Quality Changes on Cod Fish (Gadus morhua) during Desalting Process and Subsequent High-Pressure Pasteurization

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During the desalting process of salted cod, important biochemical changes occur, which will reflect on the organoleptic properties of the final product (desalted cod). Subsequent high-pressure processing with low temperature (4 °C) desalting processes can improve the physicochemical and microbial quality of desalted cod.

Abstract

During the desalting of salted cod, significant textural, histological, and biochemical changes occur. Understanding these changes is crucial for enhancing the preservation and extending the shelf life of desalted cod. This study aimed to investigate the physicochemical quality parameters and enzymatic activities during the desalting process of cod (16 h at 4 and 20 °C) and to extend the shelf life of desalted cod through high-pressure processing (HPP) at 400 and 550 MPa for 5 min. During desalting, a correlation was noted between the pH and trimethylamine content in samples desalted at 20 °C, with both parameters increasing in the initial 4 h and stabilizing thereafter. The soluble protein in cod muscle decreased over desalting time, as it dissolved into the desalting water. Enzymatic activity showed a decline in cathepsins (B and D) and acid phosphatase throughout desalting, whereas lipase activity increased, particularly at 20 °C. HPP effectively extended the shelf life of desalted cod by controlling endogenous microbial growth, enabling an extension to 14–21 days compared to the 7 days observed in untreated control samples. This study highlights quality changes during desalting, with lesser effects at lower temperatures. Subsequent HPP improved the microbiological quality of desalted cod during refrigerated storage.

1. Introduction

Codfish (Gadus morhua) is usually commercialized in the form of salted cod, and it has a significant importance in some European countries, such as Portugal and Spain [1]. Salting fresh cod is an old and traditional method that leads to a self-stable product (with low moisture and high salt contents), which allows us to store this food product for several months. This process is used to preserve several fish products by lowering their water activity (between 0.70 and 0.75), leading to an inhibition of microbial growth and enzymatic spoilage [2,3]. During the traditional salt-curing process, the water content of the cod muscle is usually reduced from approximately 82 to about 54%, and the salt content increases from 0.2% to 20% [4,5]. After salting and drying, the cod is ready to be commercialized. However, before its consumption, the product must be desalted in tap water at room temperature or under refrigeration, due to the high salt content of the salted cod. This process usually takes between 1 and 3 days, depending on the cod size, the amount of salt, and the desalting conditions [6], showing a final water content of 80–85% and a salt content of 1–3% [7].

This fact, along with the considerable lifestyle changes observed in the last few decades, caused an increase in the demand for easy processes to prepare ready-to-eat products. The adjustment of several industries to those consumers’ requirements resulted in the production of different cod products, such as frozen desalted cod, which has a relatively long shelf life (several months, being necessary to thaw before consumption), and refrigerated desalted cod. The commercialization of cod fish as a refrigerated product has many drawbacks, mostly due to its very short shelf life, between 1 and 5 days, which is caused by the favorable conditions for bacterial growth that cause its spoilage [8]. On the other hand, important changes take place in the textural, histological, and biochemical properties during desalting. The protein/cod matrix is rehydrated, and an improvement of the cod texture occurs [9], which is related to the increase in its muscle firmness [10]. The decrease in salt leads to water absorption by proteins, and consequently increases the water holding capacity [11]. Some enzymes play an important role in the post-mortem changes and degradation processes of fish, but in what concerns salted cod fish, as far as the authors are aware, there are no published data about the presence of enzymatic activity during the desalting processes.

High-pressure processing (HPP) can be an option to limit microbial growth and extend the shelf life of desalted cod. HPP, in a range of 400 to 600 MPa, is now commonly applied to extend the shelf life of food products by inactivating a variety of pathogenic and spoilage vegetative bacteria, yeasts, moulds, and viruses, with minimal impact on organoleptic properties [12]. HPP emerges as a promising solution to extend the shelf life and enhance the safety of desalted/soaked cod. There are some studies issuing the efficacy of HPP in combination with various packaging techniques. Rode and Rotabakk (2021) [13] explored different packaging solutions, revealing that HPP alone or combined with a modified atmosphere or a CO₂ emitter could extend the shelf life of rehydrated cod by at least 49 days. Also, Ferri et al. (2023) [14] focused on sustainable packaging solutions, highlighting the use of new recyclable multilayer plastic films in conjunction with HPP. The results indicate that these innovative packaging materials, when combined with HPP, can maintain high hygienic standards, and prolong the shelf life. Collectively, these findings emphasize the importance of HPP in modernizing fish processing methods, promoting sustainability, and meeting stringent quality standards in the food industry.

