1. Introduction
The European or common squid (
Loligo vulgaris) is a cephalopod found in abundance from the Atlantic waters of the North Sea to the west coast of Africa. Currently, this squid is widely exploited by commercial fishing and there is an important flow from Atlantic fisheries in Mauritania and South Africa, among others, to southern European countries, such as Spain, Portugal, Greece and Italy—traditionally the largest consumers of cephalopods [
1]. Squid are considered a part of the Mediterranean diet due to their excellent sensory and nutritional properties, including high levels of muscle proteins, ω3 fatty acids and vitamin E [
2]. Squid are also a dietary source of essential minerals, such as Ca, Mg, Na, K, P, Zn, Cu, Fe, Mn, Cr and Ni, although they may accumulate toxic heavy metals such as Cd, Hg and Pb [
3]. Pollution by As, a metalloid toxic for humans, is perceived as another health hazard linked to squid consumption [
4,
5]. Mineral accumulation depends on animal traits (species, variety, body size, age, specific tissues and organs) and environmental factors affecting animal diet, in particular, water pollution in fisheries [
6]. Squid have weak metabolic capacity [
3] because of their short life cycle, and accumulate lower levels of toxic elements than some commonly-consumed fish [
7]. Cephalopods captured from offshore sites in coastal sea areas may reach higher levels of heavy metals than others from oceanic fisheries [
5,
6], being able to accumulate more amounts of heavy metals in the viscera than in the muscle [
8] due to their ability to concentrate toxic elements in tissues owing to their carnivorous regimen [
9].
Most available data on squid minerals correspond to studies focused on polluting trace metals, without a food technological approach. Mineral content present in raw materials can be affected by treatments applied to obtain squid products (e.g., freezing-thawing, maceration and cooking). Squid caught for international trading are frozen on board as soon as possible as it can take several months for them to be transported and processed in factories. Once there, frozen squid are thawed. Exudates released by thawed squid can be replaced by maceration solutions to obtain a juicer product. Squid can be treated with sodium salts to increase water holding capacity (WHC) before cooking [
10]. Sodium chloride is used as a water holding agent, while, in addition, sodium salts of certain organic acids such as citric acid, are used as buffering agents to improve sensory properties [
11]. Maceration with sodium salts (in low concentrations) has no preservative purpose as with brined products. Whole squid can be directly macerated or eviscerated to obtain the flesh. Maceration basically provides Na, a debatable nutritional aspect, but can also contain minor quantities of other minerals from powders and tap water.
The mineral content of macerated raw squid may be affected by cooking treatment. The gain of minerals may result from water loss which occurs during cooking, or possibly from the migration of such elements from the container used, while mineral loss is likely related to their leaching into the cooking juice [
12]. Several studies agree that cooking (e.g., grilling, roasting or microwaving) often concentrates the minerals present in seafood and fish products due to dehydration, although there may be exceptions depending on the mineral and the cooking procedure [
13,
14,
15,
16,
17]. During cooking, protein denaturation promotes structural changes in muscle proteins which reduce WHC [
18]. Heating also induces changes in muscle metalloproteins, decreasing their water solubility [
19], which may affect leaching of bounded metals. In recent years, vacuum-cooking in plastic bags “
sous vide” has been increasingly introduced into the industry for manufacturing seafood products. With this method, mild temperatures are applied during more or less time depending on piece size.
Sous vide cooking at 55–65 °C has been found to be suitable in obtaining squid products of good sensory traits [
20], or to prevent microbiological risks in fish fillets kept in refrigeration [
21]. Extended cooking contributes to reducing these risks but also increases juice loss, which may negatively affect squid juiciness [
20].
