Chemical-Based Methodologies to Extend the Shelf Life of Fresh Fish—A Review

Due to its characteristics, fresh fish is a highly perishable food with a very short shelf-life under refrigeration. Several methods have been introduced to slow down its deterioration, such as by means of oxygen depletion of the food package (vacuum packaging), or by changing the natural atmosphere that is in contact with the fresh fish (modified atmosphere packaging), or by the use of chemicals generally recognized as safe: such compounds can be directly applied (by dipping or spraying) or incorporated into packaging materials and slowly migrate to the product, exerting a hurdle effect against microbial development and lipid oxidation (active packaging). This review aims to cover the most recent advances in chemical-based approaches for fresh fish preservation, applied either singly or in combination. Vacuum packaging, modified atmosphere, and active packaging preservation methodologies are presented, along with the inclusion of chemical additives, such as organic acids and natural extracts, and their combination with icing systems. Advantages and disadvantages of these methodologies and their impact on fresh fish quality and shelf-life are discussed, reaching the conclusion that both are positively influenced overall. Indeed, the contribution of chemical-based strategies for fresh fish preservation is undeniable, and is expected to be a research topic of increasing interest in the future.


Introduction
Fish is a highly consumed and nutritious food with a high protein and low saturated fat content, and is a source of high quality polyunsaturated fatty acids (PUFAs), such as omega-3 (DHA-docosahexaenoic acid and EPA-eicosapentaenoic acid, for example) that are truly relevant for cardiovascular disease prevention and exhibit anti-inflammatory properties [1,2]. Due to their nutritional characteristics and composition, fish products are highly perishable foods. Their degradation, coupled with economic pressures, results in large quantities of product waste which, even with increased public awareness and

Conventional Packaging: Vacuum-Packaging and Modified Atmosphere Packaging
The concept of VP consists of removing the air from a package containing the product and sealing it immediately. It separates the product from the external environment, limits the package volume and prevents oxidative spoilage if the films used have oxygen (O 2 ) barriers [19]. For these reasons, this low-cost technique is commonly used in the food industry [20].
The MAP process starts with the removal of the gaseous atmosphere present in the package and then inserting a mixture of selected gases before sealing the package. Typically, this mixture of gases has high levels of carbon dioxide (CO 2 ), because of its antimicrobial effect inhibiting the growth of aerobic microorganisms [19,21]. This happens because CO 2 dissolves in water and produces carbonic acid and a reduction of pH occurs. This leads to a prolongation on the lag phase and microbial growth is lowered throughout the logarithmic phase. However, the drop in pH could be minimal, and no bacteriostatic effect is noticed [22]. Nevertheless, CO 2 can be prejudicial to sensorial acceptance, affecting cellular structures from the fish, as well all color and flavor [23]. Oxygen (O 2 ) and nitrogen (N 2 ) are also used, since the former inhibits strictly anaerobic bacteria and the latter delays oxidative rancidity and limits aerobic microorganism's growth when used to replace oxygen on the package [9,21]. Ultimately, a wise combination of the abovementioned gases will result in an extension of shelf-life maintaining product quality, while it is also possible to design specific atmospheres to inhibit targeted and undesired microorganisms [21,24].
It is important to highlight that regardless the type of packaging, fish preservation will be highly dependent on multiple factors, like type and number of microorganisms present in the water of the fish's habitat, storage and transportation temperature, and handling during processing and distribution [25,26]. Furthermore, different fish species and experimental designs (type of packaging, ratio of gases, storage temperature) are Foods 2021, 10, 2300 4 of 28 studied and this could explain the differences observed on the results discussed below. Figures 1 and 2 represent the mechanism of VP and MAP technologies, respectively.
Foods 2021, 10, x FOR PEER REVIEW 4 of 30 present in the water of the fish's habitat, storage and transportation temperature, and handling during processing and distribution [25,26]. Furthermore, different fish species and experimental designs (type of packaging, ratio of gases, storage temperature) are studied and this could explain the differences observed on the results discussed below. Figures 1  and 2 represent the mechanism of VP and MAP technologies, respectively.  The most important parameter for shelf-life evaluation is the development of microorganisms during storage. This evolution will be highly dependent on original microorganisms and their tolerance to the specific gases used.
Even so, it has been observed that MAP can reduce Pseudomonas spp. counts and H2Sproducing bacteria. Likewise, atmospheres with higher CO2 content are more efficient in reducing lactic acid bacteria (LAB) counts.
Besides microbiological safety, preserving good sensorial quality is particularly important for shelf-life evaluation. Some studies [6,8,23] reported changes in color, odor, and texture along the shelf-life period with VP and MAP. Nonetheless, a study reported no sensory changes observed for a 100% CO2 atmosphere in common carp (Cyprinus carpio) [27].  present in the water of the fish's habitat, storage and transportation temperature, and handling during processing and distribution [25,26]. Furthermore, different fish species and experimental designs (type of packaging, ratio of gases, storage temperature) are studied and this could explain the differences observed on the results discussed below. Figures 1  and 2 represent the mechanism of VP and MAP technologies, respectively.  The most important parameter for shelf-life evaluation is the development of microorganisms during storage. This evolution will be highly dependent on original microorganisms and their tolerance to the specific gases used.
Even so, it has been observed that MAP can reduce Pseudomonas spp. counts and H2Sproducing bacteria. Likewise, atmospheres with higher CO2 content are more efficient in reducing lactic acid bacteria (LAB) counts.
Besides microbiological safety, preserving good sensorial quality is particularly important for shelf-life evaluation. Some studies [6,8,23] reported changes in color, odor, and texture along the shelf-life period with VP and MAP. Nonetheless, a study reported no sensory changes observed for a 100% CO2 atmosphere in common carp (Cyprinus carpio) [27]. The most important parameter for shelf-life evaluation is the development of microorganisms during storage. This evolution will be highly dependent on original microorganisms and their tolerance to the specific gases used.
Even so, it has been observed that MAP can reduce Pseudomonas spp. counts and H 2 S-producing bacteria. Likewise, atmospheres with higher CO 2 content are more efficient in reducing lactic acid bacteria (LAB) counts.
Besides microbiological safety, preserving good sensorial quality is particularly important for shelf-life evaluation. Some studies [6,8,23] reported changes in color, odor, and texture along the shelf-life period with VP and MAP. Nonetheless, a study reported no sensory changes observed for a 100% CO 2 atmosphere in common carp (Cyprinus carpio) [27].
Since VP and MAP affect microbial spoilage and the sensorial quality of foods, shelflife will also be affected. Longer shelf-lives are usually observed with MAP, followed by VP and, lastly, air packages. Normally, VP preserves the quality of the product for 2/3 days longer, compared to air packaging [11,21,25,28]. With MAP, there tends to be an increase in shelf-life of 4/5 days compared to air packs and 2/3 days compared to VP [10,11,[19][20][21]23,26]. However, some studies have reported different results (see more  Table 1). It is important to note that temperature of storage and the atmosphere used are important factors that affect shelf-life. For example, Stamatis & Arkoudelos (2007a) [28] studied chub mackerel (Scomber colias japonicus) under air packaging, VP, and MAP (50% CO 2 and 50% N 2 ) at 3 and 6 • C and observed, as expected, that the lower temperature allowed for an extension of shelf-life of 2 days for MAP and VP samples and 1 day for air samples comparing to the higher temperature. One work with sutchi catfish ((Pangasius hypophthalmus) investigated two different atmospheres: (1) 50% CO 2 and 50% N 2 ; and (2) 50% CO 2 and 50% O 2 , at 4 • C for 21 days and concluded that the second atmosphere, where O 2 was used instead of N 2 , extended the shelf-life by 2 days because of an extension of lag phase (of microbial development) with this atmosphere [25]. Another study using sea bass (Dicentrarchus labrax) with two different MAP mixtures: (1) 40% CO 2 , 50% N 2 and 10% O 2 ; (2) 60% CO 2 , 30% N 2 and 10% O 2 , at 4 • C during 21 days of storage, noticed an increase of shelf-life of about 4 or 5 days for the MAP samples with higher levels of CO 2 [9]. Notwithstanding, Lerfall et al. (2018) [24] studied the effects of four different MAP mixtures in saithe (see the details in Table 1) and observed the same shelf-life for all of them.  MAP reduced total production of ammonia, TVB-N and cadaverine. MAP enhanced the shelf-life at least twice since total mesophilic count and psychotropic microorganisms reached the upper limit of 7 log CFU g −1 on the 11th day (air-5th day; VP-7th day). [21] Red Drum (Sciaenops ocellatus)