Therefore, the aim of the present work was to study the evolution of some quality parameters of desalted cod during the desalting process at two different temperatures (4 and 20 °C). To do so, the pH, soluble protein, trimethylamine, and enzymatic activities (acid phosphatase, cathepsins B and D, and lipase) were analysed. After cod desalting, HPP was applied (400 and 550 MPa, 5 min, 15 °C) to extend its shelf life under refrigerated conditions, which was also studied by microbial quality evaluation (total aerobic mesophiles and psychrophiles, Enterobacteriaceae and yeasts and moulds).

2. Materials and Methods

2.1. Chemicals

The substrates used in enzymatic activities included the following: p-nitrophenylphosphate (p-NPP), Z-arginine-arginine-7-amido-4-methylcoumarin hydrochloride (Z-Arg-Arg-AMC HCl #C5429), hemoglobin from bovine blood, and olive oil, which were purchased from Sigma-Aldrich (Steinheim, Germany); sodium dodecyl sulfate (SDS), acetic acid, trichloroacetic acid (TCA), trizma hydrochloride (Tris-HCl), 2-bis-(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol (Bis-Tris), thylenediaminetetraacetic acid (EDTA), dithiothreitol (DTT), p-nitrophenol, and L-tyrosine, which were obtained from Sigma-Aldrich (Steinheim, Germany); other chemicals, such as thymolphtalein, sodium hydroxide (NaOH), potassium hydroxide (KOH), citric acid, trisodium citrate, silver nitrate, and ammonium thiocyanate, which were acquired from Panreac Quimica S.A.U. (Barcelona, Spain); and the ethanol had a purity grade of 99%. For microbiology analyses, the culture media plate count agar (PCA), violet red bile dextrose agar (VRBDA), and rose-bengal chloramphenicol agar (RBCA) were acquired from Merck (Darmstadt, Germany).

2.2. Sample Preparation and Cod Desalting Process

Atlantic salted cod (Gadus morhua) was acquired from a local supermarket. Loins of the salted cod, excluding skin and bones, were cut into similar-sized pieces (dimensions of 2.25′ 1.50′ 1.00 cm and an average weight of 5 g) and desalted for 16 h at two different temperatures (4 and 20 °C) using a ratio of cod/water of 1/9 (w/v), without changing the water over the desalting process.

Cod samples were collected during the process and physicochemical analyses of water/salt content, pH, soluble protein, trimethylamine, and four degradative enzymes (acid phosphatase, cathepsin B and D, and lipase) were carried out, with the analyses performed in triplicate for the samples and in duplicate for the analyses.

2.3. High Pressure Treatment and Storage Conditions

Salted cod samples desalted for 16 h at 4 °C were placed into low permeability polyamide/polyethylene bags (Ideiapack—Comércio de Embalagens Lda., Palmeira, Portugal) and were vacuum sealed. Desalted cod samples were treated using two pressure levels of 400 and 550 MPa for 5 min at 15 °C. Untreated desalted samples were also studied (as control samples). HPP treatments were carried out using 55 L high pressure equipment (Hiperbaric 55, Burgos, Spain). The microbial quality and pH variation of the HPP-treated and control samples were evaluated over 28 days under refrigerated storage conditions (4 °C).

2.4. Physicochemical Analyses

2.4.1. Water and Salt Content

The moisture content was calculated as the loss in weight, after drying at 105 °C for 24 h (ISO 6496:1999) [15] with periodical weighting and the results were expressed as g H₂O/g IS (IS—insoluble solids). IS correspond to cod components that do not change during the desalting process (approximately 30% of salted cod), excluding water and salt content.

