Antimicrobial and antioxidant effect of lyophilized Fucus spiralis addition on gelatin film during refrigerated storage of mackerel

Food Control Antimicrobial and antioxidant effect of macroalga Fucus spiralis addition on gelatin film during refrigerated storage of mackerel --Manuscript Draft-- Manuscript Number: FOODCONT-D-21-00505R1 Article Type: Research Paper Keywords: Fucus spiralis; gelatin packaging; Scomber scombrus; Refrigeration; microbial development; lipid damage Corresponding Author: Santiago P. Aubourg, Ph.D. Consejo Superior de Investigaciones Cientificas Vigo, SPAIN First Author: Marcos Trigo Order of Authors: Marcos Trigo Pedro Nozal José M. Miranda Santiago P. Aubourg, Ph.D. Jorge Barros-Velázquez Abstract: Lyophilized alga Fucus spiralis powder was incorporated into a gelatin-based film and employed as a packaging system for mackerel (Scomber scombrus) muscle portions throughout a 9-day refrigerated storage period at 4 ºC. In global terms, a progressive loss of quality could be observed in fish muscle with increasing storage time. Comparisons between batches allowed us to conclude an inhibitory effect of F. spiraliscontaining films on microbial activity (assessment of aerobes, psychrotrophs, and proteolytic bacteria) and on lipid hydrolysis (as determined by free fatty acid formation) in mackerel muscle. The presence of the lyophilized macroalga in the packaging film also led to a higher retention of primary (peroxides) and secondary (thiobarbituric acid reactive substances) lipid oxidation compounds, while the formation of fluorescent compounds (interaction compounds between lipid oxidation compounds and nucleophilic molecules present in the fish muscle) decreased. Both antimicrobial and antioxidant effects were more intense when the concentration of alga in the packaging film was increased. The preservative effect resulting from the presence of F. spiralis in gelatin-based films demonstrates the potential employment of such bioactive films to improve the retention of fish quality and enhance its commercial value. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Food Control journal Editorial Board Vigo, June 3rd 2021 Dear Ms Ichiko Charis Howells: Please find enclosed the manuscript now entitled “Antimicrobial and antioxidant effect of lyophilized Fucus spiralis addition on gelatin film during refrigerated storage of mackerel”. It has been performed according to the Reviewers’ recommendations and criticisms. Changes made are marked on the text. We would like to submit it again for publication in the Food Control journal. Yours sincerely, Prof. Santiago P. Aubourg Marine Research Institute Spanish National Research Council (CSIC) Vigo, Spain Cover Letter CHANGES MADE AND COMMENTS Reviewer #2: In this paper, the authors made an attempt to evaluate edible biofilm prepared by incorporating the the lyophilized alga Fucus spiralis into a gelatin and evaluated as a packaging system for mackerel (Scomber scombrus) fillets under refrigerated (4 ºC) storage period for 9 days. The manuscript is well written and contains some interesting information's also. However, the manuscript contains some technical drawbacks that are really in need for its improvement. The paper quality can be improved by modifying the manuscript with following suggestions 1) The treatments groups are very widely in concentrations (1:5 and 1:20 alga to gelatin ratio) why the authors are not tried intermediate concentrations Answer: As stated in sub-section 2.1., the highest alga concentration (i.e., 1:5 alga to gelatine ratio) was chosen to be employed on the basis of preliminary trials. This ratio was the highest that did not show any negative effect on sensory properties of fish muscle portions. Then, in order to cover a wide range of ratios, a relatively lower ratio (i.e. 1:20) was considered. 2) There is no information about bioactive properties of the biofilm. Answer: The bioactive properties of the biofilm constituents are now included in the revised manuscript, following the Reviewer’s advice. Thus, preservative properties of gelatin films have been described in the Introduction section of the revised manuscript. On the other side, preservative properties of F. spiralis constituents (hydrophilic and lipophilic) and their application to different kinds of seafood, this including lyophilized F. spiralis in biodegradable films (i.e. polylactic acid), are now included in the revised manuscript. In the present study, gelatin packaging was considered as control. Consequently, the Results and Discussion section shows comparison with previous research including hydrophilic and/or lipophilic constituents included in F. spiralis and other related macroalgae. 3) Which compounds are responsible for its bioactive properties? Answer: The current study focused on the direct application of this macroalga in order to evaluate the possible preservative effects. No further analytical study was accomplished on the F. spiralis composition. However, and according to previous results, both bioactive hydrophilic and lipophilic compounds are known to be present in the current macroalga as well as in related macroalgae. Accordingly, information and discussion concerning previous studies on bioactive compounds (antimicrobial and antioxidant properties) are now included in the Results and Discussion section, as suggested by the Reviewer. 