Salmo salar fish waste oil: Fatty acids composition and antibacterial activity

Introduction

Fish by-products are generally used to produce fishmeal or fertilizers, with fish oil as a by-product. Anyway, fish wastes are an enormous potential source of useful molecules, such as bioactive peptides, enzymes, antimicrobial components and polyunsaturated fatty acids (PUFA) (Ghaly et al., 2013). Despite their importance, fish wastes are still poorly explored and characterized and more studies are needed to reveal their potential.

Polyunsaturated fatty acids (PUFAs) are key constituents of the cell membranes. For this reason they are important regulators of the membrane fluidity, cell signalling, gene expression and cellular functions, and also represent important substrates for lipid mediators synthesis (Parolini, 2019). Low serum levels of ω-3 polyunsaturated fatty acids (PUFAs) have been associated to an increased incidence of cardiovascular disease and higher mortality rate. Furthermore, pre-clinical and clinical studies have shown that the addition of ω-3 PUFAs to daily diet can prevent and attenuate lipid accumulation, vascular inflammation and macrophage recruitment, which are among the major causes of the atherosclerosis plaque formation process (García-Hernández et al., 2013; Corsi, Momo Dongmo & Avallone, 2015; Yagi et al., 2017; Leshno et al., 2018). The American Institute of Medicine, Food and Nutrition Board recommended daily Adequate Intakes (AIs) of Omega-3s (Medicine, 2005) which, unfortunately, are often not met with the daily diet.

The role of PUFAs as pro- or anti-inflammatory molecules has been largely discussed (Fischer & Weber, 1983; Heidel et al., 1989; Hawkes, James & Cleland, 1992; Moreno, 2009; Simonetto et al., 2019). Lipid mediators derived from the ω-6 PUFA are involved in inflammation at different stages. For example, the ω-6 PUFA arachidonic acid is the precursor of prostaglandins, thromboxanes and pro-inflammatory leukotrienes. Instead, ω-3 PUFAs exhibit anti-inflammatory properties by competing with ω-6 PUFAs, altering the membrane phospholipid amount of arachindonic acid and reducing the production of pro-inflammatory eicosanoids. Furthermore, ω -3 PUFAs promote the resolution of inflammation through the synthesis of lipid mediators, including resolvins, protectins and maresins, which are known to be “specialized pro-resolving mediators” (SPMs) (Serhan, 2014).

PUFAs and free fatty acids (FFA) have also been studied for their antimicrobial activity which is characterized by a broad spectrum of activity and the lack of classical resistance mechanisms (Desbois & Smith, 2010; Desbois, 2012; Desbois & Lawlor, 2013). In a recent paper (Chanda et al., 2018), different hypothesis were proposed as PUFAs antimicrobial mode of action which include disruption of intercellular communication, interruption of ATP production, alteration of membrane properties, disruption of fatty acids synthesis, affecting the electron transport system and increasing the number of membrane pores (Zheng et al., 2005; Carballeira, 2008). In addition, as explained elsewhere (Desbois & Smith, 2010), FFAs can also impair Staphylococcus aureus skin colonization through stratum corneum acidification (Fluhr et al. 2001; Takigawa et al., 2005). FFAs may also impair the expression of virulence factors, which are necessary for the establishment of infection, and inhibit the cell-to-cell signalling, thus preventing initial bacterial adhesion and subsequent biofilm formation.

Marine organisms are a very important source of antimicrobial agents (Richards et al., 2001; Schillaci et al., 2010; Schillaci et al., 2013; Spinello et al., 2018; Vazzana et al., 2018; Núñez Acuña et al., 2018) and, among them, the PUFAs are normally present at high levels in Salmo salar (Linder, Fanni & Parmentier, 2005; Morais et al., 2009). Different studies have shown the antimicrobial activity of the salmon PUFAs against Gram-positive bacteria due to their specific components such as eicosapentaenoic acid (Desbois, Mearns-Spragg & Smith, 2009; Desbois & Lawlor, 2013), docosahexaenoic acid (Coonrod, 1987; Feldlaufer et al., 1993; Gladyshev et al., 2009), γ-linolenic acid (Asthana et al., 2006) and dihomo- γ-linolenic acid (Feldlaufer et al., 1993).

It is well known that among fishes farmed in Europe, the Atlantic salmon (Salmo salar) is one of the most important aquaculture species. Norway, Scotland and Ireland are the three major producers of salmon, which, in Europe, represents an important resource in terms of production and economic value. Fish waste from salmon can reach up to 40% of the total weight of the animal and is mainly composed of bones, head and offal.

Even if the most important source of fatty acids is the adipose tissue interspersed in the muscular fibers, it is still possible to obtain valuable quantities of PUFAs from the fish waste due to their large amount. Furthermore, the possibility to use the fish leftovers and to produce, from them, high value products, represents an important step in the circular economy.