The salt content was determined by the Volhard method [16]. Briefly, cod pieces were triturated using an Ultraturrax T25 at 9000 rpm for 2 min (Janke and Kunkel, Deutschland, Germany) with 100 mL of distilled water. The mixture was vacuum filtered (Whatman^® Grade 1) to remove any fine debris present in the sample. Then, 5 mL of 0.1 M silver nitrate (AgNO₃) weas added to 5 mL of cod extract, forming a silver chloride complex (AgCl). The excess of AgNO₃ was titrated with 0.1 M ammonium thiocyanate (KSCN) using an automatic micro-burette microBU 2031 (Crison instruments, Alella, Spain) and 5 mL of ferric alum saturated solution as an indicator. The solution changed its colour from yellow to orange after Fe(SCN)²⁺ complex formation. The results of the salt content were expressed as g NaCl/g IS.

2.4.2. pH Value

Initially, cod samples were homogenized with 100 mL of distilled water, using an Ultraturrax T25 at 9000 rpm for 2 min (Janke and Kunkel, Deutschland, Germany). The mixture was centrifuged (10,000× g for 15 min at 4 °C, Centrifuge 3K30, Sigma, Osterode, Germany) to remove any fine debris present in the sample. The pH of the cod samples was measured by inserting a combined glass electrode (Crison Instruments micro-pH 2000, Barcelona, Spain) in the previous extract samples prepared.

2.4.3. Trimethylamine Content

The trimethylamine (TMA) content was measured using the modified Dyer method [17]. For that purpose, 4–5 g of cod samples was homogenized with 15 mL of 7.5% (m/v) TCA, using an Ultraturrax T25 at 9000 rpm for 1 min (Janke and Kunkel, Deutschland, Germany), and the mixture was filtrated with Whatman n° 1 to remove any fine debris present in the sample. In total, 10 ml of toluene, 1 mL of 20% (v/v) formaldehyde, and 3 mL of 45% (m/v) KOH were added to 4 mL of the filtrated extract samples. The organic layer was collected and dried with anhydrous sodium sulphate. Then, 5 mL of 0.02% (m/v) picric acid in toluene solution was added to 5 mL of organic extract. The solution was measured at 410 nm using a UV–Vis spectrophotometer (PerkinElmer Instruments Lambda 35, Waltham, MA, USA). The results were expressed as mg TMA/g IS using a standard curve of TMA at concentrations ranging from 0 to 10 mg/L.

2.4.4. Soluble Protein Content

The soluble protein content was estimated by the Biuret method [18] using the same extract samples used for pH determination. The mixture of 1 mL of extract samples and 5 mL of Biuret reagent was briefly homogenized by vortex, and was placed in the dark at room temperature for 30 min. Then, the solution was measured at 540 nm using a UV–Vis spectrophotometer (PerkinElmer Instruments Lambda 35, MA, USA) and the results were expressed as mg albumin/g IS.

2.5. Enzymatic Activity

2.5.1. Preparation of Enzymatic Extract

Cod pieces were homogenized with 50 mL of ice-cold distilled water following the procedure described by Lakshmanan, Patterson, and Piggott (2005) [19], with minor modifications. Samples were homogenized with an Ultraturrax T25 at 9000 rpm for 2 min (Janke and Kunkel, Deutschland, Germany) and the homogenate was allowed to stand in ice for 30 min with occasional stirring. After 30 min, the homogenate was centrifuged at 14,600× g at 4 °C for 20 min (Centrifuge 3K30, Sigma, Osterode, Germany). The supernatant was filtered through a sterile filter (Whatman^® Grade 1) and stored at −20 °C prior to the quantification of enzymatic activities.

2.5.2. Cathepsins

The activity of cathepsin B was assayed by the methodology described by Lakshmanan, Patterson, and Piggott (2005) [19]. A mixture of 0.1 mL of enzymatic extract and 0.1 mL of substrate solution (0.0625 mM of Z-Arg-Arg-AMC in 100 mM Bis-Tris and 20 mM EDTA/4 mM DTT, pH 6.5) was incubated at 37 °C for 5 min. The reaction was stopped by the addition of 1 mL of 3% SDS (w/v) in 50 mM Bis-Tris (pH 7.0), and the fluorescence of AMC liberated was measured (excitation, 360 nm, and emission, 460 nm) using a spectrofluorometer (FluoroMax 3 Spectrofluorometer, Horiba Jovin Yvon, Piscataway, NJ, USA). The cathepsin B activity was expressed as FU/min/g IS (FU—fluorescence unit).

