Preservative Effects of a Gelatin-Based Film, Including Gelidium sp. Flour Extracted from Refrigerated Atlantic Mackerel

Citation: López, L.; Gómez, A.; Trigo, M.; Miranda, J.M.; Barros-Velázquez, J.; Aubourg, S.P. Preservative Effects of a Gelatin-Based Film, Including Gelidium sp. Flour Extracted from Refrigerated Atlantic Mackerel. Appl. Sci. 2024,14, 8817. https://doi.org/ 10.3390/app14198817 Academic Editors: Eliza Kostyra, Anna Piotrowska and Sylwia ˙ Zakowska-Biemans Received: 26 August 2024 Revised: 19 September 2024 Accepted: 24 September 2024 Published: 30 September 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). applied sciences Article Preservative Effects of a Gelatin-Based Film, Including Gelidium sp. Flour Extracted from Refrigerated Atlantic Mackerel Lucía López 1, Antonio Gómez 1, Marcos Trigo 1, JoséM. Miranda 2, Jorge Barros-Velázquez 2 and Santiago P. Aubourg 1,* 1Department of Food Technology, Marine Research Institute (CSIC), 36208 Vigo, Spain; [email protected] (L.L.); [email protected] (A.G.); [email protected] (M.T.) 2 Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, 27002 Lugo, Spain; [email protected] (J.M.M.); jorge.barr[email protected] (J.B.-V.) *Correspondence: saubour[email protected] Abstract: This research evaluated the preservative properties of flour from the alga Gelidium sp., which is a waste substrate resulting from commercial phycocolloid extraction. Gelatin-based biofilms, which included two different concentrations of red alga flour, were developed and used as packaging systems during refrigerated storage (up to 9 days at 4 ◦ C) of Atlantic mackerel (Scomber scombrus) muscle. In all batches tested, a progressive decrease in quality could be observed in the muscle of the fish as the storage time increased. Compared with the control fish, the Gelidium alga flour extract had an inhibitory effect (p< 0.05) on microbial activity (total aerobes, psychrotrophs, and proteolytic bacteria), lipid oxidation (peroxide, thiobarbituric acid, fluorescence and polyene indices), lipid hydrolysis (formation of free fatty acids) and pH increase in refrigerated mackerel muscle. In contrast, no significant effect (p> 0.05) was observed on trimethylamine formation, Enterobacteriaceae, or lipolytic bacteria counts. A preservative effect resulting from the incorporation of Gelidium alga flour into the gelatin-based biofilm was observed, indicating both quality and safety enhancement. In accordance with current global interest in the search for natural and waste sources, a novel and beneficial use of Gelidium flour for enhancing the quality of refrigerated fish has been proposed. Keywords: alga flour; aqueous extract; gelatin film; refrigerated mackerel; preservative effect 1. Introduction Marine species are reported to provide many constituents relevant to the human diet [ 1 , 2 ]. However, seafood deteriorates rapidly postmortem via several biochemical and microbial breakdown mechanisms [ 3 – 5 ]. Reducing the temperature via ice or mechanical refrigeration is the most prevalent method to delay microbial and biochemical spoilage in fish. Despite its widespread use, neither method can fully ensure the preservation of fish quality, particularly during extended storage periods or if the cold chain is not consistently maintained. Therefore, advancements in refrigeration techniques have been developed to address these challenges [6–8]. One such strategy is the use of active packaging films, which can release preservative compounds (such as antimicrobials and antioxidants) into seafood substrates [ 9 – 11 ]. In general, packaging maintains the freshness of seafood and preserves it against adverse agents, such as water vapor, microorganisms, gases, odors, dust, and mechanical shock and vibrations, during distribution and storage [ 12 , 13 ]. Notably, the release of preservative compounds extends the shelf life of packaged seafood [ 14 – 16 ]. The most commonly used packaging materials include synthetic polymers such as lowand high-density polyethylene and polyethylene [ 17 ]. However, adverse health problems (i.e., cancer development and toxicity) resulting from the persistent consumption of synthetic antioxidants (butylhydroxytoluene, butylhydroxyanisole, etc.) have led to the recommended use of natural Appl. Sci. 2024,14, 8817. https://doi.org/10.3390/app14198817 https://www.mdpi.com/journal/applsci Appl. Sci. 2024,14, 8817 2 of 16 antioxidants as alternatives to