4) What about the colour of film? Does it imparts colour to the fish fillet? Answer: As stated in sub-section 2.1, the most concentrated alga:gelatin ratio provided colourless films which did not modify the sensory properties of the muscle portions (colour, odour, or taste) 5) Sensory parameters also need to be discussed Answer: In the current study, we focused on microbial and chemical indices related to microbial breakdown and lipid damage. Sensory acceptance was not explicitly evaluated. However, a strong direct relationship between microbial counts and sensory acceptance of refrigerated fish is widely accepted. This is mentioned now in the revised manuscript. Furthermore, lipid oxidation development has also shown a direct effect on sensory acceptance. This is also included now in the Results and Discussion section of the revised manuscript. Some new references have been included now concerning the relationship between such quality parameters. Detailed Response to Reviewers Reviewer #3: General comments: This article has interesting results that can contribute for fish gelatin-based films. The research topic is interesting and the results could have interest for the food industry. However, the experimental has some week points: only one parameter to evaluate the legal limits for fish quality was used (Total viable counts). Given the potential benefice of this type of films, other parameters should be analysed to have conclusions that are more realistic and of interest to the sector. Answer: Thanks for your comment. Results on Enterobacteriaceae counts have been included now in the revised manuscript. In the previous version of the manuscript this determination was not included. It should be highlighted that, values remained in all cases below 1 log CFU/g. This microbial parameter has a widely accepted limit of 3 log CFU/g for fish. As stated in the revised manuscript now, all batches were acceptable throughout the whole study on the basis of this determination. Concerning the lipid oxidation assessment, a complete lipid oxidation analysis (three levels) was undertaken on the basis that a fatty fish species was concerned. We agree with the Reviewer in that such determinations do not provide legal limit values established for all kinds of fish products; however, the assessment of such indices are widely accepted to provide a useful information of quality loss in refrigerated fish. Additionally, sensory parameters should be considered. Answer: In the current study, we focused on microbial and chemical indices related to microbial breakdown and lipid damage. Sensory acceptance was not explicitly evaluated. However, a strong direct relationship between microbial counts and sensory acceptance of refrigerated fish is widely accepted. This is mentioned now in the revised manuscript. Furthermore, lipid oxidation development has also shown a direct effect on sensory acceptance. This is also included now in the Results and Discussion section of the revised manuscript. Some new references have been included now concerning the relationship between such quality parameters. Revision comments: - The abstract should be revised. In the Abstract, it is not clear if the authors use lyophilized alga Fucus spiralis to incorporate into a gelatin-based film or macroalga extract. Answer: The first sentence in Abstract section was modified according to the Reviewer’s comment. Later on, “macroalga extract” has been replaced with “lyophilized macroalga”. The title of the manuscript was also modified to better express that lyophilized alga was employed. - Materials and methods: Fish material, processing, and sampling This section is vague and must be improved. Answer: Description of this section has been improved. The term “fillets” has been eliminated and replaced with “muscle portions” or “fish portions”. Line 124: what type of gelatin was used? gelatin obtained from fish? Answer: Information about gelatin (i.e. from fish origin) has been included now in the revised manuscript, as suggested by the Reviewer. Line 152: please explain how the samples were sealed-packaged individually under the three above-mentioned packaging systems. Answer: Information about packaging has been improved in the revised manuscript. The size of each pack is also included now in the revised manuscript. HIGHLIGHTS - Lyophilized Fucus spiralis was incorporated into a gelatin packaging film - F. spiralis in the packaging film inhibited free fatty acid formation in mackerel - F. spiralis inhibited aerobes, psychrotrophs, and proteolytic bacteria in fish muscle - F. spiralis in the packaging film inhibited fluorescent compound formation - Superior retention of primary and secondary lipid oxidation compounds was observed Highlights (for review) 1 1 2 3 4 5 6 7 8 Antimicrobial and antioxidant effect of