Valorização sustentável e desenvolvimento de soluções inovadoras para a funcionalização nutricional e tecnológica dos produtos da pesca

Abstract

There is currently a great consumer demand for natural foods with higher nutritional value, that can provide health benefits, without chemical additives, locally sourced, sustainably produced and of easy preparation. Special attention has been given to the addition to foods of bioactive compounds, with antioxidant and antimicrobial activities, that can, both preserve the food and provide extra benefits to the consumer. Bioactive compounds have a wide range of physiological properties, including antibacterial, antiviral, anti-carcinogenic, and anti-inflammatory activities as well as acting in the prevention of cardiovascular diseases. Fish is one of the most nutritious foods yet a highly perishable product with a very short shelf-life. Portugal is the largest consumer of fish products among EU Member States, where the Portuguese fisheries sector represents a primary sector of significant socio-economic importance. In 2022 a decrease in catches occurred both on the Mainland, with 106,158 tonnes (-14.1% compared to 2021) and in the autonomous regions, with catches in the Azores (10,200 tonnes) falling by 13.9% and the 4,711 tonnes caught in Madeira representing a decrease of 9.2% The main caught species are sardines (Sardina pilchardus), chub mackerel (Scomber colias) and horse mackerel (Trachurus trachurus) with 24.1, 21, and 17.6 thousand tonnes, respectively. For the reduction recorded in the volume of catches at national level, a decisive contribution was made by the decrease occurred in marine fish (100,857 tons), 14.0% lower than in 2021. The reduction of species such as anchovy (-63.3%), with 3,533 tonnes, black horse mackerel (-51.4%), with 2,959 tonnes, and tuna (-25.4%), with 8,789 tonnes and chub mackerel (-7.8%), which did not exceed 21,144 tons. There is also a decrease for sardines, with the 24,6311 tonnes captured representing a decrease of 8.9% compared to 2021. On the other hand, some species with weight in the total volume of national fish catches registered increases, particularly horse mackerel (+6.4%) with 17,702 tonnes, black scabbard fish (+3.2%), with 4,252 tonnes and blue whiting (+61.7%), which reached a catch of 1,956 tonnes. Unlike sardines and mackerel that have a high industrial demand by the canning industry, Atlantic horse mackerel is mainly consumed in fresh. Atlantic horse mackerel stock is managed using the Maximum Sustainable Yield approach, which, together with the good condition of the stock, has allowed a successive increase in fishing opportunities in recent years. Despite the progressive increase in fishing opportunities, the national fleet does not capture the full quotas, mainly for socio-economic reasons. Recently, initiatives sponsored by the Ministry of Agriculture and Food and by organizations such as Docape Portos e Lotas, SA, the state-owned company in charge of organising the first sale of fish, sought to promote the consumption of this species among consumers, as it is a biologically sustainable fish stock, further aiming at increasing fishermen's revenue improving the household welfare of coastal fishing. Regarding pine forestry industry by-products, such as maritime pine bark from Pinus pinaster Aiton subsp. atlantica, they are sources of phenolic compounds with known antioxidant and antimicrobial activities, having a great potential for application in foods. Also, peptides extracted from marine sources such as mussels (Mytillus sp.) and microalgae (Tetraselmis sp.) have shown antimicrobial and antioxidant capacities. Only 30% of the processed mussel is finally consumed since a huge quantity is wasted at different steps of the production chain (washing and declumping shells, and mussel meat extraction stages) or by means of the rejection of mussels only due to a size characteristic criterion. Tetraselmis sp. are available wild in all marine waters and are also industrially cultivated. The smoking of foods is a long-standing practice used to preserve certain foods such as fish, meat, and dairy products. Products are directly exposed to natural smoke from combustion of sawdust or wood. Nowadays this traditional method, when applied, is mainly to give the product the characteristic colour, aroma and taste, and the preservative effect is overlooked. Also, foods can be just smoke-flavoured with smoke aromas from condensed wood smoke (liquid smoke). Using liquid smoke is easy to apply and avoids the risk of toxic and naturally occurring smoke carcinogenic particles. Although not a traditional product in Portugal, the demand for smoked fish has increased considerably