Mechanistic Insights into Fish Spoilage and Integrated Preservation Technologies

Abstract

The global fish industry faces persistent challenges due to the inherent perishability of fish, driven by enzymatic autolysis, lipid oxidation, and microbial proliferation. Although numerous studies have characterized these individual spoilage pathways and evaluated discrete preservation techniques, practitioners still lack a unified, mechanism-based framework that links spoilage chemistry to targeted interventions. This gap prevents the rational selection and optimization of preservation methods. In this review, we first synthesize recent multi-omics and microbiological findings to delineate the molecular drivers of post-harvest fish spoilage. We then critically map a suite of preservation approaches—including low-temperature treatments (refrigeration, super-chilling, freezing), high-pressure processing, modified atmosphere packaging, nanoemulsion and essential-oil coatings, pulsed electric fields, and ozonation—onto the specific mechanisms they mitigate. By comparing efficacy metrics, practical constraints, and emerging innovations, our mechanism-driven roadmap clearly defines the problems we address and offers actionable guidance for developing more effective and sustainable fish preservation strategies.

1. Introduction

The global fish industry is experiencing rapid growth and has become a crucial source of high-quality protein. With advancements in fishing and aquaculture technologies, the market now offers an increasingly diverse range of fish species, encompassing both marine and freshwater varieties to cater to the varied preferences of consumers. Fish are not only diverse in species but also highly nutritious, containing up to 20% high-quality protein by weight, which has a high biological value due to the presence of essential amino acids, unsaturated fatty acids (such as omega-3), lipid-soluble vitamins, and minerals like calcium, phosphorus, and selenium, all of which are essential for human health [1,2,3,4,5].

However, fish are highly perishable, a characteristic that presents significant challenges to the industry. Once caught, fish quickly undergo a series of complex spoilage processes, including autolytic enzymatic spoilage, oxidative spoilage, and microbial growth [6]. Their high perishability stems from intrinsic compositional and structural traits: a moisture content of approximately 75–80%, which fosters bacterial growth; low connective tissue levels that render muscle fragile and susceptible to enzymatic degradation; abundant free amino acids and non-protein nitrogen compounds that fuel microbial metabolism and produce off-flavors and toxic metabolites; and a near-neutral pH (6.0–7.0) that optimizes both enzyme activity and microbial proliferation [7,8]. This spoilage is further accelerated by the high levels of non-protein nitrogen compounds in fish flesh, low acidity (pH > 6), and the abundant resident microbiota [9]. Enzymatic and oxidative activities that occur after harvest lead to significant changes in sensory and nutritional properties, resulting in a loss of freshness, which consumers perceive as unfavorable changes in odor, flavor, and texture, raising serious food safety concerns [10]. The rate of spoilage is influenced by factors such as fish species, sanitary conditions on board, storage duration, and temperature.

Despite extensive research, the existing literature often examines fish spoilage mechanisms and preservation methods in isolation rather than integrating mechanistic insights with preservation technologies. Consequently, there remains a significant knowledge gap regarding comprehensive, integrative approaches that effectively target specific spoilage pathways. By providing a thorough mechanistic analysis of post-harvest spoilage processes—including enzymatic autolysis, lipid oxidation, and microbial proliferation—and simultaneously evaluating state-of-the-art preservation technologies designed to inhibit these pathways, this review bridges that gap. By linking spoilage mechanisms directly to targeted interventions, we offer a mechanism-based framework to guide the development of more effective and sustainable fish preservation strategies, addressing the dual imperatives of food-quality enhancement and safety assurance in the global fish industry.

2. Mechanisms of Spoilage

The spoilage of fresh fish is a multi-faceted phenomenon influenced by various interrelated systems, some of which may inhibit others [5]. The deterioration of fish quality can be attributed to three primary mechanisms: enzymatic autolysis, oxidation, and microbial proliferation [9]. Figure 1 illustrates three core fish spoilage mechanisms, including enzymatic autolysis, lipid oxidation, and microbial proliferation, and their interrelationships.

2.1. Enzymatic Autolytic Spoilage

2.1.1. Pre-Rigor Phase (T = 0 to Onset of Rigor)

As shown in Figure 2, immediately after death (T = 0), fish muscle shifts to anaerobic metabolism: ATP is rapidly hydrolyzed to ADP and AMP, lactic acid accumulates, and pH falls from about 7.0 to 6.2–6.8, triggering irreversible actomyosin cross-bridges and the onset of rigor mortis [11].

2.1.2. Rigor Mortis Phase

During rigor mortis, muscle fibers remain rigid and inextensible because insufficient ATP is available to detach myosin from actin. The timing and intensity of this phase depend on species-specific glycogen reserves, storage temperature, and ionic strength: lower temperatures delay rigor onset but prolong its duration, whereas higher temperatures accelerate both onset and resolution [12].

2.1.3. Post-Rigor Autolysis

Once rigor resolves, a cascade of endogenous enzymes degrade muscle and connective tissues. Nucleotide-degrading enzymes (ATPases, AMP deaminase, 5′-nucleotidase, nucleoside phosphorylase) convert residual ATP through ADP, AMP, and IMP into inosine and hypoxanthine, the latter imparting bitterness [13,14]. Proteases—including calpain [15], cathepsins B/D/L [13,15], collagenases, trypsin, and chymotrypsin—then cleave myofibrillar proteins (myosin, actin, tropomyosin, troponins) and collagens, weakening fiber integrity and reducing water-holding capacity [16]. Concurrently, lipases and phospholipases hydrolyze triglycerides and phospholipids into free fatty acids, which oxidize into aldehydes and ketones, producing rancid off-odors [17,18].

