Purificação e avaliação de novas lectinas como ferramenta no reconhecimento de epitopos celulares glicosilados, em células tumorais do cancro do cólon

Tese de mestrado, Controlo da Qualidade e Toxicologia dos Alimentos, Universidade de Lisboa, Faculdade de Farmácia, 2015O conhecimento sobre o valor terapêutico das plantas sempre tem acompanhado a evolução do homem através dos tempos. Várias plantas endémicas da flora Portuguesa, têm sido estudadas pela sua riqueza e elevada constituição em compostos bioactivos, não só com actividade anticancerígena, como também, na prevenção da doença. As lectinas são uns dos vários componentes extraídos das plantas e representam uma família de proteínas ubiquitárias, existentes na forma livre ou associadas às células, podendo ser encontradas na superfície ou no interior destas, exibem uma afinidade para hidratos de carbono, não apresentam actividade catalítica, nem são resultado de resposta imune. O estudo das lectinas justifica-se pela importância dos hidratos de carbono na identidade celular, promovendo mecanismos envolvidos na interacção entre células, assim como uma variedade de processos biológicos. São pontos cruciais na investigação actual, a descoberta de novas lectinas, assim como o estudo da sua especificidade de ligação ao hidrato de carbono. Para o efeito, neste estudo foram seleccionadas quatro espécies endémicas da flora portuguesa, Juniperus oxycedrus subsp. oxycedrus, Juniperus oxycedrus subsp. badia, Arbutus unedo e Corema album. A caracterização do perfil polipeptídico das espécies em estudo, a actividade hemaglutinante dos extractos obtidos, frente a grupos sanguíneos humanos e eritrócitos de coelho, além da especificidade de ligação aos hidratos de carbono, associada à detecção de proteínas glicosiladas, revelou a existência de potenciais proteínas do tipo lectina nos quatro extractos proteicos. A actividade antitumoral foi testada em linhas celulares tumorais humanas derivada de carcinoma do cólon (HT29). A incubação dos respectivos extractos com as membranas das células HT29, revelaram, por análise bidimensional (2D), ligação de péptidos com especificidade para epitopos glicosilados das membranas HT29. Esta ligação foi efectiva para as quatro espécies em estudo. O potencial antitumoral dos quatro extractos demonstraram actividades distintas na inibição da invasão celular em células HT29, ficando patente uma forte capacidade de inibição apresentada pelas espécies Juniperus oxycedrus subsp. badia e Arbutus unedo. Seguiu-se, para as duas espécies com maior poder de inibição da invasão celular, a avaliação da sua actividade inibitória para as metaloproteinases MMP9 e MMP2. Verificou-se uma inibição na produção destas enzimas, sendo um excelente prognóstico para a aplicação destas espécies na inibição da actividade tumoral.The knowledge about the therapeutic value of plants always has followed the evolution of man through the ages. Several endemic plants of Portuguese flora, have been studied for their wealth and high constitution in bioactive compounds with anticancer activity, but also in the prevention of the disease. The lectins are some of the various components extracted from plants and represent a family of ubiquitous proteins, they exist in free form or associated to the cells, which can be found on the surface or inside of these, display an affinity for carbohydrates, do not exhibit catalytic activity, nor are they a result of immune response. The study of lectins is justified by the importance of carbohydrates in cell identity, promoting mechanisms involved in the interaction between cells, as well as a variety of biological processes. Are crucial points in the current investigation, the discovery of new lectins, as well as the study of its specificity of carbohydrate binding. To that end, in this study were selected four endemic species of Portuguese flora, Juniperus oxycedrus subsp. oxycedrus, Juniperus oxycedrus subsp. badia, Arbutus unedo and Corema album. The polypeptide profile characterization of the species under study, the haemagglutinating activity of the extracts obtained against the human blood groups and rabbit erythrocytes, in addition to the binding specificity to carbohydrates, coupled with the detection of glycosylated proteins, revealed the existence of potential proteins of type lectin in the four protein extracts. The antitumor activity was tested in human tumor cell lines derived from colon carcinoma (HT29). Incubation of extracts with HT29 cells membranes by two-dimensional (2D) analysis, revealed the binding of peptides with specificity for epitopes glycosylated HT29 membranes. This results was effective for the four species under study. The potential antitumor of four distinct extracts demonstrated different activities on inhibition of cellular invasion on HT29 cells, getting a strong inhibition capacity by Juniperus oxycedrus subsp. badia and Arbutus unedo species. This was followed, for the two species with greater power of inhibition of cell invasion, the evaluation of its inhibitory activity for the Metalloproteinases MMP9 and MMP2. There has been an inhibition on the production of these enzymes, being an excellent prognosis for the application of these species in the inhibition of tumoral activity

