Preservative Effect of Aqueous and Ethanolic Extracts of the Macroalga Bifurcaria bifurcata on the Quality of Chilled Hake (Merluccius merluccius)

molecules Article Preservative Effect of Aqueous and Ethanolic Extracts of the Macroalga Bifurcaria bifurcata on the Quality of Chilled Hake (Merluccius merluccius) JoséM. Miranda 1, Bin Zhang 2, Jorge Barros-Velázquez 1and Santiago P. Aubourg 3,*   Citation: Miranda, J.M.; Zhang, B.; Barros-Velázquez, J.; Aubourg, S.P. Preservative Effect of Aqueous and Ethanolic Extracts of the Macroalga Bifurcaria bifurcata on the Quality of Chilled Hake (Merluccius merluccius). Molecules 2021,26, 3774. https:// doi.org/10.3390/molecules26123774 Academic Editor: Fátima Paiva-Martins Received: 21 May 2021 Accepted: 17 June 2021 Published: 21 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 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/). 1 Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary Sciences, University of Santiago de Compostela, Avenida Carvallo Calero, s/n, 27002 Lugo, Spain; [email protected] (J.M.M.); jorge.barr[email protected] (J.B.-V.) 2Key Laboratory of Health Risk Factors for Seafood of Zhejiang Province, College of Food Science and Pharmacy, Zhejiang Ocean University, No 1, Haida South Road, 1, Lincheng Changzhi, Zhoushan 316022, China; [email protected] 3Department of Food Science and Technology, Marine Research Institute (CSIC), c/E. Cabello 6, 36208 Vigo, Spain *Correspondence: saubour[email protected]; Tel.: +34-986-231930 Abstract: This work addressed the preservative behaviour of different icing media containing extracts from the alga Bifurcaria bifurcata. A comparative study of the antimicrobial and antioxidant effects of aqueous and ethanolic extracts of this macroalga was carried out. Whole hake (Merluccius merluccius) pieces were stored in ice containing either kind of extract and analysed for quality changes throughout a 13-day storage period. A progressive loss of microbial and biochemical quality was detected in all batches as chilling time increased. A significant inhibitory effect (p< 0.05) on microbial activity could be observed as a result of including the aqueous (lowering of psychrotrophic and lipolytic counts and pH value) and ethanolic (lowering of psychrotrophic and lipolytic counts) extracts. Additionally, both kinds of extract led to a substantial inhibition (p< 0.05) in the lipid hydrolysis rate (formation of free fatty acids), greater in the case of the batch containing ethanolic extract. Concerning lipid oxidation, a similar inhibitory effect (p< 0.05) on the formation of secondary compounds (thiobarbituric acid substances) was noticed in fish specimens corresponding to both alga extracts; however, more (p< 0.05) peroxide formation was detected in fish corresponding to the ethanolic extract batch. A preservative effect can be concluded for both kinds of extract; this effect agrees with previous studies reporting the presence of hydrophilic and lipophilic bioactive compounds in B. bifurcata. Keywords: refrigerated hake; Bifurcaria bifurcata; aqueous and ethanolic extracts; microbial activity; lipid damage; quality enhancement 1. Introduction In Asian countries the consumption of seaweeds or macroalgae dates back to ancient times. Interestingly, their consumption has increased in recent years in Western countries due to the search for new sustainable sources of healthy food and natural products [ 1 ]. Seaweeds have been shown to be a relevant source of beneficial constituents such as lipids, vitamins, trace minerals, dietary fibre, and amino acids [ 2 , 3 ]. Additionally, seaweeds have attracted great attention because they contain a profitable variety of chemical components with potential antimicrobial [4,5] and antioxidant [6,7] activity. Icing is the most common method adopted for fish preservation. However, due to the limited shelf life of marine species, it has been applied in combination with other preservation strategies such as slurry ice [ 8 ], ozone [ 9 ], packaging [ 10 ], irradiation [ 11 ], high pressure [ 12 ], and the addition of natural compounds [ 13 ]. Recently, novel icing media incorporating extracts obtained from natural sources, such as organic acids [ 14 , 15 ] or plant extracts [ 16 , 17 ] with a proven preservative effect, have been used with a view to enhancing Molecules 2021,26, 3774. https://doi.org/10.3390/molecules26123774 