The combined accumulation of ammonia, trimethylamine (TMA), hypoxanthine, volatile bases, free fatty acids, aldehydes, and small peptides leads to pronounced off-odors (fishy or ammonia-like), off-flavors (bitterness, rancidity), and progressive muscle softening, markedly compromising fish freshness, sensory quality, and safety [16,19].

The following methods can mitigate fish spoilage by targeting the enzymatic autolysis mechanism. Rapid chilling or super-chilling immediately after harvest slows enzyme kinetics by reducing temperature below activity thresholds [20]. High-pressure processing (HPP) inactivates endogenous proteases and lipases without thermal damage [21]. Modified atmosphere packaging (MAP) limits oxygen-driven reactions [20]. Edible coatings or nanoemulsions provide physical barriers to moisture and oxygen while delivering antioxidants or specific enzyme inhibitors [22,23]. Finally, chelating agents (EDTA) or natural antioxidant extracts (rosemary, green tea) sequester essential metal cofactors and scavenge free radicals, further suppressing spoilage metabolite formation [24].

2.2. Lipid Oxidation

Lipid oxidation represents a significant factor contributing to the deterioration and spoilage of pelagic fish species, such as mackerel and herring, which are characterized by their elevated oil and fat content within their flesh tissues [19,25]. This biochemical process has profound implications for the flavor, color, texture, and nutritional quality of stored fish, rendering it a critical concern within the fish industry [7]. Lipid oxidation in fish can occur via multiple pathways, including photo-oxidation, thermal oxidation, enzymatic oxidation, and auto-oxidation, with auto-oxidation being the predominant mechanism. First, auto-oxidation is a spontaneous, free-radical chain reaction initiated when molecular oxygen abstracts hydrogen atoms from unsaturated fatty acids, generating lipid radicals and hydroperoxides. Second, photo-oxidation occurs when ultraviolet or visible light activates endogenous sensitizers (chlorophyll, riboflavin, heme proteins), producing singlet oxygen that reacts directly with lipids to form peroxides. Third, enzymatic oxidation, catalyzed by lipoxygenases and other endogenous oxidases, selectively converts polyunsaturated fatty acids into lipid hydroperoxides under mild conditions. Finally, thermal oxidation is driven by elevated temperatures during processing or storage, which accelerate both radical initiation and propagation steps, exacerbating peroxide breakdown and formation of secondary oxidation products. Together, these pathways compromise fish quality by generating volatile off-odors, off-flavors, and nutritional losses [26].

As shown in Figure 3, the process of lipid oxidation is characterized by a three-stage free-radical mechanism: initiation, propagation, and termination [27,28,29,30]. The initiation phase is instigated by the abstraction of hydrogen atoms adjacent to double bonds in fatty acids, facilitated by catalysts such as heat, metal ions (Fe²⁺, Cu²⁺), and irradiation. The initiation phase involves hydrogen abstraction from methylene groups adjacent to double bonds in unsaturated fatty acids, forming lipid radicals. This leads to the generation of lipid free radicals, which subsequently react with oxygen to produce peroxyl radicals [31].

In the propagation phase, lipid radicals react rapidly with molecular oxygen (O₂), forming lipid peroxyl radicals. These radicals abstract hydrogen from other lipids, creating lipid hydroperoxides and new radicals, propagating a cyclic chain reaction. The hydroperoxides are inherently unstable and decompose rapidly into secondary oxidation products, including malondialdehyde, 4-hydroxynonenal, hydrocarbons, and volatile organic acids [32,33]. These secondary products are primarily responsible for the off-odors, undesirable flavors, and textural alterations commonly associated with spoiled fish. Secondary oxidation products—especially aldehydes (e.g., malondialdehyde, hexanal, 4-hydroxynonenal) and ketones (e.g., 2-pentanone, 2-heptanone)—exert profound effects on fish quality. Aldehydes are highly volatile and soluble in water; therefore, even at low concentrations, they dominate off-odors (green, grassy, metallic) and off-flavors (bitter, cardboard-like), drastically reducing consumer acceptance. 4-hydroxynonenal and malondialdehyde also form covalent adducts with muscle proteins, causing protein cross-linking, discoloration, and deterioration of texture and water-holding capacity. Ketones contribute sweet or fruity notes at first but quickly turn rancid, masking the fish’s natural flavor. Beyond sensory defects, these reactive carbonyls degrade essential omega-3 fatty acids and fat-soluble vitamins (A, D, E), lowering nutritional value and raising potential safety concerns due to their cytotoxicity and genotoxicity when ingested over time [17,34].

The termination phase transpires when the radicals combine to form stable, non-radical products, including dimers and polymers, ultimately slowing oxidation. However, lipid hydroperoxides can decompose further into volatile secondary oxidation products. Also, the presence of transition metals, particularly iron derived from hemoglobin, myoglobin, and cytochrome, can catalyze and expedite lipid oxidation in fish [35].

In addition to non-enzymatic pathways, lipid oxidation in fish can also occur through enzymatic mechanisms involving lipases, a process referred to as lipolysis. Lipases catalyze the hydrolysis of triglycerides into glycerol and free fatty acids, thereby contributing to rancidity and a decline in oil quality [36]. The principal lipolytic enzymes found in fish include triacyl lipase, phospholipase A2, and phospholipase B, which are located in the skin, blood, and tissues [37,38].

Furthermore, several environmental and processing factors profoundly affect lipid oxidation rates in fish. Elevated temperatures increase molecular mobility, accelerating radical initiation, hydroperoxide decomposition, and formation of secondary oxidation products. Exposure to UV and visible light activates endogenous photosensitizers (e.g., chlorophyll, riboflavin, heme proteins), generating singlet oxygen that reacts directly with unsaturated lipids. Transition metal ions (Fe²⁺, Cu²⁺) catalyze lipid hydroperoxide breakdown, producing additional free radicals and propagating the chain reaction. Finally, high salinity and ionic strength promote lipid–protein interactions and membrane destabilization, further enhancing oxidation susceptibility. Controlling these parameters, through strict temperature management, light-blocking packaging, metal chelators, and optimized salinity, can significantly mitigate oxidative spoilage and help preserve fish quality [39,40,41].