The cathepsin D activity assay used was based on the procedure described by Buckow, Truong, and Versteeg (2010) [20], with minor modifications. The enzymatic extract (0.2 mL) was mixed with 0.6 mL of substrate solution, containing 2% denatured haemoglobin (w/v) in 200 mM of citrate buffer (pH 3.7), and was incubated at 37 °C for 3 h. The reaction was stopped by the addition of 0.6 mL of 10% TCA (w/v). After vigorous stirring, the precipitate was removed by centrifugation (18,000× g for 15 min at room temperature, Centrifuge 3K30, Sigma, Osterode, Germany) and the soluble peptides were measured at 280 nm (PerkinElmer Instruments Lambda 35 UV/VIS spectrometer, MA, USA). The cathepsin D activity was expressed as µg tyrosine/min/g IS.

2.5.3. Acid Phosphatase

The acid phosphatase activity was assayed with p-NPP as a substrate following the methodology described by Ohmori, Shigehisa, Taji, and Hayashi (1992) [21], with slight modifications. The mixture of 0.250 mL of enzymatic extract and 0.225 mL of substrate solution (4 mM p-NPP in 0.1 mM acetate buffer and 1 mM EDTA, pH 5.5) was incubated at 37 °C for 15 min. The reaction was stopped by adding 1 mL of 100 mM KOH and the p-NP released was measured at 400 nm (PerkinElmer Instruments Lambda 35 UV/VIS spectrometer, MA, USA). The activity of acid phosphatase was expressed as nmol p-NP/min/g IS (ɛ of p-NP: 18,200 L/mol).

2.5.4. Lipase

The lipase activity was assayed with olive oil as the substrate following the titrimetric enzymatic assay for lipase described by Sigma-Aldrich (1999) [22]. The enzymatic extract (1.0 mL) was mixed with a substrate solution (1.5 mL of olive oil, 1.25 mL of distilled water, and 0.5 mL of 200 mM Tris-HCl buffer, pH 7.7) and incubated at 37 °C for 24 h. The reaction was stopped by adding 2 mL of 95% ethanol (v/v) and the liberated free fatty acid was titrated against 25 mM NaOH, using thymolphtalein as an indicator. The activity of lipase was expressed as µmol FFA/min/g IS.

2.6. Microbial Analyses

For microbial analyses, cod fish samples (1 g) were transferred aseptically into a stomacher bag and 9 mL of Ringer’s solution was added. The mixture was homogenized for 80 s using a laboratory blender STOMACHER 80 (Seward Laboratory Systems Inc., Davie, FL, USA). Then, decimal dilutions were prepared using Ringer’s solution.

Total aerobic mesophile (TAM) and psychrophile (TAP) counts were determined in PCA, were incubated at 30 °C for 72 h and 20 °C for 5 days, respectively, and the colonies were enumerated. Enterobacteriaceae counts were quantified in a pour plate using VRBDA, and were incubated at 37 °C for 24 h and large colonies with purple haloes were quantified. Yeast and mould (YM) counts were performed using RBCA by a spread of 0.2 mL of sample, and the counts were completed after incubation for 5 days at 25 °C. Each sample was analysed in triplicate with duplicates for analyses. Microbial loads were determined as a logarithm of colony-forming units per gram (log CFU/g).

2.7. Statistical Analysis

The data of each variable in the desalting process (desalting time and process temperature) and HPP treatments were tested with a two-way analysis of variance (ANOVA) followed by a multiple comparisons test (Tukey’s honestly significant difference, HSD). The level of significance was established at p < 0.05.

