Valorisation of codfish (Gadus morhua L.) salting processing wastewater through the extraction of high added value compounds

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. 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 mostraram actividade antioxidante em geral e de proteção do DNA contra a oxidação em particular. Para os mesmos aminoácidos, a biodisponibilidade in-vitro foi estudada usando celulas Caco-2, medindo a taxa de transporte paracelular e a resistência elétrica transepitelial para verificar a integridade das células do epitelio intestinal. Os resultados mostraram que todos os aminoácidos livres extraídos permearam através da monocamada celular intestinal, embora em taxas diferentes e dependendo da sua concentração inicial; o transporte foi superior a 90 % para todos os aminoácidos livres, excepto a creatina, cujo transporte não passou de 6 %. A presença de sal na solução teve um papel positivo sendo o cloreto de sódio entre os mais importantes osmólitos de aminoácidos em seres humanos.Despite the development of other means of preservation, salt-cured codfish (Gadus morhua L.) continues to be widely produced due to the simplicity of processing, low operating costs, and the highly appreciated sensory characteristics promoted by salt. Codfish takes salt up to ca. 20 % (w/w) during the dry-salting and drains concomitantly ca. 22 % (w/w) of its physiological water. Approximately 200 L of heavy salted wastewater are generated for each ton of fresh codfish undergoing the dry-salting process, which drives important changes in composition and structure of the muscle tissue. Water drained away through the salting process is currently treated as an ecotoxic waste due to the high content of chlorine, which can reach values as high as 160 g/L, representing a strong impact to the environment. This residual water carries important bioactive compounds, ca. 10 g/L, such as free amino acids, peptides and proteins, which although not regarded as essential nutrients, can be considered beneficial under certain circumstances. As such, the recovery of organic compounds and of food-grade marine salt used in the salting process can be considered as valuable management options for that wastewater. In this study, the chemical profile of the salting process residual water has been thoroughly examined. During the salting period the content of dry matter in wastewater increased with time. Concentration of free amino acids increased from 3.5 g/L to 6.5 g/L within 6 days probably due to proteolysis. Myofibrillar proteins concentration also increased however at a lower extent, from 3 to 3.7 g/L, a phenomenon still related to proteolysis. Creatine, aspartic and glutamic acids, arginine, glycine, methionine, lysine, taurine and tryptophan were the dominant free amino acids, which release was successfully modelled through a monomolecular or pseudo-second order kinetic, depending on the amino acid. When total nitrogen and relative fractions have been scrutinised, results showed that, by the end of the salting process, 36.6 % (w/w) of total nitrogen corresponded to small peptides (up to 20 residues) and free amino acids, 14.7 % (w/w) corresponded to peptides and the remaining 48.8 % (w/w) represented proteins. Total biogenic amines concentration in wastewater at the end of the salting process was ca. 100 mg/kg. The organic load was successfully recovered by batch sorption on a polymeric resin after a pre-treatment aimed at reducing salt concentration in wastewater and whose elevated concentration – 4.3 M – negatively affected the mechanism of adsorption of amino acids. Food-grade ethanol was used for salt precipitation from wastewater. Investigation carried out revealed however the positive effect of ionic strength – up to certain molarities – on amino acids adsorption onto resin up to certain molarities. Amberlite XAD16, a commercial macroreticular polymeric resin, neutral and non-polar, has been selected amongst all resins tested. Free amino acids and proteins were adsorbed in the same stage but desorption has been performed selectively. Parametric analysis of the adsorption process has been carried out by studying the effect of six entities, namely temperature, pH, mixing rate, ionic strength, amount of ethanol in solution and adsorbent dose. Effect of temperature and solvent nature were examined for the desorption step. Results showed that the adsorption process is controlled by temperature and ionic strength, which neutralize pH effects, and desorption is controlled by temperature and by the nature of the regenerant solvent. Acetone resulted as the best solvent for free amino acids desorption with a yield of recovery of hydrophobic and neutral amino acids of 100 %. Proteins were desorbed by a strong basic solution of sodium hydroxide in water at a rate of 4 % (w/v), with a yield of recovery of 100 %. Free amino acids extracted from wastewater showed chemical antioxidant activity; they also demonstrated biological activity by preventing DNA oxidation. In vitro bioavailability of extracted amino acids was studied using Caco-2 cell line by measuring the paracellular transport of free amino acids extracted and the transepithelial electrical resistance to verify intestinal cell monolayer integrity. Results showed that all free amino acids were transported through the intestinal monolayer, however at different rates depending on initial concentration; transport has been higher than 90 % for all free amino acids except for creatine, whose transport has been not higher than 6 %. The presence of salt in solution contributed to the paracellular transport of free amino acids being sodium chloride among the most important amino acids osmolytes in human beings