synthetic antioxidants [ 18 , 19 ]. Therefore, biodegradable and edible materials derived from plants and animals, including peptides, polysaccharides, and lipids, have proven to represent remarkable alternatives [ 20 , 21 ]. The film-forming ability of several polysaccharides, such as cellulose, chitosan, starch, pectin, and alginate, has been revised [ 22 ]. In particular, gelatin from diverse animal sources can be effectively used to develop active packaging strategies [23]. Marine macroalgae contain a wide variety of natural constituents with potential antimicrobial and antioxidant activities and are suitable for application during seafood processing and storage [ 24 – 26 ]. Among macroalgae, red varieties (i.e., Gracilaria sp. and Gelidium sp.) are chiefly recognized for their industrial application in extracting phycocolloids (such as agar, algin, furcellaran, and carrageenan) [ 27 , 28 ], which are utilized to improve the physical properties of edible films in innovative food packaging solutions [ 29 , 30 ]. Agar extraction typically involves initial alkaline pretreatment, followed by water extraction (90 and 120 ◦ C) under pressure [ 31 , 32 ]. This process generates a considerable amount of solid byproducts characterized by a moderate protein content and high polysaccharide content [33]. In the Gelidium genus, different species have been reported to contain antimicrobial and antioxidant components. Thus, a high quantity of flavonoid compounds was detected in both aqueous and ethanolic extracts of G. pusillum (Stackhouse), which was attributed to their antioxidant and antibacterial properties [ 34 ]. Similarly, a G. corneum powder–whey protein isolate was shown to have a remarkable antimicrobial effect on fish paste [ 35 ]. Sulfated polysaccharides from G. pacificum Okamura also had beneficial effects on mice with antibiotic-associated diarrhea [ 36 ]. Moreover, aqueous and ethanol extracts from G. chilense were reported to exert notable inhibitory effects on different bacterial species [ 37 ]. Recently, an aqueous extract of flour obtained from Gelidium sp. demonstrated antimicrobial activity in fish during chilled storage [38] and antioxidant properties in heated fish [39]. The present study focused on the use of the red alga Gelidium sp. flour, a waste substrate resulting from the industrial extraction of phycocolloids. The potential preservative effect of an aqueous extract of this alga flour (AF) was investigated. This flour extract was incorporated into a gelatin-based film, which was employed as a packaging medium during refrigerated storage (4 ◦ C) of Atlantic mackerel (Scomber scombrus) muscle. The evolution of microbial and chemical parameters related to quality loss was determined in fish muscle during a 9-day storage period. 2. Materials and Methods 2.1. Initial Alga Flour Composition and Aqueous Extract Preparation Commercial flour obtained from Gelidium sp. was provided by Industrias Roko S. A. (Llanera, Asturias, Spain). Its proximate composition was analyzed according to the AOAC procedure [ 40 ]. The fatty acid (FA) analysis of the Gelidium flour was carried out in accordance with the methodology presented in Section 2.5. The aqueous extract of the alga flour was obtained in agreement with previous research [ 39 ]. A mixture of 39 g of alga flour and 600 mL of distilled water was stirred (Vortex, Scientific Industries, Bohemia, NY, USA) for 30 s, sonicated (J. P. Selecta, S. A., Barcelona, Spain; 360 W, 50/60 Hz) for 30 s (22–30 ◦ C), and then centrifuged at 3500 × gfor 30 min at 4 ◦ C. The supernatant was collected, and the extraction process was repeated three more times. Finally, all four supernatants were combined and diluted to 3 L with distilled water, resulting in an alga flour concentration of 13 g·L−1. 2.2. Preparation of the Film Systems Teleostean gelatin (Sigma, Life Sciences, Steinheim, Germany) films were obtained by casting them from their film-forming solutions (FFSs) following the methods described by Trigo et al. [ 41 ]. Oxidized sodium alginate (OSA) was prepared according to the method reported by Balakrishnan et al. [ 42 ]. Two different films were prepared to investigate the effects of two different concentrations of the Gelidium flour extract. Appl. Sci. 2024,14, 8817 3 of 16 To prepare the less concentrated films, 250 mL of the Gelidium flour extract was diluted to 1 L with distilled water, and 400 mg of NaOH was added and dissolved by stirring. Then, 