Effect of Marinating Temperature of Atlantic Herring on Meat Ripening, Peptide Fractions Proportion, and Antioxidant Activity of Meat and Brine

Abstract

The temperature has a significant effect on cathepsin activity, but the effect of temperature on the ripening of marinades, and the formation of protein hydrolysis products, is less studied than other technological factors. The results of this study showed that herring marinated at 2 °C showed a higher mass yield, but lower non-protein nitrogen (NPN), peptides, and free amino acid fraction content, than after marinating at 7 and 12 °C. The higher temperature increased the free amino acid content the most, and decreased the hardness, as measured via sensory assessment, of the marinated meat. This was confirmed by the hardness measurement in the texture profile analysis. The highest activity of cathepsins D and B in the meat was found at 7 °C, while cathepsin L was found at 2 °C. Increasing the temperature by 10 °C increased the diffusion/loss of nitrogenous substances from the meat to the brine by 36%. The meat and brine showed high antioxidant activity, which depended on the marinating temperature, and originated mainly from the 5–10 or <5 kDa fraction. The meat had a higher ABTS (2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonate) activity than the brine, opposite to the DPPH (2,2-diphenyl-1-picrylhydrazyl radical) activity, while the FRAP (Ferric Reducing Antioxidant Power) capacity was similar for meat and brine. The fractionation of the meat and brine extracts increased the antioxidant potential of FRAP and ABTS only for the brine. The most hydrophobic peptides were released during marinating at 7 °C. The meat and brine were dominated by 2–4 kDa peptides, followed by 4–6 and 0.5–2 kDa. The higher temperature favored a higher proportion of <4 kDa than >4 kDa peptides in the brine.

1. Introduction

The marinating of herring has been known of since ancient times, and the process has not changed significantly to this day. The industry most often uses the classic method, which involves a single stage of marination in a solution of acetic acid and table salt [1]. Cold fish marinades are a unique food product, as they are made from wild-caught herring, which contains fatty acids with positive effects on human health. The lipids in cold marinades are not heat-treated, so they undergo minor hydrolysis and oxidation processes [2]. In addition, marinades contain complete protein and protein hydrolysis products (PHP), which are released through the activity of muscle proteases called cathepsins. In general, the quality of fish products depends on many factors, and the production process plays a special role [3]. Probably the greatest influence on the ripening of marinades is the activity of cathepsins D, B and L, which are activated in an acid-salt environment [4,5] The released PHP (peptides and amino acids), and the products of their interaction with other components of the muscle tissue, give the marinades their characteristic taste and odor. During marination, PHPs diffuse into the surrounding marinating brine, resulting in nutrient loss [6]. The PHP content of the brine is often higher than that of the marinated meat [7]. The peptides present in the brine show antioxidant activity, which has been used to develop a new method of marinating herring [8], the marinating of not herring-fish [9], and as an ingredient in the glaze during the frozen storage of fatty fish, or as an additive for minced herring tissue [10]. At present, the spent brine left over after marinating herring is a huge problem for the industry and the public, as it ends up in wastewater treatment plants, causing economic and environmental problems [11]. An increased knowledge of the brine’s composition, especially of the biologically active substances, could contribute to its faster valorization in the food industry.

In addition to their antioxidant activity, peptides also possess antibacterial, antifungal, antiviral, immunomodulatory, antiproliferative, anticoagulant, and antihypertensive functions, as angiotensin-converting enzyme inhibitors (ACE-inhibitors), and exhibit hemolytic, and opioid- and calcium-binding activity. Certain peptides, usually 2–20 amino acid residues in length, can exhibit a range of bioactivities, the activity being dependent on the sequence of amino acids [12,13].

Currently, in the industry, the ripening process of marinated herring is regulated by several basic technological factors, such as the concentration of acetic acid and salt, the marinating time, and the ratio of fish to brine, but the least studied is still the influence of the temperature [4,5,7,14]. The classical marinating of herring takes place at 7 °C, but a two-stage method is known, in which 12 °C is used for the first 2–4 days of marinating, and then the process continues at 7 °C. Nowadays, the industry uses much lower temperatures of 0–4 °C, to obtain the highest possible mass yield of marinated herring, and to reduce the proliferation of microorganisms. The temperature also affects the activity of muscle cathepsins, which in fish are highly active at refrigeration temperatures [4,5,15]. Fish cathepsins show comparable activity at 7 °C to mammalian cathepsins at 37 °C. Despite this, there is a lack of research on the effect of the temperature on the marinating process, and on the cathepsin activity, composition, and antioxidant properties of the released peptides. Until now, the effect of the temperature has only been studied as a factor in regulating the microbiological stability of marinated fish [16].

In the case of salted whole herring, in which digestive proteases with a different specificity to cathepsins are active, the content of only a few peptides in the brines is known [17]. These brines have demonstrated significant iron(II)-chelating activity, reducing power, and radical-scavenging activity; however, in addition to the peptides, the brines contain phenolic compounds from spices, which also exhibit strong antioxidant activity.

Therefore, the aim of this research was to determine the effect of different temperatures of the marinating process on the main indicators of the marinade’s quality, on the cathepsin activity, and on the profile and antioxidant activity of the peptides present in both the meat and the brine.

2. Materials and Methods

2.1. Raw Materials and Marinating Process

Atlantic herring was purchased as frozen skinless fillets (4/8 size) in 12 kg blocks packed in plastic bags stored at −18 °C. Prior to marinating, the frozen fillets in a bag were thawed in continuous circulation of water at 10 ± 2 °C. One kg of fillets was placed in 2 L glass jars, and then filled with the solution of marinating brine, including 4.5% acetic acid and 6% NaCl. The fish to brine ratio was 1.5:1.0 (w/w). The process of marinating was conducted over 7 days, at 2 ± 0.1 °C, 7 ± 0.1 °C, and 12 ± 0.1 °C. Non-iodinated rock salt and 10% spirit vinegar were used to prepare the marinating brine.