The oxidative degradation of lipids in fish is closely associated with protein denaturation, the functional breakdown of endogenous antioxidant mechanisms, and a reduction in nutritional value due to the depletion of fat-soluble vitamins. Lipid oxidation generates reactive aldehydes and radicals that interact with proteins, causing cross-linking, aggregation, and irreversible denaturation. Such interactions impair muscle protein functionality, texture, and water-holding capacity. Concurrently, oxidative stress consumes endogenous antioxidants (e.g., tocopherols, carotenoids), reducing nutritional quality by depleting essential nutrients, vitamins, and essential fatty acids, notably omega-3 polyunsaturated fatty acids [42,43].

2.3. Microbial Spoilage

2.3.1. Initial Colonization

Freshly harvested fish are immediately exposed to micro-organisms present in their aquatic environment and on processing surfaces. The initial colonizers are typically psychrotrophic, Gram-negative bacteria—including Pseudomonas, Shewanella, Vibrio, Serratia, and Aeromonas—whose prevalence depends on water microbiota, handling practices, and onboard hygiene conditions [9,44].

2.3.2. Microbial Metabolism and Metabolite Production

After colonization, specific spoilage organisms (SSOs) proliferate by exploiting muscle nutrients via distinct biochemical pathways. Proteolytic and deamination reactions—catalyzed by bacterial proteases and deaminases—break down proteins into peptides and free amino acids, releasing ammonia and biogenic amines, such as putrescine, cadaverine, and histamine [45,46,47]. Concurrently, marine SSOs (e.g., Shewanella putrefaciens, Photobacterium phosphoreum) enzymatically reduce trimethylamine oxide (TMAO) to trimethylamine (TMA), generating strong ammonia-like off-odors [47]. Sulfur-reducing bacteria further convert sulfur-containing amino acids into hydrogen sulfide (H₂S) and other volatile sulfides, producing characteristic rotten-egg aromas [48].

2.3.3. Spoilage Manifestations and Sensory Implications

The accumulation of these metabolites leads to pronounced sensory deterioration. Elevated levels of TMA, ammonia, amines, H₂S, aldehydes, and organic acids produce foul odors (fishy, ammonia-like, sulfide), off-flavors (bitter, putrid), discoloration, mucous exudation, and muscle softening [48,49]. Storage conditions modulate which SSOs dominate: chilled storage (0–4 °C) favors Pseudomonas and Shewanella; lightly salted fillets select for salt-tolerant lactic acid bacteria; and modified atmosphere packaging (high CO₂) suppresses aerobes but may permit the growth of Photobacterium phosphoreum under temperature abuse [49,50]. These changes markedly shorten shelf life and undermine fish quality and safety.

A critical spoilage marker is trimethylamine (TMA), which results from the bacterial reduction of trimethylamine oxide (TMAO), a compound fish use as an osmoregulant. Bacteria such as Shewanella putrefaciens, Aeromonas spp., and P. phosphoreum can reduce TMAO to TMA, producing ammonia-like off-flavors [47]. The production of TMA and other spoilage compounds accelerates the deterioration process, making the fish unsuitable for consumption.

Storage conditions further shape which specific spoilage organisms (SSOs) dominate fish spoilage and the speed at which it progresses. As shown in

Table 1. Overview of fish spoilage microbes and their sensory consequences.

Bacteria	Major Spoilage Metabolites	Typical Spoilage Characteristics
Pseudomonas spp.	Amines, aldehydes, ketones	Sweet, fruity odors, slimy texture
Shewanella putrefaciens	TMA, H2S, sulfides	Strong ammonia-like, rotten-egg odors
Photobacterium phosphoreum	TMA	Fishy, ammonia odors
Aeromonas spp.	Amines, organic acids	Sour, off-odors, slime formation

, under chilled storage (0–4 °C), psychrotrophic Gram-negative bacteria (primarily Pseudomonas spp. and Shewanella spp.) proliferate, exploiting low temperatures to produce trimethylamine and hydrogen sulfide that drive off-odors [48]. In salted or brining conditions, elevated NaCl reduces water activity, selectively favoring halotolerant lactic acid bacteria that generate organic acids and diacetyl, imparting sour and buttery off-flavors [49]. Meanwhile, modified atmosphere packaging (MAP) with high CO₂ concentrations suppresses aerobic spoilage organisms like Pseudomonas, but if temperatures rise above optimal levels, anaerobic SSOs such as Photobacterium phosphoreum can flourish in low-oxygen environments, accelerating trimethylamine accumulation and spoilage [50].

3. Preservation Technologies and Optimization

3.1. Physical Preservation

Physical methods slow or halt spoilage by reducing enzyme kinetics, immobilizing water, or inactivating microbes. Below, we summarize five key techniques to show how they are applied, their relative effectiveness, and suitability for various fish species, as shown in

Table 2. Comparative summary of physical preservation methods for fish.

Method	Parameter	Storage Duration	Microbial Reduction	Sensory Impact
Refrigeration	0–4 °C	3–9 days	~1 log CFU/g	Maintains fresh texture; short-term
Super-Chilling	−0.5 to −2.8 °C	10–14 days	~2 log CFU/g	Excellent texture; minimal drip loss
Freezing	≤−18 °C	6–12 months	~5 log CFU/g	Potential drip loss; texture changes
High-Pressure Processing	300 MPa, 3 min	21–28 days	>3 log CFU/g	Good texture; slight firmness change
Pulsed Electric Fields	1–3 kV/cm	7–14 days	~2 log CFU/g	Negligible sensory alterations

.