https://www.mdpi.com/journal/molecules Molecules 2021,26, 3774 2 of 14 the shelf life of fish. Based on different studies, it has been observed that the incorporation of such natural preservatives in the icing medium delays fish spoilage and enhances fish quality due to their antimicrobial and antioxidant properties. Among the recently proposed natural sources, seaweed extracts provide an interesting possibility to enhance seafood quality. According to the European Council Regulation [ 18 ], algae are considered food or food ingredients, so their use in food technology should not constitute any hazard to human health. Thus, different kinds of seaweed extract have been shown to enhance fish quality and increase fish shelf life when included in the icing [ 19 ], glazing [ 20 ], and canning [ 21 ] medium, or as a dipping treatment prior to subsequent chilled storage [ 22 ]. Previous research has shown that the extract yield and the preservative compound content obtained from algae can be influenced by the extraction procedure [ 23 , 24 ]. Thus, alcoholic solvents are better for obtaining a higher level of phenolic compounds [ 6 , 25 ], while water extraction is especially convenient for obtaining high yields of active polysaccharides, proteins, and peptides [ 26 , 27 ]. Studies concerning the comparative effects of aqueous and alcoholic seaweed extracts on seafood quality are scarce. Thus, Barros-Velázquez et al. [ 28 ] studied the storage of hake (Merluccius merluccius) in an icing medium containing aqueous and ethanolic extracts of Fucus spiralis. They observed a remarkable antimicrobial and antioxidant effect in fish corresponding to the ethanolic extract ice, while aqueous extracts led to a negligible preservative effect. It was concluded that further comparative studies of the effects of both kinds of extracts on seafood would be necessary. Among brown seaweeds, Bifurcaria bifurcata represents an interesting choice on the basis of its great availability in the Atlantic coast of France, Spain, Portugal, Ireland, and the United Kingdom [ 29 , 30 ]. Interestingly, previous studies have shown the presence of hydrophilic (phlorotannins, phenolic acids, flavonoids, alginates, polisaccharides in general) [ 31 – 33 ] and lipophilic (polyphenols and sterols) [ 34 – 36 ] preservative compounds in B. bifurcata. This work addresses the comparative preserving effects of aqueous and ethanolic extracts of this macroalga. For it, icing media containing either kind of alga extract were prepared and applied to whole hake (M. merluccius) specimens that were stored for 13 days. The evolution of microbial and chemical quality indices was comparatively analyzed throughout chilled storage. 2. Results and Discussion 2.1. Evolution of Microbial Development during Chilled Storage of Hake The results of the microbiological analysis for all three batches during refrigerated storage are presented in Table 1and Figure 1. With respect to the aerobe count, a widely used index of microbial quality, the results indicated a slight protective effect of the aqueous alga extract on fish quality. Thus, and although differences among batches were not significant (p> 0.05), the batch containing the aqueous extract exhibited better control (i.e., lower average counts) of aerobe growth at medium and advanced storage times (Table 1). Remarkably, none of the three batches reached 7 log CFU · g −1 , generally considered as a limit of acceptability for seafood products [37]. With respect to psychrotrophs, the control batch surpassed 6 log CFU · g −1 on day 9, while the counterpart batches containing alga extracts were below that value at that time (Table 1). Remarkably, the inclusion of alga extract, either aqueous (AQ batch) or ethanolic (ET batch), provided a slight protection of fish muscle with respect to psychrotrophs’ growth for the 6–13-day period (i.e., lower average counts), although this effect was found to be statistically significant (p< 0.05) only at advanced storage times (day 13). Table 1also provides the comparative analysis of Enterobacteriaceae growth in all three batches. The presence of this bacterial group was very limited in all three batches, with microbial counts being always below 2 log CFU·g−1(Table 1). Molecules 2021,26, 3774 3 of 14 Table 1. Evolution of microbial development (log CFU · g −1 ) * in chilled hake subjected to