2.2. Mass, Salt, Total Acidity, pH, Moisture, and Lipid Content Analysis

Prior to analysis, the marinated fillets and brine were transferred to a large sieve for 3 min, until the liquid and solid fractions were fully separated, and then weighed. Six fillets were randomly chosen for sensory and texture analyses, and the remaining marinated fillets and raw material were minced. The pH value was determined using a digital pH meter (F20, Mettler Toledo, Columbus, OH, USA), in water extract (1:5, w:v). The moisture (no. 950.46B) and total lipids (no. 960.39) in the meat, the total acidity (no. 935.57), and the salt (no. 937.09) content in the meat and in the brine were determined using standard AOAC analytical techniques.

2.3. Total Nitrogen and Non-Protein Nitrogen Fractions

Total nitrogen was assayed using the Kjeldahl method. In 5% TCA extracts from the meat and brine, we determined: (i) the non-protein nitrogen using the Kjeldahl method (AOAC no. 940.25); and (ii) the protein hydrolysis products of peptides fraction (PHP(R)), and the free amino acids fraction (PHP(A)), using the Lowry method, with modification by Kołakowski [18].

2.4. Cathepsins Activity

The activity of aspartyl cathepsin (D + E) and cysteine cathepsins B and L against Mca-GKPILFFRLK(Dnp)-r-NH2, with and without pepstatin-A, Z-RR-MCA, and Z-FR-MCA, respectively, was measured according to Szymczak [4]. One unit of cathepsin activity (U) was defined as 1 fluorescence unit change per minute at 37 °C. All chemicals were purchased from Peptide Institute Inc. (Osaka, Japan).

2.5. Sensory Assessment

The marinated herring fillets were analyzed by sensory profiling, performed by a sensory panel of seven people trained according to ISO 8586:2023, using a five-point unstructured scale, with 0.1 point accuracy anchored at their extremes, with minimum and maximum degrees of acceptance (ISO 11035). A higher note signifies better texture attributes (0 points for the worst/extremely disliked, 5 points for the best/extremely liked). Briefly, three skinned fillets from each sample were served on porcelain trays. Each assessor was cut into three pieces (2–3 cm width each), one from each fillet to test. The tested area of the fillets ranged from 2/10 to 6/10 fillet, with the length measured from the head side. The evaluations were performed in separate cubicles, under daylight and at ambient temperature. The assessors used water and flat bread to cleanse their palate between samples.

2.6. Texture Profile Analyses (TPA)

The TPA hardness was determined in three fillets from each sample, using a TAXTplus Texture Analyzer (Stable Micro Systems, Godalming, UK). The test included two-fold compression using a cylindrical sonde P10 (10 mm diameter), with sample deformation up to 50% of height at the speed of 5 mm∙s⁻¹. The course of the test was recorded as curves representing changes of force in time. The tests were conducted for each fillet separately (in 3–4 repetitions each). The tested area of the fillets ranged from 2/10 to 6/10 fillet, with the length measured from the head side.

2.7. ISP Extracts of Meat and Brine

The meat and marinating brine were subjected to protein removal using isoelectric solubility and precipitation (ISP). Briefly, minced fillets were mixed with water (1:5, w:v) for 15 min at pH 12.0, and centrifuged (10 min, 9000× g, 4 °C), the precipitate was discarded, and the supernatant was adjusted to pH 5.3 for 15 min; centrifuged again to discard the precipitate, and the supernatant was adjusted to pH 7.0 to obtain ISP-meat-extract. In turn, the marinating brine was filtered through 20 µm, adjusted to pH 2.0 and, after 15 min, was centrifuged to remove the precipitate; the resulting supernatant was adjusted to pH 11.0 for 15 min, centrifuged and the supernatant was adjusted to pH 7.0 to obtain ISP-brine-extract.

2.8. Antioxidant Activity of Protein Hydrolysis Products

Extracts of meat and brine obtained using the ISP method were subjected to ultrafiltration (UF) into fractions >10, 5–10 and <5 kDa using 10 and 5 kDa centrifuge filters (Amicon-Ultra, Merck Millipore, Ireland). A ferric reducing antioxidant power (FRAP) assay was measured as the reduction of ferric tripyridyltriazine complex (Fe³⁺-TPTZ). The 5 µL of unfractionated ISP extract and its UF fractions were mixed with 150 µL of TPTZ and, after 30 min, the absorbance was measured at 593 nm. We used FeSO₄ as a positive control, and to obtain a standard curve. One unit [1 U] of FRAP was expressed in mM FeSO₄ reduced by 100 g of meat or 100 mL brine in 30 min. The radical-scavenging activity (RSA) was determined against ABTS radical cation (ABTS⁺), according to Ginger at al. [17]. Ten µL of sample was mixed with 190 µL ABTS working solution and read after 5 min at room temperature at 734 nm. A DPPH free-radical-scavenging-capacity assay was measured by mixing 50 µL of DPPH solution (0.1 mM in methanol) with 150 µL of sample solution and, after 30 min at room temperature, the absorbance was measured at 517 nm. Trolox (2 mM, in methanol) was used as a positive control and a standard curve in the RSA and ABTS assays. One unit [1 U] of RSA or DPPH was expressed in µM Trolox, reduced by 100 g of meat or 100 mL of brine in 5 min. All assays were measured on a microplate spectrophotometer (Synergy 2 Multi-Mode Microplate Reader, BioTek^® Instruments, Inc., Winooski, VT, USA). As the control, distilled water was used instead of the sample.

2.9. Lyophylization Process

The ISP extracts of meat and brine were freeze-dried in a freeze-dryer (CHRIST BETA, Martin Christ, Germany) without any protective medium. The frozen ISP extracts were placed in a tray and lyophilized. As the first step of the process, the samples were loaded onto a shelf at 0 °C for 2 h and cooled to −35 °C (7 h). The primary drying was carried out in the temperature range −35 °C to 0 °C (24 h). Secondary drying was performed at 0 °C to 20 °C/11 h. The samples were unloaded at 20 °C and stored 1 week in Falcon tubes in vacuum bags before the analyses.