Influence of several oenological fining agents on ochratoxin A removal

In Europe, wine is estimated to be the second source, after cereals, of ochratoxin A (OTA), one of the most important mycotoxin found in food and feed products [1]. Its chemical structure consists of a chlorine-containing dihydro-isocoumarin linked through the 7-carboxyl group to L--phenylalanine. In wine, this fungal metabolite represents a severe risk for consumer health. According to the European Commission Regulation (EC) No. 123/2005 the maximum limit for OTA in wine is 2 g/Kg [2]. Therefore, it is important to prevent and control their occurrence in wines. With the purpose to remove this toxin, several chemical, microbiological and physical methods were described in the literature [1, 3, 4]. Consequently, the aim of this work is to understand the interaction of different types of fining agents on OTA removal from wine. To evaluate their effectiveness, eleven commercial fining agents, including mineral, synthetic, animal and vegetable proteins were used to get new approaches on OTA removal from wine. Trials were performed in wines added of OTA. Most effective fining agent in removing OTA was a commercial formulation that contains activated carbon, a well-known adsorbent of mycotoxins. Reductions between 10 and 30% were also obtained with potassium caseinate, yeast cell walls and pea protein. However, with bentonites, carboxymethylcellulose (CMC), polyvinylpolypyrrolidone (PVPP) and chitosan no considerable reduction of OTA was verify. Also, the influence of these fining agents on the physicochemical wine characteristics, namely wine color, total phenolic compounds, flavonoids and non-flavonoids were studied. Final results could provide important information to the wine industry to select treatments based on fining agents to remove OTA, in order to reduce toxicity and consequently to improve wine safety and preserving wine quality.FEDER funds through the COMPETE and by national funds through FCT , Ref. FCOMP - 01 - 0124 - FEDER - 028029 and PTDC/AGR - TEC/3900/2012, respectively. This work was also funded by IBB/CGB - UTAD and Chemical Research Centre of Vila Real (CQ- VR). Additional thanks to SAI Lda., AEB Bioquímica Portuguesa, S. A. and Enartis companies for providing fining agents

Analytical evaluation of fining treatments for white wines contaminated by Ochratoxin A

Mycotoxins are toxic secondary metabolites produced by certain molds. Ochratoxin A (OTA) is one of the most relevant. Its chemical structure is a dihydro-isocoumarin connected at the 7-carboxy group to a molecule of L--phenylalanine via an amide bond. OTA in wine is a risk to consumer health [1]. According to the Regulation No. 123/2005 of the European Commission, the maximum limit for OTA in wine is 2 µg/kg [2]. Then, it is important to control its occurrence. So, the aim of this work was to know the effect of different fining agents on OTA removal from white wine.This work was funded by FEDER funds through the COMPETE and by national funds through FCT, Ref. FCOMP-01-0124-FEDER-028029 and PTDC/AGR-TEC/3900/2012, respectively. Luís Abrunhosa received support through grant SFRH/BPD/43922/2008 from FC

Elimination of ochratoxin A from white and red wines: Critical characteristics of activated carbons and impact on wine quality

Ochratoxin A (OTA) is a mycotoxin frequently found in wines, with an allowed maximum limit of 2.0 ?g/L. To optimise OTA removal from white and red wines at high levels (10.0 ?g/L) using activated carbons (ACs), different commercial deodorising ACs were tested. OTA elimination from white wines was less dependent on ACs characteristics, with 100% removal with all but one AC with the lowest total pore volume. For red wines, only one of the ACs with higher abundance of mesopores was successful in completely removing OTA. This decrease in performance was due to the competition of anthocyanins for ACs mesopores. ACs treatment impacted positively on white wine chromatic characteristics, decreasing the yellowness-brownish colour but for the red wine, it was observed a higher but limited impact (24% decrease) on wine colour. ACs pore volume distribution, with pore sizes in the range 42.6-55.9 Å and 125.6-137.4 Å were important for efficient OTA removal. These results will allow the rational design of ACs with pore volume characteristics for OTA elimination with less impact on wine colour. Therefore, it was shown for the first time that AC with appropriate pore size distribution can remove completely OTA from white and also red wines.The authors acknowledge the financial support provided by the Fundação para a Ciência e Tecnologia (FCT, Portugal) to the project PTDC/AGR-TEC/3900/2012 and to the financial support provided to the European Investment Funds FEDER/COMPETE/POCI under Project POCI-01-0145-FEDER-007728 (CQ-VR) and Funds from the Fundaçao para a Ciência Tecnologia (Portugal) to CQ-VR (UIDB/00616/2020 and UIDP/00616/2020). This study was additionally supported by the Por tuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Elimination of aflatoxins B1 and B2 in white and red wines by bentonite fining. efficiency and impact on wine quality