different icing conditions **. Microbial Group Chilling Time (Days) Icing Condition CT AQ ET Aerobes 02.52 A (0.24) 2.52 A (0.24) 2.52 A (0.24) 22.35 aA (0.37) 2.77 aAB (0.81) 2.20 aA (0.17) 63.92 aB (0.15) 3.71 aB (0.74) 4.04 aB (0.52) 95.28 abC (0.51) 5.09 aC( (0.46) 5.95 bC (0.32) 13 5.71 aC (0.59) 5.44 aC (0.25) 5.61 aC (0.48) Psychrotrophs 03.38 A (0.41) 3.38 A (0.41) 3.38 B (0.41) 23.20 bA (0.13) 3.56 bA (0.26) 2.20 aA (0.35) 65.44 aB (0.24) 5.34 aB (0.03) 5.40 aC (0.28) 96.30 aC (0.45) 5.91 aC (0.34) 5.97 aC (0.47) 13 7.81 bD (0.35) 6.80 aD (0.23) 6.97 aD (0.46) Enterobacteriaceae *** 0 1 (0.0) 1 (0.0) 1 (0.0) 2 1 (0.0) 1 (0.0) 1 (0.0) 6 1 (0.0) 1 (0.0) 1 (0.0) 9 1 (0.0) 1.10 (0.17) 1.66 (0.58) 13 1.36 (0.32) 1 (0.0) 1 (0.0) Proteolytics 02.40 A 0.46) 2.40 A (0.46) 2.40 A (0.46) 22.20 aA 0.17) 2.42 aA (0.39) 2.00 aA (0.33) 63.44 aB (0.10) 3.32 aB (0.15) 3.55 aB (0.33) 95.29 aC (0.08) 5.27 abC (0.49) 5.64 bC (0.03) 13 6.78 aD (0.47) 6.55 aD (0.23) 6.26 aD (0.43) * Average values of three replicates (n= 3); standard deviations are indicated in brackets. For each chilling time, different lowercase letters denote significant (p< 0.05) differences as a result of icing conditions. For each icing condition, different capital letters denote significant (p< 0.05) differences as a result of chilling time. ** Icing conditions: CT (control; ice prepared without alga extract), AQ (ice containing an aqueous alga extract), and ET (ice containing an ethanolic alga extract). *** No effect (p> 0.05) of icing time or icing condition on Enterobacteriaceae counts was detected. Molecules 2021,26, 3774 4 of 14 Molecules 2021, 26, x FOR PEER REVIEW 4 of 14 Figure 1. Evolution of lipolytic counts (log CFU·g−1) * in chilled hake subjected to different icing conditions **. * Average values of three replicates (n = 3); standard deviations are indicated by bars. For each chilling time, different lowercase letters denote significant (p < 0.05) differences as a result of icing conditions. For each icing condition, different capital letters denote significant (p < 0.05) differences as a result of chilling time. ** Icing conditions as expressed in Table 1. With respect to psychrotrophs, the control batch surpassed 6 log CFU·g−1 on day 9, while the counterpart batches containing alga extracts were below that value at that time (Table 1). Remarkably, the inclusion of alga extract, either aqueous (AQ batch) or ethanolic (ET batch), provided a slight protection of fish muscle with respect to psychrotrophs’ growth for the 6–13-day period (i.e., lower average counts), although this effect was found to be statistically significant (p < 0.05) only at advanced storage times (day 13). Table 1 also provides the comparative analysis of Enterobacteriaceae growth in all three batches. The presence of this bacterial group was very limited in all three batches, with microbial counts being always below 2 log CFU·g−1 (Table 1). Proteolytic bacteria can cause the breakdown of fish muscle structure by biosynthesising and secreting extracellular proteases, whose effect on the myofibrillar protein fraction negatively affects fish texture and quality. As in the case of aerobes, the presence of alga extract in the icing medium provided, in general terms, a slight protective effect on fish muscle (Table 1). The average counts of proteolytic bacteria in the AQ batch were slightly below those determined in the control batch, especially at medium and advanced storage times (day 6 and onwards), although such differences were not significant (p > 0.05). As in the case of aerobes and psychrotrophs, the AQ extract seemed to provide better protection as compared to the ET extract. The comparative evolution of lipolytic bacteria in all three batches is displayed in Figure 1. This microbial group is able to biosynthesise and secrete extracellular lipases whose effect on triacylglycerides (TG) and phospholipids (PL) negatively affects fish quality. In our study, the inclusion of alga extract in the icing medium resulted in more limited growth of these specific spoilage organisms at different storage times. Thus, the AQ extract provided a significant (p < 0.05) reduction in lipolytic bacteria at both early (day 2) and advanced (day 13) storage times. With respect to the ET extract, this beneficial effect was significant (p < 0.05) for medium (day 6) and advanced (day 13) storage. Remarkably, neither