cottage cheese). A dominance of palmitic acid followed by oleic and capric acids was detected, being the saturated fatty acids the predominant ones. Lactose and glucose were the two detected soluble sugars; being glucose only observed in cheese incorporated with A. bisporus, probably present in the extract. Also, the incorporations did not cause any significant alterations to normal microbial growth. Despite an increase of enterobacteria in the cheeses with the incorporation of the two agents, this increase was also verified for the control cottage cheese. CaCo2 cells absorbed 43.89% of cholesterol from the control cheese, while the cheese with pure ergosterol reduced cholesterol absorption by approximately 21.1%, and cheese with A. bisporus by approximately 30.24%. These results highlight the capacity of ergosterol to reduce the absorption of cholesterol, being an interesting candidate for the development of functional foods.
- Perfil físico e estabilidade de fiambres conservados com um revestimento naturalPublication . Fernandes, Filipa A.; Carocho, Márcio; Heleno, Sandrina A.; Rodrigues, Paula; Barros, Lillian; Ferreira, Isabel C.F.R.Grande parte do plástico produzido é aplicado na conservação de alimentos, no entanto, há relatos que descrevem a migração de monómeros residuais para as matrizes alimentares, provocando efeitos nocivos para a saúde. O objetivo deste trabalho consistiu no desenvolvimento de um revestimento conservante comestível, à base de ingredientes naturais. Este revestimento foi aplicado em fiambre, sendo a sua estabilidade também comprovada através de estudos de inibição do crescimento microbiano, e avaliação de parâmetros físico (textura, humidade e cor) durante 15 dias. Todos os ensaios foram comparados com fiambre revestido com película aderente (método tradicional de embalamento), e ainda sem revestimento (controlo). Relativamente à textura, constatou-se que com o tempo a dureza das amostras sujeitas ao armazenamento com o revestimento desenvolvido aumentou; já para a humidade, o revestimento natural não conseguiu manter a mesma percentagem de água no fiambre comparativamente à película aderente, no entanto, obtiveram-se melhores resultados que na amostra sem revestimento. Para o parâmetro da cor, constatou-se que as amostras com o revestimento natural preservaram melhor a cor ao longo do tempo. De uma forma geral, em termos de carga microbiana, verificou-se que nas etapas finais de armazenamento o fiambre com o revestimento natural revelou capacidade de inibição do crescimento microbiano, enquanto que o fiambre com a película aderente favoreceu o crescimento dos mesmos pela humidade criada entre o fiambre e a película. Os resultados obtidos neste estudo mostram que a solução conservante apresenta um elevado potencial na conservação de fiambre, podendo ser também aplicado noutros produtos, sendo considerada uma solução disruptiva na conservação de alimentos.
- Nutritional enrichment of "Económicos" through the incorporation of chestnut flourPublication . Fernandes, Filipa Alexandra; Pedrosa, Mariana C.; Ueda, Jonata Massao; Ferreira, Elisabete; Heleno, Sandrina A.; Carocho, Márcio; Prieto Lage, Miguel A.; Ferreira, Isabel C.F.R.; Barros, Lillian“Económicos” are traditional Portuguese cakes from the region of Trás-os-Montes, made from inexpensive ingredients, such as flour, sugar, margarine, olive oil, eggs and brandy. Although widely consumed, the combination of ingredients does not provide any significant nutritional benefit [1]. The species Castanea sativa Mill. represents a valuable natural resource in Northern Portugal, however not all fruits are commercialized due to fruit calibre requirements [2]. There are several studies that demonstrate the nutritional importance of chestnuts [3], thus, increasing the nutritional value of "Económicos" with chestnut, which could be an opportunity to improve these cakes. Therefore, in the present work, “económicos were incorporated with chestnut flour and analyzed over 32 days of storage time. The nutritional profile analysis, including proteins, crude fat, moisture, ash, fibers, carbohydrates and energy were carried out following the AOAC official methods [4]. Soluble sugars were determined using HPLC-RI, organic acids by UPLC-DAD and fatty acids by GC-FID. Two batches of “Económicos” were used, one with 9% of chestnut flour and the other one without any incorporation, and a two-way analysis of variance was applied (storage time and type of flour as the main factors). This traditional Portuguese pastry product showed a relatively low moisture content (13.1±0.8 g/100 g fw), with carbohydrates being the macronutrient present in the highest quantity (57±2 g/100 g fw), followed by proteins (7.4±0.5 g/100 g fw), offering an energy