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- Desenvolvimento de formulações corantes de antocianinas baseadas na produção de emulsões duplas e aplicaçõesPublication . Teixeira, Liandra Gracher; Barreiro, M.F.; Ferreira, Isabel C.F.R.; Leimann, Fernanda VitóriaOs corantes são usados na indústria alimentar, química e farmacêutica, entre outras, para conferir cor, com o objetivo de tornar o produto mais atraente para os consumidores. Adicionalmente, nos últimos anos, a procura por produtos naturais seguros tem incrementado, devido à preocupação crescente dos consumidores pela adoção de um estilo de vida mais saudável. No caso concreto dos corantes, esta tendência resulta, principalmente, da imposição de doses máximas de utilização de alguns corantes sintéticos devido aos efeitos derivados do seu consumo excessivo. No entanto, do ponto de vista da aplicação, os corantes naturais apresentam algumas restrições, estando estas principalmente relacionadas com a instabilidade ao pH e temperatura. Uma das estratégias possíveis para aumentar a estabilidade e viabilizar o seu uso mais generalizado consiste na utilização de técnicas de microencapsulação ou emulsificação. Assim, o presente trabalho teve como principal objetivo estabilizar um corante natural hidrofílico baseado em antocianinas (Sambucus nigra L.) através da preparação de emulsões duplas (água/óleo/água). Com este tipo de estratégia pretendeu-se conseguir uma maior estabilidade do corante a variações de pH. Primeiramente fez-se a caracterização do extrato quanto à estabilidade da cor ao pH, temperatura (análise termogravimétrica (TGA/DTG)) e análise estrutural (Espectrocopia de Infravermelho com Transformada de Fourier (FTIR)). Posteriormente, procedeu-se à otimização da preparação da emulsão dupla, afim de obter uma formulação estável, testando diferentes variáveis como a razão água/óleo da emulsão primária, razão emulsão primária/água, sistemas emulsionantes e energia mecânica aplicada. As emulsões duplas obtidas foram avaliadas quanto à estabilidade e morfologia e capacidade corante ao longo do tempo (teste efetuado com soluções tampão na gama de pH 4-9). Face aos resultados obtidos escolheu-se a emulsão mais promissora (50/50) que foi testada em matrizes alimentares de diferentes pHs como o iogurte (pH 4,65), bebida de arroz (pH 6,01), leite (pH 6,47) e bebida de soja (pH 7,92). Estas foram observadas visualmente e caracterizadas por análise colorimétrica e comparadas com a incorporação direta do extrato livre ao longo do armazenamento (0, 3 e 7 dias). Em síntese, a dupla emulsão incorporando extrato de Sambucus nigra L. e produzida com uma razão 50/50 (emulsão primária/ fase aquosa externa) mostrou-se capaz de proteger o corante em meios ácidos e básicos, confirmando o interesse da abordagem proposta neste trabalho no campo do desenvolvimento de formulações corantes baseadas em corantes naturais. Adicionalmente, foi obtida uma coloração mais uniforme em função do pH.
- Water-in-oil-in-water double emulsions as protective carriers for Sambucus nigra l. coloring systemsPublication . Teixeira, Liandra Gracher; Rezende, Stephany; Fernandes, Ângela; Fernandes, Isabel P.; Barros, Lillian; Barreira, João C.M.; Leimann, Fernanda Vitória; Ferreira, Isabel C.F.R.; Barreiro, M.F.The use of natural colorants is needed to overcome consumer concerns regarding synthetic food colorants0 safety. However, natural pigments have, in general, poor stability against environmental stresses such as temperature, ionic strength, moisture, light, and pH, among others. In this work, water-in-oil-in-water (W1/O/W2) emulsions were used as protective carriers to improve color stability of a hydrophilic Sambucus nigra L. extract against pH changes. The chemical system comprised water and corn oil as the aqueous and oil phases, respectively, and polyglycerol polyricinoleate (PGPR), Tween 80, and gum Arabic as stabilizers. The primary emulsion was prepared using aW1/O ratio of 40/60 (v/v). For the secondary emulsion,W1/O/W2, different (W1/O)/W2 ratios were tested with the 50/50 (v/v) formulation presenting the best stability, being selected as the coloring system to test in food matrices of different pH: natural yogurt (pH 4.65), rice drink (pH 6.01), cow milk (pH 6.47), and soy drink (pH 7.92). Compared to the direct use of the extract, the double emulsion solution gave rise to higher color stability with pH change and storage time, as corroborated by visual and statistical analysis.