AQ nor ET batches reached 6 log CFU·g−1 even on day 13, while the control batch surpassed this value at that time. As was also observed for aerobes, psychrotrophs, and proteolytic bacteria, the AQ extract provided, in general terms, better control of the growth of lipolytic bacteria as compared to the ET extract. Figure 1. Evolution of lipolytic counts (log CFU · g −1 ) * in chilled hake subjected to different icing conditions **. * Average values of three replicates (n= 3); standard deviations are indicated by bars. For each chilling time, different lowercase letters denote significant (p< 0.05) differences as a result of icing conditions. For each icing condition, different capital letters denote significant (p< 0.05) differences as a result of chilling time. ** Icing conditions as expressed in Table 1. Proteolytic bacteria can cause the breakdown of fish muscle structure by biosynthesising and secreting extracellular proteases, whose effect on the myofibrillar protein fraction negatively affects fish texture and quality. As in the case of aerobes, the presence of alga extract in the icing medium provided, in general terms, a slight protective effect on fish muscle (Table 1). The average counts of proteolytic bacteria in the AQ batch were slightly below those determined in the control batch, especially at medium and advanced storage times (day 6 and onwards), although such differences were not significant (p> 0.05). As in the case of aerobes and psychrotrophs, the AQ extract seemed to provide better protection as compared to the ET extract. The comparative evolution of lipolytic bacteria in all three batches is displayed in Figure 1. This microbial group is able to biosynthesise and secrete extracellular lipases whose effect on triacylglycerides (TG) and phospholipids (PL) negatively affects fish quality. In our study, the inclusion of alga extract in the icing medium resulted in more limited growth of these specific spoilage organisms at different storage times. Thus, the AQ extract provided a significant (p< 0.05) reduction in lipolytic bacteria at both early (day 2) and advanced (day 13) storage times. With respect to the ET extract, this beneficial effect was significant (p< 0.05) for medium (day 6) and advanced (day 13) storage. Remarkably, neither AQ nor ET batches reached 6 log CFU · g −1 even on day 13, while the control batch surpassed this value at that time. As was also observed for aerobes, psychrotrophs, and proteolytic bacteria, the AQ extract provided, in general terms, better control of the growth of lipolytic bacteria as compared to the ET extract. This study has proven the antimicrobial effect on hake muscle of including aqueous and ethanolic B. bifurcata extracts in the icing media. This inhibition of microbial growth could be explained by the presence of different kinds of bioactive compounds and their antimicrobial activity due to several mechanisms, such as the inhibition of extracellular microbial enzymes, deprivation of substrates required for microbial growth, direct action on the microbial metabolism through inhibition of oxidative phosphorylation, and complexation of metal ions in the bacterial growth environment [ 4 , 38 ]. Concerning the Molecules 2021,26, 3774 5 of 14 bioactive antimicrobial components present in B. bifurcata, several previous studies have reported their presence both in aqueous and lipophilic alga extracts. Related to B. bifurcata lipophilic extracts (Table 2), previous research showed the presence of bioactive compounds such as phenols in aq. 80% ethanol extract [ 39 ] and diterpenes in ethyl ether extract [ 40 ]. Furthermore, total sterol content was analysed by FTIR spectroscopy in methanol:chloroform extracts (1:1) by Bouzidi et al. [ 35 ], fucosterol being identified as the major sterol. Additionally, a substantial antimicrobial effect was reported in methanolic extracts of this macroalga by Alves et al. [ 36 ] on the basis of its high content in polyphenolic compounds, evaluated by in vitro tests (DPPH and ORAC assays). Moreover, ethanolic extracts of the algae B. bifurcata [ 19 ] reduced the microbial counts (aerobes, psychrotrophs, proteolytic and lipolytic bacteria, and Enterobacteriaceae) in chilled megrim (Lepidorhombus whiffiagonis) muscle during storage. Table 2. Previous research related to analysis of bioactive compounds included in hydrophilic and