950 mL of the resulting solution was mixed with 100 g of gelatin (i.e., 23.75 mL flour extract · g −1 gelatin), and the gelatin was completely dissolved by soft heating (ca. 40 ◦ C for 120 min). OSA (5 g · 50 mL −1 Gelidium extract; 5% gelatin) was then added into the FFSs as a crosslinking agent. After 20 min of stirring, 30 g of glycerol was added to act as a plasticizer. The resulting suspension was stirred for 20 min, and then the FFSs were poured onto Teflon-coated trays and dried at 50 ◦ C in a convection oven for 48 h. The films were then conditioned for 48 h in a chamber at 4 ± 1 ◦ C before use. This resulting film was designated the AF-1 packaging film. To prepare the most concentrated packaging film, a similar procedure was followed but starting from 1 L of the initial Gelidium extract. The resulting film (i.e., 95.00 mL flour extract · g −1 gelatin) and subsequent batch were referred to as the AF-2 condition. A control (CTR) gelatin film without flour extract was prepared in the same way as the AF-1 and AF-2 packaging films and referred to as the CTR batch. The selection of Gelidium extracts for this work was according to previous trials conducted in our laboratory. As a result, the AF-2 film contained the highest flour concentration, which did not affect the sensory or external characteristics of the fish muscle parts (such as odor and color). Therefore, this concentration, along with a less concentrated one, was considered. All solvents and chemical reagents used in this study were of reagent grade (Merck, Darmstadt, Germany); otherwise, the source is mentioned. 2.3. Fish Processing and Sampling Thirty-three fresh Atlantic mackerel (Scomber scombrus) samples were fished in April 2024 near the Galician Atlantic coast (northwestern Spain), purchased from Vigo (Spain) harbor and transported on ice. After arrival to the laboratory, samples were measured and weighed, ranging between 33–37 cm in length and between 375–435 g in weight. Subsequently, six of the fish were analyzed as an initial sample (day 0). From among these 6 specimens, 3 different groups were taken, each including 2 fish, each of which was sampled and analyzed for dorsal white muscle (three replicates; n= 3). The remaining 27 fish samples were divided into three batches, including 9 samples per batch. From each batch, we took pieces of approximately 35 g each. Three pieces of back muscle were packaged from each sample. These pieces were then vacuum-sealed individually via a Vacuum Packaging Machine Culinary (Albipack, Águeda, Portugal) in the three specified packaging systems (CTR, AF-1, and AF-2 batches), resulting in 27 fish pieces per packaging condition. The packaged fish pieces were stored in a refrigerated room at 4 ◦ C for 9 days. Sampling and analyses were conducted on days 2, 6, and 9 of storage. These sampling times were considered appropriate in order to follow the quality evolution of the present fish species during refrigerated storage. On each of the sampling days, 9 pieces of packaged fish were extracted from each batch for subsequent analysis. These 9 pieces of fish were then divided into three groups (three pieces of packaged fish per group); white muscle was analyzed independently in each group (three replicates; n= 3). 2.4. Microbiological Analyses of Fish Muscle Ten-gram samples of fish muscle were aseptically collected and mixed with 90 mL of 0.1% peptone water. This mixture was homogenized in sterilized stomacher bags (AES, Combourg, France) as described elsewhere [ 43 , 44 ]. Subsequently, dilutions from the microbial suspensions were prepared in 0.1% peptone water. Total aerobic bacteria were investigated on plate count agar (PCA) (Oxoid Ltd., London, UK) after 48 h of incubation at 30 ◦ C. The investigation of psychrotrophs was also conducted via PCA, but the incubation period was extended to 7 days at 7–8 ◦ C. Enterobacteriaceae were determined using Violet Red Bile Agar (VRBA) (Merck, Darmstadt, Germany) after 24 h of incubation at 37 ◦ C. Specific spoilage microorganisms capable of Appl. Sci. 2024,14, 8817 4 of 16 breaking down proteins or lipids were also investigated. Micro-organisms producing extracellular proteases or lipases were detected via casein agar or tributyrin agar, respectively, after 48 h of incubation at 30 ◦C [45]. The bacterial counts were transformed into log CFU · g −1 before statistical analysis. All analyses were performed in triplicate. 