lyophilized Fucus spiralis 9 addition on gelatin film during refrigerated storage of mackerel 10 11 12 13 14 15 16 17 18 Marcos Trigo1, Pedro Nozal1, José M. Miranda2, Santiago P. Aubourg1,*, 19 and Jorge Barros-Velázquez2 20 21 22 23 24 25 26 27 28 1 Department of Food Science and Technology, Marine Research Institute (CSIC), c/ E. 29 Cabello, 6 36208-Vigo, Spain 30 2 Department of Analytical Chemistry, Nutrition and Food Science, School of Veterinary 31 Sciences, University of Santiago de Compostela, Avenida Carvallo Calero, s/n, 32 27002-Lugo, Spain 33 * Correspondent: [email protected]; +34986292762 (fax), +34986231930 (phone) 34 35 36 37 38 Manuscript Click here to view linked References 2 ABSTRACT 39 Lyophilized alga Fucus spiralis powder was incorporated into a gelatin-based film 40 and employed as a packaging system for mackerel (Scomber scombrus) muscle portions 41 throughout a 9-day refrigerated storage period at 4 ºC. In global terms, a progressive loss 42 of quality could be observed in fish muscle with increasing storage time. Comparisons 43 between batches allowed us to conclude an inhibitory effect of F. spiralis-containing 44 films on microbial activity (assessment of aerobes, psychrotrophs, and proteolytic 45 bacteria) and on lipid hydrolysis (as determined by free fatty acid formation) in mackerel 46 muscle. The presence of the lyophilized macroalga in the packaging film also led to a 47 higher retention of primary (peroxides) and secondary (thiobarbituric acid reactive 48 substances) lipid oxidation compounds, while the formation of fluorescent compounds 49 (interaction compounds between lipid oxidation compounds and nucleophilic molecules 50 present in the fish muscle) decreased. Both antimicrobial and antioxidant effects were 51 more intense when the concentration of alga in the packaging film was increased. The 52 preservative effect resulting from the presence of F. spiralis in gelatin-based films 53 demonstrates the potential employment of such bioactive films to improve the retention 54 of fish quality and enhance its commercial value. 55 56 Keywords: Fucus spiralis; gelatin packaging; Scomber scombrus; refrigeration; 57 microbial development; lipid damage 58 Running title: Alga-gelatin packaging and mackerel quality 59 60 61 3 1. INTRODUCTION 62 The consumption of marine species provides many benefits to human health, as 63 they are important sources of polyunsaturated fatty acids (PUFA), high-quality proteins, 64 minerals, and lipophilic vitamins (Tilami & Sampels, 2018). However, their high water 65 and non-protein nitrogen contents, soft muscular and skin structure, low content of 66 connective tissue, and poikilothermic nature make marine species perishable 67 commodities (Özoğul, 2010). Lowering the temperature with ice or mechanical 68 refrigeration is the most common way of retarding microbial and biochemical spoilage of 69 fish. Although widespread, neither technique can guarantee the retention of fish quality, 70 especially when relatively long storage times are applied or when the cold chain is not 71 strictly maintained. Consequently, advances in refrigeration processes have been 72 developed to circumvent these scenarios (Campos, Gliemmo, Aubourg, & Barros73 Velázquez, 2012). 74 One such preservation technology is packaging. Packaging maintains food 75 freshness and preserves foods during distribution and storage from adverse situations, 76 including water vapor, microorganisms, gases, odors, dust, and mechanical shock and 77 vibrations (Mihindukulasuriya & Lin, 2014; Dehghani, Hosseini, & Regenstein, 2018). 78 Interestingly, currently existing packaging technologies work together with a number of 79 physical, chemical, and biological processes and agents to limit both microbial activity 80 and biochemical breakdown in foods (Giménez, López de Lacey, Pérez-Santín, López81 Caballero, & Montero, 2013; Kuley, Özoğul, & Polat, 2020). 82 Biodegradable and edible materials derived from plants and animals, including 83 peptides, polysaccharides, and lipids, are profitable alternatives to synthetic packaging 84 films (Umaraw et al., 2020). Among the polysaccharides, gelatin obtained from diverse 85 animal sources (porcine, bovine, and fish) has been applied effectively in active 86 4 packaging strategies for its film-forming ability (Etxabide, Uranga, Guerrero, & De la 87 Caba, 2017). Thus, gelatin-based coating and packaging films reduce oxygen, oil, and 88 moisture transport (Pavli et al., 2018) and can be used to reduce oxidation events, preserve 89 flavor, and improve the color stability, taste, and aroma of foods (Cuter, 2006). 90 Furthermore, the combination of gelatin packaging films with different kinds of natural 91 preservative compounds from different sources, such as oregano and rosemary extracts 92 (Gómez-Estaca, Montero, Giménez, & Gómez-Guillén, 2007), oregano oil (Min & Oh, 93 2009), tea polyphenols (Feng, Ng, Mikš-Krajnik, & Yang, 2017), or a microalga protein 94 concentrate (Stejskal, Miranda, Martucci, Ruseckaite, Barros-Velázquez, & Aubourg, 95 2020a), have successfully improved the retention of seafood quality by inhibiting both 96 microbial activity and lipid oxidation events. 