• C, 29 days
Packaging: MAP (50% CO 2 and 50% N 2 ) and VP Putrescine and cadaverine were the prevalent amines and had higher counts on VP. At the end of the storage VP samples retained a slightly better appearance than MAP samples, with a firmer texture but had a stronger odor.   The spoilage of fresh fish is not exclusively determined by microbial development, but also by the formation of chemical indicators that reflect, for example, autolytic changes in the fish muscle, formation of biogenic amines, among other rancidity and oxidation associated compounds, these indicators need to be considered based on the shelf-life evaluation of fresh fish [35], although shelf life is usually limited at first instance by microbial development. Indeed, biogenic amines were evaluated in fish samples stored under different packaging conditions. Briefly and additionally, major results found in the literature regarding VP and MAP are displayed in Table 1.

Active Packaging
Active packaging systems are an innovative and alternative solution of high interest to food technologists. Food packaging and materials are categorized as active, according to the Regulation of the Commission of the European Union (EC No 450/2009), when their application aims to extend the shelf-life and maintain or improve safety and some physicochemical, quality, and sensorial properties of the packaged food due to the positive interaction between product, package, and environment [16,17,36].
Therefore, active packaging is more than just an inert barrier that protects food from external detrimental factors. In fact, this technology is based on the incorporation of active substances, components, and materials into the packaging material or within the package, designed to release or absorb substances into or from packaged food or its surrounding environment [36][37][38][39]. It is noteworthy that the main difference between active and intelligent packaging systems is the information about modifications in the quality status of the food product that is communicated to the consumer using intelligent packaging, instead of interacting with it and responding to changes in its properties, as happens with active packaging [17]. Intelligent packaging only monitors the microbiological and physicochemical modifications (like biological reactions, pH, or temperature) of packaged foods or the surrounding environment, transmitting the information to the consumer, using devices such as sensors (including biosensors, gas sensors, printed electronics, chemical sensors, and electronic nose) and indicators (including freshness indicators, leak/integrity indicators and time and temperature indicators) [16,17]. Intelligent packaging is not designed to interact with the packaged food or release active compounds as in active packaging, it merely helps to detect unsafe food [16,17].
This section is mainly focused on active packaging systems. The three main types and the most common and promising of active packaging systems of foods are: (i) gas control, including O 2 scavengers and CO 2 emitters, (ii) moisture control, and (iii) antimicrobials and/or antioxidants, which are active substances directly incorporated into the packaging material/films or sprayed on sachets, patches, or tablets [17,39]. To reduce, inhibit, or delay the microbial growth and enzymatic/oxidative reactions of food is the primary and common goal of all three methodologies [4]. Figure 3 summarizes the three main types of active packaging systems described above.
or the surrounding environment, transmitting the information to the consumer, using devices such as sensors (including biosensors, gas sensors, printed electronics, chemical sensors, and electronic nose) and indicators (including freshness indicators, leak/integrity indicators and time and temperature indicators) [16,17]. Intelligent packaging is not designed to interact with the packaged food or release active compounds as in active packaging, it merely helps to detect unsafe food [16,17].
This section is mainly focused on active packaging systems. The three main types and the most common and promising of active packaging systems of foods are: (i) gas control, including O2 scavengers and CO2 emitters, (ii) moisture control, and (iii) antimicrobials and/or antioxidants, which are active substances directly incorporated into the packaging material/films or sprayed on sachets, patches, or tablets [17,39]. To reduce, inhibit, or delay the microbial growth and enzymatic/oxidative reactions of food is the primary and common goal of all three methodologies [4]. Figure 3 summarizes the three main types of active packaging systems described above. . Active packaging technology applied to fishery products. Based on information available in [17]. Figure 3. Active packaging technology applied to fishery products. Based on information available in [17].
The gas control mechanism in active packaging technology is very similar and directly related to MAP. The substances that absorb (O 2 scavengers) or release (CO 2 emitters) atmospheric gases control the inside packaging environment. Briefly, O 2 scavengers prevent the oxidation of lipids and other sensitive compounds (like vitamins A, C, and E) and, thus, off-flavors and off-odors; and, whereas the CO 2 emitters slowdown or inhibit microbial growth. Nowadays, oxygen scavengers are the most used active packaging strategies and, as Remya et al. (2017) [40] reported, the commercial ZPT 200 EC O 2 absorber (Ageless ®® ), when applied to fresh Rachycentron canadum steaks during refrigerated storge, enhanced fish quality and shelf-life (shelf-life of 25 days compared to just 15 days for control samples). In fact, this O 2 scavenger reduced the O 2 concentration in the package to less than 0.01%, which translated into inhibition of the growth of (i) aerobic mesophilic and psychrotrophic microorganisms due to the lag phase extension and generation time, (ii) Pseudomonas sp. because they are strictly aerobic, and (iii) H 2 S-producing bacteria, as their enzymes were affected by O 2 depletion (effects observed when O 2 concentration is below 50%). So, this oxygen-reduced atmosphere promoted the growth of Gram-positive microorganisms such as LAB (which are facultative anaerobes) and significantly affected the growth of Gram-negative bacteria in fish samples. Therefore, volatile base formation values (TVB-N and TMA-N, highly dependent on microbial load) were delayed, and a similar inhibitory effect was observed for lipid oxidation.