lipophilic extracts of alga Bifurcaria bifurcata. Extraction Medium Bioactive Compound Reference aq. 80% ethanol phenols Glombitza et al. 1976 [39] ethyl ether diterpenes Culioli et al. 2001 [40] methanol:chloroform (1:1) Sterols, i.e., fucosterol Bouzidi et al. 2008 [35] methanol polyphenols Alves et al. 2016 [36] water polysaccharides, i.e., alginate Gómez-Ordóñez and Rupérez, 2011 [32] water total dietary fibre; soluble and insoluble fibre Gómez-Ordóñez et al. 2010 [31] water pholorotannins, phenmolic acids, flavonoids, fuhalols, hydroxyl-fuhalols, eckol derivatives, and rosmarinic acid Agregán et al. 2017 [33] Previous research on B. bifurcata aqueous extracts has also reported the presence of different kinds of antimicrobial components (Table 2). Thus, Gómez-Ordóñez and Rupérez [ 32 ] identified alginate as the main polysaccharide by FTIR-ATR. Later on, dietary fibre and physicochemical properties of B. bifurcata were studied by Gómez-Ordóñez et al. [ 31 ]. As a result, total dietary fibre content of this alga was 37.42% of which 14.64% was soluble, while insoluble fibres represented 22.79%; notably, the soluble fibre contained uronic acids from alginates and neutral sugars from sulphated fucoidan and laminarin, insoluble fibres being essentially made from cellulose. Recently, Agregán et al. [ 33 ] analysed the phenolic compounds in this kind of extract and identified phlorotannins as the main phenolic compounds, these being followed by phenolic acids, flavonoids, fuhalols, hydroxyfuhalols, eckol derivatives, and rosmarinic acid. Concerning the preservative effects on seafood developed by other macroalgae, ethanolic extracts of the alga U. pinnatifida [ 41 ] reduced the microbial activity (aerobes, psychrotrophs, proteolytic and lipolytic bacteria, and Enterobacteriaceae counts) in chilled megrim (Lepidorhombus whiffiagonis) muscle during storage. Remarkably, stronger inhibition of the development of microbes (aerobes, psychrotrophs, Enterobacteriaceae, and proteolytic and lipolytic bacteria) was observed in chilled hake stored in an icing system containing an ethanolic extract of Fucus spiralis as compared with a counterpart fish batch stored in ice containing an aqueous extract of this alga [28]. Previous studies have also reported on the enhancement of microbial quality in chilled fish by including aqueous or ethanolic extracts obtained from plants in the icing medium. Thus, the inclusion of ethanolic extracts of thyme (Thymus vulgaris), oregano (Origanum glandulosum), or clove (Syzygium aromaticum) led to lower average values for mesophilic aerobic and psychrotrophic bacteria in chilled anchovy (Engraulis entresols) muscle [ 42 ]. Likewise, a reduction in total viable bacteria and an increase in shelf life resulted from the inclusion of ethanolic mint (Mentha arvensis) leaf or citrus (Citrus aurantium) peel extracts in ice during the storage of Indian mackerel (Rastrelliger kanagurta) [ 43 ]. Concerning Molecules 2021,26, 3774 6 of 14 aqueous extracts, the presence in the fish storage ice of a rosemary (Rosmarinus officinalis) extract [ 17 ] provoked a reduction in total viable bacteria and an increased shelf life in chilled sardine (Sardinella aurita). Additionally, inhibition of total viable counts and increased sensory acceptance were observed in chilled mackerel (Rastrelliger kanagurta) as a result of the presence of an aqueous extract from two garcinia species (Garcinia indica and G. cambogia) [44]. 2.2. Comparative Evolution of pH Value and Free Fatty Acid (FFA) Content during Chilled Storage of Hake As shown in Figure 2, the pH value of fish muscle increased (p< 0.05) with storage time in specimens corresponding to control (CT) and ET batches. On the contrary, hake stored under AQ icing conditions exhibited negligible differences as storage time progressed. Thus, fish specimens corresponding to the AQ batch showed lower (p< 0.05) pH values than their counterparts from CT and ET batches in the 9–13-day period. Consequently, an inhibitory effect of the aqueous alga extract on the pH increase could be inferred. Increases in the pH value of fish muscle during storage have been reported to indicate the accumulation of alkaline compounds, such as ammonia, trimethylamine, and other nitrogen-containing compounds, which are mainly derived from microbial spoilage [ 4 ]. The present inhibition of a pH increase can be