3.1.1. Low-Temperature Preservation

Low-temperature preservation is an essential method for extending the shelf life of fish by significantly slowing down the enzymatic and microbial activities responsible for spoilage. This technique includes refrigeration, super-chilling, and freezing, each of which operates through distinct mechanisms to maintain the quality of fish.

Refrigeration

Refrigeration, which involves maintaining fish at temperatures just above freezing, typically between 0 °C and 4 °C, is one of the most common methods used to slow down spoilage processes. Refrigeration systems use stainless-steel tunnel or spiral chillers equipped with distributed evaporator coils and variable-speed fans to maintain uniform airflow (1–2 m/s) across trays of fish; coil spacing, airflow ducting, and insulation ensure ±0.5 °C temperature control. This method significantly inhibits microbial growth and enzymatic activity but does not stop them entirely. The growth of psychrotrophic bacteria, such as Shewanella putrefaciens, which are major contributors to spoilage, is considerably slowed at 0 °C compared to their optimal temperature, thus reducing spoilage rates. Additionally, the activity of autolytic enzymes responsible for breaking down proteins, lipids, and other molecules is reduced, preserving the texture and flavor of the fish [20]. Immediate icing of fresh fish is crucial to maximize shelf life, as delays can lead to rapid spoilage [51]. Methods like chilled seawater (CSW) and refrigerated seawater (RSW) provide uniform cooling and enhance shelf life better than ice alone due to the anaerobic conditions created.

Super-Chilling

In general, super-chilling or deep-chilling involves chilling the products to a temperature close to or just below the initial freezing point, which typically ranges between −0.5 °C and −2.8 °C for most food products [52]. In super-chilling, plate-chiller or brine-circulation tanks are engineered with integrated heat-exchanger plates and continuous mixing paddles; precise temperature sensors (±0.1 °C accuracy) and automated brine flow controls prevent localized freezing and ensure homogeneous cooling around each fillet. This method offers a balance between refrigeration and freezing, providing more effective inhibition of microbial growth and enzymatic activity without fully freezing the water in tissues, which can cause structural damage [20]. By avoiding complete freezing, super-chilling helps maintain the texture and appearance of fish, preventing the drip loss that occurs during the thawing of fully frozen fish. Combining super-chilling with modified atmosphere packaging (MAP), which reduces oxygen levels, can further extend shelf life by inhibiting oxidative reactions and microbial growth [20]. Slow cooling rates in super-chilling help avoid stress reactions in fish tissue, which can lead to undesirable textural changes.

Freezing

Frozen storage is a widely used method for preserving meat over long periods. However, it can negatively affect muscle properties by increasing free fatty acids (FFAs) and lipid oxidation products [53,54]. Freezing chambers, whether air-blast tunnels or cryogenic spray systems, feature adjustable nozzles and airflow grids to achieve rapid velocity (3–5 m/s) and uniform −40 °C conditions; in plate freezers, fish are sandwiched between refrigerated plates exerting consistent pressure for optimal heat transfer. Freezing slows microbial and enzymatic activity, preserving flavor and nutritional value better than chilling [55]. The size of ice crystals formed during freezing is crucial; larger crystals can damage texture and increase oxidation [55,56]. Faster and more uniform freezing results in smaller ice crystals [57]. Slow freezing produces larger ice crystals, causing significant muscle fiber damage [55]. Rapid freezing techniques like cryogenic freezing might cause intracellular ice crystallization and cell cracking [55]. Maintaining a stable temperature is essential to prevent ice crystal growth during storage [57]. In frozen salmon, ice crystals grow larger over time due to recrystallization, becoming more spherical [58]. Fast freezing techniques include high-pressure freezing, pressure shift freezing, cryogenic freezing, liquid immersion freezing, plate freezers, and air-blast freezers [57,59,60]. Plate freezers work well for regular-shaped fish products, operating at around −40 °C. Air-blast freezers use cold airflow, effective for various shapes, at −20 °C to −40 °C. Immersion freezing uses liquid media like brine or glycol for fast freezing [60]. High-pressure freezing creates uniform ice crystals quickly, preserving texture and microstructure better [55,57,61]. Injecting or dipping fish in antifreeze proteins helps form small ice crystals and prevent recrystallization [57]. Freezing at −40 °C minimizes unfrozen water, reducing primary oxidation [62,63].

3.1.2. High-Pressure Treatment (HPP)

High-pressure treatments are effective in preserving fish by causing lethal structural and biochemical alterations in micro-organisms, similar to high-temperature treatments. HPP vessels are cylindrical, water-filled pressure chambers constructed of thick, high-strength steel with internal diameters ranging from 200 to 500 mm; axial pumps deliver up to 600 MPa isostatic pressure, while internal temperature is maintained at 4–10 °C via heat exchangers to prevent thermal spikes [64]. Micro-organisms are inactivated under high pressure due to cell deformation and damage. However, these effects are reversible at pressures between 100 and 300 MPa and are insufficient for inactivating resistant bacterial spores, which require pressures as high as 1200 MPa [65].

High-pressure treatment at 200 MPa combined with sub-zero temperature has been shown to inhibit Listeria monocytogenes in smoked salmon, although it can also lighten color and toughen texture [21]. In vacuum-packed trout, high-pressure treatment extended shelf life from 5–6 days to 21–28 days by reducing biogenic amine content [66]. Conversely, pressure treatments caused bright and dull red appearance in species such as turbot, sheep head, cod, and carp [67,68,69].

A study on rainbow trout and mahi found that high-pressure processing at 300 MPa and 450 MPa, respectively, was optimal for controlling microbial load, lipid oxidation, and color changes [70].

