Mountain Research Centre

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Optimization and validation of two methods to determine the levels of AFM1 in milk and cheese samples using immunoaffinity columns for extraction and HPLC-FLD for quantification

Publication . Vaz, Andreia; Gomes, Francileni Pompeu; Alves, A.; Rodrigues, Paula; Venâncio, Armando

Consumption of dairy products has expanded rapidly over the past decade and constitutes an important source of dietary protein. 1 Aflatoxin M1 (AFM1) is a potent carcinogen metabolite that can be present in milk from dairy cows that consume feed contaminated with Aflatoxin B1. Even though it is less toxic than its parent compound, AFM1 is hepatotoxic and carcinogenic, and is stable during milk pasteurization, storage and preparation of various dairy products. 2,3 Due to the toxicity of this molecule, its detection and quantification is extremely important. The objective of this work was to optimize and validate two methods, according to Commission Regulation (EC) nº 401/2006 of 23 February, to determine the levels of AFM1 in milk and in cheese, using immunoaffinity columns (IAC) for extraction and HPLC with fluorescence detection for quantification.4 The method for milk samples was adapted from VICAM – the supplier of the IAC, and for cheese samples was from r-biopharm and VICAM.5,6 For both methodologies, three levels of spiking in triplicate on two different days were performed. The calibration curve was linear from 0.047 to 4.7 μg L⁻¹ and the detection and quantification limits for milk and cheese were 0.001 μg L⁻1 and 0.003 μg L⁻¹, and 0.006 and 0.02 μg kg⁻¹, respectively. For milk samples, average recoveries determined at spiking levels of 0.020, 0.050 and 0.10 μg L⁻¹ were in the range of 62 % – 87 %, with intra-day precision (RSDr) in the range of 3.4 % – 9.5 %, and inter-day precision (RSDr) in the range of 5.4 % – 6.2 %. For cheese samples, average recoveries determined at spiking levels of 0.050, 0.10 and 0.25 μg L⁻¹ were in the range of 47 % – 74 %, with intra-day precision (RSDr) in the range of 3.8 % – 7.0 %, and inter-day precision (RSDr) in the range of 3.8 % – 5.8 %. Results of the validation process indicate that, except for the recovery in cheese samples, both methods are agree with the provisions of Commission Regulation (EC) nº 401/2006. Despite the recovery for cheese, both methods are precise for the quantification of AFM1 in milk and cheese.

2019Conference object

Open access

Efeito de três embalagens e do tempo de armazenamento nas propriedades físico-químicas e qualidade microbiológica de castanhas

Publication . Ramalhosa, Elsa; Fernandes, Luana; Gomes, Auryo; Batista, Bianca; Barbosa, Larissa; Fidalgo, Maria do Céu; Galvão, Hilton; Pereira, Ermelinda

O castanheiro (Castanea sativa Mill.) é uma cultura com grande importância económica para Portugal, sendo que as maiores áreas de cultivo se encontram no Norte do País. A castanha é um fruto que apresenta alguns problemas de conservação e de manutenção da qualidade ao longo do tempo de armazenamento, visto que é um fruto muito susceptível ao crescimento de bolores e perda de peso. O uso de diferentes tipos de embalagens pode ser uma solução para a indústria da castanha. Assim, o objetivo do presente estudo foi avaliar o efeito de diferentes embalagens - atmosfera modificada (MAP) (0,3% O2 e 31,7% CO2), polietileno (PE) e vácuo (VAC) - nas propriedades físico-químicas e microbiológicas de castanhas em fresco durante o armazenamento (1, 2, 3 e 6 meses). Os resultados mostraram que as castanhas em MAP tiveram a menor variação da cor (DE*) na casca (10,9±5,8), enquanto no interior do fruto foi o controlo (7,0±2,6). O VAC e a MAP acarretaram uma diminuição nos valores de atividade de água (aw) e da força da casca, após 6 meses de armazenamento. Ao comparar os 6 meses com o tempo zero, os valores de acidez e de sólidos solúveis totais aumentaram em todas as amostras. No final do armazenamento, só se observou um aumento significativo na contagem de microrganismos totais nas amostras controlo. Em relação aos bolores e leveduras, estes aumentaram no controlo e no PE. Em conclusão, o uso de MAP e VAC inibiu a proliferação de microrganismos.

2019Conference object

Open access

Effect of cation exchange in the sorption of CO2, CH4 AND N2 and mixtures on binder-free faujasite zeolite Y

Publication . Aly, Ezzeldin; Zafanelli, Lucas F.A.S.; Freitas, Francisco A. da Silva; Silva, José A.C.