value of 417±8 kcal/100 g fw. Sucrose (28±4 g/100 g fw) was the only feee sugar present, while two organic acids were identified, with oxalic acid (0.04±0.01 g/100 g fw) standing out. Fourteen fatty acids were also found, with greater abundance of butyric (C4:0 – 11.4±1.6 g/100 g fw) and linoleic (C18:2n6c – 1.9±0.4 g/100 g fw) acids. Regarding the nutritional value of "Económicos", for moisture and ash, the interaction of both factors was significant, which did not allow for an individual classification of these factors; there was a statistically higher amount of crude fat in the control samples, but lower carbohydrates, meaning that the flour significantly influences the content of these two nutrients. Regarding the content in proteins, fibers and energy, no significant differences were verified. For the sucrose content and for the individual and total content of organic acids, a significant interaction between both factors was detected, although through the estimated marginal means, a higher amount of all these molecules was detected in the cakes with chestnut flour. Finally, concerning the fatty acid composition, the interaction of the two factors was significant, not allowing an individual classification. It could be concluded that the chestnut flour does not significantly influence the composition of the traditional "Económicos" at 9% of total flour, contributing to a market diversification and valorisation of this traditional product, while also providing higher nourishment and bioactive molecules to the traditional cakes. Sensorial analysis will be performed on the developed Económicos, as well as the bioactive analysis to understand other positive effects that chestnut flour can add to the “Económicos”.
- Nutraceutical formulations: addition of natural ingredients as preservativesPublication . Fernandes, Filipa Alexandra; Carocho, Márcio; Heleno, Sandrina A.; Rodrigues, Paula; Dias, Maria Inês; Pinela, José; Prieto Lage, Miguel A.; Simal-Gandara, Jesus; Barros, Lillian; Ferreira, Isabel C.F.R.The growing concern about the amount of chemicals added to food products has driven consumer preference towards products with less food additives, and, when possible, only with natural ones. Thus, the main objective of this work was the study of natural ingredients of recognized bioactivity (quercetin, citric acid, dry chestnut flower, and chestnut flower extract) as preservatives for a commercial nutraceutical formulation based on Aloe arborescens Mill. in substitution of the used artificial preservative (sodium benzoate). These formulations were characterized in terms of their centesimal composition, individual profile in free sugars (HPLC-RI), organic acids (UFLC-PDA), fatty acids (GC-FID) and phenolic compounds (HPLC-DAD-ESI/MS); and also in terms of their antioxidant activity (TBARS and OxHLIA). The different formulations were contaminated to understand their influence on microbial growth, having been contaminated with different microorganisms (Escherichia coli, Bacillus cereus, Aspergillus parasiticus and Zygosaccharomyces rouxii) and subsequently analyzed for the microbial loads over 45 days. All results were compared to formulations with sodium benzoate and a to negative control without any preservative. The most abundant nutrients were carbohydrates, with values between 98 and 99 g/100 g of fresh weight, followed by proteins. All nutraceutical preparations showed moisture ranging from 66 to 71 %, the fat content was 0.04 to 0.06 g/100 g, averaging an energy value of 397 kcal/100 g. Different minerals were found, with calcium being the most abundant with an average value of 105 mg/100 g fw. Three soluble sugars and two organic acids were identified, with fructose and malic acid standing out. Seventeen fatty acids were quantified with the higher abundance of palmitic (C16:0) and stearic (C18:0) acids. Regarding the phenolic compounds, the presence of 10 phenolic compounds was evident, especially alloenin (flavonoid) as the major compound. Regarding the antioxidant activity, higher activity was found in formulations containing quercetin, showing EC50 values of 0.04 µg/mL (TBARS) and IC50 of 139 µg/mL (OxHLIA - Δt = 60 min). As for the microbial stability of the formulations over the 45 days, they proved to be effective in inhibiting the growth of microorganisms, with a higher reduction in colonies when compared to the formulations with sodium benzoate, being the best of them the ones preserved with citric acid and chestnut flower. These results showed that the studied natural preservatives have little to no influence on the nutritional and chemical composition of the nutraceutical formulations, but have proved to be good alternatives to sodium benzoate in terms of their preservation.
