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Solvent Extraction of Sodium Chloride from Codfish

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Solvent Extraction of Sodium Chloride from Codfish Solvent extraction of sodium chloride from codfish (Gadus morhua) salting processing wastewater Vincenza Ferraro a,b, Isabel B. Cruz a,b, Ruben Ferreira Jorge b, Manuela E. Pintado a, Paula M.L. Castro a,⁎ a CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal b WeDoTech-Companhia de Ideias e Tecnologias, Lda./CiDEB, Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal abstract 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 one- Keywords: 2 Salting wastewater variable-at-a-time approach and then by performing a 3 fractional factorial design. The effects of pH were Codfish also assessed in absence of ethanol. The maximum amount of salt precipitated in the wastewater was ca. 33%, Free amino acids at a wastewater-ethanol ratio 1:1 (v/v), at a temperature of 0 °C and after 30 min. Proteins and free amino Protein acids present in the wastewater significantly limited salt precipitation; in a blank solution (salt in water at ca. Salt extraction 250 g/L) a higher amount of salt, ca. 37%, was precipitated in the same conditions. Ethanol and temperature Solvent extraction 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. 1. Introduction interest in lowering or removing excessive inorganic salt content from the matrix to be processed in order to avoid corrosion problems Salt-cured codfish continues to be produced in large quantities generated by salt deposits inside the process equipment, and in unit because it is highly demanded worldwide. Salt-cured codfish manu- operations such as extractive or azeotropic distillation, adsorption, facturers have to face the costly disposal of huge amounts of salted crystallisation or liquid-liquid extraction, which are salt-sensitive [3]. wastewater produced throughout the codfish salting process. During The objective of this study was the extraction of NaCl dissolved in dry salting, alternate layers of codfish and food-grade marine salt are codfish salting process wastewater, while concentrating in solution stacked in a tank for 6 days. At the end of this period, approximately proteins, peptides and free amino acids. Salt precipitation by ethanol, 200 L of heavy salted wastewater are drained away from each ton of a food-grade solvent, was chosen as an adequate strategy. Organic codfish, carrying ca. 250 g/L of salt (sodium chloride — NaCL) and ca. solvents are frequently used to precipitate inorganic species from 10 g/L of organic compounds, namely muscle proteins, peptides and aqueous solutions both in analytical or in industrial applications [4,5]. free amino acids [1]. Due to the high load of chloride, this wastewater is Pure aliphatic alcohols dissolve, in fact, inorganic salt very hardly and, regarded as ecotoxic and can not be disposed to the sea, which could for instance, just 0.18% (w/w) of NaCl can be dissolved in pure ethanol result, in the long run, in detrimental effects on the aquatic life as well at 20 °C [6]. Amino acids, proteins and peptides, although with lower as on the quality of the seawater [2]. solubility in alcohols than in water, are however much more soluble The recovery of salt and the recovery of relevant organic than sodium chloride under the same operating conditions and compounds present in such residual water are highly desirable as ethanol concentration [7,8]. part of the wastewater management process. Furthermore, it is worth In this study, the effects of wastewater–ethanol ratio, temperature, to highlight that the food processing industry has in general great pH and time on salt precipitation by ethanol addition were investigated. The effect of pH was also examined in absence of ethanol. The study was carried out first by one-variable-at-a-time 2 ⁎ Corresponding author. Tel.: +351 225580059; fax: +351 225090351. approach and then by an experimental design performing a 3 E-mail address: [email protected] (P.M.L. Castro). fractional factorial design (2 variables at 3 levels). Compared to the one-variable-at-a-time approach, the fractional factorial design sample-ethanol mixture supernatant and the supernatant arising methodology allows to study at the same time the effects of each from centrifugation of the precipitated phase were mixed and kept at factor and the effects of interaction between factors on the response ambient temperature until analysis. Similarly, both heavy phases variable. In addition, the total number of