- Development of double emulsions from Sambucus nigra L. berry extract as ph stable colorant formulationsPublication . Teixeira, Liandra Gracher; Fernandes, Isabel P.; Rezende, Stephany; Reis, Filipa S.; Barros, Lillian; Leimann, Fernanda Vitória; Ferreira, Isabel C.F.R.; Barreiro, M.F.Anthocyanins are flavonoids, one of the largest groups of water-soluble vegetable pigments, having also sweetening and antioxidant properties. They are characterized by different colors, such as red, purple, blue and violet. However, their color stability is compromised by variations in pH, temperature, moisture, and luminosity conditions. 1 The berries of Sambucus nigra L. have high contents of anthocyanins (cyanidin-3-O-sambubioside and cyanidin-3-Osambubioside-5-O-glucoside), being a promising option as food coloring agents. 2 Therefore, this work aims to stabilize the natural hydrophilic colorant obtained from S. nigra through the preparation of double emulsions (water/oil/water, W/O/W). The S. nigra berries were extracted by crushing the fruits to obtain the juice, followed by spray drying to obtain the dry extract. For the preparation of the double emulsions, the procedure was the one described by Paula D. A. et al. with some modifications. 3 Briefly, for the first emulsion, the water phase comprised 55 mg/L of the extract and 5% Polyglycerol polyricinoleate (PGPR), homogenized under stirring (20000 rpm, 5 min). This phase was dispersed into corn oil at a W/O ratio of 0.4. (Figure 1 – (A)). Thereafter, the second emulsion was prepared by dispersing the previous emulsion in a water phase comprising Tween 80 and gum Arabic, tested at different concentrations. The final W/O/W emulsion was prepared at different primary emulsion/water ratios and stirred at 6000 rpm during 2 min using an Ultra-Turrax system. The produced emulsions were analyzed by optical microscopy (OM) using a Nikon eclipse 50i microscope to assess size and morphology. This analysis showed the presence of spherical and individualized droplets with an estimated particle size between 13 and 16 μm. (Figure 1 – (B)). The role of these emulsions as pH stable colorants were tested using different buffers (pH 4-7), as shown in (Figure 1-(C)), which also presented the comparison with solutions prepared from the original S. nigra extract. The obtained results pointed out for an improved stability and color homogeneity among samples in the tested pH range.
- Soluções corantes baseadas em emulsões duplas incorporando extrato de Sambucus nigra L.: desenvolvimento e teste em diferentes matrizes alimentaresPublication . Teixeira, Liandra Gracher; Rezende, Stephany; Fernandes, Isabel P.; Reis, Filipa S.; Barros, Lillian; Barreira, João C.M.; Leimann, Fernanda Vitória; Ferreira, Isabel C.F.R.; Barreiro, M.F.As antocianinas são pigmentos vegetais hidrossolúveis, pertencentes à classe dos flavonoides apresentando propriedades edulcorantes e bioativas. São caracterizadas por conferirem cor na gama do vermelho a violeta. No entanto, para aplicações industriais, estes pigmentos apresentam problemas de variabilidade da cor com o pH, temperatura, presença de humidade e luminosidade [1]. Neste contexto, estudaram-se extratos obtidos a partir de bagas de Sambucus nigra L., reconhecidas pelo seu elevado teor em antocianinas, como soluções corantes para a área alimentar. Para o efeito, procedeu-se à estabilização do extrato através da preparação de emulsões duplas (água/óleo/água, A/O/A). A obtenção do extrato corante foi efetuada por trituração das bagas, seguindo-se da secagem do sumo obtido por spray-drying para obtenção da correspondente forma em pó. A preparação das emulsões duplas foi realizada adaptando a metodologia descrita por Paula et al. [2], podendo ser descrita como dividida nas duas etapas seguintes: i) preparação da emulsão primária, por emulsificação da fase aquosa (55 mg/L do extrato e 5% de polirricinoleato de poliglicerol (PGPR)) em óleo de milho (razão A/O 0,4) utilizando uma velocidade de homogeneização de 20000 rpm durante 5 min; e ii) preparação da emulsão dupla por dispersão da emulsão primária numa fase aquosa secundária contendo Tween 80 e goma arábica, em diferentes proporções. A emulsão A/O/A final foi preparada utilizando diferentes proporções de emulsão primária/fase aquosa secundária recorrendo a um homogeneizador a 6000 rpm durante 2 min. As emulsões produzidas foram analisadas por microscopia ótica (MO) para avaliar o tamanho e morfologia das gotículas. Adicionalmente, procedeu-se ao teste das emulsões como agentes corantes estáveis ao pH, usando para o efeito matrizes alimentares com diferentes valores de pH, nomeadamente iogurte líquido natural (pH 4,65), bebida biológica de arroz (pH 6,01), leite (pH 6,47) e bebida biológica soja (pH 7,92), tendo-se feito a comparação com o uso direto do extrato de S. nigra (Figura 1). Posteriormente procedeu-se à avaliação da cor de acordo com os parâmetros L*, a* e b*. Os resultados obtidos apontam para uma melhor estabilidade da cor e menor variabilidade desta com o pH. Em síntese, a estratégia testada revelou-se promissora tendo em vista o desenvolvimento de formulações corantes, para a área alimentar, baseadas em corantes naturais ricos em antocianinas. Esta estratégia poderá ser aplicada a outros setores industriais.