- Valorização de biorresíduos da produção de sumo de laranja: extração de ácido cítricoPublication . Fernandes, Filipa Alexandra; Pinela, José; Carocho, Márcio; Prieto Lage, Miguel A.; Heleno, Sandrina A.; Barros, LillianA laranja é um dos frutos mais consumidos mundialmente, sendo uma parcela considerável destinada à produção de sumos, operação industrial que gera uma grande quantidade de resíduos (polpa, cascas e sementes). Uma parte destes biorresíduos é utilizada na produção de ração animal; contudo, uma quantidade significativa ainda é depositada em aterros sanitários, gerando um impacto ambiental considerável [1,2,3]. As atividades biológicas da casca de laranja, como os potenciais antimicrobianos e antioxidantes foram já comprovados por diversos estudos; no entanto, são escassos os trabalhos sobre técnicas de extração eficientes para obtenção de compostos bioativos a partir destes subprodutos [4]. Assim, o principal objetivo deste trabalho foi a valorização das cascas de laranja, promovendo a sua reciclagem em ingredientes naturais, com recurso à otimização de técnicas de extração para obtenção de extratos ricos em ácido cítrico (agente antimicrobiano, acidificante e aromatizante). Para isso, foi selecionada a técnica de extração assistida por ultrassons, aplicando um delineamento experimental consistindo num desenho central composto rotativo usando a metodologia de superfície de resposta para otimização, o qual combinou cinco níveis das variáveis independentes: percentagem de etanol (0-100%), tempo (2-45 min) e intensidade ultrassónica (50-500 W). Os teores de ácido cítrico foram determinados por UFLC-PDA e usados como resposta da otimização. O software Design-Expert foi usado para modelação e análise gráfica dos dados. Os modelos teóricos desenvolvidos foram ajustados com sucesso aos dados experimentais, validados estatisticamente com base em altos valores de F e R2 e usados para prever as condições ótimas que maximizam a recuperação de ácido cítrico a partir da casca de laranja. Utilizando as condições ideais de processamento obtidas, nomeadamente intensidade ultrassónica média (119,2 W), tempo de extração médio (35,5 min.) e proporção de etanol baixa (0 %, v/v), foi prevista a recuperação de 6,4 g de ácido cítrico por 100 g de casca de laranja desidratada. A extração assistida por ultrassons revelou-se uma técnica promissora para a obtenção de ácido cítrico a partir destes biorresíduos de laranja, tornando possível a sua valorização através da obtenção de moléculas de elevado valor acrescentado com potencial aplicação em várias indústrias, como a alimentar.
- Recovery of citric acid from Citrus peels: ultrasound-assisted extraction optimized by response surface methodologyPublication . Fernandes, Filipa Alexandra; Heleno, Sandrina A.; Pinela, José; Carocho, Márcio; Prieto Lage, Miguel A.; Ferreira, Isabel C.F.R.; Barros, LillianThe production of citrus juice generates a large quantity of by-products, which are often discarded or used for animal feed. However, several studies have shown its richness in valuable compounds, namely organic acids. Thus, this work intended to valorize orange and lime peels as renewable sources of citric acid. An experimental design combining five levels of the independent variables time (2–45 min), ultrasonic power (50–500 W), and ethanol proportion (0–100%) was implemented and response surface methodology (RSM) was applied to optimize the extraction process. The UPLC-PDA analysis showed that orange peel presented a higher citric acid content than lime. For lime and orange peels, the extraction yield was maximized by sonicating at low power for 5.8 or 35.5 min, using a low ethanol proportion or only water as a solvent, respectively. Overall, optimal UAE conditions were defined for the sustainable extraction of citric acid from citrus by-products, thus contributing to its valorization and upcycling into natural food ingredients.