Moisture control is mediated by diverse kinds of absorbents (made from a micro porous polymer like polypropylene-PP-or polyethylene terephthalate-PET) such as sheets, blankets, and pads, which are strategically placed under food products (mostly fish and meat) to control their exudates [17,41]. Dri-Loc ®® (Sealed Air Corporation, Charlotte, NC 28208 USA) and Tenderpac ®® (SEALPAC, Oldenburg, Germany) are two commercially available moisture control devices based on an absorbent pad and a dual compartment system, respectively. In fact, this type of moisture absorber is widely used in the food industry to decrease water activity (an important intrinsic factor) in food and thus inhibit microbial spoilage [16].
There are two main methodologies for manufacturing antimicrobials and/or antioxidant packaging systems, which are (i) the incorporation of active compounds/substances into sachets, pads, or tablets (independent devices) that are added to the conventional inert package, and (ii) the direct incorporation of active compounds/substances in the polymer matrix or on the polymeric film surface [17,38].
Blending and coating packaging films are produced differently (as represented in Figure 4). While, with the first, the active compounds are combined with the polymeric matrix and, in the second they are coated onto the surface of the polymeric film [4,38]. These systems are defined by a controlled release (to the headspace of the package) or reaction of the active compounds onto the food surface [39]. microorganisms such as LAB (which are facultative anaerobes) and significantly affected the growth of Gram-negative bacteria in fish samples. Therefore, volatile base formation values (TVB-N and TMA-N, highly dependent on microbial load) were delayed, and a similar inhibitory effect was observed for lipid oxidation.
Moisture control is mediated by diverse kinds of absorbents (made from a micro porous polymer like polypropylene-PP-or polyethylene terephthalate-PET) such as sheets, blankets, and pads, which are strategically placed under food products (mostly fish and meat) to control their exudates [17,41]. Dri-Loc ®® (Sealed Air Corporation, Charlotte, NC 28208 USA) and Tenderpac ®® (SEALPAC, Oldenburg, Germany) are two commercially available moisture control devices based on an absorbent pad and a dual compartment system, respectively. In fact, this type of moisture absorber is widely used in the food industry to decrease water activity (an important intrinsic factor) in food and thus inhibit microbial spoilage [16].
There are two main methodologies for manufacturing antimicrobials and/or antioxidant packaging systems, which are (i) the incorporation of active compounds/substances into sachets, pads, or tablets (independent devices) that are added to the conventional inert package, and (ii) the direct incorporation of active compounds/substances in the polymer matrix or on the polymeric film surface [17,38].
Blending and coating packaging films are produced differently (as represented in Figure 4). While, with the first, the active compounds are combined with the polymeric matrix and, in the second they are coated onto the surface of the polymeric film [4,38]. These systems are defined by a controlled release (to the headspace of the package) or reaction of the active compounds onto the food surface [39].  To select the most suitable packaging film manufacturing procedure, it is important to consider the type of polymeric matrix, the active substances to be incorporated and the physicochemical characteristics of the food product [38].
The packaging films can be from synthetic or natural origin. The most used synthetic polymers are low-density polyethylene (LDPE) and polystyrene, due to their physical properties and low production cost, but their widespread use is cause for environmental concern, due to their low degradability. Barbosa-Pereira et al. (2013) [42] performed a 21-day experiment with salmon muscle, wherein the authors combined low-density polyethylene polymers (LDPE, a synthetic film) with two different formulations in tocopherols, namely natural antioxidant products C: NUTRABIOL ®® -T90 at 1 and 5% and D: TOCOBIOL ®® -PV at 5%. The active films with natural antioxidant products C and D showed an important reduction in lipid oxidation of up to 40%. Considering the Film LDPE product C at 1% (film 2) and 5% (film 3), there were some small differences between them in the first 11 days of storage, but at the end of the experiments the reduction in lipid oxidation stabilized around 40%. At 21 days of storage, Film 4 (D: TOCOBIOL ®® -PV at 5%) was able to decrease lipid oxidation in salmon by 30-35%; however, the greatest inhibition of lipid oxidation was observed for film 3, possibly due to its tocopherol composition. Therefore, it can be concluded that the inhibition of the lipid oxidation process is directly correlated with the capacity of active compounds (present in commercial antioxidant products C and D) to scavenge free radicals and peroxide radicals from the peroxidation chain reaction.
Thus, biopolymers-composed mainly of proteins and polysaccharides or their combinations (such as chitosan, agar, and starch films)-have gained more attention for being extracted from natural and renewable sources, which gives them some advantages, such as specific biological activities (antimicrobial and/or antioxidant activity, for example) and the possibility of being renewable. Natural active compounds with antimicrobial and/or antioxidant capacities can be found in extracts from spices (like rosemary, cinnamon, and oregano), herbs (such as garlic, onion, and horseradish), and from food processing by-products (for example, olive leaves and pomegranate peel), essential oils, fungal and bacterial compounds (polypeptides like nisin, natamycin, and pediocin, and some bacteriocins), and functional enzymes. These compounds can be incorporated into packaging films, as opposed to synthetic ones (like chlorine dioxide, carbon dioxide and ethanol, which are volatile antimicrobials) [4,16,17]. However, to enable the application of these natural films in food, it is necessary to add plasticizers to reduce their brittleness, and neutral lipids to increase their hydrophobicity [4,17].