with a consequent increase in their value. Mainly, fatty species, rich in omega 3-fatty acids, are used. The common smoking process of salmonids species usually occurs at temperatures below pasteurization (cold-smoking), with resulting products having a shelf-life of 5-6 weeks. Several outbreaks of the pathogen Listeria monocytogenes in these cold-smoked products have been reported and, consequently, various bio-preservation methods have been tested. For other species, such as horse mackerel, smoking temperatures used are above 60 °C (hot smoking). The oxidative deterioration of processed fatty foods generates off-flavours and odours, decreasing their organoleptic and nutritional qualities. These oxidative processes can be inhibited or delayed by various strategies such as modified atmosphere packaging and application of natural antioxidants. Consequently, as proposed in this research work, the application of natural bioactive compounds to fish products is an opportunity to develop sustainable food products adapted to current consumption trends, valorising underexploited aquatic and forestry by-products. Therefore, the research proposal encompassed multiple tasks, aiming at the valorisation of underutilized fish species through the development of a dried smoke-flavoured fish product using bioactive compounds extracted from agroforestry by-products, having antioxidant and antimicrobial properties that will contribute to improve product stability leading to higher quality of the smoke-flavoured fish products. The optimization of a liquid smoke formulation using maritime pine bark extracts, the extraction methodologies and application techniques as a proof of concept, were explored across the chapters of this thesis. Chapter 1 served as a comprehensive introduction, offering an updated review (Background and State of the Art) that covered various subjects, including increased knowledge on available sustainable undervalued fish species, the different processes used for smoking fish, the availability and properties of bioactive compounds from forest industry by-products and the methodologies for their extraction and application in such food product. The research objectives were defined in Chapter 2 and divided into specific goals. In this chapter, the thesis structure is outlined, establishing a link between the specific objectives and the investigations carried out in the subsequent results and discussion chapters. Chapter 3 provides a brief overview of the topics and the methodology used for each, with further elaboration and detail in later chapters. Chapter 4 aimed to characterize the target raw material, the Atlantic horse mackerel (Trachurus trachurus), an abundant fish species that is nutritionally very rich but undervalued and of little commercial value. Hence, the nutritional composition of muscle tissue of locally sourced Atlantic horse mackerel, as well as the fatty acid composition and the essential minerals, along with non-essential elements such as chromium (Cr), cadmium (Cd), and lead (Pb) were determined. Total phenolics content, antioxidant properties, and quality parameters such as oxidative degradation indicators, biogenic amines and microbiological safety parameters were studied. The estimated Daily Intake of the different minerals resulting from the consumption of this fish species was also evaluated. Results indicated that Atlantic Trachurus trachurus presented the following components in percentage (% w/w): moisture at 73.99±0.41, ash at 1.44±0.04, crude protein at 21.08±0.47, and crude fat at 2.87±0.02. The mineral composition of fish muscle showed phosphorus (P) as the most abundant mineral, with 2240.1 mg/kg, followed by potassium (K) at 378.2 mg/kg, and magnesium (Mg) and calcium (Ca), both at 345.3 mg/kg. Unsaturated fatty acids stood out with 69.89% of total fatty acids, with special emphasis on the omega 3 (35.54%) and omega 6 (2.02%) fatty acids. In conclusion, this research underpinned the significance of Atlantic horse mackerel (Trachurus trachurus) as a valuable source of fats and proteins, essential minerals and unsaturated fatty acids, that offer substantial health benefits to humans. In Chapter 5 the study aim was to evaluate three types of natural bioactive extracts, namely pine bark (Pinus pinaster Aiton subsp. atlantica) extracts obtained by Microwave-assisted extraction (MAE), peptides obtained from mussel (Mytilus galloprovincialis) and microalgae Tetraselmis sp., with antioxidant and antimicrobial properties, for application in dried smoke-flavoured horse mackerel fillets. The three bioactive extract solutions were applied by spraying on the dried fillet surface. After 15 days of storage at 4 °C, significant