3. Results and Discussion

3.1. Water and Salt Variation during Desalting Process

During cod desalting, rehydration of the cod matrix occurs, while sodium and chloride ions flow into the surrounding water [6]. As can be seen in Figure 1, the flow of water (H₂O) and sodium chloride (NaCl) showed a higher rate during the first hours of rehydration, starting to decline thereafter until reaching an osmotic equilibrium, which occurs after about 8 h of the desalting process. Initially, salted cod samples showed a water and salt content of 1.15 ± 0.03 g H₂O/g IS and 0.46 ± 0.03 g NaCl/g IS, respectively, which correspond to 44 and 18% of the muscle weight, respectively. These results are similar to values previously reported in the literature [4,23]. Over desalting time, the water content in muscle increased to 2.58 ± 0.12 and 2.60 ± 0.10 g H₂O/g IS, when the process was carried out at 20 and 4 °C, respectively (Figure 1A), while the salt content decreased to 0.055 ± 0.001 and 0.086 ± 0.005 g NaCl/g IS at 20 and 4 °C, respectively (Figure 1B). No significant differences (p > 0.05) were observed between desalting at 20 and 4 °C. In the literature, it is known that high temperatures increase the rate of desalting (Martínez-Alvarez et al., 2005) [10], though temperatures between 0 and 4 °C are desirable since the cod water content increases during the desalting process; consequently, the water activity increases, being less of a hurdle to microbial growth, so the cod progressively becomes more perishable. Temperature control during desalting is so particularly important owing to the rapidity of microbial growth in cod [24,25].

Since the salt content in commercially available desalted cod is usually in the range of 2–3% [6], the desalted cod samples obtained in this work had a suitable salt content for its consumption after 16 h of desalting (1.5 and 2.5% of salt content, at 20 and 4 °C of process temperature, respectively).

3.2. Physicochemical Parameters Variation during Desalting Process

3.2.1. pH Value Variation

Based on Figure 2A, it is possible to observe that the pH of the cod samples increased during the desalting process at both temperatures. Initially, salted cod showed a pH of 6.06 ± 0.06 that increased (p < 0.05) over desalting time to values of 6.53 ± 0.04 for the process at 20 °C and it was maintained (p > 0.05) thereafter at similar values. At 4 °C, the pH increased at a slower rate and to a lesser extent to a maximum value of about 6.25 after 16 h. These pH values are like values reported in the literature [26,27]. These results may be explained by microbial growth, since the main fish-contaminant microorganisms are able to produce alkaline compounds (e.g., TMA), resulting in an increase in the pH value [28]. It is also important to note that the observed pH increment was more pronounced when desalting was performed at 20 °C, where microbial growth is expected to be more considerable.

3.2.2. Trimethylamine Content Variation

Trimethylamine (TMA) is produced by the reduction in trimethylamine N-oxide (TMAO) because of microbial growth in fish muscle [29]. Figure 2B shows the results obtained for the TMA content during desalting at 4 and 20 °C. Initially, salted cod showed a TMA content of 0.043 ± 0.004 mg TMA/g IS. The TMA content increased (p < 0.05) during cod desalting time, at both temperatures, to values of 0.076 ± 0.008 mg TMA/g IS (20 °C) and 0.062 ± 0.002 mg TMA/g IS (4 °C) after 4 h of desalting, maintaining (p > 0.05) similar values thereafter.

During the first hour of desalting, the values obtained at 20 °C were statistically higher (p < 0.05) than those obtained at 4 °C, suggesting again that microbial growth is lower when cod samples were desalted at a lower temperature. In fact, these results were in accordance with those previously discussed (pH value of cod samples), since a correlation between the pH values and TMA content was observed (r² = 0.981).

Some studies in the literature correlate the TMA level with sensorial rejection, indicating 5 mg TMA/100 g as a possible sensorial limit [30]. In this study, TMA values were within the range of 1–3 mg TMA/100 g of desalted cod, showing that the TMA content in desalted cod samples was not high enough to cause a sensorial rejection.

3.2.3. Soluble Protein Content Variation

Figure 2C shows the soluble protein results obtained during cod desalting. The results showed that the soluble protein content decreased (p < 0.05) during desalting from a value of 138.8 ± 10.4 mg albumin/g IS (salted cod) to 71.3 ± 0.5 mg albumin/g IS (20 °C) and 70.3 ± 8.5 mg albumin/g IS (4 °C) after 4 h of cod desalting, maintaining (p > 0.05) similar values during the remaining desalting time. Since a similar behaviour was observed at both temperatures, it may be concluded that soluble protein variation was not affected by the desalting temperature. This pronounced decrease in soluble protein content in cod muscle may be explained by its dissolution in the desalting water (Thorarinsdottir, 2010) [7].

