Supercritical CO₂ and subcritical water technologies for the production of bioactive extracts from sardine (Sardina pilchardus) waste

Highlights

• Fractionation of sardine waste from a canning industry was investigated.

• Fish oil rich in omega-3 was obtained by supercritical fluid extraction.

• Fish protein hydrolysates were produced by subcritical water hydrolysis.

• Physical, biochemical, and bioactive properties of the extracts were studied.

• Antiproliferative effect of the extracts against adenocarcinoma cells was tested.

Abstract

The valorization of sardine (Sardina pilchardus) waste (SW) from a canning facility has been investigated within a biorefining approach. Sequential fractionation of SW into its constituents has been carried out using green solvents such as supercritical carbon dioxide (SCCO₂) and subcritical water (sCW). The lipid fraction has been isolated through supercritical fluid extraction (SFE) with SCCO₂ at 250 bar and 40 °C, yielding 20.3 ± 0.2 g oil/100 g SW with up to 17.2 %wt. omega-3 polyunsaturated fatty acids (PUFAs). Aiming at the protein fraction, sCW extraction/hydrolysis has been carried out at different temperatures (90, 140, 190 and 250 °C), using both SW and defatted sardine waste (DSW) from SFE experiments. Previous defatting increased protein recovery and purity. Bioactive properties of the fish protein hydrolysates (FPHs) obtained were affected by the extraction temperature. The highest antioxidant activity and in vitro antiproliferative effect were found in the extracts obtained at 250 °C.

Introduction

According to FAO, the world fisheries and aquaculture production was 171 million tonnes in 2016, being more than 150 million tonnes destined to human consumption [1]. Despite improvements in fish processing and distribution, fish waste generated between landing and consumption accounts for an estimated 27 % of landed fish. Processing industries discard between 20–75 % of the fish, depending on the fish species and level of processing [2]. Fish canning of oily species such as tuna, mackerel and sardine-type species is an important industrial sector worldwide and also in Portugal [3]. The large amount of fish waste generated pose important economic and environmental problems for the sector due to its high organic load, consisting mainly of protein and lipids. To date, fish waste is generally considered low value and used to produce animal feed, fish silage, and fertilizers when is not disposed of by burning or discarding in the land or sea [2]. However, studies published in the last decades have illustrated fish waste as a remarkable source of vital bioactive molecules, such as proteins, peptides, amino acids, lipids (omega-3 polyunsaturated fatty acids, PUFAs), enzymes (pepsin, trypsin), vitamins (A, D, E) and biopolymers [2,4,5]. These compounds can be used in nutraceutical, biomedical, pharmaceutical and cosmetic applications, with a much higher market value [4]. In consequence, fish waste represents a valuable resource for an integrated and product-based biorefinery process [6], obtaining different materials and building blocks with the potential to serve as renewable feedstocks for several industrial sectors.

Fish waste is considered a good source of high quality fish oil, rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two omega-3 PUFAs with applications in nutraceutical, pharmaceutics and cosmetic industries [7]. Conventional extraction of lipids from natural materials uses hazardous organic solvents and is typically energy intensive and time consuming. Supercritical carbon dioxide (SCCO₂) extraction is an environmentally friendly alternative, compatible with the label ‘natural’, since at the end of the process only recyclable CO₂ and a solvent-free extract are produced, leaving an also solvent-free and non-degraded raffinate [8]. SCCO₂ presents high diffusivity, low viscosity and low surface tension, improving mass transfer. Through manipulation of pressure and temperature, SCCO₂ density can be adjusted to allow efficient extraction and separation of the oil fraction and lipophilic bioactive solutes [[7], [8], [9], [10]].

Fish waste also contains significant amount of proteins, nutritionally superior when compared to those of plant sources and less restricted due to religious concerns and transmissible diseases than those of bovine or porcine origin [11]. Fish protein hydrolysates (FPHs) with enhanced physicochemical and biological properties can be obtained through different extraction and hydrolysis methods [2]. Chemical hydrolysis with either a strong acid or alkali is the most common, inexpensive, and simple method to produce FPHs [12]. However, the method requires of several pre-treatment steps and uses high temperatures for long times, which complicate the control of product quality and functionality. Enzymatic extraction in acidic media has emerged as an alternative due to the milder conditions and the higher specificity. However, enzyme-based methods are complicated, costly, and even more time-consuming than chemical extraction, which limits its implementation at industrial scale [13]. Advanced cost-effective processing technologies need to be developed to produce high quality FPHs with specific functionalities for specific product applications.

Subcritical water (sCW) has attracted interest as a green solvent for waste and biomass conversion [14]. sCW extraction and hydrolysis processes use water at 100−300 °C and pressure above saturation value but less than critical, just to maintain water liquid. At these conditions, polarity decreases with temperature, allowing solvent tunability for selective extraction of moderately polar to non-polar substances. High temperatures also break hydrogen bonds, facilitating sCW to penetrate into solid matrixes due to the lower viscosity and higher diffusivity. Moreover, the ionic product (K_W) is 3 orders of magnitude greater than that of water at ambient conditions. This drives the formation of hydronium and hydroxide ions and allows sCW to act as acid or base catalyst, which supports hydrolysis of proteins and amino acids [14,15] with no need of additional catalysts suchs as acids or enzymes.

Yoshida et al. [16] reported the sCW extraction/hydrolysis of fish meat from horse mackerel in a batch reactor. Analysis of the aqueous phase obtained revealed sequential production of organic acids, amino acids, and fish oil when increasing hydrolysis temperature from 200 to 400 °C; thus, a temperature-based fractionation process was proposed for industrial valorisation of fish waste. Other authors have also investigated the sCW extraction/hydrolysis of fish waste from different origins, such as white croaker [17,18], bonito [17], squid viscera [19], mackerel [20], or shrimp [13], and studied the physicochemical, biochemical, and bioactive properties of the FPHs obtained. In some cases, the sCW process was also combined with a previous SCCO₂ defatting step [21,22]. However, most of the mentioned studies were performed in batch-mode and focusing on the production of amino acids, rather than intermediate peptides. Recently, Marcet et al. [23] compared the sCW extraction and hydrolysis of proteins with the chemical and enzymatic techniques for the recovery of peptides and free amino acids from animal and vegetal food wastes, concluding that sCW allows higher efficiency and flexibility with no further addition of reagents. This is at the cost of higher equipment and energy expenditures, although energy consumption can be minimized by correct design and optimization of process parameters.

In this work, the utility and feasibility of an integrated biorefinery process for the valorisation of sardine waste from a canning facility has been evaluated. For this, the sCW extraction and hydrolysis process has been coupled with a pre-defatting step with SCCO₂, aimed at the specific recovery of the lipid fraction of sardine waste, rich in omega-3 polyunsaturated fatty acids. The sCW process was performed in a packed-bed reactor operating in semi-continuous mode since it allows the fractionation of the target bioactive compounds present in the sardine waste through continuous temperature variation. The potential effect of the sCW temperature and the prior SCCO₂ defatting on the extraction/hydrolysis yield and the extract composition was investigated, trying to assess the potential advantage of using SCCO₂ in a first step to selectively extract the lipid fraction from sardine waste, and later, to use sCW to hydrolyse its protein fraction. Furthermore, the biological activity of the extracts in terms of antioxidant and antiproliferative effects was also determined.