2.10. Chromatography and Mass Spectrometry of ISP Extracts

The brine and meat extract samples were prepared using solid-phase extraction (SPE), according to Nagai et al. [19]. The Supelclean LC-18 SPE Tube (45 µm, 60 Å, 3 mL) columns by Supelco (Bellefonte, PA, USA) was used. Samples were dissolved in 0.1% (v/v) of trifluoroacetic acid (TFA) in HPLC-suitable water (Sigma Aldrich, St. Louis, MO, USA, cat. no. 270733). The columns were initially conditioned using 6 mL 0.1% (v/v) TFA in HPLC-suitable acetonitrile (ACN) gradient grade (Sigma Aldrich, cat. no. 34851) and equilibrated using 6 mL 0.1% (v/v) TFA in water. Samples with a flow rate of 1–2 drops per second were loaded on columns, and then washed five times using 3 mL portions of 0.1% (v/v) of (TFA) in water. Elution was performed using 5 mL of 0.1% TFA in acetonitrile. Eluates were evaporated using a vacuum concentrator (Martin Christ, Osterode am Harz, Germany). The remaining pellets were dissolved in water, lyophilized, and stored at −86 °C before further analysis.

Samples were dissolved in 300 µL of buffer with pH 6.6, containing 0.1 M BIS-TRIS and 4 M urea. Samples were reduced by the addition of 20 µL of 2-mercaptoethanol, followed by shaking for 1 h at room temperature, 1000 × min⁻¹ (ThermoMixer^®, Eppendorf, Hamburg, Germany). The reaction was stopped by the addition of 680 µL of 6 M urea in water (pH 2.2 adjusted using TFA), and samples were centrifuged (10 min, 4 °C, 10,000× g). The final concentration was 1 mg × mL⁻¹ for brine samples, and 3 mg × mL⁻¹ for meat samples [20].

RP-HPLC analyses were carried out using the Shimadzu (Tokyo, Japan) assembly-containing controller CBM-20A, the DGU-20A5 degasser, the SIL-20AC HT autosampler, two LC-20AD pumps, the CTO-10AS thermostat, and the SPD-M20A photodiode array detector. The assembly was equipped with the Jupiter Proteo (Phenomenex, Torrance, CA, USA) C18 column, 250 × 2 mm, with a particle size of 5 µm, and pore size of 300 Å. The mobile phase consisted of solvents A and B—0.075% (v/v) TFA in water, and 0.1% (v/v) TFA in ACN, respectively, to minimize baseline drift [21]. The following gradient of solvent B was applied: analysis: 5–20% from 0.00 to 30.00 min; 20–70% from 30.01 to 60.00 min; column washing: from 70–100% from 60.01 to 61.00 min; 100% from 61.01 to 75.00 min; column equilibration: 100–5% from 75.00 to 76.00 min; 5% from 76.01 to 90.00 min. The injection volume was 50 µL, flow rate: 0.2 mL × min⁻¹, and column temperature: 30 °C. Chromatograms were acquired at wavelength 220 nm. Data were collected and processed using Lab Solution (LC Solution) software (Shimadzu). RP-HPLC analyses were replicated twice.

Samples were reduced using the same procedure as previously. Before analysis, samples were purified using C18 ZipTips (Merck Millipore, Burlington, VT, USA) with bed capacity of 0.6 µL, according to provider instruction. The resin was washed twice using 10 µL ACN (wetting solution), and twice using 10 µL of 0.1% (v/v) TFA in water (equilibration solution). The samples were loaded by passing them ten times through ZipTip. Then, the ZipTip was washed five times with equilibration solution. Finally, 1.5 µL of 50% acetonitrile in water with 0.1% TFA addition (v/v) in clear tubes was aspirated and dispensed through the ZipTip for sample elution.

Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-ToF) mass spectrometry was carried out using MALDI-7090 (Kratos, Shimadzu Group, Manchester, UK), according to provider instruction. Samples were loaded onto target (Kratos) using the matrix pre-coated targets method according to the provider instruction, as follows: 0.5 µL of matrix solution containing 5 mg/mL of sinapinic acid (Sigma-Aldrich) in ACN containing 0.1% TFA for proteins analysis, was spotted and left for a few seconds. The surplus was removed using a pipette and left to dry. Next, 0.5 µL of desalted sample was spotted and, immediately, 0.5 µL of matrix solution was added. The spotted target plate was left for several minutes at room temperature, until samples were dried. Next, the target was inserted into the instrument. The TOF-mix kit (LaserBio Labs, Valbonne, France) was used for internal calibration. The samples were analyzed using positive linear mode within the range of 100–300 000 Da (proteins). The 20 laser shots per sample with a frequency of 1000 Hz were used to generate each mass spectrum. The number of profiles was 109. Laser power of 140 arbitrary units for proteins analysis was used. The spectra were processed using the MALDI Solutions v. 2.6 software (Kratos-Shimadzu).

2.11. SEM Analysis

Lyophilized ISP extracts of meat and brine were analyzed using a scanning electron microscope (SEM). The microscopic analysis was performed using a Vega 3 LMU microscope (Tescan, Brno-Kohoutovice, Czech Republic). The experiments were necessary to compare the morphology of the lyophilizate particles of meat and brine. An analysis was performed at room temperature with a tungsten filament, and an accelerating voltage of 10 kV was used to capture SEM images. All specimens were viewed from above.

2.12. Statistical Analysis

If not mentioned, all analyses were performed in three replications. Results were analyzed statistically (Statistica 13.1, Tulsa, OK, USA) using one-way analysis of variance (ANOVA), p value was set at 0.05, and the differences between treatments were examined using the post hoc Tukey’s honestly significant difference test (p < 0.05) [22].

3. Results and Discussion

3.1. Mass Yield, Moisture, Salt, and pH of Meat

Atlantic herring, the most used in marinade production, was used in the study, and contained 17.5 ± 0.5% lipids, and an average activity of cathepsins D, B, and L (

Table 1. Basic composition, nitrogen fractions content, and cathepsin activity in the raw meat and marinated meat; TN—total nitrogen, NPN—non-protein nitrogen, PHP(R)—peptides fraction, PHP(A)—free amino acids fraction; ^abcd means within the same row with different lowercase letters differing significantly.