97 Marine macroalgae have been reported to contain a wide variety of chemical 98 constituents with potential antimicrobial and antioxidant activities (Sandsdalen, Haug, 99 Stensvag, & Styrvold, 2003; Gupta & Abu-Ghannam, 2011), potentially applicable to 100 seafood processing. Thus, a wide variety of bioactive compounds, such as polyphenols, 101 pholorotannins, terpenes, chlorophylls, and carotenoids, have been isolated from different 102 algae species. Among them, Fucus spiralis, a brown macroalga living on the littoral shore 103 of the Atlantic coasts of Europe and North America, has shown promising preservation 104 potential (Farvin & Jacobsen, 2013; Tierney, Smyth, Hayes, Soler-Vila, Croft, & 105 Brunton, 2013a; Andrade et al., 2013). Thus, its presence in a polylactic acid-based film 106 employed for megrim (Lepidorhombus whiffiagonis) refrigerated storage (García-Soto et 107 al., 2015) and in the icing medium employed during hake (Merluccius merluccius) 108 (Barros-Velázquez, Miranda, Ezquerra-Brauer, & Aubourg, 2016) storage leads to 109 substantial quality enhancement of fish specimens. In the present study, lyophilized F. 110 spiralis was incorporated into a gelatin-based packaging film and employed for the 111 11 side, hydrophilic compounds such as sulfate polysaccharides, proteins, peptides, 261 glycosides, low-molecular organic acids and salts have been reported to be present in 262 seaweed and to exhibit potential preservative properties (Kuda & Ikemori, 2009; Pereira, 263 Amado, Critchley, Van de Velde, & Ribeiro-Claro, 2009). Interestingly, water extracts of 264 F. spiralis include preserving phenolic acids such as chlorogenic acid, vanilic and caffeic 265 acid (Farvin & Jacobsen, 2013). On the other side, lipophilic compounds such as terpenes 266 and polyphenols (Sandsdalen et al., 2003), halogenated alkane and alkenes, alcohols, 267 aldehydes, hydroquinones, and ketones (Smit, 2004) and oligomeric phlorotannins 268 (Serrano, Puupponen-Pimia, Dauer, Aura, & Saura-Calixto, 2009) have also been 269 reported to be present in macroalgae and to exhibit preservative properties. This 270 preservative effect has been explained on the basis of their role in several mechanisms, 271 such as the inhibition of extracellular microbial enzymes, deprivation of the substrates 272 required for microbial growth, direct action on microbial metabolism through the 273 inhibition of oxidative phosphorylation, and complexation of metal ions in the bacterial 274 environment (Sandsdalen et al., 2003; Smit, 2004). 275 Previous studies accounted for an inhibitory effect of F. spiralis bioactive 276 compounds on microbial activity in fish muscle during refrigerated storage. Thus, 277 lyophilized F. spiralis, when included in a polylactic-based packaging film, inhibited the 278 growth of aerobes, psychrotrophs, and Enterobacteriaceae in megrim (Lepidorhombus 279 whiffiagonis) fillets stored at 4 ºC for 11 days (García-Soto et al., 2015). Furthermore, the 280 presence of a F. spiralis extract in the icing medium employed for the chilled storage of 281 hake (Merluccius merluccius) (Barros-Velázquez et al., 2016) and megrim 282 (Lepidorhombus whiffiagonis) (Miranda, Trigo, Barros-Velázquez, & Aubourg, 2016) 283 allowed for better control of aerobic, psychrotrophic, proteolytic, and lipolytic bacteria. 284 12 The inclusion of other algae or algae extracts in biofilms has also been reported to 285 provide antimicrobial activity. This is the case of the red macroalga Gelidium corneum, 286 whose presence in an edible film, also including persimmon peel and grape fruit seed 287 extracts, improved the physical properties and provided antimicrobial activity (Jo, Song, 288 Lee, & Song, 2014). Furthermore, the presence of polyhydroxybutyrate and phenolic 289 compounds extracted from microalga Spirulina platensis led to a marked inhibitory effect 290 of microbial activity when included in an edible packaging system (Goettems Kuntzler, 291 Araujo de Almeida, Vieira Costa, & Greque de Morais, 2018). Remarkably, recent studies 292 have reported microbial quality enhancement of refrigerated (4 ºC) hake (M. merluccius; 293 Stejskal et al., 2020a) and mackerel (S. scombrus; Stejskal, Miranda, Martucci, 294 Ruseckaite, Aubourg, & Barros-Velázquez, 2020b) when a protein concentrate from S. 295 platensis was included in the gelatin-based packaging film. 296 297 3.2 Determination of lipid hydrolysis development 298 FFA formation was found to be negligible (p>0.05) in all batches after 2 days of 299 refrigerated storage (Figure 3). However, remarkable lipid hydrolysis was detected in all 300 mackerel batches after 6 days of storage, this being followed by a further increase 301 (p<0.05) at advanced storage times. A batch comparison showed an inhibitory effect on 302 FFA formation (p<0.05) in fish specimens corresponding to the F-2 batch for the 2–9-day 303 storage period. Notably, no significant effect (p>0.05) was concluded for the less 304 concentrated packaging film (F-1), although this batch showed lower average values for 305 the 6–9-day period when compared with the control batch. 