Chemical-bromatological characterization of canned tuna processing sidestreams as a potential source of compounds of high added value

Introduction The increase of side-streams generated by the fish supply chain represents a huge economic and environmental problem [1]. Since discarded by-products and co-products are potential sources of compounds of high added value, their valorisation is the aim of EcoeFISHent [2], a European Union’s Horizon project (H2020-LC-GD-2020-3, https://cordis.europa.eu/project/id/101036428/it), “Demonstrable and replicable cluster implementing systemic solutions through multilevel circular value chains for eco-efficient valorization of fishing and fish industries side-streams”. UniGe, as one of EcoeFISHent's 34 partners, focused on green extraction, biological testing and formulation of nutraceutical and cosmetic ingredients from the canned tuna supply chain and aquaculture collateral streams (FSS: Fishery Side Streams). As one of the first steps in their enhancement, the chemical-bromatological characterization was mandatory. In detail, two types of FSS originating from canned tuna industrial processing were examined, both dehydrated by a patented process (EFP: Eco-FISH-powder) in order to stabilize this highly perishable biomass. This preliminary characterization heavily influenced the design of the subsequent extraction steps. Materials and Methods Tuna Yellowfin Tuna Yellowfin FSS have been obtained from two different steps of canned tuna processing performed at Generale Conserve SpA- ASdoMAR, an industrial partner of the project: 1) Raw tuna samples (RAW) are made up of the discarded fillet scraps, fishbones, fins, heads and viscera (two batches of about 75-100 kg); 2) Cooked tuna samples (CKD) are obtained during the processing of tuna fillets, are made up of discarded fillets’ part (red meat & bones) after a cooking treatment performed to obtain the commercial product (two batches of about 80-100kg). Both RAW and CKD samples have been dehydrated by a patented process in a modified atmosphere obtaining a powdered material (EFP) with a low residual moisture in order to stabilize the biomass without using high temperatures. Moisture content: Termogravimetric Analysis The residual moisture (M%) has been evaluated with a Sartorius thermogravimetric humidity analyser (Massachusetts, USA) and data have shown to be comparable with those obtained by applying the less fast official drying-oven method (AOAC Official Method 934.01) Protein content: Kjeldahl Method Protein fraction (P%) has been evaluated with the official Kjeldahl method using a Kjeldahl automatic distillation system K-360 BUCHI (Büchi Labortechnik AG, Flawil, Switzerland) adapting the method to the specific type of sample and considering 6.25 as a conversion factor. Autumn School in Food Chemistry – Pavia October 2022 Ashes content: Oven and Muffle Furnace Method Ashes (A%) have been determined using a muffle furnace (Nabertherm, Lilienthal, Germany) following the AOAC official method (942.05). Lipid content Hara-Radin method [3] for cold lipid extraction has been performed to replace Folch method, in order to avoid halogenated solvents, by using a 3:2 mixture of hexane:isopropanol followed by a wash of the extract with aqueous sodium sulfate to remove non-lipid contaminants and lipid fraction (L%) has been gravimetrically determined. Fatty acids profile The fatty acids (FA) profile has been determined, by derivatization of FA to fatty acid methyl esters (FAME) and then analysing FAME by gas chromatography-mass spectrometry (GC-MS). The SFA, MUFA, PUFA, ω3- PUFA are summarised in Figure 1. Oxidation status monitoring The spectrophotometric analysis of the conjugated dienes (CD) has been performed together to the peroxide value (PV) test for the primary oxidation products evaluation, since conjugated dienes exhibit an intense absorption at 232 nm and conjugated trienes (CT) at 270 nm (in isoctane) [4]. The TBARS (Thiobarbituric Acid Reactive Substances) test [5] directly applied on food samples, avoiding the lipid extraction step, has been proposed to evaluate the secondary oxidation products. Results are expressed as mmol of malonaldehyde (MDA)/kg of sample; MDA represents one of the main products of PUFA oxidation and it forms a pink complex with Thiobarbituric acid that shows high absorption at 532nm. Results, presented as range since non-disclosure agreement, are shown below in Table 2. The analytical determinations suggested by Codex Alimentarius [6], such as Peroxide value (Table2) and p Anisidine value (AV) to evaluate primary and secondary oxidation products respectively and their combination in the Total oxidation value (ToTox), are currently in progress with CDR FoodLab®, an analysis system that allows to test easily and rapidly the key parameters for fish oil quality control. Results and Discussion Preliminarily, chemical-bromatological analysis of the following EFPs: A1) RAW non-dehydrated, A2) RAW dehydrated, B1) CKD non-dehydrated and B2) CKD dehydrated, have been carried out to evaluate their proximate composition in moisture, protein, ash, lipid and carbohydrates contents. Samples have been milled with Grindomix GM200, Retsch, Haan, Germany, to get homogeneous materials. All measurements have been performed by two different laboratories (UNIGE and MICAMO labs) on two different batches (batch1 and 2) of EFPs both in duplicate, and results are summarized in Table 1 as range, since non-disclosure agreement and because the composition of these side streams has an intrinsic variability from batch to batch. Carbohydrates (C%) have always been obtained by difference. Their values are not reported for tuna samples, since they are negligible. Despite the variability of these typologies of samples, characterized by non-homogenous and non-constant composition due to their specific origin, results of different laboratories and on different batches show compatible ranges of proximate composition, although the ash content is the most susceptible to the inhomogeneity of the samples since some bones are difficult to homogenize without risking heating the samples too much during grinding. In all the tuna side-streams taken into account the protein content was judged suitable to continue the extraction process, both in RAW and in CKD, and therefore suitable to continue with their valorisation as planned in EcoeFISHent project. As far as the lipids are concerned, the ω3-PUFA content is suitable to be valorised too. Nevertheless, this fraction presents oxidation parameters quite high, for this reason, at present, a previous refining step is recommended to valorised it as edible ingredient. To attain the aim of EcoeFISHent Horizon Project further analysis on the quality and safety of the different fractions and on the extraction of added value compounds are in progress. References 1) Alfio VG, Manzo C, Micillo R. From Fish Waste to Value: An Overview of the Sustainable Recovery of Omega- 3 for Food Supplements, Molecules, 2021 Feb 13;26(4):1002 doi: 10.3390/molecules26041002 2) EcoeFISHent H2020-LC-GD-2020-3, https://cordis.europa.eu/project/id/101036428/ 3) Atsushi Hara, Norman S. Radin, Lipid extraction of tissues with a low-toxicity solvent, Analytical Biochemistry, Volume 90, Issue 1, 1978, Pages 420-426, ISSN 0003-2697 4) Shahidi F et al. Bailey's Industrial Oil and Fat Products, 2005, 10(4):1103-1112. DOI:10.1002/047167849x.bio050.pub2 5) Hu Z et al. Food Chemistry, 2010, 123(3):794-799. DOI: 10.1016/j.foodchem.2010.05.012 6) Codex Alimentarium Commission Standard for fish oils Codex Stan, 2017. Acknowledgment EcoeFISHent project (Demonstrable and replicable cluster implementing systemic solutions through multilevel circular value chains for eco-efficient valorisation of fishing and fish industries side-streams) Grant agreement