Analyses	Fresh Herring	Marinating Temperature
		2 °C	7 °C	12 °C
Mass yield (%)	100	89.5	87.2	81.5
pH	6.52 ± 0.01 a	4.15 ± 0.01 b	4.16 ± 0.01 b	4.17 ± 0.01 b
NaCl (%)	0.30 ± 0.01 d	2.41 ± 0.01 b	2.43 ± 0.01 b	2.54 ± 0.01 a
Total acidity (%)	0.22 ± 0.01 d	1.56 ± 0.02 c	1.62 ± 0.01 b	1.67 ± 0.01 a
Moisture (%)	66.6 ± 0.29 a	59.9 ± 0.6 d	62.0 ± 0.3 b	61.3 ± 0.3 c
TN (g/100 g)	2.30 ± 0.00 b	2.45 ± 0.01 a	2.43 ± 0.03 a	2.44 ± 0.02 a
NPN (mg/100 g)	233.6 ± 3.16 b	218.8 ± 1.6 c	237.9 ± 1.6 b	280.5 ± 0.0 a
PHP(R) (mg/100 g)	6.4 ± 1.8 d	208.6 ± 3.5 c	228.3 ± 2.3 b	252.4 ± 6.8 a
PHP(A) (mg/100 g)	12.9 ± 0.2 d	35.2 ± 0.9 c	42.3 ± 0.2 b	55.2 ± 0.4 a
Cathepsin D (U)	3259 ± 32 d	6077 ± 22 c	6315 ± 45 a	6118 ± 22 b
Cathepsin B (U)	214.7 ± 1.2 a	136 ± 1.5 d	190 ± 4.8 b	168 ± 2.3 c
Cathepsin L (U)	390 ± 3.2 a	188 ± 1.6 b	168 ± 0.5 c	131.1 ± 2.8 d

). The fillets were from fresh fish, as they had a pH of 6.52, and a low content of PHB(R) and PHB(A) fractions. The herring were probably caught during the feeding season, before spawning. A higher marinating temperature had a significant effect on the lower mass yield of the marinades, which decreased according to an exponential function by 0.5–1.1 percentage points for each 1 °C (Table 1). The herring marinated at 2 °C (89.5%) had the highest mass yield, followed by the herring marinated at 7 °C (87.2%), and the lowest using 12 °C (81.5%). The lower mass of marinades was the result of several phenomena simultaneously. The higher temperature increased the acid and salt content of the marinades, so the higher moisture may have been due to the higher pH of the meat (Table 1), but the differences were not so great as to significantly reduce the yield of the fatty herring marinades [14]. In addition, the small differences (about 0.1%) in the acid and salt content of the marinades were not related to their content in the brine (

Table 2. Basic composition, nitrogen fractions content, and cathepsin activity in the marinating brine; TN—total nitrogen, NPN—non-protein nitrogen, PHB(R)—peptides fraction, PHB(A)—free amino acids fraction; ^abc means within the same row with different lowercase letters differing significantly.

Analyses	Marinating Temperature
	2 °C	7 °C	12 °C
Mass yield (%)	111.3	114.6	125.2
pH	4.28 ± 0.00 a	4.27 ± 0.00 b	4.26 ± 0.00 c
NaCl (%)	1.73 ± 0.00 a	1.73 ± 0.00 a	1.73 ± 0.00 a
Total acidity (%)	1.98 ± 0.00 b	1.98 ± 0.00 b	2.04 ± 0.00 a
TN (mg/100 mL)	355.4 ± 4.18 c	396.3 ± 1.36 b	444.9 ± 4.7 a
NPN (mg/100 mL)	326.4 ± 0.8 c	362.2 ± 0.0 b	429.9 ± 0.8 a
PHB(R) (mg/100 mL)	194.0 ± 3.5 b	200.9 ± 4.2 ab	200.9 ± 3.4 a
PHB(A) (mg/100 mL)	33.1 ± 0.2 c	41.4 ± 0.0 b	61.7 ± 0.9 a
Cathepsin D (U)	1817 ± 52 a	1196 ± 32 b	609 ± 41 c
Cathepsin B (U)	1194 ± 30 c	1363 ± 21 b	1463 ± 58 a
Cathepsin L (U)	1411 ± 48 b	1689 ± 35 a	1374 ± 22 b

). Therefore, the higher acidity of the brine at a higher temperature may be due to a greater diffusion of acidic substances (free amino acids, free fatty acids) from the meat into the brine, or a greater proliferation of lactic acid bacteria. Shenderyuk and Bykowski [1] found an effect of higher temperature (3 °C vs. 13 °C) on faster salt and acid penetration, mainly in the first days of marinating, and lower marinating yield, mainly using a higher (2:1 vs. 1.5:1) proportion of fish to brine. In turn, Rodger et al. [23] found that an increase in marinating temperature for herring from 2.3 °C to 5.0 °C accelerated acid penetration (from 75 h to 51 h) more than salt penetration (from 110 h to 94 h), while an increase from 5.0 °C to 10.3 °C accelerated salt penetration (from 94 h to 45 h) more than acid penetration (from 51 h to 48 h).

3.2. Nitrogen Indicators of Ripening

The higher marinating temperature did not determine the total protein content, but significantly increased the NPN (326 vs. 430 mg/100 g), peptide fraction (194 vs. 201 mg/100 g), and amino acid content (33 vs. 62 mg/100 g) in marinated meat (Table 1). Zamojski [24] found that the ripening of herring at 18 °C was 35% faster than at 10 °C, obtaining 500 and 440 mg of NPN in 100 g of marinated meat, respectively. In our study, increasing the temperature by 10 °C increased the content of the amino acid fraction 2–3 times more than that of the peptide fraction, whereby the peptide content increased by the same amount in both the 2–7 °C and 7–12 °C ranges, while the amino acid content increased twice as much in the 7–12 °C range as in the 2–7 °C range. On the one hand, this may indicate that exopeptidases are more sensitive to temperature change than endopeptidases, and therefore an increase in temperature is more conducive to the release of amino acids. On the other hand, a higher exopeptidase activity depends on more peptides being released by higher endopeptidase activity. A more accurate assessment of protein proteolysis in marinades requires analysis of both the meat and the brine, into which large amounts of nitrogenous substances diffuse [6]. The use of a higher temperature significantly increased the NPN content of the brine by 10.4 mg for every 1 °C, while the peptide and amino acid fractions increased by 1.4 mg and 2.9 mg, respectively (Table 2). Thus, using a higher marinating temperature, the NPN and amino acid content increased more in the brine than in the meat, while the opposite was the case for peptides (Table 1 and Table 2). The sum of the nitrogen fractions in the meat and the brine showed that the higher temperature reduced the proportion of amino acids in the meat to the brine from 1.59 to 1.34, while for peptides, the proportion increased from 1.61 to 1.88. This means that the higher temperature was more conducive to amino acid loss than peptide loss, which may be related to the described greater increase in the amino acid content of the meat. The results may also indicate that an increase in temperature does not have a major effect on endopeptidase activity, in contrast to an increase in exopeptidase activity, confirming Zamojski’s [24] results that high temperature caused an increase in mainly free amino acids. Both the quantitative and qualitative composition of the amino acids and peptides released during cathepsin activity have important effects on the sensory quality of marinades [7,14].