2.5. Chemical Analyses Related to Quality Loss The pH values of the mackerel muscle were monitored throughout the storage period via a 6 mm diameter insertion electrode (Crison, Barcelona, Spain). Trimethylamine (TMA) formation was measured via the picrate spectrophotometric method (410 nm) (Beckman Coulter, DU 640; London, UK), as described by Tozawa et al. [ 46 ]. This involved the preparation of a 5% trichloroacetic acid extract from fish white muscle (10 g in 25 mL), with the results expressed as mg TMA-N·kg−1muscle. Lipids were extracted from mackerel white muscle via the Bligh and Dyer [ 47 ] method, which involves single-phase solubilization with a chloroform–methanol (1:1) mixture. The results were calculated as g lipid · kg −1 muscle. Lipid quantification followed the Herbes and Allen [48] method, with the lipid content expressed as g·kg−1muscle. Free fatty acid (FFA) content was determined from the lipid extract of fish muscle via the Lowry and Tinsley [ 49 ] method on the basis of complex formation with cupric acetate–pyridine and subsequent spectrophotometric assessment at 715 nm. The results are expressed as g FFAs·kg−1lipids. The peroxide value (PV) of the lipid extract was measured spectrophotometrically at 520 nm, following the method described in previous research [ 50 ]. The results are expressed as meq. active oxygen·kg−1lipids. The thiobarbituric acid (TBA) index (TBA-i) was determined following the methodology described by Vyncke [ 51 ]. The content of TBA-reactive substances (TBARSs) was determined by spectrophotometry at 532 nm, and the results obtained were expressed as mg malondialdehyde·kg−1muscle. The formation of fluorescent compounds was determined in the lipid extract obtained from fish muscle using an LS 45 fluorimeter (Perkin Elmer España; Tres Cantos, Madrid, Spain), proceeding to its reading at wavelengths of 393/463 nm and 327/415 nm, as described in a previous work [ 52 ]. Relative fluorescence (RF) was determined following the formula: RF = F/F st , where F is defined as the fluorescence measured at each excitation/emission wavelength pair, and F st represents the fluorescence intensity of a quinine sulfate solution (1 µ g-mL −1 in 0.05 M H 2 SO 4 ) at the corresponding wavelength pair. The results obtained were presented as the fluorescence ratio (FR), which was calculated from the ratio between the two RF values: FR = RF393/463 nm/RF327/415 nm. To determine the lipid profile of the samples, FA methyl esters (FAMEs) were previously obtained from the lipid extracts using acetyl chloride in methanol. Subsequently, the FAMEs obtained were analyzed by gas chromatography (Perkin–Elmer 8700 chromatograph, Madrid, Spain) [ 53 ]. An SP-2330-fused silica capillary column (0.25 mm i.d. × 30 m, Supelco, Inc., Bellefonte, PA, USA) was used for these determinations, using temperature adjusted to increase from 145 ◦ C to 190 ◦ C at 1.0 ◦ C min −1 and from 190 ◦ C to 210 ◦ C at 5.0 ◦ C min −1 , maintained at 210 ◦ C for 13.5 min. Nitrogen was used as a carrier gas at 10 psig, and detection was performed with a flame ionization detector at 250 ◦ C. A programmed temperature vaporizing injector was used in split mode (150:1) and heated from 45 ◦C to 275 ◦C, increasing 15 ◦C min−1. Peaks corresponding to FAMEs were identified by means of comparing their retention times with those of standard mixtures (Qualmix Fish and Supelco 37 Component FAME Mix, Supelco, Inc., Bellefonte, PA, USA). The peak areas were automatically integrated, using C19:0 as an internal standard for quantitative purposes. The content of each FA was calculated as g FA · 100 g −1 total FAs. The polyene index (PI) was calculated as the FA ratio: C20:5ω3 + C22:6ω3 to C16:0. Appl. Sci. 2024,14, 8817 5 of 16 2.6. Statistical Analysis The study was carried out in triplicate (n= 3). Data from all microbiological and chemical analyses were subjected to ANOVA to examine differences due to the packaging system and refrigeration time. Special attention was given to the comparison of treated fish batches with respect to the control batch. Mean comparisons were performed via the least-squares difference (LSD) method. All statistical comparisons were conducted via PASW Statistics 18 software for Windows (SPSS Inc., Chicago, IL, USA), with differences considered significant at the 95% confidence level (p< 0.05). 