Thus, the aim of the present research was to characterize, by qualitative point of view, and to investigate, for the first time, the antimicrobial effects of the fish oil extracted from the market salmon waste through an unbiased approach regard fishing or farming source and condition. Furthermore the potential use of these compounds, for treating Gram + and Gram- bacterial infections, was evaluated through the determination of minimum inhibitory concentrations against S. aureus and Pseudomonas aeruginosa, two relevant pathogens cause of polymicrobial infections (Serra et al., 2015) and included in the global priority list of antibiotic-resistant bacteria from WHO/OMS (Tacconelli et al., 2017). In addition, we tried to understand if the antimicrobial activity could be due to any chemical contaminants present in the samples.

Materials and Methods

Results

Discussion

Despite its potential value, fish waste actually represents a significant cost for fish industries and markets. This huge mass is normally discarded but could represent an important source of bioactive compounds for pharmaceutical, cosmetic, nutrition and biotechnological applications. Molecules such as proteins (i.e., enzymes and collagen), lipids, protein hydrolysates, astaxanthin, chitin (Caruso, 2015) can be extracted and utilized. Fish oil, enriched in Polyunsaturated Fatty Acids (PUFAs), is still another important source of high quality bioactive molecules that could be extracted from the fish waste.

Salmo salar fish waste can represent up to 30–50% of the total weight of the animal (Torrissen et al., 2011; He, Franco & Zhang, 2011; Opheim et al., 2015; Dinh et al., 2018). It is well known that the salmon oil has important features in terms of PUFAs and is a rich source of omega-3 fats. In our study we have confirmed the presence of PUFAs in the fish waste both in the oil extracted from head and soft tissues and we have shown how these PUFAs are characterized by the presence of omega-3 and omega-6 fatty acids (Tables 1A and 1B). The most abundant omega-3 found in the fish waste oil was the linolenic acid, which represent 5.91% in head oil and 4.46% in soft tissue oil. The α-Linolenic acid is really important in the human diet due to its role as substrate for the synthesis of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which confer unique biophysical properties to cell membranes and are necessary for tissue functions (Burdge & Calder, 2006). Linoleic acid was found to be the most abundant omega-6 fatty acid both in head oil (15.43% ± 0.47%) and in soft tissue oil (14.56% ± 0.19%). This fatty acid is known for its important physiological role, as a constituent of acylglycosyl ceramides, in maintaining the integrity of the water permeability barrier of the skin (Sanders, 2016). The omega-6 linoleic acid is also the precursor of the arachidonic acid, which, in turn, is the major precursor of the eicosanoids, such as prostaglandins, thromboxanes, prostacyclins, leukotrienes and anandamides, which control a large number of physiological processes. Furthermore, arachidonic acid regulates the membrane fluidity, modulates the function of specific membrane proteins involved in cellular signalling and act maintaining cell and organelle integrity and vascular permeability (Tallima & El Ridi, 2018). Arachidonic acid also affects neuronal excitability and synaptic transmission through acting on voltage-gated ion channels and the accumulation of unesterified arachidonic acid compromises cell survival via induction of apoptosis. Omega-9 oleic acid was the most abundant fatty acid found both in the head oil (53.58% ± 0.72) and in soft tissue oil (39.47% ± 2.57%). This molecule, that is a monounsaturated fatty acid, has been shown to exert many biological functions such as regulation of plasma lipid and lipoprotein concentrations, modification of coagulation properties, improvement of glucose homeostasis, and attenuation of inflammation and oxidative stress (Lopez et al., 2010). Furthermore oleic acid has been shown to inhibit tumour cell proliferation in a dose- and time-dependent manner, inducing cell cycle G0/G1 arrest, increasing the apoptosis and the expression of p53 and cleaved caspase-3, and decreasing the expression of CyclinD1 and Bcl-2 (Jiang et al., 2017).

Fatty acids also exert an important role against microorganism infections. Pathogenic agents can cause infections in different ways, such as through the production of virulence factors and biofilms formation, (Beceiro, Tomás & Bou, 2013; Schroeder, Brooks & Brooks, 2017). In vitro and in vivo studies have demonstrated that omega-3 fatty acids, and in particular linolenic acid and its derivatives, used alone or in combination with conventional antibiotics, possess antimicrobial properties (Chanda et al., 2018). Omega-6, -7, -9 fatty acids, such as γ-linolenic, linoleic, arachidonic, palmitoleic and oleic acids, their ethyl esters and methyl esters, were also shown to be effective against various microorganisms (Huang, George & Ebersole, 2010). The mechanism of action that would explain the antimicrobial properties of fatty acids could be the alteration of cell membrane hydrophobicity, charge and integrity, which result in electron leakage and subsequent cell death (Desbois & Smith, 2010; Lopez-Romero et al., 2015; Calo et al., 2015). Furthermore fatty acids can contribute to bacterial death through cell lysis, inhibition of enzyme activity, impairment of nutrient uptake and the generation of lethal oxidation products (Desbois & Smith, 2010).