The percentage of soluble protein loss by the cod muscle was 65 and 58% at 4 and 20 °C, respectively, compared to the initial value. Thorarinsdottir et al. (2002) [27] monitored the total protein content (insoluble and soluble) in cod muscle and observed a protein loss of 32% after 110 h of desalting at 3 °C. This value is lower than that observed in this study; however, it is important to note that this value corresponds to the total protein content (soluble and insoluble).

3.3. Enzymatic Activity Variation during Desalting Process

3.3.1. Cathepsins Activity Variation during Desalting Process

In fresh fish, the autolytic activity of major muscle endogenous proteases induces the hydrolysis of key myofibrillar proteins, and thus contributes to muscle textural deterioration. The main proteolytic systems involved in the hydrolysis of myofibrillar proteins during the post-mortem storage of fish muscles are the lysosomal cathepsins [31]. Although cathepsins’ activity has been found to decrease with the increasing salt concentration during the salting process [32], no information was found in the literature concerning cathepsins’ activity during the desalting of salted cod.

Salted cod showed cathepsins’ B and D activities of 1.53 ± 0.19 FU/min/g IS and 26.3 ± 0.7 µg tyrosine/min/g IS, respectively, at day 0 in Figure 3A,B. During desalting, cathepsins’ activities significantly decreased (p < 0.05) in cod fish muscle to an activity of ≈0.48 FU/min/g IS (for cathepsin B at both temperatures) and 9.3 µg tyrosine/min/g IS (for cathepsin D after 8 h of desalting) with no further changes. This decrease corresponds to an activity reduction around 70 and 65% of the initial activity for cathepsin B and D activities, respectively. This reduction in cathepsin B and D activities might be due to the loss of soluble protein to the desalting water. The temperature of desalting showed statistically significant effects in the lysosomal cathepsins’ activity (p < 0.05) during the first hour, in the case of cathepsin B, and during 4 h in the case of cathepsin D, with no effect afterwards.

3.3.2. Acid Phosphatase Activity Variation during Desalting Process

In many studies of marine food, phosphatase activities in fish muscles are reported to cause the breakdown of adenosine triphosphate (ATP) and the subsequent formation of its by-products (adenosine diphosphate, adenosine monophosphate, and inosine monophosphate), which are related to the K-value, an index of fish freshness [33]. However, the behaviour of these enzymes in desalted fish and during the desalting process is not well known. As depicted in Figure 4A, salted cod showed an activity of 161.4 ± 7.1 nmol p-NP/min/g IS, with a decrease (p < 0.05) in this activity during desalting at both temperatures (4 and 20 °C). However, a different behaviour was observed between the two temperatures. When the desalting process was carried out at 4 °C, the initial activity decreased (p < 0.05) to an average value of 85 nmol p-NP/min/g IS after 4 and 8 h, which corresponds to an initial activity reduction of 50%. At 20 °C, a significant and more pronounced decrease (p < 0.05) in activity was observed when compared to 4 °C. Specifically, after 1 h of desalting at 20 °C, the acid phosphatase activity decreased (p < 0.05) to a value of 82.2 ± 0.7 nmol p-NP/min/g IS, corresponding to approximately 50% of the initial activity. After that, a slight decrease (p < 0.05) was observed at a value of 24.0 ± 0.6 nmol p-NP/min/g IS at 8 h of desalting, corresponding to an 85% reduction in the initial activity.

On the other hand, at both temperatures, a slight increase in activity was observed between 8 and 16 h of desalting, which can possibly be related to microbial growth leading to an increase in microbial enzymes’ content in the cod muscle. At the end of desalting (16 h), desalted cod muscle showed an acid phosphatase activity of 112.4 ± 8.6 p-NP/min/g IS (4 °C) and 43.4 ± 1.2 nmol p-NP/min/g IS (20 °C), which corresponds to 30 and 70% of the initial activity.

