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Valorisation of Codfish (Gadus Morhua L.) Salting Processing Wastewater Through the Extraction of High Added Value Compounds

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Valorisation of Codfish (Gadus Morhua L.) Salting Processing Wastewater Through the Extraction of High Added Value Compounds Valorisation of codfish (Gadus morhua L.) salting processing wastewater through the extraction of high added value compounds Thesis submitted to the Universidade Católica Portuguesa to attain the degree of PhD in Biotechnology – with specialisation in Environmental Engineering By VINCENZA FERRARO September 2011 Valorisation of codfish (Gadus morhua L.) salting processing wastewater through the extraction of high added value compounds Thesis submitted to the Universidade Católica Portuguesa to attain the degree of PhD in Biotechnology – with specialisation in Environmental Engineering By VINCENZA FERRARO Under the academic supervision of Prof. Paula Maria Lima Castro Under the co-supervision of Prof. Maria Manuela Estevez Pintado September 2011 To my Family and Faustino Without whose encouragement, understanding and support, I could not have finished my PhD course. Thank you so much Preface Preface The research work published in this PhD dissertation has been developed at WeDoTech -Companhia de Ideias e Tecnologias, Lda., spin-off enterprise of the College of Biotechnology of Catholic University of Portugal, in Porto, through an Early Stage Research grant inside the InSolEx-RTN Programme (Innovative Solution for Extracting high value natural compounds-Research and Training Network) of Marie Curie Actions, under the 6th Frame Programme of the European Research Area. VI Acknowledgements Acknowledgements I would like to express my gratitude to my academic supervisors Prof. Maria Paula Lima Castro and Prof. Maria Manuela Estevez Pintado, whose expertise, understanding and patience added considerably to my graduate and professional experience. I really appreciate not only their vast knowledge and skills in many areas but also the human relationship they guided me with through the very intense PhD research period at the College of Biotechnology of the Catholic University of Portugal. I express a special gratefulness to WeDoTech Lda., in the persons of Dr. Isabel Braga da Cruz and Dr. Ruben Ferreira Jorge, for giving me the opportunity to develop my PhD course in their company. I am grateful not just for the technical support and direction they provided me but also for the encouragements, motivation and friendship. I acknowledge Marie Curie Actions of European Research Area for the doctoral grant inside the InSolEx–RTN programme (Innovative Solution for Extracting high value natural compounds – Research and Training Network) under FP6. I am grateful to the programme co-ordinator Prof. Andrew Livingston of Imperial College London (UK), for the scientific supervision provided during the InSolEx–RTN programme’s meetings. I would also like to thank all the InSolEx–RTN network partners – MET, Membrane Extraction Technology, Ltd., London (UK); Dynamic Extraction, Ltd., London (UK); Denomega Nutritional Oils, SA, Sarpsborg (Norway); Due Miljø, SA, Oslo (Norway); Brno University of Technology (Czech Republic); Technical University of Crete (Greece); John Frantzeskakis and BROS, SA, Crete (Greece) – for the interesting debates, points of view and suggestions provided along the InSolEx–RTN programme’s meetings. I still express my gratefulness to Dr. Anita Estevez Pintado and Eng. Cristina Santos for having teaching me important analytical and technical concepts on chromatographic analysis, and to Dr. Teresa Brandão for having helped me in statistical data treatment. VII Acknowledgements The cod fishing and processing Company Pascoal & Filhos, S.A., Aveiro (Portugal), is acknowledged for having provided samples and for technical support. Finally, I would like to say thanks to all my researcher colleagues who worked at the CBQF (Centro de Biotecnologia e Química Fina) Laboratory of Escola Superior de Biotecnologia along with me, for their friendship, encouragement, exchange of knowledge and assistance, which consistently contributed enrich my life and research experience. VIII Scope of the Thesis Scope of the Thesis The studies reported in this PhD thesis aimed at the valorisation of the wastewater arising from the dry salting processing of codfish (Gadus morhua L.). The residual water object of this research has been collected at Pascoal & Filhos, S.A., a cod fishing and processing company based in Aveiro (Portugal). Salting processing wastewater has been selected among all liquid waste effluents – salting, drying and re-hydrating water – based on its composition. Codfish salting processing wastewater is currently treated as an ecotoxic effluent, due to the high load of chloride, and according to the Portuguese environmental regulation. Extensive characterisation has shown that 10 