3. Results 3.1. Alga Flour Composition The alga flour exhibited the following proximate composition (%): 12.2 (moisture), 31.5 (protein), 0.2 (lipids), 14.3 (ash), and 42.8 (total carbohydrate). The following compositions for individual FAs were observed (g · 100 g −1 total FAs): 6.69 ± 0.08 (C14:0, myristic acid), 0.96 ± 0.02 (C15:0, pentadecanoic acid), 66.85 ± 0.45 (C16:0, palmitic acid), 2.43 ± 0.04 (C16:1 ω 7, palmitoleic acid), 0.60 ± 0.01 (C17:0, margaric acid), 3.41 ± 0.07 (C18:0, stearic acid), 7.82 ± 0.09 (C18:1 ω 9, oleic acid), 1.88 ± 0.03 (C18:1 ω 7, vaccenic acid), 0.62 ± 0.05 (C18:2 ω 6, linoleic acid), 0.47 ± 0.03 (C20:1 ω 9, gondoic acid), 0.15 ±0.04 (C20:2 ω 6, eicosadienoic acid), 2.91 ± 0.16 (C20:4 ω 6, araquidonic acid), 0.20 ±0.02 (C22:1ω9, erucic acid). 3.2. Evaluation of Microbial Growth in Atlantic Mackerel Muscle under Different Packaging Systems The evolution of microbial development in fish corresponding to the AF-treated (AF-1 and AF-2 for low and high concentrations of AF) and CTR batches is depicted in Table 1. Table 1. Determination of Enterobacteriaceae, psychrotrophs, and lipolytic bacterial counts (log CFU·g−1muscle) * in refrigerated mackerel subjected to different packaging conditions **. Microbial Group Packaging Condition Refrigeration Time (Days) 0 2 6 9 Enterobacteriaceae CTR 1.00 ±0.00 a 1.86 ±0.52 Ab 2.33 ±0.35 Ab 4.21 ±0.65 Ac AF-1 1.00 ±0.00 a 1.53 ±0.20 Ab 1.73 ±0.88 Ab 3.23 ±0.35 Ac AF-2 1.00 ±0.00 a 1.75 ±0.64 Ab 2.28 ±0.87 Ab 3.73 ±0.41 Ac Psychrotrophs CTR 2.82 ±0.24 a 4.68 ±0.79 Bb 6.32 ±0.32 Bc 8.78 ±0.15 Bd AF-1 2.82 ±0.24 a 2.93 ±0.35 Aa 5.16 ±0.32 Ab 7.83 ±0.30 Ac AF-2 2.82 ±0.24 a 4.68 ±0.39 Bb 6.44 ±0.15 Bc 7.54 ±0.55 Ad Lipolytic bacteria CTR 2.00 ±0.00 a 2.26 ±0.24 Aa 3.13 ±0.41 Ab 4.95 ±1.09 Ac AF-1 2.00 ±0.00 a 2.66 ±0.58 Aab 2.43 ±0.51 Aab 3.64 ±1.05 Ab AF-2 2.00 ±0.00 a 2.16 ±0.28 Aab 3.00 ±0.89 Abc 3.63 ±0.46 Ac * Average values ± standard deviations (n= 3). In each row, different lowercase letters denote significant differences (p< 0.05) with refrigeration time; in each column, different capital letters denote significant differences (p< 0.05) as a result of the packaging conditions. ** Packaging conditions: CTR (Control; gelatin films prepared without alga flour extract); AF-1 and AF-2 correspond to low and high concentrations of alga flour extracts, respectively, in gelatin films. In terms of Enterobacteriaceae, the two batches containing algal extracts presented lower average values than did the gelatin CTR batch. Thus, the presence of the algal extract in the packaging films led to lower average values at all the sampling times. Remarkably, the AF-1 batch presented lower average values than its counterpart, the AF-2 batch. These results indicate that the incorporation of algal extracts into the gelatin films resulted in Enterobacteriaceae numbers nearly ten times lower in the AF-1 batch than in the pure gelatin CTR batch. Appl. Sci. 2024,14, 8817 6 of 16 Table 1shows the evolution of psychrotroph counts in all three batches during refrigerated storage. Similar to the results observed for Enterobacteriaceae, the AF-1 batch, which included a lower concentration of alga extract, provided better microbial control than the other two batches did. Thus, the AF-1 batch presented significantly (p< 0.05) lower pyschrotroph counts than did the two other batches at intermediate storage times (days 2 and 6). However, at the most advanced storage time, all three batches presented microbial numbers above seven log units, indicating that the protection exerted by the bioactive compounds present in the alga extracts concerning the development of psychrotrophic bacteria was not as relevant on day 9. The comparative evolution of total aerobes in all three batches was also evaluated in this work, and the obtained results are shown in Figure 1. Similar to the effects observed for other microbial groups, the incorporation of algal extracts into the gelatin films was associated with slower microbial growth than the CTR batch. This effect was observed at early (day 2) and advanced (day 9) storage times, and the greatest differences between the alga-treated and CTR batches were close to 1 log unit. Remarkably, these differences were statistically significant (p< 0.05). Appl. Sci. 2023, 13, x FOR PEER REVIEW 7 of 17 Figure 1. Determination of aerobic counts (log CFU·g−1 muscle) in refrigerated mackerel subjected to different packaging conditions. Average values ± standard deviations (n = 3). The packaging conditions are expressed in Table 1. Different lowercase letters denote significant differences (p < 0.05) with chilling time; different capital letters denote significant differences (p < 0.05) as a result of the packaging condition. Figure 2. Determination of proteolytic counts (log CFU·g−1 muscle) in refrigerated mackerel subjected to different packaging conditions. Average values ± standard deviations (n = 3). The packaging conditions are expressed in Table 1. Different lowercase letters denote significant differences (p < 0.05) with chilling time; different capital letters denote significant differences (p < 0.05) as a result of the packaging condition. 