3.3.3. Lipase Activity Variation during Desalting Process

Lipid enzymatic hydrolysis, or lipolysis by lipases hydrolyses triglycerides forming free fatty acids, which are then more susceptible to oxidation that results in the common off-flavour, is frequently referred to as rancidity [34]. The lipase activity in the cod muscle during the desalting process is presented in Figure 4B. The salted cod showed a lipase activity of 6.4 ± 1.6 × 10² µmol free fatty acids (FFA)/min/g IS. The temperature of the cod desalting process affected the lipase activity, since this enzyme activity was considerably increased (p < 0.05) at 20 °C in the first 4 h to a value of 25.3 ± 2.7 × 10² µmol FFA/min/g IS (4-fold), which was maintained (p > 0.05) with similar values occurring until 16 h of desalting. In the case of desalting at 4 °C, the activity was maintained at similar values (p > 0.05) during the first 8 h of the desalting process, but it increased 3-fold after 16 h (p < 0.05).

The results indicated that the lipase activity in the desalting process at higher temperatures was higher than in the one carried out at refrigerated temperatures. It has been demonstrated that lipids in fresh fish are hydrolysed mainly by microbial enzyme activity, such as lipases and phospholipases, and non-microbial enzyme activity (i.e., natural lipase present in the fish muscle) [35]. However, in salted and hydrated cod fish, the contribution of endogenous enzymes is probably very low, with microbial growth being the main cause of higher lipase enzymes content and, consequently, a higher lipid hydrolysis rate. On the other hand, a prolonged period of desalting caused an increase in lipase activity, leading to an increase after 8 h of desalting at 4 °C, attributed to the increase in microbial lipases in cod muscle.

3.4. Quality of High Pressure Treated-Desalted Cod

3.4.1. pH Value Variation in Treated-Desalted Cod

Table 1. Variation of pH on control desalted and treated by high-pressure processing (HPP) cod samples during 28 days of storage at 4 °C. Different letters denote significant differences (p < 0.05) between storage times (a,b) and processing conditions (A,B).

Storage Time (Days)	Control Desalted Cod	HPP Desalted Cod
		400 MPa	550 MPa
0	6.33 ± 0.01 bB	6.53 ± 0.01 aA	6.54 ± 0.01 aA
7	6.49 ± 0.10 aA	6.53 ± 0.09 aA	6.59 ± 0.10 aA
14	6.50 ± 0.04 aA	6.53 ± 0.06 aA	6.58 ± 0.01 aA
21	6.50 ± 0.07 aA	6.59 ± 0.06 aA	6.51 ± 0.04 aA
28	6.54 ± 0.01 aA	6.59 ± 0.09 aA	6.60 ± 0.05 aA

shows the pH variation in desalted cod samples treated by HPP (400 MPa and 550 for 5 min) and in the control (unprocessed) samples during 28 days of storage at 4 °C. The pH of desalted cod muscle increased slightly (p < 0.05) immediately after HPP treatments from 6.33 ± 0.01 to 6.53 ± 0.01 and 6.54 ± 0.01 at 400 and 550 MPa, respectively, maintaining similar values (p > 0.05) during all of the different periods of storage. In the control samples the pH value increased after 7 days of storage at 4 °C until the 28th day to a value of 6.49 ± 0.10, with no significant differences (p > 0.05) until the 28th day of storage, showing values between 6.50 and 6.54. As can be seen previously, there was an increase in microbial load until the seventh day of storage, which is like the behaviour for the pH value. Increasing the microbial load increases the formation of basic metabolites, which may explain the observed pH increase [36].

3.4.2. Microbial Activity Variation in Treated-Desalted Cod

The results of the microbial loads of the control (untreated) and HPP-treated samples are shown in Figure 5. The initial microbial load of the control sample was 2.76 ± 0.05, 2.99 ± 0.16, 1.15 ± 0.15, and 1.85 ± 0.01 log CFU/g for TAM, TAP, Enterobacteriaceae, and YM, respectively. Fernandez-Segovia et al. (2003) [24] verified similar initial values for TAP counts (2.8 log CFU/g, respectively) but higher YM counts (3.2 log CFU/g). In this study, 6.0 log CFU/g of TAM counts were used as the limit to evaluate microbial spoilage acceptability for consumption [37].

During storage at 4 °C, TAM (Figure 5A) and TAP (Figure 5B) counts in untreated desalted cod increased (p < 0.05) to 7.15 ± 0.21 and 7.62 ± 0.09 log CFU/g at the seventh day of storage, respectively, exceeding the acceptable limit for the consumption of 6 log CFU/g. Enterobacteriaceae (Figure 5C) and YM (Figure 5D) counts showed a similar behaviour, with a progressive increase (p < 0.05) over the storage time, reaching values of 5.79 ± 0.11 and 4.56 ± 0.17 log CFU/g, respectively, at the 14th day. For all microorganisms, counts were maintained at similar values (p > 0.05) after the 14th day of storage.