runs in factorial design is generated by centrifugation of sample–ethanol mixture and the much less as compared to the one-variable-at-a-time method [9]. The precipitate were mixed and kept at ambient temperature until behavior of proteins and amino acids in the wastewater-alcohol analysis. solution was also assessed by determining their concentration in Each combination of parameters was tested in duplicated: 12 wastewater before and after addition of ethanol. mixtures (6 for wastewater and 6 for blank) for 3 different pH in absence of ethanol, 12 mixtures (6 for wastewater and 6 for blank) for 2. Materials and methods 3 different pH value in the presence of ethanol, 28 mixtures (14 for wastewater and 14 for blank solution) for 7 different times, 28 2.1. Wastewater, blank solution and ethanol mixtures (14 for wastewater and 14 for blank solution) for 7 different amounts of ethanol added to sample, 16 mixtures (8 for wastewater Wastewater was supplied by Pascoal & Filhos, S.A. (Aveiro, and 8 for blank solution) for 4 different temperatures, and 8 mixtures Portugal), as generated throughout the salting process of Atlantic for factorial design (for wastewater only) were prepared, giving a codfish (Gadus morhua) caught in the Northern Sea. Wastewater was total of 104 experiments. Amount of salt precipitated was calculated collected from a tank where 800 kg of codfish had gone through the as the percentage ratio of the difference between initial and final NaCl salting process for 6 days at 17 °C. Before sampling, wastewater was concentration in sample-ethanol mixture and the initial NaCl homogenized by stirring, and after sampling was centrifuged at concentration in the sample-ethanol mixture. 5000 rpm, 4 °C, 10 min in order to precipitate suspended solids, such as fish flesh, skin and bonds residues. 2.3. Determination of salt and dry matter A blank solution of 254 g/L NaCl (resembling concentration in wastewater) was prepared with food-grade marine salt, the same used Total salt content, as NaCl, was determined by titration according for the codfish salting, supplied by Pascoal & Filhos, S.A. An amount of to the Mohr method [10]. Dry matter was quantified as described in 319 g of salt was added to 1 L deionized water and the mixture was EN 12880:2000 [11]. Determinations were carried out in triplicate. placed in an ultrasound bath to accelerate salt dissolution. Absolute ethanol (99.9% purity) of food-grade used for salt 2.4. HPLC–UV/Vis analysis of free amino acids precipitation was supplied by Aga (Porto, Portugal). Chromatographic analysis were performed using a Beckman&Coulter 2.2. Sodium chloride precipitation procedure 168 series HPLC system interfaced with a Photo Diode Array UV/Vis detector (PDA 190–600 nm) (Beckman&Coulter; Fullerton, CA, USA). The following procedure was carried out for both wastewater and Data acquisition and analysis were accomplished using Karat32 software. blank solution. The effects of pH and of ethanol addition on salt All eluents were filtered through a 0.45 μm cellulose membrane precipitation were assessed. Effects of pH were studied in presence (Millipore-Interface; Amadora, Portugal) and degassed in an ultrasound and in absence of ethanol. For the sample-ethanol mixtures, beside bath (Millipore-Interface) for 15 min prior to be used as mobile phases. the effects of pH, the effects of settling time, temperature and ethanol All determinations were carried out in triplicate. in solution were also studied. The effect of pH changes in both blank and wastewater were 2.4.1. Taurine and creatine analysis assessed by acidifying samples down to pH 2, using 1 M hydrochloric Taurine and creatine were analysed using a Waters Nova-Pack RP- acid (Sigma-Aldrich; Sintra, Portugal), and by alkalinizing up to pH 9 HPLC, C18 column (150×3.9 mm, 4 μm) (ViaAthena; Lisbon, Portugal). and pH 11, using 5 M sodium hydroxide (Sigma-Aldrich). Taurine analysis was carried out using the method described by Orth A volume of sample of 100 mL was transferred into a 250 mL cone [12], with a slight modification in the gradient elution and in the column. shaped funnel. Then, acid or alkali was mixed with the sample in order Standard solutions for calibration, covering the range of 10–80 μg/ml, to study the effect of pH. When the effect of ethanol was investigated were prepared from a stock solution containing 100 μg/ml taurine (without changing the original sample pH), an aliquot of that solvent (Sigma-Aldrich; Sintra, Portugal) dissolved in ultrapure water.
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