explained on the basis of the above-mentioned antimicrobial compounds (Section 2.1; Table 2) present in both kinds of B. bifurcata extracts. Figure 2. Evolution of pH value * in chilled hake subjected to different icing conditions **. * Average values of three replicates (n= 3); standard deviations are indicated by bars. For each chilling time, different lowercase letters denote significant (p< 0.05) differences as a result of icing conditions. For each icing condition, different capital letters denote significant (p< 0.05) differences as a result of chilling time. ** Icing conditions as expressed in Table 1. A different result was reported by Barros-Velázquez et al. [ 28 ] when comparing the effects of two ice systems containing aqueous and ethanolic extracts of the alga F. spiralis, respectively. In their study, no differences in pH value were found between batches of chilled megrim (L. whiffiagonis) muscle; in addition, no differences were observed as compared with fish specimens of the control batch which did not contain any alga extract. In contrast, the results obtained in another study allowed the conclusion that including ethanolic B. bifurcata extract in the icing system for megrim (L. whiffiagonis) muscle led to better maintenance of the pH value as compared with the control batch during chilled storage [19]. In agreement with the current results, previous studies have reported an inhibition of the pH increase in chilled fish as a result of using other natural preservative compounds in the icing medium. These studies used aqueous extracts obtained from rosemary (R. of- Molecules 2021,26, 3774 7 of 14 ficinalis) and applied to chilled sardine (S. aurita) [ 17 ], and from garcinia (G. indica and G. cambogia) employed for mackerel (R. kanagurta) refrigeration [ 44 ]. Notably, previous research on plant extracts has also shown an inhibition of an increased pH value of chilled fish specimens when using ethanol extracts from thyme (T. vulgaris), oregano (O. glandulosum), or clove (S. aromaticum) on anchovy (E. encrasicolus) [ 42 ] and from mint (M. arvensis) leaf and citrus (C. aurantium) leaf extracts during the chilled storage of Indian mackerel (R. kanagurta) [43]. A substantial increase (p< 0.05) in FFA content was detected in all batches as storage time progressed (Table 3). A comparison among batches showed lower average values in specimens corresponding to the ET batch; differences were found to be significant (p< 0.05) throughout the whole experiment as compared with the control batch and for the 2–9-day period as compared to the AQ batch. Notably, FFA formation was also inhibited in fish corresponding to the AQ batch at days 2, 6, and 13. Table 3. Assessment of lipid damage * related to quality loss in chilled hake subjected to different icing conditions **. Chemical Parameter Chilling Time (Days) Icing Condition CT AQ ET Free fatty acids (mg·kg−1 muscle) 064.38 A (6.52) 64.38 A (6.58) 64.38 A (6.56) 278.88 cB (1.98) 66.75 bA (1.36) 58.41 aA (2.07) 694.70 cC (3.41) 87.58 bB (2.86) 70.21 aB (1.79) 9113.67 bD (7.19) 102.72 bC (5.44) 95.97 aC (1.89) 13 139.50 bE (6.17) 105.88 aC (7.97) 93.23 aC (3.95) Peroxide value (meq. active oxygen·kg−1 lipids) 00.14 A (0.00) 0.14 A (0.00) 0.14 A (0.00) 22.28 aB (0.15) 2.69 aB (0.55) 3.75 bB (0.50) 62.50 aB (0.07) 2.45 aB (0.71) 3.50 bB (0.18) 93.80 aB (2.62) 2.66 aB (1.05) 7.28 bC (1.14) 13 4.37 aB (2.67) 3.69 aB (0.95) 7.24 bC (0.57) Thiobarbituric acid index (mg malondialdehyde·kg−1 muscle) 00.13 A (0.09) 0.13 A (0.09) 0.13 A (0.09) 20.35 bB (0.09) 0.17 aA (0.06) 0.26 abAB (0.06) 60.36 aAB (0.15) 0.35 aB (0.10) 0.48 aD (0.05) 90.37 aB (0.05) 0.51 a (0.07) 0.45 aCD (0.05) 13 0.70 bC (0.07) 0.41 aB (0.07) 0.36 aBC (0.05) * Average values of three replicates (n= 3); standard deviations are indicated in brackets. For each chilling time, different lowercase letters denote significant (p< 0.05) differences as a result of icing conditions. For each icing condition, different capital letters denote significant (p< 0.05) differences as a result of chilling time. ** Icing conditions as expressed in Table 1. Molecules 2021,26, 3774 8 of 14 Both endogenous and microbial lipases have been reported to be responsible for the formation of FFA during the chilled storage of fish [ 8 ]. Before the end of the microbial lag phase (about 6–9 days), endogenous enzyme activity should be predominant; after