Ion-exchange was performed on commercial binder-free NaY zeolite with alkali metal and alkaline earth metal cations to produce binder-free beads containing 23, 58 and 95% of potassium, as well as 56 and 71% of calcium exchanged from the bare samples. These cation-exchanged faujasites were studied by adsorption of carbon dioxide (CO2), methane (CH4), and nitrogen (N2) through single, binary, and ternary fixed bed breakthrough experiments, covering the temperature range between 308 and 348 K and pressure up to 350 kPa. The single and multi-component breakthrough apparatus that was used to study the fixed bed adsorption of CO2, CH4, and N2 and their binary/ternary mixture, is illustrated in Figure 1. The dynamic equilibrium loading is calculated by integrating the molar flow profiles of the breakthrough curves, as explained in previous works. The adsorption equilibrium data was then modelled by the extended dual-site Langmuir model, and the breakthrough curves were numerically simulated using Aspen Adsorption v10. Adsorption equilibrium measurements of CO2 on each of the studied material can reveal different behaviours and trends based on the modification of the intracrystalline environment through ion-exchange. Factors such as cation size, surface basicity, number and location of exchangeable cations, and strength of electric field can all have a great impact on the performance of the adsorbent. Figure 2 shows a comparison of the CO2 isotherms between NaY, K(23)Y, K(58)Y, K(95)Y, Ca(56)Y, and Ca(71)Y, collected at 308 K. A trend in the order of adsorption at low pressure (between 0 and 50 kPa) is observed: Ca(71)Y < Ca(56)Y < NaY < K(23)Y < K(58) < K(95)Y. As the exchange rate from Na+ to K+ increases, the CO2 adsorption capacity increases; the opposite is observed (decrease of adsorption uptake) when the rate changes from Na+ to Ca2+. At 25 kPa, the loading of binder-free Na(100)Y is equal to 4.05 mol/kg, compared to 4.29 for K(23)Y, 4.57 for K(58)Y, 4.97 for K(95)Y, 2.63 for Ca(56)Y and only 2.02 mol/kg for Ca(71)Y. This indicates a good response between the acidic CO2 to the basic properties of the zeolites containing larger monovalent cations at low pressure. Bigger cations such as K+ exhibit strong interaction with CO2, since they are both preferentially exchanged in the supercages; while smaller cations such as Na+ have less molecular interaction with the adsorbate molecules, since they are spread around the zeolite framework accessing narrow locations such as the sodalite cages, where CO2 cannot reach due to its size. Moreover, the CO2 loading of Ca(71)Y is significantly lower than all the rest (around half of that of NaY), which is due to the decrease of the amount of exchangeable cations between the divalent Ca2+ cations and the adsorbate molecules. For partial pressures above 200 kPa, K(23)Y and NaY are characterized with the highest adsorption capacity followed by K(95)Y and K(58)Y, then Ca(56) and finally Ca(71)Y, as shown in Figure 2. These trends are explained by the reduction of the basic strength and the electropositivity of exchangeable cations in larger ions, since they accept less charge transfer from the neighboring lattice oxygen atoms when compared to smaller cations. This leads to the weakening of the electric field induced by the exchangeable cations and so the adsorption capacity is reduced. It is also explained by the volume occupied by the large cations, which reduces the space available for adsorption of CO2 when the pores are reaching saturation. The studied binary experiments consist of 15% CO2 / 85% N2 (vol.%) mixture, representing a typical post-combustion stream. Figure 3a shows the adsorption breakthrough curves in binder-free K(95)Y for the binary mixture at 313 K. Figure 3b displays the breakthrough curves for ternary mixtures feeds of CO2 /CH4/ N2 (20/20/20 vol.% balanced with He) on binder-free zeolite KY, under conditions in the range used for biogas upgrading regarding the removal of CO2. As can be seen in Figure 3c, the binary experiment show a selectivity of CO2 over N2 around 105 at 313 K; the ternary system resulted in a selectivity of CO2 over CH4 and over N2 of around 14 and 32 at 313 K, respectively. These results indicate that binder-free K(95)Y works best in the low-pressure region and therefore, is a promising adsorbent for the recovery of CO2 from post-combustion streams. Numerical simulations were performed with a model implemented in Aspen Adsorption simulator, allowing to predict accurate breakthrough curves for dynamic experiments carried out in a fixed bed adsorption system, as shown in Figure 3. Briefly, most of the studied ion-exchanged materials show a lot of potential for the capture of CO2 from CO2/N2 and CO2/CH4/N2 mixtures. Nevertheless, each adsorbent differs from one another and can only reach its full potential under specific conditions. Therefore, it is possible to tune the adsorptive properties of zeolites by ion exchange, to optimize the most suitable material that enriches substantially the CO2 adsorption for a specific process.