Valorisation of canned sardines and mackerel residues through extraction of bioactive compounds

Healthful and valuable compounds can be recovered from the fish canning residues and employed in high-priority fields such as medicine and food, or in other areas such as agrochemical and animal feedings. Proteins, lipids, biopolymers, amino acids and enzymes can be recovered either from wastewaters or from solid residues (head, viscera, skin, tails and flesh) generated along the canning process of sardine and mackerel, throughout the salting, cooking and filleting stages. Sardine and mackerel scales were processed for the recovery of collagen and its hydrolysed derivatives, either enzymatically, such as collagen peptides, or thermally, such as gelatine. The hydroxyapatite and calcium phosphate-based materials were extracted from bones throughout calcination, while anti-hypertensive peptides were recovered from flesh or cooking wastewater through enzymatic hydrolysis of muscle proteins. Peptides obtained from hydrolysis showed bioactivity namely high anti-hipertensive property. Collagen and collagen peptides could be employed in cosmetics and biomedicine, while gelatine could be used in low-fat food formulations, due to its fat-like melting properties which can contribute to a smooth and creamy mouth-feel. Hydroxyapatite and calcium phosphate could be used for the development of biocompatible bone cement for craniofacial, oral-maxillofacial and orthopaedic defect repair, and coating for femoral components; the ion-exchange properties of hydroxyapatite could make it also suitable for wastewater treatment (heavy metals removal). Finally, anti-hypertensive peptides from flesh residues, as well as collagen peptides (<3000 Da) could be employed in the development of functional foods and drinks formulations. This research shows the opportunities for the valorisation of bioactive compounds from sardine and mackerel canning residues. These are amongst the most consumed fish in the Mediterranean area; moreover, canning is one of the most important and applied methods of preservation. The large quantities of by-products generated have great potentials of valorisation and the extraction of bioactive compounds will also contribute to reduce their impact on the environment.info:eu-repo/semantics/publishedVersio

Solvent extraction of sodium chloride from codfish (Gadus morhua) salting processing wastewater

Codfish is worldwide mostly consumed salt-cured due to the highly appreciated sensory characteristics promoted by salt. During the salting process huge amounts of salted wastewater are generated – approximately 22% w/w of the codfish – carrying ca. 250 g/L of sodium chloride and ca. 10 g/L of organic compounds, namely proteins and free amino acids. In this study, the salt load of wastewater generated during the salting process of codfish (Gadus morhua) was successfully reduced by ethanol extraction. The effects of time, sample:ethanol ratio, pH and temperature on salt extraction by ethanol addition were assessed by a onevariable-at-a-time approach and then by performing a 32 fractional factorial design. The effects of pH were also assessed in absence of ethanol. The maximum amount of salt precipitated in the wastewater was ca. 33%, at a wastewater-ethanol ratio 1:1 (v/v), at a temperature of 0 °C and after 30 min. Proteins and free amino acids present in the wastewater significantly limited salt precipitation; in a blank solution (salt in water at ca. 250 g/L) a higher amount of salt, ca. 37%, was precipitated in the same conditions. Ethanol and temperature showed a linear effect on salt precipitation for both wastewater and blank solution however ethanol was the driving factor. Changes in pH did not result in salt precipitation either in absence or presence of ethanol. During salt extraction, no precipitation of free amino acids occurred in the wastewater while ca. 1.4% of protein fell into the precipitated phase along with the salt

Valorisation of natural extracts from marine source focused on marine by-products: A review

The wide chemical and biological diversity observed in the marine environment makes the ocean an extraordinary source of high added value compounds (HAVC) which can be employed in many applications. Minerals, lipids, amino acids, polysaccharides and proteins from marine sources have unique features and, surprisingly, their highest concentration is often found in parts of marine organisms that are commonly discarded. Fish heads, viscera, skin, tails, offal and blood, as well as seafood shells possess several HAVC suitable for human health applications, yet most end up as residues throughout the raw material processing. This review updates information on this issue and conveys critical analysis of the chief methodologies to carry out extraction, purification and eventual transformation, with a focus on their actual and potential applications.info:eu-repo/semantics/publishedVersio