- Photodynamic inactivation of Staphylococcus aureus by ecological antibacterial solutions associating LED (ʎ 450 ± 10 nm) with curcumin and olive leaf extractsPublication . Crugeira, Pedro Jorge Louro; Almeida, Heloísa H.S.; Teixeira, Liandra Gracher; Barreiro, M.F.Antimicrobial resistance is a problem in contemporary society, with Staphylococcus aureus standing out as a threat due to its ability to colonize, its pathogenicity, and its expression of several virulence factors. In this context, antimicrobial photodynamic inactivation (aPDI) emerges as an alternative to conventional microbicidal or microbiostatic systems, enabling numerous and successive applications without developing side effects and microbial resistance. In this context, an aPDI system against cultures of S. aureus based on a water-in-oil (W/O) emulsion incorporating curcumin as the photosensitizer (PS), with and without olive leaf extract (OLE), was developed and the antibacterial efficacy evaluated under LED activation (lambda 450 +/- 10 nm) by depositing an energy density of 14 J/cm(2). The produced emulsified systems showed no significant differences in the droplet size and morphology, remaining stable along the tested period of 30 days. The bacterial reduction achieved after the first aPDI application for the emulsions added with curcumin and curcumin combined with the OLE was 5 log10 CFU. mL(-1) and 6 log10 CFU.mL(-1), respectively, revealing a significant difference between the two groups (p < 0.0001). After the second aPDI application, an increased microbial reduction (7 log10 CFU.mL(-1)) was observed for both studied groups even with a low significant difference (p < 0.05). The PS loading through an emulsified system for aPDI obtained a bactericidal action against S. aureus, increased by applying two aPDI, showing a significant synergy between photodynamic inactivation, OLE delivery and antibacterial activity. In addition, the developed solutions were produced using natural products by an ecologically correct process.
- Development of colloidal lignin particles through particle design strategies and screening of their Pickering stabilizing potentialPublication . Colucci, Giovana; Santamaria-Echart, Arantzazu; Silva, Samara Cristina; Teixeira, Liandra Gracher; Ribeiro, Andreia; Rodrigues, Alírio; Barreiro, M.F.Colloidal lignin particles (CLPs) have increased interest as green and sustainable materials for Pickering stabilizers, with particle design being an important step towards their effective use. In this context, the antisolvent precipitation method was selected to conduct a study aiming at understanding the effect of process variables (initial lignin concentration, antisolvent pH, final ethanol concentration, and antisolvent addition rate) on particle size, zeta potential, color parameters, and contact angle. Moreover, their Pickering stabilizing potential was preliminarily screened. The evaluation using a Fractional Factorial Design revealed that the particle size is significantly influenced by the initial lignin concentration (as it increases, larger particles are obtained) and the final ethanol concentration (as it increases, smaller sizes result). The zeta potential is significantly affected by the antisolvent pH and the initial lignin concentration; the increase in both parameters results in higher negative values. The color is significantly dependent on the used initial lignin concentration (as it increases, particles become lighter and the yellowish accentuates) and the antisolvent pH (as it increases, particles become darker). Both initial lignin concentration and final ethanol concentration increase promote hydrophobicity, whereas increasing the antisolvent pH and its addition rate turns particles more hydrophilic. Through this strategy, it was possible to achieve CLPs with promising Pickering stabilizing potential, putting in evidence the importance of understanding the production process to design effective particles for target applications.