Aflatoxins B1 and B2 are two highly toxic mycotoxins that have been sometimes found in wines. Currently, no technological solution is available to reduce or eliminate aflatoxins from wines when they are present. Therefore, this work aims to study the efficiency of already approved wine fining agents like activated carbon, potassium caseinate, chitosan, and bentonite for aflatoxins B1 and B2 removal from white and red wines. It was observed that the fining agents efficiency in removing aflatoxins was dependent on the wine matrix, being higher in white than in red wine. Bentonite was the most efficient fining agent, removing both aflatoxins (10 g/L total) from the white wine and 100% of aflatoxin B1 and 82% of aflatoxin B2 from red wine. The impact of bentonite on white wine chromatic characteristics was low (color difference, E* = 1.35). For red wine, bentonite addition caused a higher impact on wine chromatic characteristics (E* = 4.80) due to the decrease in total anthocyanins, although this decrease was only 1.5 points of color intensity. Considering the high efficiency of bentonite in aflatoxins B1 and B2 removal and despite the impact on red wine color, bentonite is a very good technological solution for aflatoxin removal in white and red wines.This research was funded by the Portuguese Foundation for Science and Technology (FCT) through the project PTDC/AGR-TEC/3900/2012 and the financial support provided to the European Investment Funds FEDER/COMPETE/POCI under the Project POCI-01-0145-FEDER-007728 (CQ-VR) and the funds from the FCT to CQ-VR (UIDB/00616/2020 and UIDP/00616/2020). This study was additionally supported by the FCT under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Effect of the application of oenological products on fumonisin B2 (FB2) reduction/removal in contaminated red and white wines

ICFC 2017 - International Conference on Food Contaminants (Book of Abstracts)Fumonisins are mycotoxins produced by species of Fusarium, mainly F. verticillioides and F. proliferatum. Fumonisins have hepatotoxic and nephrotoxic effects in various animals and are also associated to human esophageal cancer [1] thus being classified by the IARC in Group 2 [2]. Chemically, fumonisins are characterized by a 19- or 20-carbon aminopolyhydroxyalkyl chain that is diesterified with propane-1,2,3-tricarboxylic acid groups [3]. Sharing a basic structure, several related groups of fumonisins have been isolated and identified (A, B, C and P). Fumonisins B (FBs) are the major forms found in most food products. Recently they were detected in grapes, musts and wines around the world. It was established an association between Aspergillus niger and the presence of FB2 in grape must and wine. WHO has recommended a maximum tolerable daily intake of 2 mg/kg of body weight to FB1, FB2 and FB3, alone or in combination [4], therefore it is important to prevent and control its occurrence, as well other mycotoxins, in wines [5]. The main objective of this work was to evaluate the ability of different oenological products on FB2 removal of white and red wines. For this purpose, ten commercial oenological fining agents (mineral, synthetic and organic - proteins of animal and vegetable origin) were studied to remove FB2 in white and red wines artificially contaminated with FB2. In addition to the FB2 removal, the effect of these products on wine physicochemical characteristics, namely, flavonoids, non-flavonoids and total phenolic compounds were evaluated. In red wine all products showed low reduction on FB2 removal, being the highest value (30%) achieved by calcium bentonite. The results obtained by the action of these products may be considered as a pioneer approach on wine FB2 reduction/removal, with relevance for wine industry, in order to select the best fining agent to reduce toxicity and consequently to improve wine quality and safety.info:eu-repo/semantics/publishedVersio

Avaliação da eficiência de diferentes carvões ativados comerciais na remoção de Ocratoxina A de vinho