differences in properties were observed. Moisture and salt had an inverse relationship, with decreasing moisture and increasing total dissolved solids. Oxidation levels remained acceptable, although sensory quality was affected by storage. Microbiological analysis revealed high contamination levels in certain samples at specific sampling points in time, although no pathogens such as Salmonella spp. or Listeria monocytogenes were detected. While the microalgae extract demonstrated the most powerful antioxidant capacity, its performance was hampered by the poor sensory scores. On the other hand, the pine bark extract was the most acceptable from a sensory point of view and also revealed some antimicrobial inhibition. Thus, the pine bark extract was selected for subsequent studies. Chapter 6 aimed to characterize the pine bark (Pinus pinaster Aiton subsp. atlantica), an abundant timber industry by-product, collected in the Minho region of northwest Portugal. The research extensively examined the barks chemical and thermal characteristics, including ash content, extractives, lignin, cellulose, hemicellulose, fatty acids, and mineral composition. Additionally, various analytical techniques like FTIR, SEM, DSC, DTG, and XRD were used to observe chemical structure differences. The results reveal that the Pinus pinaster bark primarily consists of lignin and holocellulose, with extractives mainly soluble in tolue ethanol, followed by water, and a small amount of them are soluble in ethanol. The bark contained around 0.44% ash, and heavy metals such as Cd and Pb were not found. During degradation, Pinus pinaster experienced a 10% mass loss at 140 °C. In terms of crystallinity, holocellulose and cellulose showed similar percentages at approximately 25.5%, while -cellulose displayed the highest crystallinity index at 41%. Total extractive materials with a higher value, approximately 10% w/w of the sample by mass, showing potential use in a biorefinery concept. The four subsequent chapters, Chapters 7, 8, 9 and 10, include the research studies carried out on maritime pine bark extracts. In Chapter 7 the aim was to identify the ideal conditions for the extraction of bioactive compounds from pine bark, employing two distinct extraction methods, exploring inner, outer, and whole pine bark layers, and using water, ethanol and a hydroethanolic mixture as solvents. Eighteen different extracts were obtained and tested. The study unveiled that the microwave-assisted extraction method was the most effective approach for extracting bioactive compounds from pine bark, consistently surpassing the traditional Soxhlet technique across all assessed parameters. Among the bark layers investigated, both the inner and whole bark displayed higher levels of extractable compounds. Furthermore, using a 50% water-ethanol solvent generally resulted in superior outcomes, than ethanol or water alone. Employing solvent mixtures that combine organic solvents like ethanol with water tended to be more cost-effective for the recovery of these compounds. The highest amount of condensed tannins was obtained using the microwave-assisted method and water:ethanol as solvent and the whole bark, this last finding allowing the full use of the pine residue. Chapter 8 aimed to compare the pine bark hydroethanolic extracts obtained from MAE and Soxhlet in terms of inhibitory effects against known food pathogens. In the current study, the antimicrobial activity of Pinus pinaster Aiton subsp. atlantica extracts was screened against gram-positive bacteria (Bacillus cereus, Clostridium perfringens, Listeria monocytogenes, Staphylococcus aureus), gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Salmonella enterica serovar Enteritidis) and the fungus Aspergillus brasiliensis ATCC 16404. The hydroethanolic extracts obtained by Microwave-assisted extraction (MAE) at 1600 W and 110 ºC for 30 minutes, were compared to the Soxhlet extracts. Hydroethanolic pine bark extracts were prepared at 0.25, 0.50 and 80.00 mg/mL concentrations. The extraction yield and the total phenolic content were determined MAE enabled extracts with higher extraction yield and higher total phenolic content (20.4±0.7% (w/w) and 71.1±3.4 mg of gallic acid equivalent (GAE) per g of sample, respectively) than corresponding Soxhlet extracts (17.6±0.2% (w/w) and 54.9±2.6 mg GAE/g sample, respectively). The pine bark extracts presented inhibition halos ranging from 7 to 17 mm against the gram-positive bacteria, namely C. perfringens ATCC 13124 and B. cereus NCTC 11143. In terms of extraction method, the MAE extracts presented larger inhibition halos than the Soxhlet counterparts. Regarding the tested concentrations, only C. perfringens ATCC 