- Applying Response Surface Methodology to Phenolic Compounds from Arbutus unedo: Case Studies with Ultrasound and Dynamic Maceration ExtractionPublication . Pedrosa, Mariana C.; Derbassi, Nabila Ben; Fernandes, Filipa Alexandra; Dias, Maria Inês; Heleno, Sandrina A.; Carocho, Márcio; Barros, LillianArbutus unedo L. is a small Mediterranean plant, being fgound mostly in southern Europe, north eastern Africa, Ireland, Palestine and the Canary Islands, and have been studied for its chemical composition.
- Effect of natural preservatives on the nutritional profile, chemical composition, bioactivity and stability of a nutraceutical preparation of aloe arborescensPublication . Fernandes, Filipa A.; Carocho, Márcio; Heleno, Sandrina A.; Rodrigues, Paula; Dias, Maria Inês; Pinela, José; Prieto Lage, Miguel A.; Simal-Gandara, Jesus; Barros, Lillian; Ferreira, Isabel C.F.R.Citric acid, quercetin, dried chestnut flowers and an aqueous extract of chestnut flowers were screened as candidates for preserving a drinkable nutraceutical preparation for 45 days. The assays encompassed antioxidant and antimicrobial activities, nutritional and chemical profiles, and individual profiles of fatty acids and mineral composition, all of which in comparison with a sodium benzoate, a synthetic preservative. The centesimal composition of the nutraceutical formulation was mainly composed of carbohydrates, followed by proteins and fat, with moisture levels between 66% and 71%. Palmitic and stearic acid were the most abundant fatty acids, while calcium and magnesium where the minerals in higher amount. Anthroquinones, followed by flavonoids where the most abundant groups of phenolic compounds. In terms of the preserving e ects of the extracts, the chestnut flowers and the citric acid were the most e ective natural preservatives, which better preserved phenolic compounds. Furthermore, these two ingredients also revealed the strongest capacity to control the microbial growth in the formulation by inhibiting the growth of food contaminants. In general, these ingredients revealed higher preservation capacity than sodium benzoate, while not altering the nutritional and fatty acid profile. The chestnut flowers and citric acid could be used to preserve foods, food supplements, and nutraceutical formulations after passing the required regulatory procedures for food additives.
- Dragon fruits as an alternative source of colorants: evaluation of antibacterial activity and colouring capacityPublication . Roriz, Custódio Lobo; Dias, Maria Inês; Reis, Filipa S.; Pires, Tânia C.S.; Alves, Maria José; Fernandes, Filipa A.; Morales, Patricia; Barros, Lillian; Ferreira, Isabel C.F.R.Betalains are a group of secondary metabolites named chromoalkaloids that are synthesized from tyrosine. These compounds have gained some attention in the last few years mainly due to their interesting bioactive potential, namely antioxidant, antimicrobial, and other bioactive properties [1]. Their strong and vibrant colours are also one of the characteristics by which these compounds have gained visibility in the food and pharmaceutical industries [2]. Betalains can be divided in two groups regarding the colour range: betaxanthins in the orange to yellow range, and betacyanins in the purple to pink range. Thereby, these compounds can be used as natural colouring agents, providing alternatives to the massively used artificial counterparts [3]. Although there are already some natural options in the market, these are not enough to meet the needs of the food industry, due to the growing concern of consumers regarding what they eat. Thus, the objectives of this work were to: i) obtain bioactive extracts and with strong colouring capacity from the epicarp of two pitaya varieties, white-fleshed pitaya Hylocereus undatus (Haworth) Britton & Rose (WFP) and red-fleshed pitaya Hylocereus costaricensis (F.A.C.Weber) Britton & Rose (RFP); ii) chemically characterize the betalains’ content through HPLC-DAD-ESI/MS; iii) evaluate the bioactive properties of the extracts, namely antimicrobial activity. The extracts were obtained through a dynamic maceration assisted by heat, and the betacyanins’ profile was characterized by HPLC-DAD-ESI/MS. The antibacterial capacity was determined against a panel of Gram-negative and Gram-positive bacteria using the colorimetric method of rapid detection with p-iodonitrotetrazolium chloride (INT), and the responses obtained were expressed as minimum inhibitory (MIC) and minimum bactericidal (MBC) concentrations. By the chromatographic analysis of the extracts it was possible to identify six compounds in both samples. For the WFP the major compounds identified were 6'-O-malonylbetanin (phylocactin), followed by 4'-malonyl-betanin, while for the RFP the major compound found was phylocactin. In the evaluation of the antibacterial activity, both extracts showed MIC and MBC values that ranged from 10 to 20 mg/mL. Gram-positive bacteria showed to be more susceptible to both extracts then the negative strains. Further studies need to be conducted to better understand the correlation between the bioactive potential and the betacyanins’ composition. Nevertheless, these natural matrixes proved to be viable alternatives for obtaining colouring extracts with antimicrobial properties.
- Optimization of the ultrasound-assisted extraction process to obtain a citric acid-rich extract from citrus peelsPublication . Fernandes, Filipa Alexandra; Heleno, Sandrina A.; Carocho, Márcio; Pinela, José; Prieto Lage, Miguel A.; Ferreira, Isabel C.F.R.; Barros, LillianCitrus fruits belong to the most consumed group of fruits worldwide and, in most cases, their peels are discarded as by-product, used for the manufacture of animal feed or simply disposed of as waste.1,2 However, scientific studies have shown that these by-products are rich in high added-value compounds, including phenolic compounds and organic acids.1,3 Fernandes et al. (2020) demonstrated the effectiveness of citric acid as a natural preservative in a nutraceutical product, concluding that it has a strong potential to be used in the food industry as an alternative to artificial preservatives.4 The objective of this work was to optimize the ultrasound-assisted extraction of citric acid from citrus (orange, lemon and lime) peels using the response surface methodology (RSM). Orange, lemon, and lime peels were frozen, lyophilized and reduced to ~20 mesh particle size. For the extraction optimization, a central composite design coupled with RSM was implemented by combining five levels of the independent variable’s ethanol concentration (0–100%, v/v), time (2–45 min) and ultrasonic power (50–500 W). The UAE was performed using a sonicator system with a titanium probe. The citrus peel samples (~1 g) were placed in a beaker with 50 mL of solvent and processed according to the experimental design at room temperature. The levels of citric acid were determined by UFLC-PDA and used as a response variable. Design-Expert software was used for regression and graphical analysis of the data. The developed theoretical models were successfully fitted to the experimental data, statistically validated based on high F-values and R2 values and used to predict the optimal conditions that maximize the recovery of citric acid from the citrus peels. Citric acid was identified and quantified in two by-products, orange and lime peels, with the optimal processing conditions yielding about 10 and 8 g of this compound per 100 g of dry peels, respectively. Thus, to obtain a higher extraction yield, the citric acid from the orange peels must be extracted with short times, high ultrasonic powers and the extraction solvent must contain a low ethanol concentration. For lemon peels, citric acid was not detected in some runs of the experimental design, so it was not possible to construct a predictive model. It was concluded that, with this extraction technique and applied methodology, citrus peels, particularly of orange, are a good source of citric acid. The present work contributes to the valorization of citrus by-products through their recycling into a natural food ingredient. However, a deeper analysis of these ingredients is necessary to allow the industries to implement sustainable extraction techniques and to use citric acid recovered from citrus as a food preservative.