In our research, the antimicrobial properties of the salmon head and soft tissue waste oils were tested against two important Gram positive and Gram negative pathogens and the results showed a MIC of 25% and 12.5% (v/v) respectively (Table 2). These data seem to indicate that the fish oil extracted from the waste is still active against tested microorganisms and can act independently from the bacterial wall type. Furthermore, the antimicrobial activity of the waste oil extracted from the salmon soft tissue seems to be more efficient than head oil against the two bacterial strains utilized. This difference could be probably due to the different fatty acid composition of the two tissue sources (Tables 1A and 1B). In fact, comparing the fatty acids composition in the two samples, SUFAs, MUFAs and PUFAs were highest in the oil extracted from soft tissue. These data seem to be in accordance with similar results obtained in other fishes (Li, Sinclair & Li, 2011; Hong et al., 2015).

Our research was performed through an unbiased approach regard fishing or farming source and condition. However, it is legitimate to ask whether farming conditions, and in particular fish diet, could alter the fish oil composition. Recently studies have shown that alternative salmon feeds can increase the salmon oil concentration but not, at least completely, its composition (Ruyter et al., 2019; Bruni et al., 2020)

Almost the totality of the Salmon sold in Italian fish markets is produced through aquaculture methods. The importance of food safety is crucial for fish farm and the potential hazards include dangerous chemicals and microorganisms. The former could be accumulated by the fish, especially in their fat tissues, from the aquaculture environment, the feed and residues from veterinary prophylaxis, the latter are represented by parasites, viruses and bacteria that may be harmful for humans (Fairgrieve & Rust, 2003; Estévez et al., 2018; Ben Hamed et al., 2018; Gjessing et al., 2019; Feist et al., 2019). In addition seafood produced through aquaculture can be contaminated by different types of toxic substances from natural and/or anthropogenic origins due to both indirect and direct pollution from continental human activities (Fremy & Bordet, 2002; Chiesa et al., 2019; Quiñones et al., 2019; Heldal et al., 2019). Aquaculture fish management practices can represent an important stressing factor for fishes, in particular for salmon, and could result in high mortalities leading to significant economic loss for producers (Wilson et al., 2009; Sudheesh et al., 2012; Overton et al., 2019). In fact, stressors like handling, stripping of brood stock, antimicrobial treatments, vaccination, temperature, crowding, starvation and transport can result in an increase of a number of diseases evaluable through the measure of the levels of cytokines, heat shock proteins (HSP), corticosteroid hormones, immunoglobulin and immune cells levels, haematological parameters (Gabriel & Akinrotimi, 2011; Cordero et al., 2016; Rehman et al., 2017; Parisi et al., 2017; Chiaramonte et al., 2019; Cammilleri et al., 2019a; Inguglia et al., 2020; Vazzana et al., 2020). Moreover, salmon, which are usually farmed in crowded conditions, are easily targeted by infective pathologies (Poppe, Barnes & Midtlyng, 2002; Håstein, 2004; Bang-Jensen, Gu & Sindre, 2019). Over the past years, fish farms, and in particular that of salmon, have increased their productivity in parallel with the growth of the use of substances used to prevent and treat microbial and bacterial disease, such as antibiotics (Miranda, Godoy & Lee, 2018). We must say that other solutions are being tried, such as vaccines and immunostimulants (Eslamloo et al., 2017; Meza et al., 2019; Xue et al., 2019; Chalmers et al., 2020), in order to limit the use of antibiotics (Gravningen, Sorum & Horsberg, 2019) but they still remain a largely utilized solution expecially in Non-European countries. Specific data about antibiotics in the aquaculture industry are not easy to report due to the different current laws of the involved countries. For this reason data are often unavailable or unattainable (Heuer et al., 2009; Romero, Gloria & Navarrete, 2012; Miranda, Godoy & Lee, 2018). However, based on available data, the qualities and quantities of the used antibiotics are variable. For example, among the world countries with the highest rates of aquaculture antibiotic use (Van Boeckel et al., 2015) there are Chile and Vietnam. In Norway, the antibiotics amount used in aquaculture, decreased enormously in the last years but still present, is of 1g/ton of farmed Salmon while in Vietnam the utilized amount is of 700 g/ton of farmed shrimp (Smith, 2008; Bang-Jensen, Gu & Sindre, 2019).

Considering this information, we performed the LC-MS/MS analysis to exclude the involvement of aquaculture contaminants in the biological antibacterial activity of the fish waste oil (Table 3). The analysis has shown the presence of traces of molecules that, anyway, are considerably under the maximum residue limits indicated by European law (REGULATION, 2009; Commission Regulation (EU), 2009). Furthermore, the small drug amounts highlighted by the experiment would not seem explain the MIC values observed. However, we cannot completely exclude the hypothesis of a contribute of this molecules to the antibacterial properties of the fish waste oil and further analysis are needed to totally exclude this possibility.

Conclusions

The present research have shown, through GC-MS analysis, the specific composition of the fish waste oil extracted from different discarded parts of the Salmo salar present in Italian fish markets. The analysis has also highlighted the oil enrichment in polyunsaturated fatty acids and, among them, in omega-6, -7 and -9 fatty acids. In addition, the MIC experiments have revealed the antibacterial activity of the extracted Salmon waste oil

These data confirm that the fish waste is still quantitatively and qualitatively an important source of available biological properties that could be extracted and utilized representing an important strategy to counteract infective diseases in the context of the circular economy.

Supplemental Information