A presença de micotoxinas em alimentos é uma preocupação ao nível da segurança alimentar. Contudo, os vinhos não são exceção podendo também estar contaminados com micotoxinas, especialmente ocratoxina A (OTA) [1]. Como estas substâncias tóxicas não podem ser completamente removidas da cadeia alimentar, muitos países definiram limites legais nos alimentos, com o intuito de aumentar a segurança alimentar. Assim, o limite máximo aceitável de OTA em vinhos é de 2,0 µg/kg de acordo com o regulamento da Comissão nº 1881/2006 [2]. A fim de reduzir os níveis de OTA para valores mais seguros, vários produtos enológicos foram estudados. Foi objetivo deste estudo avaliar a eficácia de produtos compostos por carvão ativado na redução de OTA presente em vinhos, bem como o seu impacto nas características finais do vinho. Foram testados oito carvões ativados diferentes, sendo um deles um produto composto por uma formulação comercial de gelatina, bentonite e carvão ativado, na dose máxima recomendada pelo fabricante. Os ensaios foram realizados em vinhos artificialmente suplementadas com OTA numa concentração final de 10 µg/L. Os resultados mostraram que a mistura comercial composta por gelatina, bentonite e carvão ativado reduziu 73% da concentração inicial de OTA em vinho branco. Contudo, verificou-se que todos os carvões ativados, exceto um, reduziu a concentração inicial de OTA em 100%. Estes resultados podem fornecer informações valiosas para os produtores de vinho, permitindo-lhes escolher o carvão ativado mais adequado para a remoção de OTA, aumentando a segurança e qualidade do produto final.Agradecimentos: Este trabalho foi financiado por fundos FEDER através do Programa Operacional Factores de Competitividade - COMPETE e por fundos nacionais através da Fundação para a Ciência e a Tecnologia -FCT, ref. FCOMP-01-0124-FEDER-028029 e PTDC/AGR-TEC/3900/2012, respetivamente. Luís Abrunhosa recebeu apoio através da bolsa Incentivo/EQB/LA0023/2014 do ON.2 O Novo Nort

Influence of different activated carbons on Ochratoxin A decrease in wines

The presence of mycotoxins in foodstuff is a matter of concern for food safety. Wines can also be contaminated with these toxicants. Several authors have demonstrated the presence of mycotoxins in wine, especially ochratoxin A (OTA) [1]. As these toxicants can never be completely removed from the food chain, many countries have defined levels in food in order to attend health concerns. The maximum acceptable level of OTA in wines is 2.0 µg/kg according to the Commission regulation No. 1881/2006 [2]. Although, higher levels of OTA have been detected in several wine samples. In order to reduce OTA to safer levels, several oenological products can be used in wine; including activated carbons, as shown in previous experiments. Regarding this, the aim of present study was to evaluate the effectiveness of several activated carbons for reducing the amount of OTA present in white and red wines as well as to evaluate their effect on wines physicochemical characteristics. Wine samples were artificially supplemented with OTA at a final concentration of 10.0 µg/L. The different activated carbons were applied at the concentration recommended by the manufacturer in order to evaluate their efficiency in reducing OTA levels. A mixture composed by gelatine, bentonite and activated carbon reduced 80% of OTA concentration in white wine. The same mixture was however less efficient in red wine, achieving only a reduction of 55%. Thereafter, the effect of activated carbon was evaluated in a red wine, achieving reductions of 66%. Considering these results more assays are being performed with other commercial activated carbons, in order to evaluate their efficiency. These results may provide valuable information for winemakers. Knowing the effect of commercial activated carbons they may choose most appropriate products to remove OTA, thus enhancing wine safety and quality.This work was funded by FEDER funds through the COMPETE and by national funds through FCT, Ref. FCOMP-01-0124-FEDER-028029 and PTDC/AGR-TEC/3900/2012, respectively. Luís Abrunhosa received support through grant Incentivo/EQB/LA0023/2014 from ON.2 O Novo Norte

Removal of ochratoxin A from contaminated white and red wines using oenological fining agents