3.3. Cathepsins Activity

Increasing the temperature of the marinating process from 2 to 7–12 °C increased cathepsin D activity by only 1–4% (Table 1). In the case of cathepsin B, activity increased by 23–40% (most at 7 °C), while the highest increase in cathepsin L activity of 28–43% occurred in marinades ripened at the lowest temperature. Refrigeration temperature strongly decreases protease activity, but fish cathepsin activity remains at around 20% [25]. Even the application of negative temperatures (superchilling) does not inhibit cathepsin activity. Cooling close to 0 °C was found to contribute to an increase in cathepsin L activity in the sarcoplasm, but also a decrease in activity in the cellular structure, because of the tight tangle of more muscle fibers [26]. Szymczak [4] noted that a decrease in temperature from 12 to 5 °C reduced the total proteolytic activity (GPA) in marinades, and the proportion of cathepsin D in GPA, particularly up to day 7. The cathepsins tested are endopeptidases, with cathepsin B (B1) also showing dipeptidase activity, which may be related to its higher activity in meat and brine under higher marinating temperatures. In optimal conditions, cathepsin D releases very short tetrapeptides and, together with cathepsin B, acts on myofibrillar proteins, while cathepsin L and, to a lesser extent, cathepsin B are specific to collagen, which has a greater effect on meat texture than myofibrillar proteins.

In contrast, the effect of temperature on the activity of cathepsins D and L in the brine was different from that in the meat. At 12 °C, cathepsin D activity was 67% lower compared to 2 °C (Table 2). Cathepsin B activity increased from 1194 to 1463 U when the temperature was increased by 10 °C, while cathepsin L had the highest activity at 7 °C. Cathepsins are present in the brine because of their diffusion from the muscle tissue, which increases using frozen-thawed raw material and a high concentration of salt [5].

3.4. Sensory Assesment of Marinades

The sensory quality of the marinades ripened at higher temperatures was evidently the best, but the increase in the overall sensory acceptability score from 4.45 to 4.84 points was not statistically significant (

Table 3. Sensory assessment and TPA hardness of marinated meat; ^ab means within the same row with different lowercase letters differing significantly.

Analyses	Marinating Temperature
	2 °C	7 °C	12 °C
Appearance	4.95 ± 0.09 a	4.91 ± 0.10 a	4.79 ± 0.23 a
Odor	4.60 ± 0.51 a	4.65 ± 0.41 a	4.79 ± 0.25 a
Flavor	4.30 ± 0.42 a	4.39 ± 0.53 a	4.78 ± 0.23 a
Texture	4.37 ± 0.35 b	4.42 ± 0.23 b	4.91 ± 0.07 a
Overall sensory evaluation	4.45 ± 0.40 a	4.51 ± 0.37 a	4.84 ± 0.17 a
TPA hardness	10.49 ± 0.95 a	8.03 ± 0.57 b	7.40 ± 1.14 b

). The fillets marinated at 12 °C reached consumer ripeness, while the other samples only reached technological ripeness, and included fragments of less-ripened tissue, which significantly reduced the texture score from 4.91 to 4.37–4.42 points. In addition, the TPA hardness analysis showed a significant increase in hardness by 2–3 N (20–30%) for marinades obtained at the lowest temperature (Table 3), indicating a reduced activity of cathepsins D and B (Table 1). The marinades obtained at 12 °C had the highest rating for taste and odor, which were harmonized, and characteristic of ripened marinades, in contrast to the other marinades, which were dominated by the taste of acetic acid and salt. This indicates that the higher marinating temperature favored the activity of alanine aminopeptidase and cathepsins A and C, which release sweet amino acids. The lower temperature and slower ripening only had a beneficial effect on the appearance of the marinades (Table 3), mainly for less dissection of the myomeres throughout the length of the fillets, and their bright color.

3.5. Chromatography and Mass Spectrometry of ISP Extracts

The RP-HPLC chromatograms of samples of brine and meat ISP extracts are presented in Figure 1 in the main text, and in Figures S1–S6 in the Supplementary Materials. Figure 1 presents chromatograms within the time interval up to 60 min. Chromatograms presented in this figure show peaks with retention times between 15 and 60 min. Entire chromatograms are presented in the Supplementary Materials (Figures S1–S6). Our resignation from the interpretation of the injection peaks may appear controversial, due to the fact that short and hydrophilic peptides may be not retained at the column, but on the other hand, absorbance at the injection peak apex is c.a. 4, and is too high to provide predictable dependence between the composition of the solution and the output from the detector. The peaks at the start of the chromatogram contain many low-molecular compounds belonging to various classes. It is impossible to estimate the contribution of peptides in this fraction. In our previous works [27,28,29], interpretation of the area of injection peaks did not provide valuable information, in contrast to fractions with retention times exceeding 15 min. A time interval exceeding 60 min contains so-called system peaks, artifacts that are useless from the point of view of information about the sample [30]. The sample concentration of the meat extracts was three times higher than the concentration of the brine samples. The retention times of peptides depend on their hydrophobicity and length. More hydrophobic and longer chains have longer retention times than those that are shorter and more hydrophilic. Reversed-phase HPLC does not provide a simple correlation between retention time and molecular mass [31,32], but experiments using proteins from herring [33] and other sources [27,28,34,35,36,37] may serve as examples, illustrating the fact that retention times of proteolysis products are usually shorter than those of entire proteins. Thus, RP-HPLC may serve as a tool for the comparison of the proteolysis degree if it is not determined directly.