- Developing High-Coloring Natural Systems Using Double Emulsions with Daucus carota L. Extract to Meet High-Performance RequirementsPublication . Teixeira, Liandra Gracher; Silva, Samara Cristina; Colucci, Giovana; Santamaria-Echart, Arantzazu; Peres, António M.; Dias, Madalena M.; Barreiro, M.F.Daucus carota L. extract is attracting interest as a natural colorant alternative. However, the presence of anthocyanins (ACNs), which are sensitive to pH changes, limits its application. To tackle this issue, water-in-oil-in-water (W1/O/W2) double emulsions are emerging as innovative solutions. Nevertheless, the problem of reaching robust colorant systems for industrial use still needs to be overcome. One important target is to reach a high coloring power, minimizing its impact on the final product. In this context, the effect of colorant concentration and the volume of the primary emulsion, two routes to increase the colorant power, on color attributes and stability, an important feature to reach a marketable product, was studied. The optimal experimental design was conducted to two optimal solutions, whether through heightened colorant concentration or primary emulsion volume: a 41/59 (W1/O)/W2 ratio with 11 wt.% colorant, and a 48/52 (W1/O)/W2 ratio with 6 wt.% colorant, respectively. A subsequent assessment of color and physical emulsion stability over 30 days pointed out the solution with the lower colorant concentration (6 wt.%) as the one with better performance (L*: 44.11 ± 0.03, a*: 25.79 ± 0.01, D4;3: 9.62 ± 0.1 μm, and CI: 14.55 ± 0.99%), also minimizing the permeability of the colorant to the outer aqueous phase. Overall, these optimized emulsions offer versatile coloring solutions suitable for various industrial applications, such as food matrices and functional cosmetics.
- Optimisation of stability and colouring power of double emulsion systems loaded with Daucus carota L.Publication . Teixeira, Liandra Gracher; Silva, Samara Cristina; Colucci, Giovana; Santamaria-Echart, Arantzazu; Peres, António M.; Dias, Madalena M.; Barreiro, FilomenaAnthocyanins (ACNs) are water-soluble bioactive flavonoids found in flowers, roots, leaves, seeds, and stalks. ACNs cover a wide palette of colours, from blue and purple through orange to red1,2. They are used in the food industry (food colourant with E163 code), namely in fruit juice concentrates, nectars, jellies, yoghurts, marmalades, potato chips, ice creams, and soft drinks, replacing the synthetic Red 40 colourants. They also have diversified pharmaceutical and cosmetic uses due to their colouring and antioxidant attributes. However, their instability due to pH, light, and temperature is a major concern. This study used a double water-in-oil-in-water (W1/O/W2) emulsion technique to prevent rapid colour loss and colour variability with pH of a commercial black carrot extract (Fig. 1). A Central Composite Rotatable Design (CCRD) 22 was used to examine the effects of colourant concentration (wt%) and emulsion ratio ((W1/O)/W2) on stability parameters (droplet size (D4,3), creaming index (CI), and colourimetric values (L*, a*). The main objective was to find an optimised formulation in terms of colourant potential and emulsion stability. Measurements were taken at 1, 7, 15, and 30 days. The optimal experimental design revealed two optimal solutions: 1) 48/52 (W1/O)/W2 ratio with 6 wt.% colorant, and 2) 41/59 (W1/O)/W2 ratio with 11 wt.% colourant. These formulations showed lower creaming index and droplet sizes with higher colourant potential. After accounting for the effect of time, the optimal condition was 6 wt% colourant concentration and 48/52 v/v (W1/O)/W2 emulsion ratio. A stable colour was maintained after 30 days (L*: 44.11±0.03, a*: 25.79±0.01, D4;3: 9.62 ± 0.1 µm, and CI: 14.55±0.99%). These findings are promising and might be applicable to produce novel colourants to be applied in several industries, such as food and cosmetics.
- Optimisation of stability and colouring power of double emulsion systems loaded with Daucus carota L.Publication . Teixeira, Liandra Gracher; Silva, Samara Cristina; Colucci, Giovana; Santamaria-Echart, Arantzazu; Peres, António M.; Dias, Madalena M.; Barreiro, FilomenaAnthocyanins (ACNs) are water-soluble bioactive flavonoids present in flowers, fruits and vegetables, ACNs cover a wide palette of colors, from blue and purple through orange to red.