that time, microbial activity should gain importance and be mostly responsible for the development of lipid hydrolysis. The results of the present study show a constant and gradual increase in FFA content with chilling time, so it can be concluded that both mechanisms occurred. Moreover, the results obtained can be considered the result of two opposite reactions. On one side, both endogenous and microbial enzymes (namely, lipases and phospholipases) can hydrolyse high-molecular-weight lipids such as PL and TG. On the other, and as they are low-molecular-weight molecules, FFA are likely to be rapidly oxidised or broken down during storage due to their greater accessibility to oxygen and other pro-oxidant molecules as compared to TG and PL [ 45 ]. On the basis of the marked formation of FFA, the first factor has been shown to be more important. The current inhibitory effect on FFA formation in hake muscle stored under both ice batches can be explained on the basis of the presence of antimicrobial compounds present in both kinds of B. bifurcata extracts. Consequently, the above-mentioned bioactive compounds (Section 2.1; Table 2) can be involved in such a preserving effect. In agreement with the current results, inhibition of FFA formation derived from the inclusion of an ethanolic extract of B. bifurcata in the icing medium was observed in chilled megrim (L. whiffiagonis) by Miranda et al. [ 19 ]. A similar inhibitory effect on FFA formation was observed when an ethanolic extract of U. pinnatifida was employed for the chilled storage of megrim (L. whiffiagonis) [ 37 ]. Furthermore, FFA formation showed to be diminished during the storage of hake (M. merluccius) when an icing system containing the alga F. spiralis was employed [ 28 ]; remarkably and according to the current study, a lower FFA formation was observed in chilled hake stored in ice containing an ethanolic alga extract as compared to its counterpart kept in an icing system containing an aqueous extract. In agreement with the inhibition of FFA formation reported in the present study, previous research has shown the effect of including plant extracts in the icing medium of fish. Thus, a lower FFA content was detected in anchovy (E. encrasicolus) due to the presence of an ethanolic thyme (T. vulgaris), oregano (O. glandulosum), or clove (S. aromaticum) extract [ 40 ] in the icing medium. A similar situation concerning FFA formation was observed in Indian mackerel (R. kanagurta) due to the presence of ethanolic mint (M. arvensis) leaf and citrus (C. aurantium) leaf extracts [ 43 ] in the icing medium. In agreement with the results of the present study, Quitral et al. [ 16 ] also reported the inhibition of FFA formation due to the inclusion of aqueous oregano (O. vulgare) or rosemary (R. officinalis) extract in the ice employed for storage of Chilean jack mackerel (Trachurus murphyi). 2.3. Lipid Oxidation Development during Chilled Storage of Hake Progressive formation of peroxides (p< 0.05) was observed in all batches as storage time progressed (Table 3). Nevertheless, the values detected were in all cases below 7.5, which can be considered a relatively low level for refrigerated fish [ 8 ]. A comparison among batches showed higher (p< 0.05) values in fish specimens corresponding to the ethanolic extract batch at all storage times. Notably, fish specimens belonging to the AQ batch exhibited lower average values than the counterpart CT batch (6–13-day period), although differences were not significant (p> 0.05). A significant trend (p< 0.05) of an increase in the content of secondary lipid oxidation compounds (i.e., thiobarbituric acid reactive substances, TBARS) was observed for fish specimens corresponding to CT and AQ batches (Table 3). Concerning the ET batch, average values showed an increase up to day 6, this being followed by a decrease until the end of the storage time. A comparison among batches showed an inhibitory effect (p< 0.05) of the aqueous alga extract (AQ batch) at days 2 and 13, while this effect was only detected at day 13 in fish belonging to the ET batch. It is concluded that the inclusion of a water extract of B. bifurcata in the icing medium employed for the chilled storage of hake led to the inhibition of lipid oxidation events (i.e., Molecules 2021,26, 3774 9 of 14 the formation of secondary compounds) in hake muscle. This effect can be