3.3. Evolution of the pH and TMA Values A progressive increase (p < 0.05) in the pH value was detected in all batches as the storage time increased (Table 2). An increase in pH was derived from the presence of the Figure 1. Determination of aerobic counts (log CFU · g −1 muscle) in refrigerated mackerel subjected to different packaging conditions. Average values ± standard deviations (n= 3). The packaging conditions are expressed in Table 1. Different lowercase letters denote significant differences (p< 0.05) with chilling time; different capital letters denote significant differences (p< 0.05) as a result of the packaging condition. Time 0 corresponds to initial fish. The results obtained in this study concerning the ability of lipolytic bacteria to produce extracellular lipases with activity against triacylglycerols (TAGs) and phospholipids (PLs) in fish muscle are also displayed in Table 1. Thus, the incorporation of algal extracts into the gelatin packaging film led to restrictions in the growth of lipolytic bacteria during storage compared to the CTR batch. However, although the greatest difference among batches was 1.30 log units on day 9 (AF-1 batch with respect to the CTR batch), such differences were not statistically significant (p> 0.05). Similar results were observed at intermediate storage times (day 6). These results indicate that the incorporation of algal extracts into the gelatin films exerted slight microbial control over lipolytic bacteria, but the effect was not very intense. Appl. Sci. 2024,14, 8817 7 of 16 Figure 2shows the results of the comparative evolution of proteolytic bacteria in Atlantic mackerel muscle in all three batches. As with other microbial groups, the incorporation of the bioactive alga extracts into the gelatin films was associated with better control of proteolytic bacteria, which was especially relevant in the AF-1 batch compared with the CTR batch. Accordingly, the results indicated statistically significant (p< 0.05) differences derived from the more limited growth of proteolytic bacteria, especially in the AF-1 batch. These differences were especially relevant at intermediate (day 6) and advanced (day 9) storage times, reaching a maximum of 0.92 log units on day 6. Appl. Sci. 2023, 13, x FOR PEER REVIEW 7 of 17 Figure 1. Determination of aerobic counts (log CFU·g−1 muscle) in refrigerated mackerel subjected to different packaging conditions. Average values ± standard deviations (n = 3). The packaging conditions are expressed in Table 1. Different lowercase letters denote significant differences (p < 0.05) with chilling time; different capital letters denote significant differences (p < 0.05) as a result of the packaging condition. Figure 2. Determination of proteolytic counts (log CFU·g−1 muscle) in refrigerated mackerel subjected to different packaging conditions. Average values ± standard deviations (n = 3). The packaging conditions are expressed in Table 1. Different lowercase letters denote significant differences (p < 0.05) with chilling time; different capital letters denote significant differences (p < 0.05) as a result of the packaging condition. 3.3. Evolution of the pH and TMA Values A progressive increase (p < 0.05) in the pH value was detected in all batches as the storage time increased (Table 2). An increase in pH was derived from the presence of the Figure 2. Determination of proteolytic counts (log CFU · g −1 muscle) in refrigerated mackerel subjected to different packaging conditions. Average values ± standard deviations (n= 3). The packaging conditions are expressed in Table 1. Different lowercase letters denote significant differences (p< 0.05) with chilling time; different capital letters denote significant differences (p< 0.05) as a result of the packaging condition. Time 0 corresponds to initial fish. 3.3. Evolution of the pH and TMA Values A progressive