There has been an increase in available literature featuring the combination of biopolymers and natural active compounds to formulate new films to be used as active packaging materials. Rocha et al. 2018 [43] carried out a detailed experiment to evaluate the antimicrobial effect of protein hydrolysate (PH, a by-product obtained from Argentine croaker-Umbrina canosai-which has a high protein content) and/or clove essential oil (CEO) combined with agar film in prolonging the shelf-life of flounder (Paralichthys orbignyanus) fillets. A 15-day assay at 5 • C, showed that the PH-agar film with lower molecular weight (<10 kDa) has a higher content of hydrophobic amino acids (~50.0%) which, due to their positive charge, allowed the formation of hydrophobic bounds with the negatively charged bacterial cell membrane, leading to its disruption and, proving to be an effective film against to H 2 S-producing microorganisms, lactic acid bacteria, and total aerobic mesophiles. CEO-agar film also extends the shelf-life of flounder fillets, in a slightly superior manner compared to PH-agar film, due to its recognized antimicrobial properties-its hydrophobicity allows the disruption of bacterial cell membrane lipids and mitochondria, leading to loss of cytoplasm, and, in addition, phenolic compounds can interact with bacterial enzymes, promoting lesions that lead to loss of cell viability. So, these CEO/PH-agar films performed well in extending the shelf-life of flounder fillets by microbial inhibition and enhanced their properties. This study also evaluated the mechanical properties of films such as thickness, tensile strength (TS), elongation break (EB), water vapor permeability (WVP), solubility, color, opacity, and transparency, which are very important parameters for evaluating the applicability of these active films in foods. The PH-agar film showed the best properties when compared to the CEO-agar film, with similar thickness and transparency to the control-agar film, but with an increase in WVP and EB values and a reduction in the TS values, due to the presence of short chain hydrolysate peptides that act as effective plasticizers in the protein films, by decreasing intramolecular attractive forces and increasing chain mobility and free volume (EB and WVP parameters) and by preventing the polymer-polymer interactions (TS parameter). The main disadvantage of this PH-agar film is related to the greater water solubility due to the weak interaction between the short PH peptides and the agar matrix, which limits its application in fish preservation.
Regarding biopolymer enriched active compounds and their advantages for the environment compared to synthetic ones, advances in active packaging technology have been made to meet consumers' demands for healthier, greener, and more sustainable packaging materials. In this context, Kakaei & Shahbazi (2016) [44], among many other studies, investigated the effect of chitosan-gelatin films enriched with ethanolic red grape seed extracts (GSE, at 1-2%) and Ziziphora clinopodioides essential oil (ZEO, at 1-2%), on minced rainbow trout fillet. After 11 days of storage experiments (at 4 • C) the control samples (wrapped only with chitosan-gelatin film) faced an increase of about 9.0 log CFU/g for total viable counts (TVC) and psychrotrophic total counts (PTC), with a higher microbial reduction verified for the film with the combination of 2% ZEO + 2% GSE, where TVC increased up to 6.5 log CFU/g and PTC reached about 6.0 log CFU/g. The same behavior was observed for the other microorganisms tested, namely Pseudomonas spp., Pseudomonas fluorescens, Enterobacteriaceae, LAB, and Listeria monocytogenes where the same combination of 2% ZEO + 2% GSE showed the lowest counts after 11 days of storage (ranging between~3.5 and 5.0 log CFU/g), when compared to the other ZEO+GSE combinations. The microbial reduction for the 2% ZEO + 2% GSE films was even more relevant when compared to the control samples (on day 11 counts were in a range between~7.0 and 8.0 log CFU/g for control samples). Therefore, these results showed the high effectiveness of the chitosan-gelatin films in retarding and inhibiting the growth of aerobic microorganisms, as they act as an oxygen barrier around the fish and additionally block access to essential nutrients and metals by bacteria. Furthermore, the presence of ZEO and GSE compounds in the films causes a significant antibacterial effect due to their high content of phenolic compounds (ZEO has mainly thymol and carvacrol and GSE has mainly resveratrol, gallic acid, caffeic acid, and hydroquinone), which, when combined, exert a synergistic antimicrobial effect, such as the inhibition of bacterial enzymes and the establishment of bonds with cell membrane components that promote the loss of cell viability.
Physicochemical analyses, namely of pH value, peroxide value (PV), and total volatile base nitrogen (TVB-N) content, were also performed and it was concluded that the films with 2% ZEO + 2% GSE obtained the best results. In the case of pH, the final pH value, at 11 days of storage, was 7.4 in contrast to~6.7 for the best film, because of the bacterial reduction and due to decomposition of nitrogenous/alkaline compounds. Considering the PV values, the control samples exceeded the legal limit for fresh fish, showing a value of 1.9 meq peroxide/1000 g lipids (legal value for fresh fish is between 0.04-0.06 meq peroxide/1000 g lipids) at a higher rate when compared to active films samples. Finally, the TVB-N content values reinforce the effectiveness of chitosan-gelatin films with ZEO and GSE, because it was found that the TVB-N content of control samples significantly increases compared to the acceptability value of 25 mg N/100 g of fresh fish, in contrast with ZEO and GSE, where the values remained below the limit-the possible explanation being the lower microbial load or the low capacity of the remaining bacterial load to carry out the oxidative deamination reaction of the non-protein nitrogenous compounds.
Active packaging systems have been investigated to preserve perishable foods, namely fish, pork/bovine/poultry meat products and their derivatives, as well as for fruits, vegetables, and dried products, among others [4,16,38]. In fact, it is for fish and fishery products that this technique is most promising, due to its benefits for foods (safety, quality and shelf-life), industry, and consumers [4,39]. However, the adoption of active film packaging systems depends on factors such as production cost and legislative approval of active substances and polymer matrices for use in contact with food, even though they are useful to extend the shelf-life, improve safety, and improve or maintain quality and sensorial parameters of fish and fishery products [16,39].
Given the diversity of matrix polymers and active compounds, it is necessary to choose the most appropriate combination, based on the characteristics of fish and fishery products [4]. By adjusting the film system, the release rate of active compounds must be defined to retain a specific concentration in the fish-based products and to compensate for biochemical reactions during storage [4,37]. Examples of active packaging films where natural active compounds have been incorporated into natural/synthetic polymers in fish and fishery products are represented in Table 2.
There are already some commercially available antimicrobial packaging systems such as Biomaster ®® and Agion ®® for active packaging systems, but also O 2 scavengers and moisture control devices [16].