3.1.3. Pulsed Electric Field (PEF)

Pulsed electric field (PEF) technology involves delivering short, high-power electrical pulses to a product placed in a treatment chamber between electrodes. PEF installations consist of treatment chambers with parallel stainless-steel electrodes (spacing 1–3 cm) surrounded by cooling jackets; pulse generators deliver 1–3 kV/cm square-wave pulses at controlled frequencies (100–500 Hz), and real-time voltage and current monitoring ensures uniform electroporation without overheating [71]. This process causes minimal thermal increases and primarily damages the cell membranes of biological cells, including those of microbes, plants, and animals [72]. The most accepted theory, introduced by Zimmermann, posits that the cell membrane acts as a capacitor with a low dielectric constant. Exposure to an electric field increases the transmembrane potential until it exceeds a critical value, causing the membrane to collapse, forming pores, and increasing permeability. This can result in either reversible or irreversible damage, depending on the intensity of the PEF treatment [73].

Low-intensity PEF treatments create small, reversible pores, while higher-intensity treatments result in irreversible cell membrane damage [72]. This process, known as electroporation, has been applied in various fields, including food processing, due to its ability to alter cell permeability without significant thermal effects [74]. For example, mild PEF treatment improved the texture and microstructure of salmon fillets [75]. However, the same benefits were not observed in the tenderness of mussels and mollusks [76].

PEF technology is also effective in improving the extraction of high-value compounds from fish by-products, such as proteins from mussels and abalone viscera, and calcium and chondroitin sulfate from fish bones [77,78,79,80]. More recently, PEF has been explored as a preservation method due to its ability to inactivate micro-organisms while maintaining the nutritional and sensory quality of raw fish [81,82,83]. Combining PEF with other non-thermal technologies like UV irradiation, microwaves, high-intensity light pulses, and high hydrostatic pressure further enhances microbial inactivation [84,85].

Although primarily used for liquid foods, PEF has shown promising results in fish preservation. For instance, a study on Pacific white shrimp (Litopenaeus vannamei) treated with PEF every second day during 10 days of storage at 4 °C found significant reductions in microbial load [86]. The treatment conditions varied in intensity, with higher densities leading to greater microbial inactivation.

3.2. Chemical and Biological Preservation

Chemical and biological preservation strategies complement physical methods by directly targeting microbial growth and oxidative reactions at the fish surface and within muscle tissues. These approaches include the application of nanoemulsion-based coatings, ozonation treatments, and essential-oil formulations, each designed to deliver antimicrobial and antioxidant compounds in a controlled manner. Figure 4 illustrates the general workflows for coating application, ozone exposure, and antioxidant diffusion, providing a visual roadmap for the processes described below.

3.2.1. Nanoemulsions

Nanoemulsions are kinetically stable oil-in-water colloidal systems with droplet sizes of 20–200 nm, typically composed of bioactive oils (e.g., cumin, thyme), water, surfactants, and emulsifiers, such as chitosan or whey protein [22,23]. Their nanoscale dimensions create a large interfacial area that enhances the solubility, stability, and bioavailability of hydrophobic, antimicrobial, and antioxidant compounds, while the moisture-proof and gas-barrier properties of the colloidal matrix protect these actives from environmental degradation [89].

Nanoemulsions inhibit fish spoilage through three complementary mechanisms. First, antimicrobial constituents—cuminaldehyde and γ-terpinene from cumin, thymol and carvacrol from oregano and thyme, and eugenol from clove—insert themselves into bacterial lipid bilayers, increasing membrane permeability, collapsing proton gradients, and causing leakage of intracellular contents, ultimately leading to cell death [90,91]. Second, the released bioactives bind to and inhibit endogenous spoilage enzymes (e.g., ATPases, proteases, dehydrogenases), delaying the autolytic breakdown of myofibrillar and connective-tissue proteins and preserving muscle texture [92,93]. Third, the encapsulated antioxidants scavenge free radicals and interrupt radical-propagation chains, reducing thiobarbituric-acid-reactive substances (TBARS) and peroxide values (PVs), thereby delaying lipid oxidation and maintaining flavor, color, and nutritional quality [24,87,94].

In refrigerated trials, nanoemulsion-loaded coatings have demonstrated significant preservation effects. Chitosan-based nanoemulsions containing cumin oil reduced Pseudomonas spp. and Shewanella putrefaciens counts by over 2 log CFU/g and lowered TBARS by 60% in sardine fillets over 10 days at 4 °C [90,95]. Similarly, thyme-oil nanoemulsions achieved a 1.5 log CFU/g reduction in aerobic bacteria and extended the shelf life of rainbow trout by 8 days under the same conditions [92,93]. Curcumin- and rosemary-extract nanoemulsions also decreased PV in trout, further demonstrating their antioxidant efficacy [87,94].

Nanoemulsions are applied through dipping, spraying, or integration into edible films and coatings. For example, 0.1% nanoemulsion sprays in chitosan or whey-protein films provide a dual barrier against oxygen and moisture while delivering actives in a controlled manner [89,95]. Optimization of droplet concentration, emulsifier type, and active-loading levels is essential to maximize antimicrobial and antioxidant effects without introducing off-flavors or off-odors [24]. This balance makes nanoemulsions a versatile tool for preserving both high-fat species (e.g., salmon, mackerel) and lean fish (e.g., cod, trout) in refrigerated storage.

3.2.2. Ozonation

Ozonation applies ozone gas or ozonated water, typically at 0.5–2.0 ppm, to sanitize seafood. Ozone is generated by feeding oxygen or air through a corona-discharge reactor or exposing it to ultraviolet light. In practice, either ozone gas is introduced into cold-storage rooms and packaging headspace, or ozonated water is used in immersion or slurry-ice systems to deliver both cooling and microbial control [22].