13124 and B. cereus NCTC 11143 were sensitive to the 0.25 and 0.50 mg/mL concentrations. At 80 mg/mL all the gram-positive bacteria were sensitive to the pine bark extracts obtained by both extraction methods. On the other hand, the extracts did not show inhibitory effect against the tested gram-negative bacteria and A. brasiliensis. This study reveals that MAE is a fast and efficient method for obtaining Pinus pinaster Aiton subsp. atlantica extracts with antimicrobial activities against Gram-positive bacteria. In Chapter 9 the aim was to optimize the MAE conditions for whole pine bark extracts production regarding the type of solvent, extraction temperature and process duration on antioxidant properties, for subsequent application to the fish product. This study systematically examined the impact of solvents (water, ethanol and a 50% ethanol-water mixture), extraction time (15 or 30 minutes), and extraction temperature (90°C, 110°C, or 130°C) on phenolic recovery from pine bark, utilising a one-variable-at-a-time experimental approach. High-Performance Liquid Chromatography (HPLC) profiling was employed to analyse the polyphenol composition of the extracts. All extraction parameters, including solvent ethanol/water concentration, extraction time, and extraction temperature, exhibited a significant effect on phenolic compounds' extraction efficiency and on Pinus pinaster subsp. atlantica extracts antioxidant capacities. Results underscored the critical role of the extraction solvent choice, revealing that ethanolic and hydroethanolic extracts exhibited potent antioxidant activity. Microwave-assisted extraction proved effective in increasing the extraction of antioxidant phenolic compounds without compromising the integrity of the pine bark extracts under all tested conditions. Considering the economic factors (time-consumption), the yield of phenolic compounds, and the extract's antioxidant activities evaluated by three methods, DPPH Free Radical Scavenging Effect (DPPH), ABTS Radical Cation Scavenging Effect (ABTS), Oxygen Radical Absorbance Capacity (ORAC), the optimal extraction temperature and time were determined to be 130 °C for 15 minutes using the hydroethanolic solvent mixture. Chapter 10 focuses on the comparison between two extraction methodologies ultrasound-assisted extraction (UAE) and the optimized MAE conditions. In this study the following parameters were evaluated: extraction yield, total phenolic compounds, antioxidant activity (DPPH, ABTS, ORAC, Thiobarbituric Acid Reactive Substances (TBARs), Cellular Antioxidant Activity (CAA) and Oxidative Haemolysis Inhibition Assay (OxHLIA)), antimicrobial activity, antiproliferative activity, anti-inflammatory activity and phenolic compounds profile. MAE was the technique that most favoured extraction, as reflected in the phenol content of the extracts, as well as in their antiradical capacity, which was higher than the extract obtained by UAE. However, in the determination of antioxidant capacity by ORAC and OxHLIA, no statistically significant differences were observed. Both extracts showed very good ability to inhibit lipid peroxidation. The extraction method influenced the antiproliferative activity of the extracts, as statistically significant differences were observed in all cell lines, except for Caco-2 cells. In the three tumour cell lines for which statistically significant differences were observed, the GI50 values were lower in the case of the extract obtained by UAE, so its anti-proliferative potential is higher. No evidence of anti-inflammatory activity within the tested concentration range was found. In the individual quantification of phenolic compounds in the extract obtained by MAE, a total of 230.02 mg/g of extract was obtained, while the extract obtained by UAE contained 86.95 mg/g of extract. Although both extracts did not show any activity against Gram-negative strains, they showed effective inhibition against Gram-positive bacterial strains. This highlights the potential use of these extracts for their antimicrobial capabilities. Both techniques are simple, fast, and useful methods to extract bioactive phenolic compounds from P. pinaster bark, but findings suggest the applicability of MAE technique more promising for pine bark samples. Thus, MAE extracts were selected for subsequent application in the fish product development studies of Chapters 13, 14 and 15. Chapters 11, 12, 13 and 14 have as major goals the identification of the best processing conditions, namely drying process parameters, salt, smoke flavour and pine bark extracts concentrations, as well as the most appropriate methods for applying the extracts to the fish fillets, conditions that will be selected