Chromatograms of samples with an unknown composition may be subjected to simple interpretation by comparison of the relative area of peaks within various retention time intervals [27,28,29]. The relative areas of peaks within the intervals 15–34.99 min (shorter) and 35–60 min (longer) are presented in

Table 4. Relative areas of peaks in the chromatograms of brine and meat ISP extracts, measured using RP-HPLC.

Sample	Relative Area of Chromatographic Peaks (%) 1
	Retention Time Interval 15–34.99 min	Retention Time Interval 35–60 min
Meat, 2 °C	52.32	47.68
Meat, 7 °C	60.24	39.76
Meat, 12 °C	51.41	48.59
Brine, 2 °C	69.72	30.28
Brine, 7 °C	71.22	28.78
Brine, 12 °C	93.36	6.63
All meat extracts 2,3	54.66 ± 4.86 a	45.34 ± 4.86 a
All brines 2,3	78.10 ± 13.23 b	21.90 ± 13.24 b

¹ Area of peaks within the retention time interval between 15 and 60 min was considered as 100%; ² Mean ± SD; ^{3 ab} means within the same row with different lowercase letters differing significantly.

. It is possible to discriminate the chromatographic patterns of the brine and meat extracts. The brine extracts contain more material within a shorter retention time interval, compared with the meat extracts. This suggests the predominant extraction of hydrophilic or short peptides during marinating. The extracts from meat, obtained after marinating, contained a relatively higher content of hydrophobic or long peptides than brine. The brine, after marinating at a temperature of 12 °C, contained larger amounts of compounds forming peaks within a shorter retention time segment, than other brines did. This may indicate a larger extent of hydrolysis of herring proteins at 12 °C than in lower temperatures. This agrees with previous results revealing that proteolysis progress causes an increase in the relative area of peaks within shorter retention time intervals [27,28].

The MALDI-ToF mass spectra of brine and meat ISP extracts within the m/z (mass to charge ratio) range up to 10 kDa are presented in Figure 2. Entire MALDI-ToF mass spectra are presented in Figures S7–S12 in the Supplementary Materials. MALDI-ToF serves mainly as a tool for the qualitative analysis of peptides, proteins, and their mixtures [38,39]. Quantitation of individual proteins or peptides, although possible, is difficult, and requires many repeats to obtain a significant correlation between the amount of compound and the peak intensity [38]. On the other hand, mass spectrometry measurements are not affected by hydrophobic or any other interactions (in contrast to chromatography) and provide a precise mass to charge ratio. The last value measured using MALDI-ToF mass spectrometry should also be interpreted carefully. Proteins form not only singly protonated ions (M + H)⁺, but also may be doubly protonated or appear as aggregates containing two or three analyte molecules with one proton [39,40,41].

Taking the above into account, we decided to interpret the MALDI-ToF mass spectra in a similar way to chromatograms, i.e., to compare relative areas of peaks within particular m/z segments. For the purpose of comparison, a few samples were obtained and analyzed in the same way we could use relative peak areas within defined m/z intervals. We did not utilize m/z lower than 500 Da. The matrix (in this experiment, sinapinic acid) clusters predominantly possess masses below 500 Da, although they may also reveal higher masses [42,43]. Relative areas of peaks within various m/z segments are presented in

Table 5. Relative areas of peaks on the MALDI-ToF MS spectra of meat and brine ISP extracts.

Sample	Relative Area of Peaks (%) 1
	m/z Range 0.5–2 kDa	m/z Range 2–4 kDa	m/z Range 4–6 kDa	m/z Range 6–8 kDa	m/z Range 8–10 kDa	m/z Range >10 kDa
Meat, 2 °C	14.99	47.31	22.29	9.68	3.47	2.26
Meat, 7 °C	11.95	43.71	26.21	12.03	3.74	2.36
Meat, 12 °C	14.08	45.40	23.01	11.27	3.59	2.64
Brine, 2 °C	14.95	44.64	21.11	9.05	5.89	4.36
Brine, 7 °C	14.57	45.83	18.04	8.28	5.92	7.36
Brine, 12 °C	18.26	60.88	14.49	4.48	1.31	0.59
All meat extracts 2,3	13.68 ± 1.56 a	45.47 ± 1.80 a	23.83 ± 2.09 a	10.99 ± 1.20 a	3.60 ± 0.14 a	2.42 ± 0.20 a
All brines 2,3	15.93 ± 2.03 a	50.45 ± 9.05 a	17.88 ± 3.31 b	7.27 ± 2.45 b	4.37 ± 2.66 a	4.10 ± 3.39 a
Ratio meat to brine, 2 °C	1	1.06	1.06	1.07	0.59	0.52
Ratio meat to brine, 7 °C	0.82	0.95	1.45	1.45	0.63	0.32
Ratio meat to brine, 12 °C	0.77	0.75	1.59	2.52	2.74	4.47

¹ Area of all peaks with mass to charge (m/z) ratio exceeding 500 Da are considered as 100%; ² Mean ± SD; ³ Difference between values denoted by the same letter in column is statistically insignificant at p < 0.05 as judged by Student t-test; ^ab means within the same row with different lowercase letters differing significantly.

.