explained by the presence of relatively polar compounds (namely, hydrophilic molecules), which have been reported to play a crucial role in the inhibition of oxidation [ 46 , 47 ]. With respect to the ethanol extract, no definite effect could be inferred, as increased peroxide formation was accompanied by a decrease in TBARS. Previous research has accounted for the presence of antioxidant compounds in the aqueous extract of B. bifurcata (Table 2). Thus, the dietary fibre and physicochemical properties of B. bifurcata were studied by Gómez-Ordóñez et al. [ 31 ]. As a result, total dietary fibre content was 37.42% of which 14.64% was soluble, insoluble fibres representing 22.79% of the content; notably, the soluble fibre contained uronic acids from alginates and neutral sugars from sulphated fucoidan and laminarin, while insoluble fibres were essentially made from cellulose. Furthermore, Gómez-Ordóñez and Rupérez [ 32 ] identified alginate by FTIR-ATR as the main polysaccharide in B. bifurcata aqueous extract. Agregán et al. [ 33 ] concluded that B. bifurcata aqueous extracts could be used as sources of antioxidant phenolic compounds on the basis of the presence of phlorotannins, phenolic acids, flavonoids, fuhalols, hydroxyfuhalols. The same research group [ 48 ] showed antioxidant activity (DPPH, FRAP, and ORAC assays) and also an antioxidant effect on canola oil during storage at 60 ◦ C by the presence of the aqueous B. bifucata extract. In agreement with the results of the present study, less peroxide formation was also observed in hake (M. merluccius) muscle subjected to an ice system containing an aqueous extract of F. spiralis as compared with its counterpart treated with an ethanolic extract [ 28 ]. Furthermore, ethanolic extracts of B. bifurcata [ 19 ] led to higher levels of peroxides in chilled megrim (L. whiffiagonis), while the TBARS content was not modified as compared to the control fish batch. Neither did an ethanolic extract of U. pinnatifida provide significant differences in lipid oxidation rates when included in an icing medium for the chilled storage of megrim (L. whiffiagonis) [ 41 ]. However, greater rancidity stability was detected in a fish oil system in the presence of different kinds of ethanolic macroalga extracts when compared to their counterpart aqueous extracts [ 25 ]. Concerning thermally treated fish, an inhibitory effect of aqueous alga extracts was also detected by Ortiz et al. [ 21 ] in canned fish. Thus, aqueous extracts of various algae (Durvillaea antarctica,Ulva lactuca,Pyropia columbina, Macrocystis pyrifera, and Gracilaria chilensis) were included in the packaging medium during the canning of Atlantic salmon (Salmo salar); as a result, remarkable rancidity stabilisation (i.e., p-anisidine value) was observed throughout an accelerated canned storage study (up to 140 days at 40 ◦C). Previous research has also shown the inhibition of lipid oxidation in fish muscle derived from the presence of aqueous or ethanolic plant extracts. Thus, less formation of peroxides and TBARS was detected in mackerel (R. kanagurta) in the presence of aqueous garcinia (G. indica or G. cambogia) extracts in the icing medium [ 44 ], as well as in Chilean jack mackerel (Trachurus murphyi) due to the presence of aqueous oregano (O. vulgare) or rosemary (R. officinalis) extract in the icing medium [ 16 ]. Moreover, a substantial reduction in both primary and secondary lipid oxidation events was observed when ethanolic extracts of thyme (T. vulgaris), oregano (O. glandulosum), or clove (S. aromaticum) were used for the chilled storage of anchovy (E. encrasicolus) [ 42 ]. A similar result was observed in chilled Indian mackerel (R. kanagurta) when stored in the presence of ethanolic extracts of mint (M. arvensis) leaf and citrus (C. aurantium) leaf [43]. 3. Materials and Methods 3.1. Starting Macroalga B. bifurcata and Preparation of Icing Systems The lyophilised alga B. bifurcata was provided by Porto-Muiños (Cerceda, A Coruña, Spain). Two kinds of alga extract were prepared. As a first preparation, 15 g of lyophilised alga were distributed into 3 tubes (5 g in each) and mixed with distilled water (40 mL in each), stirred for 30 s, centrifuged at 2500 × gfor 10 min at 4 ◦ C, and the supernatants recovered. Then, the remaining alga sediments were extracted a second time with the same water quantities. Finally, all super-