306 Lipid hydrolysis itself does not lead to nutritional losses. However, a direct 307 relationship between FFA formation and sensory acceptance was detected (Özoğul, 2010; 308 Campos et al., 2012). Thus, the accumulation of FFA may imply detrimental sensory 309 13 properties and negatively affect the consumers’ acceptability of seafood (Sikorski & 310 Kolakowski, 2000). Among such negative modifications, texture changes and the 311 development of off-odor and off-taste can be highlighted. Furthermore, a marked direct 312 effect of FFA formation on lipid oxidation has been reported, this effect being explained 313 on the basis of a lower oxidative stability of FFA as compared with their corresponding 314 triacylglycerols and phospholipids as a result of lower steric hindrance to oxidative 315 reactions (Aubourg, 2001). FFA formation in fish muscle during refrigerated storage has 316 been explained as a result of endogenous and microbial enzyme activities (Campos et al., 317 2012). Before the end of the microbial lag phase, FFA formation should be mostly caused 318 by endogenous enzyme activity (i.e., lipases and phospholipases); later on, microbial319 based extracellular lipases should be the predominant mechanism of FFA generation. On 320 the basis that strong development of FFA formation was observed in the present study at 321 extended storage times (6–9-day period), microbial activity seemed to be the most 322 relevant mechanism responsible for FFA formation. Consequently, the inhibition of FFA 323 formation in the F-1 and F-2 batches can be explained on the basis of the above-mentioned 324 inhibition of microbial growth. This inhibition could be justified by the presence in the 325 lyophilized alga of preservative hydrophilic and lipophilic bioactive compounds, as stated 326 in the previous sub-section. 327 Previous studies reporting the effects of algae extracts on FFA development have 328 led to opposite results. Thus, enhanced lipid hydrolysis events were observed in chilled 329 minced Atlantic mackerel (S. scombrus) previously treated with an aqueous extract of 330 Polysiphonia fucoides (Babakhani, Farvin, & Jacobsen, 2016). Similar results were 331 observed in chilled hake (M. merluccius) exposed to ice including a F. spiralis water 332 extract (Barros-Velázquez et al., 2016). However, when an ethanol extract of F. spiralis 333 alga was considered, no significant effect on FFA formation was observed in hake (M. 334 14 merluccius) (Barros-Velázquez et al., 2016) and megrim (L. whiffiagonis) (Miranda et al., 335 2016) during chilled storage. In agreement with the results obtained in the current study, 336 Taghavi Takyar, Haghighat Khajavi, and Safari (2019) reported the inhibitory effect of 337 ethanol extracts of S. platensis on lipid hydrolysis in rainbow trout (Oncorhynchus 338 mykiss) fillets packaged in polyethylene bags and kept at 4 ºC for up to 16 days. Recently, 339 an inhibitory effect of a crosslinked-gelatin film including a spirulina protein concentrate 340 on FFA formation was observed in lean (hake, M. merluccius; Stejskal et al., 2020a) and 341 fatty (mackerel, S. scombrus; Stejskal et al., 2020b) fish muscle. 342 343 3.3 Determination of lipid oxidation development 344 The stability of packaged mackerel muscle against rancidity was studied by means 345 of primary (peroxide value), secondary (TBA-i), and tertiary (FR) oxidation compound 346 formation. 347 No significant formation of peroxides (p>0.05) was detected after 2 days of 348 storage in any of the batches under study (Table 2). In contrast, a general increase 349 (p<0.05) in the peroxide content was observed after 6 days, this increase being especially 350 relevant in the case of the F-2 batch. At the end of the storage period, significant peroxide 351 formation (p<0.05) was detected in fish specimens corresponding to the CT and F-1 352 batches, while no differences (p>0.05) were observed in the F-2 batch. The presence of 353 the lyophilized alga in the packaging film led to higher average peroxide values in 354 specimens corresponding to the F-2 batch throughout the whole storage period; notably, 355 differences were found to be significant (p<0.05) at day 6, as compared with their 356 counterparts belonging to the CT and F-1 batches. 357 A progressive formation (p<0.05) of TBARS was detected in all batches 358 throughout the storage period (Table 2). Increasing average TBARS values were observed 359 15 as the presence of alga in the packaging film increased. Thus, fish portions corresponding 360 to the control batch exhibited the lowest average values at all sampling times. 361 Interestingly, fish muscle corresponding to the F-2 batch showed higher TBARS levels 362 (p<0.05) than the counterpart CT batch throughout the storage period. Furthermore, fish 363 samples corresponding to the F-1 batch exhibited higher TBARS concentrations (p<0.05) 364 at days 2 and 9, as compared with their control counterparts. 