g/L of valuable organic compounds, such as free amino acids and proteins, are present in codfish salting wastewater along with ca. 250 g/L of sodium chloride. The nutritional, medical and also technical value of both essential and not essential free amino acids and of proteins found in codfish salting processing wastewater corroborated the potential of valorisation of the effluent. The release of free amino acids and proteins has been studied in order to understand the mechanisms of their liberation from codfish muscle tissue along time. Free amino acids and proteins released at the end of salting have been selectively extracted from the wastewater by sorption using a commercial resin. Free amino acids extract has shown to have high intestinal bioavailability, antioxidant capacity and protection against oxidation of DNA, supporting the possibility of application as a nutritional or medical supplement. IX Resumo Resumo Apesar do desenvolvimento de outros métodos de preservação, a salga do bacalhau (Gadus morhua L.) continua a ser uma realidade devido a um conjunto de factores, entre os quais a simplicidade e o baixo custo do processo bem como as características sensoriais do produto final, muito apreciadas pelos consumidores. Ao longo do processo de salga o bacalhau incorpora sal até 20% do seu peso e liberta concomitantemente cerca de 22% da sua água fisiológica; assim, aproximadamente 200 litros de água residual salgada são gerados para cada tonelada de bacalhau fresco. Esse efluente é actualmente tratado como resíduo tóxico devido ao alto teor de cloro, que pode atingir valores de concentração de cerca de 160 g/L. A libertação desta água trás como consequência alterações significativas na composição e na estrutura do tecido muscular do bacalhau, levando à perda de compostos bioativos importantes, entre os quais aminoácidos livres, peptídeos e proteínas, nutrientes que, embora não essenciais, podem ser benéficos em certas circunstâncias. Assim, a recuperação de compostos orgánicos e do sal marinho de grau alimentar utilizado no processo de salga pode contribuir para a gestão integrada da água residual em questão. Com este objetivo, o perfil químico da água libertada foi avaliado. No final do período de salga o conteúdo de matéria seca na água libertada atinge o valor de ca. 10 g/L. A concentração de aminoácidos livres aumentou de 3.5 g/L a 6.5 g/L em 6 dias, devido a fenómenos de proteólise. Creatina, ácidos aspártico e glutâmico, arginina, glicina, metionina, lisina, taurina e triptofano, foram os aminoácidos livres predominantes e cuja libertação demonstrou-se obedecer a uma cinética monomolecular ou de pseudo- segunda ordem, dependendo do aminoácido. Embora em menor escala, a concentração de proteínas miofibrilares também aumentou com o tempo – de 3 para 3.7 g/L – fenómeno este ainda atribuível à proteólise. A análise do azoto total e as suas fracções mostraram que, no final do processo de salga, 36.6% (w/w) de azoto total corresponde a peptídeos curtos com até 20 residuos e a aminoácidos livres, 14.7% (w/w) aos peptídeos com mais de 20 residuos e os restantes 48.7% (w/w) às proteínas. A concentração total de aminas biogénicas na água no final do processo de salga foi de ca. 100 mg/kg. X Resumo Proteínas, peptídeos a aminoácidos foram recuperados com sucesso por meio de um processo de sorção em batch e após um pré-tratamento com etanol de grau alimentar de forma a reduzir a concentração de sal da água, cuja elevada concentração, ca. 4.3 M, demonstrou afectar negativamente o mecanismo de adsorção dos aminoácidos. A presença do sal em certas concentrações demonstrou-se no entanto ser positiva, devido ao efeito da força iónica sobre a adsorção de aminoácidos. A resina polimérica comercial Amberlite XAD16, uma resina neutra e não-polar, foi seleccionada para prosseguir com estudos de adsorção. Aminoácidos livres, proteínas e peptideos foram adsorvidos no mesmo estágio. O processo de recuperação foi efectuado recorrendo a solventes de grau alimentar. Foi efectuada uma análise paramétrica do processo de adsorção examinando o efeito de diferentes parâmetros, nomeadamente temperatura, pH, percentagem de etanol adicionado a água residual, agitação, força iónica da solução, quantidade de adsorvente. O volume de solvente eluente e a temperatura foram os parâmetros avaliados na etapa de desadsorção. Os resultados mostraram que o processo de adsorção é controlado pela temperatura e pela força iónica, a qual neutraliza os efeitos do pH, e que a desadsorção é controlada pela temperatura e pela natureza do solvente eluente. A acetona resultou como o melhor solvente para desadsorção de aminoácidos livres, com um rendimento de recuperação de aminoácidos hidrofobicos e neutros de 100 %. As proteínas foram desadsorvidas por uma solução básica de hidróxido de sódio em água ao 4% (w/v), com um rendimento de recuperação de 100 %. Os aminoácidos livres extraídos da água residual
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