Chemical Additives
The aforementioned preservation strategies relied on either changing the natural atmosphere wherein fresh fish was packaged or by the impregnation of packaging materials with chemicals that slowly migrate to fresh fish, exerting an antioxidant and/or antimicrobial effect. Nevertheless, such chemicals can be applied directly to fresh fish, either by dipping or spraying prior to packaging, providing an additional hurdle to the refrigeration processes. Green tea films effectively reduced microbial growth and some spoilage indicators such as TVB-N, TMA-N, and pH value, in hake. Probiotic films have been able to extend the shelf-life of hake and transmit some probiotic bacteria to fish. [46] Rainbow trout (Oncorhynchus mykiss) Chitosan-gelatin Ethanolic red grape seed extract (GSE) and Ziziphora clinopodioides essential oil (ZEO) Reduction of lipid oxidation and bacterial growth, increasing the shelf life of rainbow trout at refrigerated storage. This section will focus on the main chemicals (single or in combination with icing systems) to preserve fresh fish, as well as natural extracts, bacteriocins, among others, which have different effects on fish quality and shelf-life. Figure 5 summarizes the main chemicals and application methods used to preserve fresh fish.

Organic Acids
Organic acids are known to have antimicrobial and antioxidant properties, are generally recognized as safe (GRAS) [48], and their use as food preservatives has been extensively reviewed and forbidden in some cases, as is the case for benzoic acid, whose use is being gradually reduced/eliminated in several countries due to toxicity issues [49].
The organic acids action against microorganisms depends on the carbon chain length and degree of unsaturation, but overall, the pKa of the acid influences its antimicrobial mechanism of action [50]. The organic acids exist in a pH-dependent equilibrium between the undissociated and dissociated state, and the first one is the primarily responsible for the antimicrobial activity [51,52]. At low pH, they have an optimal inhibitory effect, which is a result of the undissociated organic acid being able to freely cross the plasma membrane to enter the bacterial or fungal cell. Once inside the cell, the molecule confronts a higher pH and will then dissociate, releasing charged anions and protons [52]. These accumulating anions have been found to be toxic and able to inhibit metabolic reactions [50].
Other mechanisms have been proposed, as the case of membranes disruption and stress on intracellular pH homeostasis [53,54]. According to the available literature, fresh fish preservation by organic acids/salts may follow two different strategies, namely by dipping fish samples for a certain period, followed by packaging and refrigeration, or by using organic acids to prepare flake ice for cooling fresh fish.
mention the scientific name of the species.

Chemical Additives
The aforementioned preservation strategies relied on either changing the natural atmosphere wherein fresh fish was packaged or by the impregnation of packaging materials with chemicals that slowly migrate to fresh fish, exerting an antioxidant and/or antimicrobial effect. Nevertheless, such chemicals can be applied directly to fresh fish, either by dipping or spraying prior to packaging, providing an additional hurdle to the refrigeration processes.
This section will focus on the main chemicals (single or in combination with icing systems) to preserve fresh fish, as well as natural extracts, bacteriocins, among others, which have different effects on fish quality and shelf-life. Figure 5 summarizes the main chemicals and application methods used to preserve fresh fish.

Organic Acids
Organic acids are known to have antimicrobial and antioxidant properties, are generally recognized as safe (GRAS) [48], and their use as food preservatives has been extensively reviewed and forbidden in some cases, as is the case for benzoic acid, whose use is being gradually reduced/eliminated in several countries due to toxicity issues [49].
The organic acids action against microorganisms depends on the carbon chain length and degree of unsaturation, but overall, the pKa of the acid influences its antimicrobial mechanism of action [50]. The organic acids exist in a pH-dependent equilibrium between the undissociated and dissociated state, and the first one is the primarily responsible for the antimicrobial activity [51,52]. At low pH, they have an optimal inhibitory effect, which is a result of the undissociated organic acid being able to freely cross the plasma membrane to enter the bacterial or fungal cell. Once inside the cell, the molecule confronts a

Fresh Fish Dipping
The first known report, as the authors are aware, regarding the use of a dipping process with organic acids in fresh fish belongs to Martinez & Gilderb (1988) [55], who showed the possibility of reducing anchovy (Engradis encrasicholus) degradation by pH depletion from 8 to 5, followed by cold storage at 1 • C in sea water for 35 h. The pH depletion allowed partial inhibition of the activity of proteolytic enzymes. Some factors may be considered when applying the dipping strategy, such as the concentration of organic acid, dipping time, and air-availability within the package (air or vacuum package) as well as the storage temperature and draining time after dipping. For example, Kim, et al. (1995) [56] evaluated the effects of lactic acid (2 and 3%) dipping (1 and 5 min) on catfish fillets (Silurus glanis) and observed that, for the lowest lactic acid concentration, the dipping time influenced microbial development over 9 days of storage (at 4 and 10 • C), with microbial loads being quite similar for the concentration of 2% with 5 min of dipping time and 3% concentration with 1 min of dipping time. Manju et al. (2008) [57] evaluated the effects of sodium acetate and potassium sorbate, both at a concentration of 2%, on pearl spot (Etroplus suratensis) dipped for 30 min and stored under refrigerated conditions (1-2 • C) in combination with vacuum-and airpackaging. The authors reported a shelf-life extension, in both packaging methods, of up to 16 days when compared to control samples (7 days without organic acid salts). The addition of sodium acetate and potassium sorbate yielded considerable reductions in TVB-N and TMA-N, improved textural properties, and slowed down microbial development, especially for vacuum-packed samples, which correlated well with the lower levels of TVB-N and TMA-N found in samples packed at such conditions, with the authors stating that this could be due to microbial development control, as well as the decrease of bacteria's ability to perform oxidative deamination of non-protein nitrogen. In addition, the authors observed that samples treated with potassium sorbate presented lower values of TVB-N compared to those treated with sodium acetate, which was thought to be due to the greater inhibition of the first salt on Gram-negative bacteria. The susceptibility of Gram-negative bacteria to acidity was also noticed by Kim et al., (1995) [56], who stated that undissociated organic acids may penetrate within the lipid membranes of microorganisms, leading to the protonation of anionic components (as is the case for phosphate and carbonyl groups) of such structures, weakening molecular interactions, leading to membrane disruption [58]. Organic acids may also penetrate within microbial cytoplasm, where they usually dissociate and tend to donate protons (H + ), forcing microorganisms to expel the excess of H + , which is an energy consuming process, thus limiting microbial proliferation. Consequently, the cytoplasm of the microorganisms reaches pH levels that are unbearable for microbial development [59].
This could be an interesting strategy to inhibit pathogens such as L. monocytogenes, Escherichia coli [60], and Vibrio spp. [61] and reduce bacterial loads responsible for fish spoilage, as is the case for Pseudomonas spp., LAB, H 2 S-producing bacteria, and Enterobacteriaceae [62]. These are the main bacterial groups responsible for fish spoilage and foodborne illnesses caused by the consumption of raw or undercooked fish. Additionally, pathogens such as L. monocytogenes and E. coli could also be inhibited [60].
While the dipping strategy seems to have quite beneficial effects on the shelf-life of fresh fish, high dipping times and organic acid concentrations may lead to fish muscle digestion and undesirable sensorial attributes, especially of color parameters (which can be minimized with the cooking process) [57]. Usually, during fish dipping, the fish pH tends to decrease, depending on the organic acid concentration and dipping time, which will have a major impact on fish texture, and in water-holding capacity (WHC), as when the pH of fish reaches the isoelectric point, proteins tend to aggregate or denature, leading to the reduction of WHC [63]. In addition, the pH of fish tends to increase during cold storage due to microbial deamination and protein breakdown, so dipping fish in organic acids can slow down microbial proliferation (as previously described) and inhibit some enzymatic reactions [64] given these effects on proteins. Some other examples of studies covering fresh fish dipping and spraying are displayed in Table 3. In addition to the effects of fish dipping previously described, one interesting effect of this technique is the reduction of heavy metals levels in fish from fresh and sea waters, for instance, a reduction of heavy metals was verified in freshwater fish, Tilapia nilotica (Oreochromis niloticus), pre-treated with acetic acid, which could be due to the formation of insoluble acetate salts of these metals [65]. Briefly, Elnimr (2011) observed a reduction of 41.6 and 51.9% of cadmium and lead in Basa fish (Pangasius hypothalmus) after dipping in acetic acid solution (5%) for 15 min. Bolti fish (Oreochromis niloticus) Acetic and citric acid (1 and 3%, respectively, 5 min) and a mixture of both 4 • C, air packed, for 12 days Slower microbial proliferation, along with catalase and protease activity decrease in dipped fish, especially when the mixture of both acids was used.
[64]  Lower microbial loads, pH, and peroxide values in fish fillets sprayed with the combination of both organic acids, along with improved sensorial characteristics. [74] * The authors did not mention the scientific name of the species.