for the development of the final prototype in Chapter 15. Chapter 11 focuses on reducing the fish fillets salt content, studying both brining immersion times, salt concentration and drying times. Eight formulations comprising different salt concentrations, immersion times and drying times were developed and evaluated aiming at a balance of salt content reduction and organoleptic acceptance of a product with reduced salt content. Determination of pH, water activity (aw), moisture content and NaCl levels, as well as total phenolic content and antioxidant activities (DPPH, ABTS and ORAC) were performed. The effect of the fish fillets immersion in brine were analysed by determination of chloride content, pH, conductivity, total dissolved solids and antioxidant activity of the brines before and after fillets immersion. Sensory studies of the dried fish fillets were carried out using quantitative descriptive analysis - QDA® to evaluate the tasters' perception of the salt reduction in this type of products. Formulations containing 5% brine for 30 min with 2 or 3 hours of drying and 5% brine for 60 min with 2 hours of drying, allowed to obtain a reduction of NaCl in the range of 43%, 28% and 35%, respectively, compared to a commercial-like product (product on the market with the lowest NaCl content: 3.1 g/100g of product). Thus, for these three prototypes, results allowed the potential endorsement of a claim for a product labelled as "reduced-salt" since they demonstrated a reduction higher than 25% in sodium content compared to commercial-like products. Overall, the most desirable attributes of dried horse mackerel fillets were obtained using a 5% NaCl brine for 30 min, followed by drying at 70 ºC for 3 hours. Chapter 12 aimed at identifying the best liquid smoke brine concentration regarding the desired antioxidant and sensory properties of fish fillets. In this sense, the primary aim of this current study was to create smoke-flavoured dried fish fillets of Atlantic horse mackerel (Trachurus trachurus) using liquid smoke. This study involved studying the best concentration of liquid smoke to be applied to a brine solution by evaluating several physicochemical characteristics and antioxidant properties of brines and fish samples, followed by sensory analysis of final products. Fresh fish samples were immersed in the previously selected (Chapter 11) brine, brining and drying conditions. Five batches of fish fillets were produced: FF Fresh fish fillets (control); S5 Dried fillets without smoke flavour; LS5 Dried fillets with 5% (v/v) liquid smoke; LS10 - Dried fillets with 10% (v/v) liquid smoke; and LS20 - Dried fillets with 20% (v/v) liquid smoke. The results showed that as the concentration of liquid smoke in the brine step increased the concentration of phenolic compounds and the antioxidant activity also increased; furthermore, increased sensory preference was also noted. This study highlighted that applying liquid smoke to Atlantic horse mackerel drying process could have notable effects on its phenolic content and antioxidant activity and on sensory attributes, including colour, flavour perception, and texture. The decrease in NaCl in the dried fillets with increasing liquid smoke concentrations is also a promising finding for the production of these type of products with reduced salt content without interfering with consumers acceptability. Prototypes incorporating 5% liquid smoke, 10% liquid smoke, and 20% liquid smoke (LS20), if in the market, could be endorsed with the potential nutrition claim "reduced-salt," as they demonstrate a reduction exceeding 25% in sodium content when compared to a similar commercially available product. Overall, data analysis of results allowed us to determine that the product preference fell on horse mackerel fillets brined with 20% liquid smoke that presented higher phenolic content, greater antioxidant activity and less NaCl content. Chapter 13 aimed to study and select the best pine bark extracts concentrations to be added in the production of dried smoke-flavoured fish fillets. Therefore, the best concentration of pine bark extract to be added to a previously tested brine solution was assessed by evaluating several physicochemical characteristics and antioxidant properties of brines and final products. Seven batches of fish fillets were produced: a batch of control fillets in the standard brine and liquid smoke formulation selected in the previous Chapter 12 (5% NaCl and 20% liquid smoke) and six batches of fillets now with added bark extracts at different concentrations (100, 120, 140, 160, 200 and 220 g of extract/mL of brine), chosen taking into account the limit of toxicity of the extract (Caco-2 GI50, 228 