increase (p< 0.05) in the pH value was detected in all batches as the storage time increased (Table 2). An increase in pH was derived from the presence of the alga extract in the packaging medium. Thus, higher average pH values were detected in samples corresponding to the CTR batch than in their fish counterparts subjected to the preservative extract; the differences were found to be significant (p< 0.05) on day 2 (AF-2 batch) and on day 9 (AF-1 batch). With respect to TMA formation, an increase (p< 0.05) with storage time was observed in all three batches (Table 2). This increase was remarkably high for the 6–9-day period. The presence of the flour extract in the packaging medium did not lead to significant differences (p> 0.05); however, lower average values were detected at the end of the storage time in the fish samples corresponding to batches, including the flour extract (AF-1 and AF-2 batches). Appl. Sci. 2024,14, 8817 8 of 16 Table 2. Evolution of the pH and trimethylamine (TMA; mg TMA-N · kg −1 muscle) values * in refrigerated mackerel subjected to different packaging conditions **. Quality Index Packaging Condition Refrigeration Time (Days) 0 2 6 9 pH CTR 6.25 ±0.02 a 6.51 ±0.02 Bb 6.58 ±0.15 Ab 6.59 ±0.02 Bb AF-1 6.25 ±0.02 a 6.39 ±0.10 ABab 6.40 ±0.16 Aab 6.42 ±0.08 Ab AF-2 6.25 ±0.02 a 6.29 ±0.08 Aa 6.44 ±0.11 Aab 6.50 ±0.03 ABb TMA CTR 1.60 ±0.42 a 1.87 ±0.47 Aa 12.80 ±1.7 Ab 61.50 ±15.7 Ac AF-1 1.60 ±0.42 a 2.60 ±0.78 Aa 11.57 ±2.3 Ab 52.67 ±23.6 Ac AF-2 1.60 ±0.42 a 2.80 ±0.79 Aa 11.00 ±4.6 Ab 50.20 ±13.1 Ac * Average values ± standard deviations (n= 3). In each row, different lowercase letters denote significant differences (p< 0.05) with refrigeration time; in each column, different capital letters denote significant differences (p< 0.05) as a result of the packaging conditions. ** Packaging conditions as expressed in Table 1. 3.4. Assessment of Lipid Hydrolysis Development Lipid hydrolysis was assessed via the FFA value (Figure 3). The formation of this catabolic end product significantly increased (p< 0.05) over the storage period across all batches. An inhibitory effect (p< 0.05) on FFA formation was noted at the end of the storage period in the fish samples, where the alga flour extract was included in the packaging medium. However, no significant differences (p> 0.05) were detected between batches containing the alga flour. Appl. Sci. 2023, 13, x FOR PEER REVIEW 9 of 17 Figure 3. Determination of the free fatty acid (FFA; g·kg−1 lipids) content in refrigerated mackerel subjected to different packaging conditions. Average values ± standard deviations (n = 3). The packaging conditions are expressed in Table 1. Different lowercase letters denote significant differences (p < 0.05) with chilling time; different capital letters denote significant differences (p < 0.05) as a result of the packaging condition. 3.5. Determination of Lipid Oxidation Evolution The progression of lipid oxidation was evaluated by analyzing the formation of primary (PV), secondary (TBA-i), and tertiary (FR) compounds, as well as by assessing the PI. Special attention was given to comparisons between the treated and CTR batches. Peroxide formation in this study was minimal [38,54]. Across all batches, the peroxide content ranged from 0.68 to 5.03 meq·kg−1 lipids throughout the study (Table 3). No significant trend with storage time was observed (p > 0.05), although the highest average values were recorded at the end of the storage period in all batches. An inhibitory effect (p < 0.05) on peroxide formation was noted on day 9 due to the presence of flour extract in the packaging medium; this effect was more pronounced (p < 0.05) with higher concentrations of the alga extract. Table 3. Determination of lipid oxidation evolution * in refrigerated mackerel subjected to different packaging conditions **. Quality Index *** Packaging Condition Refrigeration Time (Days) 0 2 6 9 PV CTR 0.98 ± 0.47 a 1.24 ± 0.43 Aa 3.05 ± 1.70 Aab 5.03 ± 1.61 Cb AF-1 0.98 ± 0.47 a 0.72 ± 0.35 Aa 2.11 ± 1.66 Aab 2.27 ± 0.15 Bb AF-2 0.98 ± 0.47 ab 1.57 ± 0.44 Aab 0.68 ± 0.46 Aa 1.60 ± 0.33 Ab TBA-i CTR 0.42 ± 0.12 a 0.91 ± 0.17 Ab 2.86 ± 1.07 Ac 4.77 ± 0.76 Bd AF-1 0.42 ± 0.12 a 1.13 ± 0.45 Ab 3.25 ± 1.39 Ac 3.10 ± 1.77 ABc AF-2 0.42 ± 0.12 a 1.18 ± 0.42 Ab 1.84 ± 0.42 Ab 1.77 ± 0.27Ab FR CTR 3.69 ± 1.56 a 4.85 ± 0.77 Aa 6.39 ± 0.65 Ab 7.22 ± 0.29 Cc AF-1 3.69 ± 1.56 a 4.89 ± 0.17 Aa 5.40 ± 0.61 Aab 6.16 ± 0.58 Bb AF-2 3.69 ± 1.56 a 