Fresh Fish Organic Acid-Icing
The organic acid-icing process consists in preparing ice flakes from aqueous solutions of organic acids that will be directly in contact with the fish surface, thus allowing a slow migration of such compounds, as well the possibility of keeping fresh fish under chilling conditions near 0 • C.
In this sense, Rey et al. (2012) [75] assessed the performance of an icing-system containing ascorbic, citric, and lactic acids, with concentrations of about 400 and 800 mg/kg (C-400 and C-800) of ice under refrigeration conditions (4 • C) to preserve hake (Merluccius merluccius), megrim (Lepidorhombus whiffiagonis), and angler (Lophius piscatorius). Generally, it was verified that the C-800 icing-system retarded microbial development, along with lower levels of TMA-N formation for 12 days of storage, and with a good overall acceptance by the sensorial panel, which attributed a sensorial shelf-life between 8 (megrim) and 12 (hake and angler) days, especially concerning muscle odor and taste.
The effects of organic acid-icing (like the one described above) on fish lipid oxidation (primary, secondary, and tertiary) and hydrolysis were assessed by García-Soto et al. (2011) [76], using hake, megrim, and angler as case studies. The results showed that lipid oxidation was partially inhibited in the three studied fishes, especially for the icing-system containing the highest organic acid concentration (C-800). The authors attributed this effect to the antioxidant properties of the organic acids used. Lipid hydrolysis (free fatty acids content) was also generally inhibited, especially in megrim and angler, by the C-800 icingsystem. The hydrolysis of lipids is, in a first phase, mainly due to endogenous enzymatic reactions carried out by lipases and phospholipases, and by microbial activity in a second phase. So, it is noteworthy that the pH of fish samples can be depleted by the icing system, resulting in partial enzymatic reaction inhibition (as explained before for fish dipping), as well as by inhibition of microbial development in lean fish. This behavior was validated in a fatty fish (mackerel, Scomber scombrus) by Sanjuás- Rey et al. (2012) [77], who observed microbial development inhibition and lower values of TMA-N and TVB-N by using an icing-system consisting of 0.05% of citric, acetic, and lactic acids.

Ozone
The first report of ozone usage in the food sectors dates from 1936, when it was used in France to treat shellfish, and was recognized as a secondary direct food additive to destroy food pathogenic microorganisms in 2001 by the Food and Drug Administration (FDA). From this date onwards, several industries have been using ozone as a surface decontaminant, especially for fruits, vegetables, eggs, seafood, meat, sausage, and dairy industries [78].
The application of ozone to fresh fish follows a strategy of ozone dissolution in water, with the fish individuals or processed fish being dipped for a certain period, or it can be combined with icing systems, where it is stored at temperatures slightly below 0 • C, remaining in contact with the ozone (dissolved and entrapped in ice) during the storage period. In this section, both techniques will be presented and then discussed in the next section.

Ozonized Water Dipping
As previously stated, this strategy consists of dipping fresh fish in ozonized water for a certain period. Several variables are to be considered when performing this method, such as ozone concentration, exposure time (dipping time), and ozonized-water temperature.
When it comes to ozone concentration, it is expected that higher concentrations may yield higher microbial load inactivation/injuries, as reported by Silva & Gonçalves (2017) [79], who treated Nile tilapia fillets (Oreochromis niloticus) with ozonated water (0.5, 1.0 and 1.5 mg/L for 0 to 15 min) and observed a reduction of about 3 and 4 log units of total aerobic mesophiles in the whole tilapia-surface at an ozone concentration of 1 and 1.5 mg/L and an exposure time of 10 and 15 min. Regarding tilapia fillets (Oreochromis niloticus), lower microbial loads reductions were observed, with both concentrations of 1.0 and 1.5 mg/L and exposure times of 10 and 15 min having very similar effects. Nevertheless, the inactivation rates decreased by increasing the exposure time, with the authors attributing this effect to the decreasing oxidizing effect of ozone due to its instability and consequent dissociation, releasing oxygen to water. Lipid oxidation increased with exposure time and ozone concentration, which can also be explained by the dissociation of ozone and release of oxygen, promoting lipid oxidation, increasing the formation of peroxides and/or products resulting from their decomposition. The proposed mechanism of lipid-enhanced oxidation by ozone relies on the generation of free O 2 and HO . radicals that have a high oxidationreduction potential. Generation of these radicals in water (H 2 O − can be formed by reacting with O 2 − and HO) enhances lipid peroxidation and protein denaturation [80]; thus, both concentration and exposition time are to be carefully controlled to avoid undesirable changes in the fish surface. Some examples of the effects of ozone dipping and spraying are displayed in Table 4. Table 4. Literature regarding the effects of dissolved ozone dipping and spraying on fresh fish.        [86]

Concentration
Trout * 0.1 mg/L, 2 h, ** 5 • C, air packed, for 9 days Shelf-life extension of ozonized trout from 4 to 6 days compared to control samples, along with lower values of TVB-N and peroxides' index. No major differences in protein content for ozonized samples compared to controls. [87] TVB-N: Total volatile basic-nitrogen; TMA-N: Trimethylamine-nitrogen; TBARS: Thiobarbituric acid reactive substances; TBA: Thiobarbituric acid. * The authors did not mention the scientific name of the species. ** The authors did not mention the water temperature.
Ozone-washing can also be used as a tool to remove muddy-flavors (such as those derived from geosmin), as reported by Zhang et al. (2016) [88], who was able to remove 69.2% of geosmin after an ozuone-flotation treatment for 15 min, against 54.3% for conventional ozone water washing for the same treatment time.