Maceration offers a good chance to adjust the amount of essential and undesirable minerals in cooked squid products. However, it is unclear how different minerals may simultaneously spread among the squid flesh and the saline solution upon different concentration gradients. Vacuum-cooking may also result in both minerals leaching into their own juices as minerals concentration by dehydration. Accumulative effects of maceration and vacuum-cooking on squid minerals may be antagonistic and should be elucidated. Technological treatments that lead to the loss or gain of minerals can modify the nutritional value and toxicological risks related to mineral intake through squid products.
The aim was to study how the mineral content of squid flesh is affected by maceration and further vacuum-cooking. Dietary intake for minerals and toxicological risks for As and heavy metals through the consumption of squid flesh were also determined.
4. Discussion
Fishery origin led to some differences in Na, K, Mg and other squid minerals. As mentioned, the quantities of minerals accumulated in cephalopods may vary depending on animal (species, variety, body size, age, specific tissues and organs) and environmental factors (animal diet and water pollution in fishery) [
6]. Among these, animal diet would be particularly important, since squid is a carnivorous species with a low metabolic capacity and a certain tendency to accumulate minerals [
3,
9]. Therefore, it is expected that the composition of raw materials from distant fisheries may vary despite squid size being standardized. Na was by far the most abundant mineral in squid flesh, as others have reported [
3,
25], due to sodium chloride being the predominant salt in marine waters. Squid flesh also contained relevant levels of P and S, two elements that integrate several salts, proteins, phospholipids and other molecules. Marine waters and sediments may also contain other salts, so squid can accumulate little quantities of other minerals, such as Ca, Mg, Zn, Cu, Fe or Mn, among others [
3].
Minerals can be removed from raw material through thawing exudates, maceration effluents and/or cooking juices. The freezing–thawing cycle may cause some denaturation of muscle proteins [
35], diminishing WHC which favors the exudation of liquids containing dissolved minerals. In the present study, thawed squid were drained to control microbial loads, and exudates were removed. Maceration with sodium salts improved WHC in squid flesh by way of an osmotic retention mechanism. Sodium chloride acts as the main water holding agent [
10,
36], while sodium citrate is a buffering agent which is able to move the pH away from the isoelectric point of muscle proteins, which facilitates their rehydration [
37]. In fact, the pH slightly increased when squid was macerated, likely due to the buffering action of sodium citrate and the formation of trimethylamine from protein hydrolysis [
38]. Maceration mainly increased Na content, favoring water retention. As seen, Na and moisture contents of raw squid flesh were increased by 0.41 g/100 g and 4.2 g/100 g, respectively. This fact evidences the relevance of maceration with sodium salts for the resulting WHC in this product. Guldas and Hecer [
11] reported weight increases of 4.0–4.7% when macerated squid flesh with citric acid (3.5% w:w) and NaCl (2% w:w). Na diffusive behavior was a logical exception compared to those seen for other minerals that had lower concentrations in the maceration medium than in squid flesh. Maceration increased squid wet base and favored minerals leaching. The result was a decrease in the levels of most macro and micro squid minerals with respect to the thawed raw material. For example, Zn level decreased from 11.63 mg/kg (untreated) to 8.23 mg/kg (macerated) in raw squid treated with a solution containing 5.50 mg Zn per kg. This was the most common diffusive pattern for squid minerals, although with some exceptions, such as for Al, which was more concentrated in the medium than in squid flesh. In any case, the presence of minor minerals in the maceration sodium salts does not seem to play a relevant role in the mineral content of macerated raw squid. Squid flesh minerals have been studied in Chinese (
Loligo chinesis), Pacific (
Ommastrephes bartramii and Dosidicus gigas), Atlantic (
Illex argentines), Mediterranean (
Loligo vulgaris) and Japanese squid [
5,
14,
15,
39]. Despite existing differences in body weight and origin, from these studies it can be extracted that Zn (7.1–14.8 mg/kg), Fe (6.0–10.0 mg/kg) and Cu (0.8–5.5 mg/kg) are the predominant microminerals in squid flesh, as also determined in the present study. Concentrations (mg/kg) of toxic metals reported by the above authors for raw squid were Cd (0.02–1.28); Hg (0.01–0.05), Pb (0.03–0.10) and As (2.1–22.0). The Atlantic squid analyzed in the present study would be at the bottom of these ranges. Available data are scarce on how squid minerals are affected by maceration with sodium salts. There are basically two maceration methods, immersion in a tank appropriate for small pieces, and injection is used in large pieces, as with giant squid, due to the transference ratio for sodium salts being more efficient. A product vacuum-cooked in its own juice will require more intense maceration than one cooked in a salted broth. In addition, agents that improve juice retention, such as phosphates, carrageenan and others, can be used [
11]. In a previous trial [
25], a maceration solution containing 13 g sodium salts per kg (3.1 g Na/kg) was injected into giant squid arms that were then cooked in a salty broth. As a result, injected raw arms only reached 0.15 g Na/100 g, a lower Na level than those reached in the present study. Both trials agree that raw flesh from Atlantic (macerated or not) and Pacific (macerated) squid had a similar micromineral profile: a predominance of Zn over other metals and a scarce presence of toxic metals.