Some results of the MALDI-ToF MS confirmed our interpretation of the RP-HPLC chromatograms. The brine after marinating at 12 °C contained the most peptides with m/z below 4000 Da, and the least compounds with m/z above 4000 Da, characteristic of polypeptides and intact proteins. This result is consistent with the RP-HPLC, revealing a lower amount of material with long retention times, compared to other samples. On the one hand, this suggests higher activity of endoproteases and exoproteases at the higher temperature of 12 °C, as compared with lower temperatures. On the other hand, temperature changes may affect not only proteolysis, but also the extraction of oligo- and polypeptides from meat to brine during marination. Changes in the brine composition cannot be thus considered as results of proteolysis only. The 2–4 kDa fraction dominated the meat and the brine, followed by 4–6 kDa, and 0.5–2 kDa (Table 5). An increase in the marinating temperature had no effect on the proportion of fractions > 8 kDa in meat, while a temperature of 7 °C favored a higher proportion of 4–6 and 6–8 kDa fractions, at the expense of 2–4 and 0.5–2 kDa fractions, than using 2 and 12 °C. All brines contained fewer polypeptides with a mass to charge ratio within the range 4–8 kDa than meat extracts did. There was no significant difference between the brine and meat extracts in their content of material within other segments of m/z, annotated in Table 5. In contrast, the meat to brine ratio for the fractions <4 kDa decreased with an increasing marinating temperature from 1.0–1.06 to 0.75–0.77, in contrast to fractions >4 kDa, where the meat to brine ratio changed from 0.52–1.06 to 1.59–4.47. This means that the higher marinating temperature promotes both higher protease activity, and a greater diffusion of peptides from the meat into the brine. Søtoft et al. [44], using the SDS-PAGE method, found that the brine was dominated by the 10–20 kDa protein fraction, but after membrane fractionation of the brine, a higher total amino acid content was found in the nanofiltration fraction than in the ultrafiltration fraction.

All samples contained only a slight amount of material with a high molecular mass (m/z exceeding 10 kDa). This may suggest that the main compounds extracted to the brine during marinating, as well as during post-marinating extraction, were proteolysis products. RP-HPLC and MALDI-ToF mass spectrometry may be recommended as tools for monitoring proteolysis, as alternatives to polyacrylamide gel electrophoresis (SDS-PAGE), which is more commonly used for this purpose [45]. Both HPLC and MS may serve for simultaneous detection compounds in a broad range of molecular masses, from oligopeptides to intact proteins. SDS-PAGE serves as a method for the analysis of polypeptides and proteins, but not oligopeptides.

3.6. Antioxidant Activity

To further characterize the ripening process, three in vitro assays were used to determine the potential for antioxidant activity of protein hydrolysis products in meat and brine (

Table 6. Antioxidant activity of ISP extracts from meat and brine before (unfractionated) and after fractionation (>10, 5–10, and <5 KDa); ^a–prs means within the same column with different lowercase letters differing significantly.

Sample	Fraction	FRAP [U]	TEAC [U]	DPPH [U]
Meat, 2 °C	Unfractionated	34.9 ± 3.9 ab	135.7 ± 1.8 a	281.1 ± 2.2 f
	>10 kDa	4.0 ± 0.0 k	8.6 ± 0.2 o	38.3 ± 1.7 m
	5–10 kDa	20.3 ± 1.4 d	78.5 ± 3.7 d	70.7 ± 3.9 k
	<5 kDa	10.9 ± 0.9 i	64.9 ± 5.1 ef	168.5 ± 9.7 h
Meat, 7 °C	Unfractionated	30.4 ± 0.8 bc	106.9 ± 5.3 c	312.7 ± 18.1 e
	>10 kDa	4.6 ± 0.5 j	13 ± 0.8 n	22.6 ± 1.4 p
	5–10 kDa	26.4 ± 3.2 c	61.9 ± 4.7 fg	35.2 ± 0.5 o
	<5 kDa	10.5 ± 3.0 i	55.7 ± 3.8 g	113.7 ± 12.3 i
Meat, 12 °C	Unfractionated	33.7 ± 2.0 a	124.2 ± 3.8 b	251.5 ± 14.3 g
	>10 kDa	4.3 ± 0.1 j	6.9 ± 0.1 p	36.2 ± 0.1 n
	5–10 kDa	13.3 ± 1.5 ghi	60.5 ± 3.2 fg	41 ± 3.9 m
	<5 kDa	16.1 ± 0.9 ef	71.1 ± 4.1 de	138.9 ± 17.4 ij
Brine, 2 °C	Unfractionated	31.2 ± 1.3 ab	35.7 ± 0.1 i	615.5 ± 38.9 b
	>10 kDa	1.8 ± 0.0 l	1.7 ± 0.0 r	35.2 ± 1.2 no
	5–10 kDa	15.7 ± 0.9 fg	29.3 ± 0.8 k	128.6 ± 7.8 ij
	<5 kDa	14.1 ± 0.4 h	24.0 ± 0.9 m	325.3 ± 22.4 e
Brine, 7 °C	Unfractionated	26.6 ± 1.5 c	34.3 ± 0.3 j	638.4 ± 19.3 b
	>10 kDa	1.6 ± 0.1 l	1.5 ± 0.0 s	57.3 ± 5.4 l
	5–10 kDa	15.5 ± 0.6 f	27.4 ± 0.9 l	121.3 ± 1.3 j
	<5 kDa	17.6 ± 1.0 e	27.5 ± 0.9 l	410.6 ± 15.5 c
Brine, 12 °C	Unfractionated	29.1 ± 1.0 bc	36.6 ± 0.1 h	679.9 ± 15.3 a
	>10 kDa	1.8 ± 0.1 l	1.5 ± 0.0 s	12.8 ± 1.2 r
	5–10 kDa	20.3 ± 0.7 d	34.9 ± 0.9 ij	372.5 ± 9.3 d
	<5 kDa	18.3 ± 1.3 de	27.8 ± 1.5 kl	392 ± 8.8 c

). Marinades obtained at 2 and 12 °C showed the highest FRAP reduction capacity, 10% higher than using marinades at 7 °C. FRAP activity in meat was mostly from the 5–10 kDa or <5 kDa fraction for herring marinated at 2 or 12 °C, respectively. The ability to inhibit lipid peroxidation in unfractionated brine ISP extracts was 26.6–31.2 mM FeSO₄/100 mL, which was as high as in marinated meat 30.4–34.9 mM FeSO₄/100 g. FRAP activity in the brine was derived almost entirely equally from the two fractions 5–10 and <5 kDa. The fractionation of ISP extracts did not significantly affect the sum of FRAP-reducing capacities, indicating that fractionation does not alter the synergistic action of peptides of marinades and/or the proportion of lysine and sulfur-containing amino acids in peptides of different molecular weights [46].