4.44 ± 0.94 Aa 5.84 ± 1.32 Aa 4.86 ± 0.16 Aa PI CTR 2.27 ± 0.16 a 2.04 ± 0.23 Aa 1.97 ± 0.21 Aa 1.88 ± 0.47 Aa AF-1 2.27 ± 0.16 a 2.20 ± 0.16 ABa 1.98 ± 0.10 Aa 1.94 ± 0.53 Aa Figure 3. Determination of the free fatty acid (FFA; g · kg −1 lipids) content in refrigerated mackerel subjected to different packaging conditions. Average values ± standard deviations (n= 3). The packaging conditions are expressed in Table 1. Different lowercase letters denote significant differences (p< 0.05) with chilling time; different capital letters denote significant differences (p< 0.05) as a result of the packaging condition. Time 0 corresponds to initial fish. 3.5. Determination of Lipid Oxidation Evolution The progression of lipid oxidation was evaluated by analyzing the formation of primary (PV), secondary (TBA-i), and tertiary (FR) compounds, as well as by assessing the PI. Special attention was given to comparisons between the treated and CTR batches. Appl. Sci. 2024,14, 8817 9 of 16 Peroxide formation in this study was minimal [ 38 , 54 ]. Across all batches, the peroxide content ranged from 0.68 to 5.03 meq · kg −1 lipids throughout the study (Table 3). No significant trend with storage time was observed (p> 0.05), although the highest average values were recorded at the end of the storage period in all batches. An inhibitory effect (p< 0.05) on peroxide formation was noted on day 9 due to the presence of flour extract in the packaging medium; this effect was more pronounced (p< 0.05) with higher concentrations of the alga extract. Table 3. Determination of lipid oxidation evolution * in refrigerated mackerel subjected to different packaging conditions **. Quality Index *** Packaging Condition Refrigeration Time (Days) 0 2 6 9 PV CTR 0.98 ±0.47 a 1.24 ±0.43 Aa 3.05 ±1.70 Aab 5.03 ±1.61 Cb AF-1 0.98 ±0.47 a 0.72 ±0.35 Aa 2.11 ±1.66 Aab 2.27 ±0.15 Bb AF-2 0.98 ±0.47 ab 1.57 ±0.44 Aab 0.68 ±0.46 Aa 1.60 ±0.33 Ab TBA-i CTR 0.42 ±0.12 a 0.91 ±0.17 Ab 2.86 ±1.07 Ac 4.77 ±0.76 Bd AF-1 0.42 ±0.12 a 1.13 ±0.45 Ab 3.25 ±1.39 Ac 3.10 ±1.77 ABc AF-2 0.42 ±0.12 a 1.18 ±0.42 Ab 1.84 ±0.42 Ab 1.77 ±0.27Ab FR CTR 3.69 ±1.56 a 4.85 ±0.77 Aa 6.39 ±0.65 Ab 7.22 ±0.29 Cc AF-1 3.69 ±1.56 a 4.89 ±0.17 Aa 5.40 ±0.61 Aab 6.16 ±0.58 Bb AF-2 3.69 ±1.56 a 4.44 ±0.94 Aa 5.84 ±1.32 Aa 4.86 ±0.16 Aa PI CTR 2.27 ±0.16 a 2.04 ±0.23 Aa 1.97 ±0.21 Aa 1.88 ±0.47 Aa AF-1 2.27 ±0.16 a 2.20 ±0.16 ABa 1.98 ±0.10 Aa 1.94 ±0.53 Aa AF-2 2.27 ±0.16 a 2.40 ±0.10 Ba 2.25 ±0.02 Ba 2.14 ±0.12 Aa * Average values ± standard deviations (n= 3). In each row, different lowercase letters denote significant differences (p< 0.05) with refrigeration time; in each column, different capital letters denote significant differences (p< 0.05) as a result of the packaging conditions. ** Packaging conditions as expressed in Table 1. *** Abbreviations and units: PV (peroxide value; miliequivalents · kg −1 lipids), TBA-i (thiobarbituric acid index; mg malondialdehyde·kg−1muscle), FR (fluorescence ratio) and PI (polyene index). A significant increase (p< 0.05) in TBARS formation was observed in all batches during the 0–6-day period (Table 3). However, by the end of the storage period, a decrease in the average value was noted in samples from batches containing the alga extract. On day 9, an inhibitory effect (p< 0.05) on TBARS formation was observed in the AF-2 batch compared with the CTR batch. A significant increase (p< 0.05) in FR values with storage time was detected in all batches (Table 3). Additionally, an inhibitory effect (p< 0.05) on fluorescent compound formation was observed on day 9 due to the inclusion of alga flour extract in the packaging medium (AF-1 and AF-2 batches). This effect was more pronounced (p< 0.05) with higher concentrations of the alga extract. A progressive decrease in the PI was detected in most cases due to refrigerated storage (Table 3). Throughout the refrigeration period, higher average values were observed in the fish samples treated with the flour extract. Significant differences (p< 0.05) were found after 2 and 6 days when the high-concentration (AF-2) packaging conditions were considered. 4. Discussion 4.1. Antimicrobial Activity The results of microbiological analyses revealed that incorporating bioactive algal extracts into gelatin packaging films inhibited the growth of all five microbial groups investigated in Atlantic mackerel stored for 9 d under refrigeration conditions. 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