Ozonized Icing-Systems
The ozone-icing process consists in preparing ice flakes with ozone that will be directly in contact with the fish surface, allowing fresh fish to be kept under chilling conditions near 0 • C, in a similar process described previously for organic acid icing-systems.
For example, Campos, Losada, Rodríguez, Aubourg, and Barros-Velázquez (2006) [89] evaluated the effects of ozonated slurry ice (−1.5 • C), and ozone concentration of 0.2 mg/L to preserve turbot (Psetta maxima) stored at 2 • C for 35 days, and observed that lipid hydrolysis and oxidation slowed down in the presence of ozone in the slurry ice, in addition to the lower microbial loads in fish individuals (nevertheless, parameters such as nucleotide degradation and TMA-N were not influenced by the presence of ozone). The authors also reported a sensorial shelf-life for ozonated slurry ice of 14 days, compared to 7 days for control samples. Comparable results were obtained by Aubourg et al. (2006) [90] for megrim (Lepidorhombus whiffiagonis) preserved by ozonated slurry ice (at a concentration of 0.08 and 0.16 mg/L), with megrim individuals presenting improved sensorial attributes after 14 days of on-board storage when compared to un-ozonated slurry ice, showing that on-board ozonated slurry ice systems are an interesting approach to preserve fresh fish with minimal impact on fish quality and safety. Another study from Chen et al. (2016) [91] with bighead croaker (Collichthys niveatus) discovered that ozonated slurry-ice can retard the degradation of myofibrillar proteins and reduce the deterioration of fish microstructure. In addition, this methodology proved to have inhibitory effects on formation of TVB-N, peroxide index, and thiobarbituric acid reactive substances (TBARS) values.

Natural Extracts
The application of natural preservatives in foods has caught the attention of the general public. Foods containing natural preservatives will be chosen over those containing synthetic preservatives [2]. According to Gyawali & Ibrahim (2014) [92], natural preservatives should have a broad action against bacteria and fungi, be non-toxic, and be active in low concentrations, not change the color and flavor of foods, have no pharmaceutical applications, be clean-label, and cost-effective. Most natural preservatives are derived from microorganisms, animals, and plants [93].

Extracts from Plants
Plants, especially their secondary metabolites, present a wide range of applications, especially in pharmaceutical and food sectors. These extracts usually show antimicrobial activity, attributed to polyphenolic compounds which come into contact with bacterial membranes and lead to their disruption and cell leakage, along with the formation of hydroperoxides [2]. In addition, these extracts may also present antioxidant activity, which may be interesting for fish preservation by means of slowing down lipid oxidation. Table 5 presents the effects of some plant extracts on fresh fish preservation.   Similar to organic acid dipping, the effects of plant extracts in fresh fish are dependent upon extract concentration, dipping time, and temperature, since excessive dipping can result in undesirable physicochemical and sensorial effects.
The origin of the extracts and extraction solvent may influence the evolution of fish degradation rates during refrigerated storage. For example, Yazgan et al. (2020) [100] evaluated the feasibility of water and ethanol extracts of propolis (0.4 and 0.8%) in sardine (Sardine aurita) and observed microbial load inhibition for both extracts, which was more pronounced for ethanolic extracts. The authors also reported that, despite ethanolic extracts providing the best sensorial scores, chemical parameters such as TBARS and peroxide values were lower for water extracts when compared to ethanolic extracts, showing that the extract composition (influenced by the extraction solvent) plays a significant role in antioxidant activity of the compounds extracted [101], and, consequently, on the feasibility of the extracts to preserve fresh fish. The fact that the sardine samples were vacuum-packed also inhibited some oxygen-dependent reactions.
In this sense, the addition of natural extracts can even be combined with other hurdles to microbial development to further improve the shelf-life of fresh fish. Houicher et al. (2015) [102] combined VP with ethanolic extracts of mint and artemisia on sardine (Sardina pilchardus) kept under refrigeration for 21 days. Both extracts, at 1% concentration, yielded lower values of histamine, tyramine, and cadaverine, while mint extracts were more effective to decrease biogenic amines production. Nevertheless, artemisia extracts were more effective to inhibit histamine-forming bacteria.
The application of natural extracts in icing-systems was assessed by Miranda et al. (2018) [103], who evaluated the combined effect of ethanolic quinoa extracts (at a concentration of 0.05 and 0.20%) and icing-system to preserve Atlantic chub mackerel (Scomber colias). The results showed that lipid hydrolysis was lower during the 13 days of storage, which correlated well with lipolytic bacteria growth slow down. In addition, the peroxide, TBARS and fluorescent compound values observed for fish individuals in the icing-system with 0.20% quinoa extracts were lower than those without quinoa extracts. These results were attributed to the phenolic compounds and other hydrophobic compounds with antimicrobial activity.
Indeed, the use of natural extracts in combination with other hurdles (such as MAP and vacuum packaging, refrigeration, etc.) can enhance the shelf-life of fresh fish even further, as an additional hurdle (provided by the natural extracts) is being added to the system. The combination of multiple natural extracts is also reported in the literature, although it must be carefully studied to avoid loss of fresh fish sensorial attributes [104]. Table 6 summarizes some examples of the use of the multi-hurdle concept for fish preservation, and the main effects on the fish products.  The combination of extracts and MAP allowed for better microbial growth control and lower melanosis formation compared to MAP alone. [108] TVB-N: Total volatile basic-nitrogen; TMA-N: Trimethylamine-nitrogen; TBARS: Thiobarbituric acid reactive substances; GFSE: Grapefruit seed extract. * The authors did not mention the scientific name of the species. ** The authors did not mention the dipping temperature.
In a circular economy approach, the use of plant-based agri-food wastes with antimicrobial activity may be an interesting approach to valorize these wastes, allowing reduction of food waste. For example, Shinde et al. (2015) [109] reported a 2-fold shelf-life of Indian mackerel (Rastriliger kanagurta) dipped in 0.5 and 1% of pomegranate peel extract, which also resulted in lower values of TMA-N and TVB-N and lower lipid hydrolysis for both concentrations. As expected, higher concentrations of extract allowed improved control of microbial growth. Pomegranate peels are very rich in flavonoids and tannins that are known to have high antioxidant activity, along with other agri-food wastes, such as potato and apple peels, rice bran, and grape seeds, among others, which may be an interesting research topic to evaluate in coming years.
Plant-based extracts can be combined with other natural compounds, such as bacteriocins, which will provide an amplified antimicrobial effect and improved physicochemical stability of fresh fish. In this sense, Gao et al. (2014) [97] combined rosemary extracts with nisin on pompano fish (Trachinotus ovatus) and observed that the combination of extracts resulted in lower values of TBARS, peroxide indexes, TVB-N, TMA-N and improved sensorial and texture parameters, along with microbial development slow down.