Characterisation of soluble nitrogen and muscle proteins in wastewater throughout the salting process of Codfish (Gadus morhua)

In Portugal, Atlantic codfish (Gadus morhua) is usually consumed after dry salting; this process is carried out by mixing deboned codfish with food–grade marine salt followed by stacking in a tank for 6 days. Along the salting process, codfish incorporates salt as well as it is partially dried by the release of water - up to 22 % (w/w). Currently, this wastewater is treated as a residue not being further valorized. However, the presence of a significant amount of valuable compounds in this rest, such as amino acids and proteins, may allow for valorization opportunities not yet explored. The present work focuses on the identification of the nitrogen-containing compounds present in such wastewater. Total nitrogen (WSN), trichloroacetic acid-soluble nitrogen (TCASN) and phosphotungstic acid-soluble nitrogen (PTASN) were evaluated by the micro-Kjeldahl method; Biuret method was used for total protein determinations and SDS-Page was performed for protein molecular weight screening. The results revealed an increase of WSN, TCASN and PTASN with time, with corresponding values of 3.17 g/L (WSN), 1.62 g/L (TCASN) and 1.16 g/L (PTASN) by the end of the process; the evolution of WSN versus the released water was approximately constant during the salting process, as well as the ratios of TCASN/WSN and PTASN/WSN with values of 51.25 and 36.55 % (w/w) at equilibrium, respectively.info:eu-repo/semantics/publishedVersio

Antigen Detection Tests for SARS-CoV-2: a systematic review and meta-analysis on real world data

Background and aim Rapid antigen detection (RAD) tests on nasopharyngeal specimens have been recently made available for SARS-CoV-2 infections, and early studies suggested their potential utilization as rapid screening and diagnostic testing. The present systematic review and meta-analysis was aimed to assess available evidence and to explore the reliability of antigenic tests in the management of the SARS-CoV-2 pandemic

Protective effects of lemon nanovesicles: evidence of the Nrf2/HO-1 pathway contribution from in vitro hepatocytes and in vivo high-fat diet-fed rats

The cross-talk between plant-derived nanovesicles (PDNVs) and mammalian cells has been explored by several investigations, underlining the capability of these natural nanovesicles to regulate several molecular pathways. Additionally, PDNVs possess biological proprieties that make them applicable against pathological conditions, such as hepatic diseases. In this study we explored the antioxidant properties of lemon-derived nanovesicles, isolated at laboratory (LNVs) and industrial scale (iLNVs) in human healthy hepatocytes (THLE-2) and in metabolic syndrome induced by a high-fat diet (HFD) in the rat. Our findings demonstrate that in THLE-2 cells, LNVs and iLNVs decrease ROS production and upregulate the expression of antioxidant mediators, Nrf2 and HO-1. Furthermore, the in vivo assessment reveals that the oral administration of iLNVs improves glucose tolerance and lipid dysmetabolism, ameliorates biometric parameters and systemic redox homeostasis, and upregulates Nrf2/HO-1 signaling in HFD rat liver. Consequently, we believe LNVs/iLNVs might be a promising approach for managing hepatic and dysmetabolic disorders

The cl2/dro1/ccdc80 null mice develop thyroid and ovarian neoplasias

We have previously reported that the expression of the CL2/CCDC80 gene is downregulated in human papillary thyroid carcinomas, particularly in follicular variants. We have also reported that the restoration of CL2/CCDC80 expression reverted the malignant phenotype of thyroid carcinoma cell lines and that CL2/CCDC80 positively regulated E-cadherin expression, an ability that likely accounts for the role of the CL2/CCDC80 gene in thyroid cancer progression. In order to validate the tumour suppressor role of the CL2/CCDC80 gene in thyroid carcinogenesis we generated cl2/ccdc80 knock-out mice. We found that embryonic fibroblasts from cl2/ccdc80(-/-) mice showed higher proliferation rate and lower susceptibility to apoptosis. Furthermore, cl2/ccdc80(-/-) mice developed thyroid adenomas and ovarian carcinomas. Finally, ret/PTC1 transgenic mice crossed with the cl2/ccdc80 knock-out mice developed more aggressive thyroid carcinomas compared with those observed in the single ret/PTC1 transgenic mice. Together, these results indicate CL2/CCDC80 as a putative tumour suppressor gene in human thyroid carcinogenesis