Changes in the mineral content of squid flesh due to heating are closely related to minerals leaching with the cooking juice and/or flesh dehydration [
12]. Vacuum cooking at mild temperatures for long periods aims to retain as much juice as possible and to reach a suitable gelatinization of collagen to obtain an edible texture. A culinary study where
sous vide squid was cooked at 45 °C, 53 °C, and 72 °C for 20 min found that intermediate conditions are sufficient to obtain a product with good sensory traits [
20]. This may be due to denaturization of myosin and collagen occurring at mild temperatures (50 °C and 57 °C, respectively) in squid muscle [
40]. Microbiological risks would also be controlled in refrigerated
sous vide products. For example, vacuum-cooking at 65 °C for 5 min was sufficient to inhibit pathogen bacteria (e. g.
S. aureus, B. cereus, C. perfringens and
L. monocytogenes) in salmon fillets kept at 2 °C for 45 days [
21]. Thus, the cooking conditions used in the present study (65 °C for 20 min) should ensure microbial quality in a cooked-frozen squid product. In fact, the microbial quality of cooked squid was checked (data not shown) in the factory. In addition,
Sous vide cooking conditions moderated juice loss. For example, a giant squid arm may lose around 70% of its raw weight when cooked in polypropylene bags at 100 °C for 10 min [
41]. As seen (
Figure 1), cooking decreased the concentration of macrominerals but, in contrast, did not affect the concentration of microminerals in the untreated squid. This suggests that squid microminerals leach with some difficulty with cooking juice, perhaps because product–juice diffusion gradients are less effective as they are present in microquantities.
Maceration and cooking would have associated effects on the mineral profile of squid flesh. During vacuum-cooking, a part of Na passed from squid to the cooking juice, remaining in the bag. This fact would be proportional to the quantity of Na retained by squid. As seen, Na content decreased less in the untreated (0.07 g/100 g) than in the macerated (0.26 g/100) cooked squid. Despite this, maceration fullfed its technological aim: moisture content (juice retention) increased by 2.7 g/100 g in the ready-to-eat product when macerated. On the other hand, it can be deduced that a part of the macro (K, Mg, P and S) and microminerals (As, B, Cr, Mn and others) whose levels decreased with cooking, ended up in the cooking juice. For example, the noticeable loss of P and S reveals that a part of the muscle component was removed with the cooking juice. In contrast, other squid minerals such as Zn or Cd were concentrated with cooking. This different behavior (dilution of concentration) might be due to several causes. Heating coagulates muscle proteins, which favors juice exit and increases dry basis [
8]. This may concentrate squid minerals or not, depending on the quantity of minerals lost with the cooking juice. Moreover, cooking decreases the solubility of metalloproteins and may induce different chemical reactions [
19], therefore coagulated proteins would have more difficulty in passing to the juice released by squid. Cd levels in squid mantle may considerably increase by roasting and industrial canning [
42]; this behavior is associated with changes in metallothioneins during squid processing, while, in contrast, Pb content is not dependent on processing or associated with metallothioneins. Thus, processing operations may affect Cd and Pb content differently due to the specific metal bioaccumulation and the chemical features of each heavy metal type. This would explain why the levels of some metals can increase or not with cooking. Macerated or not, any change in the mineral content of squid flesh will depend upon cooking conditions (time, temperature and cooking procedure) [