Extracts from Algae
Algae have received a lot of attention lately due to their abundance, being natural sources of terpenes, phlorotannins, phenolic compounds, fatty acids, steroids, halogenated ketones and alkanes, acrylic acid, polysaccharides, and cyclic polysulphides that have potent antioxidant and antimicrobial capacity [2]. Considering this, Wang et al. (2009) [110] reported that phlorotannins can inhibit oxidative phosphorylation and binding to bacterial membranes, proteins, and enzymes, leading to cell lysis. In addition, Amorim et al. (2012) [111] suggested that some sulphated polysaccharides from algae may bind to cell wall compounds and cytoplasmic membrane and penetrate within the cell and bind to DNA, which will increase cytoplasmatic membrane permeability, thus slowing down microbial development. As an example, Miranda et al. (2018) [103] included ethanolic extracts from Bifurcaria bifurcata to evaluate their potential to extend the shelf-life of megrim (Lepidorhombus whiffiagonis). During the 14 days of storage, it was observed that indigenous microorganisms were inhibited by the algae extracts, and TMA-N values and lipid hydrol-ysis were lower for fish samples with algae extracts. Oucif et al. (2018) [112] combined ethanolic-water extracts of the algae Cystoseira compressa with an icing system to preserve horse mackerel (Trachurus trachurus) and observed that microbial development slowed down during 11 days of storage. The addition of algae extracts also resulted in lower lipid hydrolysis rates, along with lower values of TMA-N and reduced lipid oxidation (primary, secondary, and tertiary) compared to control samples (where no extract was added).

Latic Acid Bacteria and Bacteriocins
According to Huss et al. (1995) [116], candidate protective cultures for fish preservation must fulfil four essential criteria, as follows: (1) present no threats to health; (2) have the ability to grow and compete with other cultures under refrigeration conditions (5 • C); (3) must present a consistent and continuous antimicrobial activity; (4) must not spoil fish (by means of off-flavors and odor production, slime, etc.).
Bacteriocins are usually produced by LAB in order to create a more competitive environment for nutrient uptake, i.e., while bacteriocins inhibit other microbial consortia proliferation, LAB take advantage of such inhibition and can uptake the available nutrients [117]. Several studies have demonstrated the effectiveness of bacteriocins (peptides containing between 30 and 60 amino acids), especially those produced by LAB against fish pathogens and degradative microorganisms [2]. Nisin, lacticin, pediocins, and reuterin (an organic compound derived from D-ribose) are, by far, the most studied bacteriocins for preserving fresh fish. Nevertheless, this strategy relies on direct application of LAB cultures by fish dipping/spraying, or by application of the isolated bacteriocins [2].
In this sense, Cao, Liu, Chen, Yang, and Li, (2015) [118] evaluated the effects of Lactobacillus plantarum 1.19 dipping of tilapia (Oreochromis niloticus) fillets and observed a lower increase in K-value (freshness index of fish flesh, whereas high K-values are related with fish spoilage) and total aerobic mesophiles during refrigerated storage (4 • C), as well as a better sensorial score when compared to undipped samples. In another study, Anacarso et al. (2014) [119] sprayed fresh Atlantic salmon (Salmo salar) with Lactobacillus pentosus 39 and observed its activity against Aeromonas hydrophila (a Gram-negative foodborne pathogenic bacteria that is quite resistant to sanitizing agents and able to thoroughly reproduce under refrigeration conditions) and L. monocytogenes, suggesting that, under refrigeration conditions, Lact. pentosus 39 was able to reduce the counts of both aforementioned microorganisms, in addition to the total psychrophilic bacteria. Although, the success of this technique requires a good control of the cold-chain, as a simulated breakage of the cold-chain (for 12 h) resulted in the development of both pathogenic microorganisms.
When it comes to the direct application of bacteriocins, Sarika and colleagues [120] studied the effects of three different concentrations of enterocin CD1 (0.1, 1 and 10% v/v) on the preservation of reef cod fish (Epinephelus diacanthus) and reported a 2-log units reduction of total aerobic microorganisms for the highest concentration of enterocin CD1 after 28 days of storage at 4 • C, with the authors also reporting that this bacteriocin performed better than the conventional sodium benzoate to extend the shelf-life of cod. The regulatory status on the use of LAB and bacteriocins for food preservation must be carefully considered. When it comes to regulatory status, nisin is the only bacteriocin licensed as a biopreservative. To overcome this limitation, in situ production by lactic acid bacteria is a common approach and several bacteriocinogenic cultures are commercially available. To be used in food production, LAB species must be granted the FDA GRAS designation or be included in the European Food Safety Authority's (EFSA's) list of qualified presumption of safety (QPS) recommended biological agents [121,122].
Further research is needed to evaluate the impact of bacteriocins and lactic acid bacteria on fish quality and safety, as well as to meet regulatory requirements to ensure their safe application in the food industry.

Conclusions
Refrigeration and freezing are common methods used to preserve fresh fish, which is perishable due to its rich nutritional composition and physicochemical characteristics. However, these low temperature-based technologies cause some detrimental quality changes, such as off-odors, off-flavors, rancidity, and loss of texture; therefore, several technologies have been introduced to improve the overall preservation of the product.
Vacuum and modified atmosphere packaging technologies are characterized by the manipulation of the atmosphere of the package, offering an additional hurdle to the physical barrier protecting the fishery products. This allows the slowdown of microbial deterioration and the rate of degradation reactions and, consequently, an increase in the shelf-life. Overall, increases in the shelf-life of fresh fish by MAP of 2/3 days and 4/5 days, when compared with VP and air packages, respectively, along with better sensorial quality have been reported.
By adding active compounds with antimicrobial and/or antioxidant capacities to synthetic or natural packaging films, active packaging technology has a positive impact on the shelf-life and quality characteristics of fresh fish. As in active packaging, chemical additives (like organic acids and ozone) and natural extracts make it possible to slow down microbial proliferation and decrease TVB-N and TMA-N values, both important parameters related to the shelf-life and sensorial quality of fresh fish.
Despite the advantages of the use of chemical-based strategies for fresh fish preservation here presented and discussed, both regulatory and consumer approval challenges must be overcome until real commercial applications can be safely used in the industry. Moreover, an intense validation procedure must be performed to fully elucidate and convince regulatory authorities on the safe use of natural extracts as food preservatives, alongside with raising consumer awareness of the advantages of such procedures in the food industry. Nevertheless, even though some of the presented preservatives exemplified here come from natural sources, their toxicity is yet to be evaluated, as well as the interactions that such compounds can have either with the fish products or packaging.
In conclusion, these chemical-based strategies are proven to successfully impact fresh fish preservation and the interest in their application will certainly warrant new developments in research in the future.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author, Jorge A. Saraiva, upon reasonable request.