The antioxidant activity against the ABTS (the TEAC method) ranged from 107 to 136 µM TE/100 g of the marinades; the highest was in marinades ripened at 2 °C, and the lowest at 7 °C (Table 6). Using the TEAC method, activity was mainly formed by the 5–10 kDa fraction for marinades at 2–7 °C, and the <5 kDa fraction for marinades at 12 °C, similar to the FRAP method, but the differences in activity between these fractions were much smaller. In the brine, the antioxidant activity was 34.3–36.6 µM TE/100 mL; however, the sum of the activities of the three fractions was almost two times higher than the activity in the brine before fractionation. This may be due to (i) the release of substances with antioxidant properties, similar to the phenomenon of cathepsin release from lysosomes [5]; (ii) the removal of enzymes with oxidative activity [17], which remained in the >10 kDa fraction and reduced its activity almost completely (Table 6); and/or (iii) an increase in the synergistic effect of individual peptides after purification/fractionation. A synergistic effect, and a 68% increase in antioxidant activity, was also described for the cod hydrolysate and its RP-HPLC fractions [46].

The free-radical-scavenging capacity of the unfractionated ISP extract and its UF fractions were also tested by their ability to scavenge the stable DPPH radical. The DPPH radical scavenging activity in the meat was 251.5–312.7 µM TE/100 g, while in the brine, it was 615.5–679.9 µM TE/100 ml. (Table 6). The highest DPPH activity was using marination at 7 °C for the meat, and 12 °C for the brine. The DPPH activity in the meat consisted mainly of the <5 kDa fraction, while in the brine, in addition to the <5 kDa fraction, the 5–10 kDa fraction contributed significantly, especially as the marinating temperature became higher. Increasing the marinating temperature increased the DPPH activity in the unfractionated ISP extract of the meat more than the activity of the sum of the three UF fractions, while the opposite was the case for the brine.

Despite the methodological differences, similar relationships in activity between methods were found when herring was marinating in reused brine [8]. There was no statistically significant effect of non-protein nitrogen content, peptide and amino acid fractions, or cathepsins activity on antioxidant activity. The >10 kDa fraction contained mainly proteins and had the lowest antioxidant activity in the three in vitro analyses. This confirms that antioxidant activity is mainly possessed by peptides and free amino acids. Moreover, in the brine after the salting of the herring, higher antioxidant activity was shown in the <10 kDa fraction than in the >10 kDa fraction [17]. Similar relationships have been shown for the hydrolysis products of proteins of milk and plants. Girgih et al. [46] showed that the DPPH activity depended on the elution time (25–37 > 14–25 > 0–14 = 37–48 min), and the hydrophobic amino acid content, as more hydrophobic peptides interact with hydrophobic radicals. Similarly, in our study, the highest positive correlation (r² = 0.883) was found between the DPPH activity and the peak area with elution times of 15–35 min. In turn, the ABTS activity in the meat correlated (−0.995, p = 0.02) with the proportion of the peptides fraction 4–10 kDa in the MALDI-Tof assay.

3.7. SEM Analyses

To investigate the effect of the marinating temperature of Atlantic herring on the microstructure of freeze-dried ISP extracts, SEM (200×) micrographs were determined. No cryoprotectants, which might modify the growth of the ice crystals during the freezing process, thus forming a large or small number of small ice crystals, were added to the ISP extracts before the freeze-drying. All the samples were freeze-dried in the same conditions, meaning that all differences that were visible on the SEM micrographs were caused by the marinating temperature, and not by the freeze-drying process. The results of scanning electron microscopy showed that the ISP extracts from meat contained oblong microstructures similar to myofibrils and showed the effect of the marinating temperature on differences in the three-dimensional microstructure. As shown in Figure 3a, the myofibrils ISP-extracted from the marinades at 2 °C were tightly connected, and arranged in parallel, and the spaces between the myofibrils that were observed indicated a well-organized structure of the herring muscle. The SEM image of the ISP extract from the marinades-7 °C demonstrated shorter myofibrils, which formed small aggregates of fibers that began to appear, indicating that the well-organized structure of the muscle tissue was destroyed (Figure 3b). The results of SEM of ISP extract from the marinades at 12 °C showed a loss of myofibril structure, visibly dense aggregates, and a low population of small cavities, unevenly distributed (Figure 3c). It is tempting to suggest that a lower ripening temperature decreased cathepsins activity, which to a lesser degree destroyed the myofibrils’ structure, that can be extracted from meat to water, despite the precipitation process. The ISP technique does not obtain 100% efficiency in protein precipitation, and short myofibrils treated by proteases probably precipitated at a lower degree. The SEM analysis of the ISP extracts from the brine samples indicated that neither myofibril structure was observed. The globular particles and their aggregates (Figure 3d–f) visible on SEM micrographs of the brine samples were larger than the myofibrils visible on the micrographs of the meat samples, suggesting that these globular particles were NaCl particles. The globular proteins extracted from the myofibrils (cut or dissolved) would have been lower than the fibers they were extracted from. Additionally, seven days of marinating in pH 4.3, close to the isoelectric point of herring proteins, probably precipitated most of the proteins diffused to the brine.

4. Conclusions

The marinating temperature between 2 and 12 °C had a significant effect on the ripening of the marinades, and especially on the composition of the meat and the marinating brine. On the one hand, the use of the lowest temperature had advantages, such as the highest marinating yield, the lowest nitrogenous substance loss, the highest cathepsin L activity, the best surface appearance of the marinated fillets, and the highest antioxidant activity of the TEAC. On the other hand, the lowest temperature also had disadvantages, such as the slowest ripening dynamics, the lowest-rated texture of the marinades, the lowest cathepsin B and D activity, and the lowest content of protein hydrolysis products (PHP), especially free amino acids, which give the characteristic flavor of marinades.

The results show that the marinating brine contained high amounts of PHPs, which have strong antioxidant properties in vitro, as evidenced by their strong radical-scavenging and metal-binding activities. The fractionation of PHP released during the marinating of herring, on the basis of molecular weight, had a negative effect, by reducing antioxidant synergy with respect to DPPH in the meat, while it had a positive effect with respect to FRAP, ABTS, and DPPH activity in the brine. This shows that brine PHPs should be industrially fractionated and used separately in food technology. Chromatography and mass spectrometry may serve as tools for monitoring the effect of simultaneous proteolysis, and the extraction of peptides and proteins, from meat to brine. The RP-HPLC results correlate with the antioxidative activity of the extracts.