43]. Different studies reported that mineral content increases with cooking in fish and seafood products [
13,
14,
15,
16,
17]. There is consensus that Zn, Fe and Cu were the most abundant trace metals in cooked squid flesh. A previous trial on macerated squid arms also revealed that cooking (in a salted broth with 0.56 g Na/100 g) increases levels of Na (from 0.15 to 0.37 g/100 g), Zn (from 13.7 to 21.4 mg/kg), As (from 0.48 to 0.81 mg/kg) and Cd (from 0.02 to 0.09 mg/kg) [
25]. Evidently, the different cooking conditions tested (cooking in salted broth, microwaving, grilling or pan-frying) may explain these findings. In the present study, macerated squid was cooked at a mild temperature (65 °C), which moderated juice loss. Concentrated or not, the levels of Cd, Hg and Pb determined for the ready-to-eat product were well below the maximum levels for Cd (1 mg/kg) and Pb (0.3 mg/kg) authorized in cephalopods flesh and for Hg (0.3 mg/kg) authorized in fishery products [
26]. As content was also below the maximum limits permitted for fish and shellfish in Australia (1–2 mg/kg) [
44].
The consumption of cooked squid covered a relevant percentage (>10%) of the DRI established for Na, P, Zn, Mg and Mn. Other studies agree that cooked squid is a relevant dietary source of Na and Zn [
14,
19]. As expected, Na intake was higher (up to 28% DRI) when cooked squid was previously macerated. This percentage can even be increased if vacuum-cooked squid is consumed with its own juice. In a previous trial, macerated-cooked giant squid arms provide less dietary Na (16% DRI) [
25]. Na intake associated to the consumption of macerated-cooked squid poses a health risk and could negatively affect its consumption. The close relationship between hypertension and dietary sodium intake is widely recognized and supported by several studies [
45]. Regarding toxic metals, squid flesh from the FAO 34 and 47 fishing zones can be consumed with a wide margin of safety. The %PTWIs calculated in the present study for Cd, Hg and Pb were below recommendations established by the EFSA [
31,
32,
33]. Similarly, their levels of inorganic As were below the PTWI (15 μg/kg b.w.) and the range (0.3–8 μg/kg b.w.) of benchmark dose lower confidence limit published by the EFSA [
30]. Both evisceration and the possibility that offshore marine waters are less polluted may support this finding. However, it has been noted that the health risk linked to the intake of toxic metals also depends on other factors affecting their bioaccessibility (e.g., denaturation and de-methylation of muscle metalloproteins) [
46]. Other studies [
14,
15,
38] agree that the consumption of squid flesh does not pose any health risk to consumers; the % PTWI reported in these studies range from 7.2 to 35.2% (Cd), 0.2 to 0.3% (Pb) and 0.1 to 2.9% (Hg). It is also known that cephalopods tend to accumulate more Cd than Hg or Pb [
46]. The results obtained support the idea that consuming products based on squid flesh does not have to be associated with the intake of toxic metals and semi-metals. The whole technological treatment (evisceration, maceration, cooking, etc.) applied to the squid leads to a reduction in the intake of mineral pollutants, which might even be modulated by modifying the processing conditions (adjusting sodium salts concentrations and/or replacing sodium salts by other water holding agents in maceration media; using agents to retain cooking juices). Sodium intake through the consumption of squid products could also be reduced by applying some of these strategies.