g/mL) previously evaluated (Chapter 10). The effects of the different pine bark extracts concentrations in the final products were evaluated, namely, the incorporation of phenolic compounds with antioxidant capacity into fish fillets during immersion in brine (maximum - 71%) and the average weight loss of the fillets during the drying process (25%). The results showed that as the concentration of extract in the brine step increases, the concentration of phenolic compounds as well as the antioxidant activity in the fillets also increase. Overall, the analysis of the results allowed to determine that the extract concentration that best suited the objectives proposed was 200 g extract/mL of brine, as it presented a high phenolic content and high antioxidant activity, allowing the fillets to be referenced as a safe product. Chapter 14 study intends to ascertain if the methodology used in previous chapters for incorporation of the pine bark extract (directly in the brine before drying) is the best choice by comparison with another technique of spraying the extract directly on the fish fillets surface after the drying process, regarding the antioxidant and organoleptic properties of smoke-flavoured horse mackerel fillets. Three types of microwave pine bark extracts were applied (direct; evaporated; lyophilized) and two methods of application to the fish fillets were tested: before drying by immersion of the fillets in a brine containing the extracts or after drying by spraying the extracts on fish fillets surfaces. Sample immersed in brine with direct extract application showed a lower salt content and a higher content of total polyphenols and antioxidant activity. The preference of the sensory panel also indicated this sample as the one that came closest, in terms of colour and flavour, to a typically smoked fish product. Therefore, the best application method for pine bark extracts in horse mackerel fillets appeared to be through immersion in brine containing the direct microwave extract formulation. Chapter 15 aims at the development and characterization of the final prototype, that is, the vacuum-packed horse mackerel fillets enriched with pine bark extracts utilizing the process conditions selected in previous chapters, that is, a standard brine of 5% NaCl, with 20% liquid smoke (v/v) and 200 µg pine bark extract/mL brine, brining for 30 min, followed by drying at 70 °C for 3 h. Smoked fish fillets were assessed for their proximate composition, nutritional properties, total phenolic content, antioxidant properties, in vitro gastrointestinal bioacessibility and quality parameters, such as oxidative degradation indicators (Peroxide value, TVBN, TBARs). Histamine content, as well as microbiological hygiene and safety indicators were also evaluated. The functional stability of the vacuum-packed fish fillets during storage at 4 °C was studied. The nutritional analysis of the final prototype, comprising lipid content, total protein, carbohydrates and salt according to well-established AOAC methods, as well as lipid and mineral profile analysis were performed. The decrease in antioxidant activity during in vitro gastrointestinal digestion was approximately 50% in all tests performed. Colour measurement of the final prototype took place with a Minolta CR-400 set, recording L*, a*, and b* values for lightness, redness and yellowness. A descriptive sensory analysis and an acceptability test of the final prototype were carried out. Results indicated that the application of pine bark extracts as a brine ingredient together with liquid smoke, to produce dried horse mackerel fillets under the process conditions stated above, extended the product storage life to 85 days. Combining the intrinsic raw material nutritional characteristics, and the ones arising from the liquid smoke, there is a net added value that comes from the incorporation of pine bark bioactive compounds having shown antimicrobial and antioxidant properties, increased the product's lifespan and gave the product not only technological functionality, but eventually biological functionality. Chapter 16 is a general discussion of all results obtained in the thesis and compared with the existing literature. The general conclusion (Chapter 17) is the accomplishment of the research proposal through the multiple studies and achieved results, aiming at the valorisation of an underutilized fish species, through the development of a dried smoke-flavoured fish product, using bioactive compounds extracted from agroforestry by-products, and giving to the product not only technological functionality, but eventually biological functionality since the analysed extracts still retained considerable antioxidant potential.