365 A significant formation of fluorescent compounds (p<0.05) was observed at day 366 2 in all batches, this formation being of special relevance in the control batch (Table 2). 367 Analysis of interaction compound formation revealed a further formation at day 6 in all 368 batches; at this time, the highest increase (p<0.05) was observed in mackerel muscle 369 corresponding to the F-1 batch. An additional increase of the average FR value was 370 depicted in all batches at the end of the experiment. The highest average FR values were 371 determined in mackerel samples corresponding to the control batch throughout the whole 372 storage period; differences among batches were found to be significant (p<0.05) at day 2 373 with respect to the F-1 batch and in the 29-day period with respect to the F-2 batch. 374 Lipid oxidation has been recognized as a multi-step process where different 375 molecular species are subsequently produced. Those produced in the earliest stages (i.e., 376 peroxide compounds) are reported to be more unstable and susceptible to breakdown and 377 to produce lower-molecular-weight compounds (i.e., carbonyl compounds). Finally, at 378 the advanced stages of lipid oxidation, both peroxide and carbonyl compounds are 379 susceptible to reactions with other molecules (mostly of the nucleophilic-type including 380 groups like -NH2, or -SH) present in the fish muscle, thus leading to the formation of 381 fluorescent compounds (namely, tertiary lipid oxidation compounds) (Pokorný, 1981; 382 Aubourg, 1999). In the present study, higher peroxides and TBARS contents were 383 observed in fish samples corresponding to the F-1 and F-2 batches, these contents 384 16 increasing as the presence of lyophilized alga in the packaging film increased. However, 385 the fact that the content of advanced lipid oxidation compounds (detected by FR) was 386 lower in fish corresponding to the F-2 batch allowed us to conclude an inhibitory effect 387 of F. spiralis on the breakdown and reactivity of primary and secondary lipid oxidation 388 compounds. As a result, a decreasing effect on the FR value was detected, this effect 389 being more important in the F-2 batch, including the highest concentration of alga. 390 Remarkably, fluorescent compound formation can be considered an indicator of protein 391 damage, which negatively affects sensory descriptors such as texture and juiciness 392 (Özogul, 2010; Rustad, 2010). 393 Algae in general have been described as important sources of antioxidant 394 compounds (Gupta & Abu-Ghannam, 2011; Farvin & Jacobsen, 2013). According to their 395 photosynthetic role, algae are exposed to a strong combination of light and oxygen. 396 Consequently, their natural content on antioxidant substances has been reported to be 397 responsible for the lack of structural damage in their organs (Smit, 2004). In the case of 398 F. spiralis, an antioxidant capacity has already been proved in different in vitro tests 399 (DPPH and FRAP analyses) (Cérantola, Breton, Gall, & Deslandes, 2006; Andrade et al., 400 2013; Peinado, Girón, Koutsidis, & Ames, 2014), due to its marked content of 401 polyphenols (Tierney et al., 2013a) and α-tocopherol (Paiva et al., 2014). The 402 identification of active antioxidant compounds supported the assumption that 403 phlorotannins were also present in this macroalga (Tierney, Smyth, Rai, Soler-Vila, Croft, 404 & Brunton, 2013b). Additionally, and as stated above, Farvin and Jacobsen (2013) 405 reported the presence of antioxidant phenolic acids such as chlorogenic acid, vanilic and 406 caffeic acid in the current alga (Farvin and Jacobsen, 2013). 407 In agreement with the present results, no effect on TBARS content and a higher 408 PV was detected in chilled megrim (L. whiffiagonis) subjected to ice including a F. 409 17 spiralis extract (Miranda et al., 2016). Likewise, an inhibitory effect on fluorescent 410 compound formation was also observed. Similarly, the inclusion of F. spiralis extracts in 411 the icing medium did not exert a definite effect on peroxide and TBARS formation in 412 chilled hake (M. merluccius) (Barros-Velázquez et al., 2016). Remarkably, the 413 incorporation of the alga extract in the ice led to an inhibitory effect on fluorescent 414 compound formation. Closely related to the current study, the presence of lyophilized F. 415 spiralis in a polylactic acid packaging film also showed a marked inhibitory effect on 416 fluorescent compound formation during refrigerated storage (11 days at 4 ºC) of megrim 417 (L. whiffiagonis) fillets (García-Soto et al., 2015). Additionally, no effect on peroxide 418 content was detected. 419 Previous research also accounted for an antioxidant effect derived from the 420 inclusion of extracts from other algae in biofilms. Thus, alginate-based films prepared 421 from a red macroalga (Sargassum fulvellum) provided antioxidant properties (ABTS and 422 DPPH assays) to a biofilm also including black chokeberry (Kim, Back, & Song, 2018). 