2022Conference object

Open access

Efeito da torra e temperatura de conservação nas características físico-químicas e sensoriais de amêndoa Cv. Duro Italiano

Publication . Rodrigues, Nuno; Antunes, Rogério Costa; Oliveira, Catarina Isabel; Rodrigues, Sandra; Podence, Morgane; Pereira, J.A.; Ramalhosa, Elsa

O nosso País tem um património rico em variedades tradicionais de amendoeira (Prunus dulcis) que interessa conhecer e valorizar. No sentido de contribuir para a caracterização, valorização e consumo de uma variedade tradicional de Trás-os-Montes, estudou-se o efeito da torra e do tempo de conservação, nas características físico-químicas e sensoriais de amêndoas da Cv. Duro Italiano. Assim, foram constituídos seis lotes de miolo de amêndoa de aproximadamente um kg cada, sendo três deles foram sujeitos a torra, à temperatura de 180 ºC durante 20 minutos, enquanto os restantes três não sofreram qualquer tratamento. De cada lote, constituíram-se amostras de 100 g de miolo que foram colocadas em cuvetes de polietileno próprio para indústria alimentar simulando as condições de mercado. Metade das cuvetes foram colocadas à temperatura ambiente e expostas à luz, enquanto as restantes foram colocadas à temperatura de 50 ºC . Ao longo de quatro semanas, foi retirada uma cuvete de cada lote, ou seja a T0 (Tempo inicial), T1 (1 semana), T2 (2 semanas), T3 (3 semanas) e T4 (4 semanas), onde foram avaliados os parâmetros físico-químicos: cor, atividade da água (aw), teores de humidade e de gordura, índice de peróxidos, coeficientes de extinção específica (K232 e K268) no ultravioleta, e parâmetros sensoriais, no que diz respeito à dureza, crocância e ranço. Os resultados obtidos, indicam não ter havido alteração na aw , tanto nas amêndoas cruas como nas torradas, quando armazenadas à temperatura ambiente ao longo das quatro semanas, com valores médios de 0,47, enquanto nas amêndoas colocadas a 50 ºC, verificou-se um valor inferior (0,21), tanto nas cruas como nas torradas. No teor de humidade apenas se registou uma diminuição ligeira com o tempo de armazenamento em ambos os tratamentos. Na cor observaram-se algumas alterações, nomeadamente com uma diminuição dos parâmetros L*, b*, C* e h* de T0 para T1, e um ligeiro aumento da coordenada a*, indicativo de escurecimento e de tons avermelhados. A avaliação sensorial mostrou uma ligeira diminuição da dureza e da crocância observando-se o aparecimento de ranço em T4 quando armazenadas a 50 ºC. Os resultados indicam que o miolo desta variedade tradicional apresenta uma grande resistência à oxidação quando à temperatura ambiente, contudo, temperaturas elevadas aceleram o processo de oxidação.

2019Conference object

Open access

Valorisation of a threatened plant species through in vitro culture elicitation of phenolic compounds

Publication . Ayuso, Manuel; Dias, Maria Inês; Pinela, José; Gallego, Pedro Pablo; Barreal, M. Esther; Barros, Lillian; Ferreira, Isabel C.F.R.

Abstract: Ex situ conservation strategies, such as in vitro culture, have been suggested for the recovery of threatened plants close to extinction. In vitro culture or micropropagation is a plant-multiplication technique that allows to obtain a large number of clones in a short period of time and limited space, which can be used in reintroduction programs and for research purposes (1). This biotechnological tool also allows increasing the production yield of secondary metabolites, such as phenolic compounds, thus emerging as an efficient system to obtain high added-value bioactive compounds (2). This approach allows valorising the culture of threatened plants for their commercial use in different industrial sectors interested in bioactive compounds, such as the food and pharmaceutical industries. Therefore, the aim of this research was to study the phenolic compounds elicitation by cytokinins in roots and aerial parts of the in vitro cultured endangered plant Eryngium viviparum J. Gay (Fam. Apiaceae). E. viviparum explants were cultured in vitro in a solidified MS medium. A 3-level factorial design was implemented to study the effect of two cytokinins, 6-benzylaminopurin (BAP) and kinetin (KIN), at 0, 1 and 2 mg L-1, on the elicitation of phenolic compounds. The obtained plant material was lyophilized, reduced to powder, and submitted to a solid-liquid extraction with ethanol:water (80:20, v/v) to obtain combined extracts (3). Then, phenolic compounds were identified and quantified by HPLC-DAD-ESI/MS (3) and the main phenolic compounds with known bioactivities were correlated with both cytokinins. Seventeen phenolic compounds were identified in all E. viviparum hydroethanolic extracts, corresponding to 12 phenolic acids and 5 flavonoids. Rosmarinic and chlorogenic acids were the main bioactive phenolic acids. Regarding flavonoids, tectorigenin-O-glucuronide was identified and quantified only in the aerial parts. In the roots, the increase of phenolic acids was correlated with the presence of KIN in the culture media. Concerning the aerial parts, cytokinins did not correlate with phenolic acids, but KIN correlated with the increase of tectorigenin-O-glucuronide. In addition, the roots had a higher phenolic composition content than the aerial parts and the combination of both cytokinins promoted a doubling of the phenolic compounds concentration in some samples. Therefore, this study demonstrated the efficacy of in vitro culture in adding value to the threatened plant species E. viviparum, as the levels of valuable secondary metabolites were increased by elicitation, thus being a sustainable approach that can help save and valorise other endangered plants.

2019Conference object

Open access