Mycotoxins are toxic secondary metabolites produced by certain moulds, being ochratoxin A (OTA) one of the most relevant. Its chemical structure is a dihydro-isocoumarin connected at the 7-carboxy group to a molecule of L--phenylalanine via an amide bond. OTA contamination of wines might be a risk to consumer health, thus requiring treatments to achieve acceptable standards for human consumption [1]. According to the Regulation No. 1881/2006 of the European Commission, the maximum limit for OTA in wine is 2 µg/kg [2]. Therefore, the aim of this work was to know the effect of different fining agents on OTA removal, as well as their impact on white and red wine physicochemical characteristics. To evaluate their efficiency, 11 commercial fining agents (mineral, synthetic, animal and vegetable proteins) were used to get new approaches on OTA removal from white and red wines. Trials were performed in wines artificially supplemented (at a final concentration of 10 µg/L) with OTA. The most effective fining agent in removing OTA (80%) from white wine was a commercial formulation that contains gelatine, bentonite and activated carbon. Removals between 10-30% were obtained with potassium caseinate, yeast cell walls and pea protein. With bentonites, carboxymethylcellulose, polyvinylpolypyrrolidone and chitosan no considerable OTA removal was verified. In red wine, removals between 6-19% were obtained with egg albumin, yeast cell walls, pea protein, isinglass, gelatine, polyvinylpolypyrrolidone and chitosan. The most effective fining agents in removing OTA from red wine were an activated carbon (66%) followed again by the commercial formulation (55%), being activated carbon a well-known adsorbent of mycotoxins. These results may provide useful information for winemakers, namely for the selection of the most appropriate oenological product for OTA removal, reducing wine toxicity and simultaneously enhancing food safety and wine quality.This work was funded by FEDER funds through the COMPETE and by national funds through FCT, Ref. FCOMP-01-0124-FEDER-028029 and PTDC/AGR-TEC/3900/2012, respectively. Luís Abrunhosa received support through grant Incentivo/EQB/LA0023/2014 from ON.2 O Novo Norte

Food safety in wine: removal of ochratoxin A in contaminated white wine using commercial fining agents

The presence of mycotoxins in foodstuff is a matter of concern for food safety. Mycotoxins are toxic secondary metabolites produced by certain molds, being ochratoxin A (OTA) one of the most relevant. Wines can also be contaminated with these toxicants. Several authors have demonstrated the presence of mycotoxins in wine, especially ochratoxin A (OTA) [1]. Its chemical structure is a dihydro-isocoumarin connected at the 7-carboxy group to a molecule of L--phenylalanine via an amide bond. As these toxicants can never be completely removed from the food chain, many countries have defined levels in food in order to attend health concerns. OTA contamination of wines might be a risk to consumer health, thus requiring treatments to achieve acceptable standards for human consumption [2]. The maximum acceptable level of OTA in wines is 2.0 g/kg according to the Commission regulation No. 1881/2006 [3]. Therefore, the aim of this work was to reduce OTA to safer levels using different fining agents, as well as their impact on white wine physicochemical characteristics. To evaluate their efficiency, 11 commercial fining agents (mineral, synthetic, animal and vegetable proteins) were used to get new approaches on OTA removal from white wine. Trials (including a control without addition of a fining agent) were performed in white wine artificially supplemented with OTA (10 µg/L). OTA analysis were performed after wine fining. Wine was centrifuged at 4000 rpm for 10 min and 1 mL of the supernatant was collected and added of an equal volume of acetonitrile/methanol/acetic acid (78:20:2 v/v/v). Also, the solid fractions obtained after fining, were centrifuged (4000 rpm, 15 min), the resulting supernatant discarded, and the pellet extracted with 1 mL of the above solution and 1 mL of H2O. OTA analysis was performed by HPLC with fluorescence detection according to Abrunhosa and Venâncio [4]. The most effective fining agent in removing OTA (80%) from white wine was a commercial formulation that contains gelatine, bentonite and activated carbon. Removals between 10-30% were obtained with potassium caseinate, yeast cell walls and pea protein. With bentonites, carboxymethylcellulose, polyvinylpolypyrrolidone and chitosan no considerable OTA removal was verified. Following, the effectiveness of seven commercial activated carbons was also evaluated and compared with the commercial formulation that contains gelatine, bentonite and activated carbon. The different activated carbons were applied at the concentration recommended by the manufacturer in order to evaluate their efficiency in reducing OTA levels. Trial and OTA analysis were performed as explained previously. The results showed that in white wine all activated carbons except one reduced 100% of OTA. The commercial formulation that contains gelatine, bentonite and activated carbon (C8) reduced only 73% of OTA concentration. These results may provide useful information for winemakers, namely for the selection of the most appropriate oenological product for OTA removal, reducing wine toxicity and simultaneously enhancing food safety and wine quality.Acknowledgements This work was funded by FEDER funds through the COMPETE and by national funds through FCT, Ref. FCOMP-01-0124-FEDER-028029 and PTDC/AGR-TEC/3900/2012, respectively. Luís Abrunhosa received support through grant Incentivo/EQB/LA0023/2014 from ON.2 O Novo Nort