Once formed, ozone decomposes rapidly to generate reactive oxygen species (ROS), such as hydroxyl radicals (•OH), superoxide anions (O₂•⁻), and singlet oxygen (¹O₂). These ROS attack microbial cells by abstracting hydrogen atoms from unsaturated fatty acids in membrane lipids, initiating lipid peroxidation. They also oxidize membrane proteins and intracellular thiol groups, disrupting enzyme function and compromising membrane integrity. The combined damage to membranes, proteins, and DNA leads to microbial inactivation and cell lysis [22,89]. Unfortunately, the same ROS and ozone molecules can react with fish tissue lipids, accelerating hydroperoxide breakdown and generating secondary oxidation products, such as aldehydes and ketones, which raise thiobarbituric-acid-reactive substances (TBARS) and can produce off-flavors [23,89].

In one study, whole freshwater tilapia immersed in 1.5 ppm ozonated water for 15 min exhibited an 88.3 percent reduction in total microbial load without altering the pH or color. When tilapia fillets were treated at 1.0 ppm and 1.5 ppm, microbial counts decreased by 77.2 percent and 79.5 percent, respectively, although TBARS values increased, indicating enhanced lipid oxidation [23]. Optimized ozone treatments can achieve up to a 3 log CFU/g reduction in spoilage organisms, but exposure parameters must be carefully controlled to balance sanitization and lipid stability [22].

Commercially, ozonation is integrated into slurry-ice systems for species such as cod and tilapia to combine effective temperature control and microbial reduction while minimizing drip loss. Ozone gas is also applied in controlled atmospheres within cold-storage rooms and packaging headspaces to suppress surface contamination on fillets. To counteract oxidative side effects, ozonation is often followed by antioxidant interventions, such as dipping fish in polyphenol-rich extracts or applying antioxidant-releasing edible coatings, thereby preserving sensory quality while maintaining microbial safety.

3.2.3. Essential Oils

Essential oils (EOs) are volatile, plant-derived mixtures of bioactive compounds—including eugenol and methyleugenol in bay, cinnamaldehyde and eugenol in cinnamon, eugenol and β-caryophyllene in clove, citral (geranial + neral) in lemongrass, terpinen-4-ol and γ-terpinene in marjoram, carvacrol and thymol in oregano, thujone and camphor in sage, and thymol and p-cymene in thyme—that have long been used for medicinal purposes and are now gaining traction as natural preservatives in fish products [22,29]. Their complex volatile organic compounds (VOCs) confer both antimicrobial and antioxidant properties, and they can be applied directly via dipping or spraying or incorporated into edible polymer matrices for controlled release.

The primary antimicrobial mechanism of EOs involves disruption of microbial cell membranes and inhibition of essential enzymatic functions. Phenolic constituents, such as thymol, carvacrol, and eugenol, insert themselves into lipid bilayers, increasing membrane permeability, causing ion leakage, and collapsing proton gradients; aldehydes like cinnamaldehyde and citral react with membrane and cytoplasmic proteins to inactivate ATPases and dehydrogenases, leading to energy depletion and cell death. Additionally, certain VOCs interfere with quorum-sensing pathways, preventing biofilm formation and further enhancing microbial control [23,91].

Numerous studies demonstrate the efficacy of EOs in extending shelf life and reducing spoilage indicators. For instance, oregano oil at 0.05% (v/w) under modified atmosphere packaging (2 °C) extended the shelf life of cod (Gadus morhua) fillets from 11–12 days to 21–26 days, achieving a ~3 log CFU/g reduction in Photobacterium phosphoreum counts [23]. In common carp (Cyprinus carpio), carvacrol and thymol treatments doubled freshness—extending the shelf life from 4 to 12 days at 5 °C—by reducing total viable counts by over 2 log CFU/g. Blends of oregano, thyme, and star anise oils also delayed the accumulation of TVB-N and putrescine in grass carp (Ctenopharyngodon idella), prolonging acceptable quality from 6 to 8 days [89].

In practice, EOs are often formulated into edible films and coatings to combine their antimicrobial action with barrier properties. Cinnamon-oil–whey-protein coatings extended Beluga sturgeon (Huso huso) shelf life by approximately 8 days by retarding lipid oxidation and microbial growth [96]. Chitosan–gelatin films incorporating clove oil significantly reduced spoilage bacteria and TBARS in Atlantic cod (Gadus morhua) [97], while tarragon-oil–whey-protein coatings delayed microbial proliferation in brook trout (Salvelinus fontinalis) [98]. These polymer–EO combinations harness both physical and chemical preservation mechanisms, although their sensory impacts and cost implications necessitate further optimization of EO concentration, polymer matrix, and application method [90,91,92,93,95].

3.2.4. Antimicrobial Polymer and Biopolymer Coatings

Antimicrobial coatings are thin films applied to fish surfaces that combine film-forming polymers, such as poly(lactic acid) (PLA), polyethylene–vinyl alcohol (EVOH), or natural biopolymers, including chitosan, alginate, and gelatin, with active agents like metal nanoparticles or essential oils to inhibit spoilage micro-organisms [22,23].

Synthetic polymer coatings typically incorporate inorganic nanoparticles (for example, silver or zinc oxide) into a PLA or EVOH matrix. These nanoparticles continuously release antimicrobial ions and act as an oxygen barrier. Biopolymer coatings exploit chitosan’s intrinsic antimicrobial properties or embed nanoemulsified essential oils. Once applied, these coatings adhere to the fish surface, control moisture and oxygen transfer, and provide sustained release of antimicrobial compounds that disrupt microbial cell membranes and inhibit enzyme activity.