423 Moreover, Carissimi, Flôres, and Rech (2018) reported the antioxidant properties of a 424 starch-based film including an ethanolic extract of microalgae Heterochlorella 425 luteoviridis and Dunaliella tertiolecta; this effect, determined as a TBA-i decrease, was 426 observed in salmon fillets stored at 6±2 ºC for 6 days. Also related to a microalga, the 427 presence of a protein concentrate from S. platensis in the packaging film led hake (M. 428 merluccius) muscle to a higher PUFA retention rate during refrigerated storage (4 ºC) 429 (Stejskal et al., 2020a). Finally, a lower formation of fluorescent compounds was detected 430 in mackerel (S. scombrus) muscle packaged in a gelatin film including a protein 431 concentrate from S. platensis stored at 4 ºC (Stejskal et al., 2020b). 432 433 434 18 4. CONCLUSIONS 435 A gelatin-based film including lyophilized alga F. spiralis was tested as a novel 436 packaging method for the preservation of mackerel muscle under refrigeration. The 437 inclusion of lyophilized alga in the packaging films exerted a remarkable inhibitory effect 438 on microbial activity (aerobes, psychrotrophs, and proteolytic bacteria) and on lipid 439 hydrolysis events (free fatty acid formation). Concerning lipid oxidation, the presence of 440 the macroalga in the packaging film led to a higher retention of primary (peroxides) and 441 secondary (TBARS) lipid oxidation compounds, while the formation of fluorescent 442 compounds (interaction compounds between such lipid oxidation compounds and 443 nucleophilic molecules present in the fish muscle) decreased. Consequently, a 444 preservative effect on lipid oxidation development was concluded on the basis of the 445 inhibition of oxidation compounds produced at advanced deteriorative stages in fish 446 muscle (i.e., inhibition of tertiary lipid oxidation formation). The preservative effect was 447 concluded to be more intense in the batch containing a higher alga concentration in the 448 packaging film. In global terms, a preservative effect derived from the incorporation of 449 F. spiralis in the gelatin-based film was concluded, as a consequence of both 450 antimicrobial and antioxidant activities. According to the direct relationship of microbial 451 and chemical quality indices with respect to sensory assessment, a favourable effect on 452 sensory acceptance could be implied for the packaging system evaluated in this work. 453 This study opens the door to the potential employment of lyophilized F. spiralis 454 in bioactive gelatin films for enhancing the quality and the potential commercial value of 455 refrigerated fish species. Further research focused on optimization of the current biofilm 456 preparation (i.e., the alga/gelatin ratio, fish species concerned, refrigeration time and 457 temperature, etc.) should be considered to apply this active packaging strategy to a wide 458 variety of fish species. Furthermore, sensory acceptance evaluation would be necessary 459 19 in order to fulfil consumers’ demand for high-quality fresh products subjected to minimal 460 processing and not including chemical preservatives. 461 462 463 Acknowledgements 464 Lyophilized alga F. spiralis was provided by Porto-Muiños (Cerceda, A Coruña, 465 Spain). This work was supported by the Xunta de Galicia (Galician Government, Spain) 466 through the research project INNOVA-PEMES (IN848D), 026‒IN848D‒2020‒1119362. 467 468 20 REFERENCES 469 Andrade, P., Barbosa, M., Pedro Matos, R., Lopes, G., Vinholes, J., Mouga, T., & 470 Valentão, P. (2013). Valuable compounds in macroalgae extracts. Food 471 Chemistry, 138, 1819‒1828. 472 Aubourg, S. P. (1999). Review: Recent advances in assessment of marine lipid oxidation 473 by using fluorescence. Journal of the American Oil Chemists’ Society, 76, 409‒ 474 419. 475 Aubourg, S. P. (2001). Fluorescence study of the prooxidant activity of free fatty acids 476 on marine lipids. Journal of the Science of Food and Agriculture, 81, 385‒390. 477 Babakhani, A., Farvin, K., & Jacobsen, C. (2016). Antioxidative effect of seaweed 478 extracts in chilled storage of minced Atlantic mackerel (Scomber scombrus): 479 effect on lipid and protein oxidation. Food and Bioprocess Technology, 9, 352‒ 480 364. 481 Barros-Velázquez, J., Miranda, J. M., Ezquerra-Brauer, J. M., & Aubourg, S. P. (2016). 482 Impact of icing systems with aqueous, ethanolic and ethanolic-aqueous extracts 483 of alga Fucus spiralis on microbial and biochemical quality of chilled hake 484 (Merluccius merluccius). International Journal of Food Science and Technology, 485 51, 2081‒2089. 486 Bligh, E., & Dyer, W. (1959). A rapid method of total extraction and purification. 487 Canadian Journal of Biochemistry and Physiology, 37, 911‒917. 488 Campos, C., Gliemmo, M., Aubourg, S. P., & Barros-Velázquez, J. (2012). Novel 489 technologies for the preservation of chilled aquatic food products. In A. 490 McElhatton, & P. Amaral Sobral (Eds.), Novel Technologies in Food Science (pp. 491 299‒323). New York, USA: Springer (chapter 13). 