Initially (day 0), HPP treatments effectively reduced (p < 0.05) TAM and TAP to values very close to 1.00 log CFU/g, which corresponds to a reduction of about 1.53 and 1.75 log units, respectively. Concerning Enterobacteriaceae and YM, counts in the HPP samples were below the detection limit in both cases (<1.00 and <1.70 log CFU/g, respectively) during the total storage time (28 days).

Compared to the control samples, a slower TAM and TAP growth rate was observed along storage in HPP-treated samples, showing similar profiles until the day 14 with no significant differences (p > 0.05) between the HPP conditions. Afterwards, TAM and TAP counts increased to values above 6.00 log CFU/g (acceptable limit) at the 21st day of storage, being observed as slower growth (p < 0.05) occurred in the samples treated at 550 MPa, rather than on those treated at 400 MPa.

These contrasting effects of HPP treatment between microbial groups can be attributed to the difference in resistance levels of these organisms to HPP. Enterobacteriaceae (which regards gram-negative bacteria) and YM are more susceptible to HPP due to their weaker cellular structures, which likely explains why their counts remained below the detection limits. On the other hand, TAM and TAP, particularly mesophiles and psychrophiles, may include other bacteria, especially gram-positive bacteria, which are known to be more pressure-resistant than gram-negative bacteria and YM due to the thick peptidoglycan layer [38].

Similarly, Rode and Rotabakk (2021) [13] study the effect of different packaging methods (vacuum, MAP, and CO₂ emitter) alone or in combination with HPP for the shelf life extension of rehydrated clipfish and saltfish. The authors highlight an increase in bacterial load in samples processed at lower pressures (500 MPa, 5 min) after 15 days of storage. However, samples treated at the highest pressure (600 MPa, 5 min) showed no significant increase in bacterial levels throughout the 49 days of storage. This aligns with our observation of slower growth occurring in samples treated at a higher pressure of 550 MPa compared to 400 MPa (also for 5 min).

Despite the very promising results, this study aimed to provide the first insights on the use of HPP for the nonthermal pasteurization of codfish; still, some additional analyses should be made to deepen the knowledge on the effects of HPP on other quality parameters, such as colour, texture and, most importantly, sensorial analyses. Additionally, an in-depth cost-benefit analysis for the potential industrialization of this process should be made to access the economic viability of HPP for the nonthermal pasteurization of cod, despite the fact that there are some fish products pasteurized by HPP already available in the market.

4. Conclusions

Due to the unpalatable high salt concentration (approximately 16 to 20% w/w) in salted cod fish muscle, salt-cured and dried salt-cured cod must be desalted before consumption. During this process, several changes occur in the physicochemical quality parameters and enzymatic activities. Cod pieces desalted over 16 h at two different temperatures (4 and 20 °C) showed an increase in water content and a decrease in salt content, as expected. After 16 h, the desalted cod reached 75 and 2% of water and salt content, respectively. The pH and TMA content increased with the desalting time during the first 4 h but remained constant thereafter. However, soluble protein in the cod muscle decreased over the desalting time, which can be explained by its dissolution in the desalting water. Regarding endogenous enzymes activities, cathepsins’ (B and D) and acid phosphatase activities decreased during desalting, while lipase activity increased significantly to similar values when desalting was carried out at 4 and 20 °C, but this occurred faster for the latter temperature.

Afterwards, the desalted cod quality after high-pressure processing (400 and 550 MPa for 5 min) was evaluated by the study of microbiological and physicochemical parameters during 28 days of refrigerated storage (4 °C). Both pressure levels were effective on the inactivation of the studied deteriorative microorganisms in desalted cod, allowing a shelf life extension from less than 7 days to between 14 and 21 days. Overall, these results report the usefulness of HPP to extend the microbial shelf life of desalted cod, although it is still necessary to evaluate other quality parameters, such as textural and sensorial analyses. Furthermore, investigating the effects of HPP on the physicochemical quality of desalted cod would be interesting.