In cod fillets, PLA films containing zinc oxide nanoparticles reduced counts of Pseudomonas and Shewanella by 3 log CFU/g over 10 days while remaining transparent and mechanically stable [99]. Chitosan coatings with thyme-oil nanoemulsions achieved a 2.5 log CFU/g reduction in psychrotrophic bacteria and a 50% decrease in TBARS in trout over 12 days at 4 °C [95]. Alginate coatings loaded with silver nanoparticles extended carp fillet shelf life by 8 days by inhibiting both aerobic and anaerobic spoilage organisms [90].

Future research should focus on adjusting polymer molecular weight and cross-link density to fine-tune antimicrobial release rates, developing biodegradable nanocarriers such as starch or cyclodextrin inclusion complexes for safer delivery, and designing multi-layer films that integrate oxygen scavengers, moisture regulators, and freshness indicators to further enhance efficacy and consumer acceptance.

Figure 5 shows that each chemical or biological intervention offers a distinct balance between oxidative protection and microbial control. Nanoemulsions deliver the greatest shelf-life extension (≈10 days) by forming deep, oxygen-barrier coatings rich in antioxidants, but they achieve only moderate microbial kill (≈1.5 log CFU/g). Essential-oil treatments provide an intermediate profile, extending freshness by ≈8 days while reducing spoilage bacteria by ≈2 log CFU/g through combined radical scavenging and membrane disruption. Ozonation excels at rapid microbial inactivation (≈3 log CFU/g) yet yields the smallest shelf-life gain (≈7 days) due to its propensity to generate secondary oxidation products. Polymer coatings match ozonation’s microbial reduction (≈3 log CFU/g) but also deliver a stronger barrier effect, extending shelf life by ≈8 days through reduced oxygen ingress and controlled release of embedded antimicrobials. These trends highlight the importance of selecting or combining methods according to the dominant spoilage pathway: nanoemulsions for high-fat, oxidation-sensitive species; ozonation or polymer coatings for lean, microbe-susceptible fish; and essential-oil systems when balanced control of both oxidation and microbial growth is needed.

3.3. Modified Atmosphere Packaging (MAP)

Modified atmosphere packaging (MAP) extends the shelf life of refrigerated fish by altering the headspace gas composition—substituting ambient air (78% N₂, 21% O₂, 0.03% CO₂) with specific mixtures of CO₂, N₂, and O₂—to inhibit lipid oxidation and suppress aerobic spoilage bacteria [22]. CO₂ exerts bacteriostatic and fungistatic effects by dissolving into fish tissues, lowering surface pH, and disrupting microbial cell membranes and enzyme activities; N₂ serves as an inert filler to prevent package collapse; and O₂ levels can be adjusted to maintain desirable flesh color in certain species [22].

After fillet preparation (cleaning, grading, and pre-cooling), fish are placed in gas-impermeable trays or pouches on a MAP line. Ambient air is evacuated and replaced by flushing with a controlled gas blend—commonly 40–60% CO₂, 30–50% N₂, and 0–5% O₂—using precise gas injectors before hermetic sealing. Packages are stored at 0–4 °C to maximize CO₂ solubility and minimize package deformation; material selection ensures low gas permeability to maintain the target atmosphere over the storage period [22,100].

In sea bass (Dicentrarchus labrax), MAP blends of 40% CO₂/60% N₂, 50% CO₂/50% N₂, and 60% CO₂/40% N₂ extended shelf life from 4 days under air to 11–14 days, with the highest CO₂ level yielding the best microbiological and sensory scores [23]. For farmed cod (Gadus morhua), a 63% O₂/37% CO₂ mixture reduced total viable counts and suppressed H₂S-producing bacteria, maintaining freshness beyond that possible with air or vacuum packaging [89]. Vacuum-packaged fish (e.g., Lethrinus atkinsoni at –18 °C) showed improved peroxide and free fatty acid profiles compared to non-vacuum controls [95], but MAP generally outperformed vacuum conditions in quality retention [90].

MAP is often combined with complementary preservation methods. For example, coupling MAP with super-chilling (−1.3 °C) extended cod and turbot shelf lives by an additional 5–7 days compared to either treatment alone [91,92]. Controlled atmosphere packaging (CAP) systems, which continuously monitor and adjust headspace gases, further stabilize CO₂ levels and prevent quality loss due to temperature fluctuations [90]. Incorporating active packaging elements—such as CO₂-scavenging films or UV-C treatment prior to MAP—can enhance microbial control and oxidative stability, offering a versatile, scalable solution for high-value fish products.

As shown in

Table 3. Effects of MAP and combined preservation techniques on fish shelf life.

Species	MAP Conditions	Additional Technique(s)	Shelf-Life Extension	References
Atlantic cod (G. morhua)	63% O2/37% CO2	—	4–14 days	[101]
Cod loins	60% CO2/40% N2	Super-chilling at –1.3 °C	16 days	[102]
Turbot (S. maximus)	80% CO2/20% N2	Super-chilling	18 days	[103]
Nile tilapia (O. niloticus)	50% CO2/50% N2	—	10 days	[88]
Salmon (S. salar)	60% CO2/40% N2	HPP (300 MPa) + Ultra-cooling (–2.8 °C)	7–14 days	[70]

, for Atlantic cod (Gadus morhua), packaging fillets under a 63% O₂/37% CO₂ atmosphere suppressed Photobacterium phosphoreum and minimized H₂S production, extending shelf life from 4 days in air to 14 days at 0 °C [101]. When combined with super-chilling at −1.3 °C and a 60% CO₂/40% N₂ mixture, fresh cod loins maintained freshness for up to 16 days by synergistically inhibiting microbial growth and enzymatic autolysis [102]. High-CO₂ MAP (80% CO₂/20% N₂) plus super-chilling likewise prolonged turbot (Scophthalmus maximus) shelf life to 18 days, effectively delaying biochemical and flavor deterioration [103]. In Nile tilapia (Oreochromis niloticus), MAP at 50% CO₂/50% N₂ reduced psychrotrophic counts by 2 log CFU/g and improved physicochemical stability over 10 days versus air or vacuum storage [88]. Finally, a multi-hurdle system combining MAP, high-pressure processing (300 MPa), and ultra-cooling at −2.8 °C doubled the shelf life of salmon (Salmo salar) by jointly suppressing microbial activity and enzymatic degradation [70].