492 27 FIGURE LEGENDS 634 635 Figure 1: Development of aerobe counts (log CFU·g-1 muscle)* in refrigerated mackerel 636 stored under different packaging conditions** 637 * Average values of three replicates (n=3); standard deviations are indicated in bars. For 638 each refrigeration time, values accompanied by different lowercase letters denote 639 significant differences (p<0.05) as a result of the packaging system. For each 640 packaging system, values accompanied by capital letters denote significant 641 differences (p<0.05) as a result of the refrigeration time. 642 ** Packaging conditions: CT (control packaging), F-1 (lowest-concentrated alga film 643 packaging), and F-2 (highest-concentrated alga film packaging). 644 645 646 Figure 2: Development of psychrotroph counts (log CFU·g-1 muscle)* in refrigerated 647 mackerel stored under different packaging conditions** 648 * Average values of three replicates (n=3); standard deviations are indicated in bars. For 649 each refrigeration time, values accompanied by different lowercase letters denote 650 significant differences (p<0.05) as a result of the packaging system. For each 651 packaging system, values accompanied by capital letters denote significant 652 differences (p<0.05) as a result of the refrigeration time. 653 ** Packaging systems as indicated in Figure 1. 654 655 656 28 Figure 3: Determination of free fatty acid (FFA) content (mg·kg-1 muscle)* in 657 refrigerated mackerel stored under different packaging conditions** 658 * Average values of three replicates (n=3); standard deviations are indicated in bars. For 659 each refrigeration time, values accompanied by different lowercase letters denote 660 significant differences (p<0.05) as a result of the packaging system. For each 661 packaging system, values accompanied by capital letters denote significant 662 differences (p<0.05) as a result of the refrigeration time. 663 ** Packaging conditions as indicated in Figure 1. 664 665 Figure Click here to access/download;Figure;Figure 1.tif Figure Click here to access/download;Figure;Figure 2.tif Figure Click here to access/download;Figure;Figure 3.tif TABLE 1 Development of proteolytics, lipolytics and anaerobes counts (log CFU·g-1 muscle)* in refrigerated mackerel stored under different packaging conditions** Microbial group Packaging condition Refrigeration time (days) 0 2 6 9 Proteolytics CT 2.00 A (0.02) 2.16 aA (0.28) 2.05 aA (0.03) 3.53 bB (0.68) F-1 2.00 A (0.02) 2.33 aAB (0.58) 2.07 aA (0.06) 2.72 aB (0.10) F-2 2.00 A (0.02) 2.10 aA (0.17) 2.02 aA (0.06) 3.15 abB (0.83) Lipolytics CT 2.00 A (0.01) 2.00 aA (0.00) 2.00 aA (0.00) 3.13 aB (0.31) F-1 2.00 A (0.01) 2.00 aA (0.00) 2.00 aA (0.00) 2.72 aB (0.10) F-2 2.00 A (0.01) 2.00 aA (0.00) 2.00 aA (0.00) 2.74 aB (0.13) Anaerobes CT 2.62 A (0.28) 2.42 aA (0.39) 2.36 aA (0.39) 2.92 aA (0.35) F-1 2.62 A (0.28) 2.39 aA (0.35) 2.30 aA (0.30) 2.60 aA (0.21) F-2 2.62 A (0.28) 2.30 aA (0.10) 2.46 aA (0.56) 2.52 aA (0.24) * Average values of three replicates (n=3); standard deviations are indicated in brackets. In each column, values accompanied by different low-case letters denote significant differences (p<0.05) as a result of packaging. In each row, values accompanied by capital letters denote significant differences (p<0.05) as a result of refrigeration time. ** Packaging conditions: CT (control packaging), F-1 (lowest-concentrated alga packaging) and F-2 (highest-concentrated alga packaging). Table Click here to access/download;Table;Tables.docx TABLE 2 Determination of lipid oxidation* in refrigerated mackerel stored under different packaging conditions** Chemical index Packaging condition Refrigeration time (days) 0 2 6 9 Peroxide value (meq active oxygen·kg-1 lipids) CT 1.95 A (0.78) 2.34 aA (0.41) 7.24 aB (1.40) 11.82 aC (0.93) F-1 1.95 A (0.78) 1.31 aA (0.63) 7.98 aB (1.33) 11.98 aC (1.24) F-2 1.95 A (0.78) 2.44 aA (0.45) 11.05 bB (0.88) 12.32 aB (2.13) Thiobarbituric acid index (mg malondialdehyde·kg-1 muscle) CT 0.03 A (0.03) 0.31 aB (0.08) 2.59 aC (0.28) 3.65 aD (0.70) F-1 0.03 A (0.03) 0.55 bB (0.03) 3.11 abC (1.03) 5.76 bD (1.12) F-2 0.03 A (0.03) 0.86 cB (0.20) 3.81 bC (0.47) 8.64 cD (1.01) Fluorescence ratio CT 2.34 A (0.90) 11.14 bB (1.10) 14.19 bBC (2.11) 16.86 bC (1.71) F-1 2.34 A (0.90) 6.31 aB (0.94) 13.82 bC (3.72) 16.45 bC (3.27) F-2 2.34 A (0.90) 5.82 aB (0.57) 6.45 aB (1.52) 9.48 aC (1.03) * Average values of three replicates (n=3); standard deviations are indicated in brackets. In each column, values accompanied by different low-case letters denote significant differences (p<0.05) as a result of packaging. In each row, values accompanied by capital letters denote significant differences (p<0.05) as a result of refrigeration time. ** Packaging conditions as expressed in Table 1. Conflict of interest The authors declare no conflict of interest. Conflict of Interest Form 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 CREDIT AUTHOR STATEMENT Author contributions: Conceptualization (SPA and JBV), methodology (MT, PN, and JMM), data curation (MT, PN, JMM, and SPA), writing-original draft (SPA and JBV) and writingreview and editing (SPA and JBV). All authors have read and agreed with the revised version. Credit Author Statement 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65