3.4. Future Preservation Techniques and Development Trend

Future research and industrial practice in fish preservation will hinge on three intertwined trends, each accompanied by distinct challenges. Nanotechnology-enhanced coatings and films—such as nanoemulsion-loaded biopolymer layers—offer highly targeted delivery of antimicrobial and antioxidant agents, significantly prolonging shelf life in high-fat species; however, unresolved questions regarding nanoparticle migration, long-term toxicity, and environmental persistence necessitate the development of standardized safety assessment protocols and GRAS-certified nanocarriers [104]. Synergistic multi-hurdle systems that combine non-thermal processes (PEF, HPP), controlled atmospheres (MAP), and active coatings can achieve additive or even synergistic inhibition of enzymatic, oxidative, and microbial spoilage—often doubling shelf life without compromising sensory quality—but their widespread adoption is limited by the high capital and energy costs of batch-mode reactors and the technical complexity of integrating multiple processes, underscoring the need for continuous-flow designs and modular processing platforms [105]. Finally, intelligent and active packaging solutions, including colorimetric freshness indicators, RFID-based sensors, and oxygen-scavenging films or sachets, promise real-time quality monitoring and controlled-release preservation; yet, ensuring sensor accuracy under variable storage conditions, obtaining food-contact regulatory approval, and building consumer trust in technology-embedded packaging remain critical barriers [106].

To bridge these gaps, coordinated efforts are required to establish robust toxicological and migration testing for nanomaterials, engineer cost-effective continuous-flow PEF/HPP equipment, define clear regulatory pathways for active and smart packaging, and leverage predictive modeling and machine-learning tools to tailor multi-hurdle preservation protocols to the specific spoilage profiles of different fish species.

4. Conclusions

The integration of preservation technologies presents both opportunities and challenges in addressing the multi-factorial mechanisms of fish spoilage. While single-method approaches exhibit inherent limitations—such as incomplete enzymatic inhibition in low-temperature preservation or gas ratio dependency in modified atmosphere packaging—synergistic combinations demonstrate amplified efficacy. For instance, coupling ultra-cooling (−2.8 °C) with high-pressure treatment (300 MPa) extends salmon shelf life by 100% compared to standalone methods, attributable to complementary mechanisms of enzymatic denaturation and microbial suppression. However, such integration introduces technical complexities, including ice-crystal-induced non-uniformity during pressure application, necessitating optimized protocols for cooling rates and treatment sequences. Similarly, pulsed electric field (PEF) technology, though promising for microbial inactivation, faces scalability barriers due to uneven electric field distribution in solid matrices. Innovations like dual-frequency pulses (10 kHz/1 MHz) enhance penetration depth by 37%; yet, prohibitive equipment costs (5× traditional freezing systems) underscore the need for cost-effective engineering solutions. These findings highlight the imperative to balance synergistic gains with operational feasibility in multi-technology frameworks.

The industrialization of emerging technologies is further complicated by economic and environmental trade-offs. Nanoemulsions, despite their enhanced antimicrobial activity, encounter nanoparticle agglomeration during mass production, which microfluidic mixing partially mitigates by controlling particle size (50–100 nm). However, energy-intensive processes limit accessibility for small-scale producers. Concurrently, environmental sustainability concerns arise: high-pressure processing generates 2.3× higher carbon emissions than conventional freezing, while biodegradable chitosan/nano-ZnO coatings, despite reducing emissions by 18%, pose unresolved risks of nanoparticle leaching. This duality necessitates a dual-axis evaluation system prioritizing both technological efficacy and ecological impact. Meanwhile, microbial community regulation via probiotics like Lactobacillus plantarum PS128 offers a green alternative, achieving 85% colonization rates and 40% TVB-N reduction in cod. Yet, strain specificity and consumer skepticism toward “live preservation” demand rigorous metagenomic screening and sensory validation, emphasizing the need for tailored biological solutions.

Advancements in intelligent monitoring systems are reshaping preservation paradigms. Blockchain-integrated RFID sensors enable real-time freshness tracking via ATP degradation detection, reducing supply chain losses from 12% to 5%. However, sensor data variability (±20% threshold discrepancies) underscores the urgency for standardized international protocols. Future efforts must focus on integrating smart technologies with multi-omics insights—elucidating enzyme–lipid–microbe interactions—to design precision preservation agents. Furthermore, the development of stimuli-responsive packaging materials and hybrid non-thermal processing systems could bridge gaps between scalability, sensory quality, and environmental stewardship.

In conclusion, overcoming the limitations of current fish preservation strategies requires a paradigm shift toward synergistic, intelligent, and sustainable systems. By harmonizing multi-technology integration with molecular-level insights into spoilage mechanisms, the field can achieve targeted inhibition of enzymatic, oxidative, and microbial degradation pathways. Future research should prioritize scalable innovations, such as cost-optimized PEF systems and eco-functional nanomaterials, while establishing unified standards for smart monitoring and environmental impact assessment. Ultimately, a holistic approach balancing technological advancement, ecological responsibility, and consumer acceptance will be critical to ensuring the safety, quality, and sustainability of global aquatic food systems.