Circular valorisation of fish processing by-products: integrated protease recovery and functional protein hydrolysate production

1 Introduction

As the global fishing industry expands, large volumes of fish-processing by-products, including heads, skin, viscera, bones, and scales, are generated. While these residues pose environmental challenges, they also represent valuable sources of bioactive compounds that can be recovered through sustainable valorisation strategies (Coppola et al., 2021; Ramakrishnan et al., 2024).

Circular economy approaches provide an important framework for converting fish-processing by-products into marketable products while reducing environmental impact, supporting the broader goals of the blue bioeconomy such as sustainable resource use and waste minimisation (Fotodimas et al., 2025; Desai et al., 2023).

Among the available valorisation pathways, FPH have gained increasing attention due to their bioactive properties, including antioxidant, antihypertensive, and cardioprotective activities (Iñarra et al., 2023; Vázquez et al., 2024). These hydrolysates are typically produced through enzymatic hydrolysis using commercial enzymes or endogenous fish proteases, enabling efficient conversion of protein-rich by-products into high-value biomolecules.

Structural proteins such as collagen and gelatin, abundant in fish skin and scales, also serve as important substrates for enzymatic hydrolysis and contribute to the generation of bioactive peptides (Manni et al., 2024). In addition, recent advances in membrane fractionation, non-thermal extraction, and other mild processing techniques have improved the efficiency of by-product valorisation while preserving functional properties (Li, 2022; Pires et al., 2024; Ghalamara et al., 2024).

This review highlights current strategies for converting fish-processing by-products into high-value biomolecules, with particular emphasis on the role of proteases and enzymatic hydrolysis in sustainable bioconversion.

2 Recovery and valorisation of fish-processing by-products into high-value protein-based products

Fish-processing by-products are rich in diverse proteins that can be efficiently recovered and transformed into high-value biomolecules. Structural proteins such as collagen and gelatin are primarily obtained from skin, scales, and bones (Bisht et al., 2021; Blanco et al., 2019), whereas viscera serve as important sources of endogenous enzymes, including proteases and lipases (Klomklao et al., 2007; Camargo et al., 2021). Controlled hydrolysis of these protein substrates further generates FPH and bioactive peptides with demonstrated functional and biological activities (Balti et al., 2010; Batista et al., 2022). The subsequent sections discuss strategies for protein recovery, purification approaches, and the potential applications of these biomolecules.

2.1 Collagen and gelatin

Collagen and gelatin derived from fish skin, scales, bones, and swim bladders remain in high demand in the food, cosmetic, pharmaceutical, and biomedical industries due to their biocompatibility, biodegradability, and functional properties (FAO, 2016; Jimenez-Champi et al., 2024; Blanco et al., 2019). Valorising these by-products into biomaterials facilitates waste avoidance while contributing to sustainable economic growth (Jafari et al., 2020).

The bioconversion of fish-processing waste into collagen and gelatin typically involves pretreatment, extraction, purification, and recovery processes. Pretreatment removes non-collagenous proteins, lipids, and minerals to improve extraction efficiency (Gaikwad and Kim, 2024). Acid-soluble collagen (ASC) extraction employs weak organic acids, whereas enzymatic methods using proteases such as pepsin selectively hydrolyse telopeptide regions to increase solubility without disrupting the native triple-helix structure (Bhagwat and Dandge, 2016; Wang et al., 2021). Emerging green technologies, including ultrasound-assisted extraction (UAE), deep eutectic solvent (DES) systems, and supercritical fluid extraction (SFE), have demonstrated improved efficiency while reducing environmental impact (Bai et al., 2017; Sousa et al., 2020; Bisht et al., 2021).

Following extraction, purification steps such as centrifugation, salting-out, dialysis, ultrafiltration, and chromatographic separation are applied to remove residual salts and low-MW impurities, yielding high-quality collagen suitable for food-grade and biomedical applications (Brodsky and Ramshaw, 1997; Yu et al., 2018; Tan and Chang, 2018). Gelatin, produced through controlled partial hydrolysis of collagen, functions as a gelling, stabilising, and encapsulating agent in food and pharmaceutical formulations (Bisht et al., 2021).

Collagen and gelatin represent major structural protein fractions. Their modification highlights the importance of proteolytic systems in fish by-product valorisation. Proteases help in catalysing the generation of smaller peptides and hydrolysates, establishing a direct link to enzymatic bioconversion strategies. Gelation at concentrations of 2–10% function as a gelling and encapsulating agent.

2.2 Enzymes from fish by-products

Fish processing generates substantial quantities of viscera, heads, skins, and residual muscle tissues that are rich sources of endogenous enzymes (Halim et al., 2016; Bougatef et al., 2008; Klomklao et al., 2007). Among these, viscera serve as primary reservoirs of digestive proteases and lipases, which exhibit unique biochemical characteristics such as activity at low temperatures and tolerance to saline environments (Roslan et al., 2014; Abraha et al., 2018).

Recovery of these enzymes generally involves homogenisation in cold buffer systems, followed by filtration, centrifugation, precipitation, and chromatographic purification (Sila and Bougatef, 2016; Binsi et al., 2016). Ion-exchange chromatography (IEC) and gel filtration chromatography (SEC) are commonly applied to obtain purified protease fractions (Roslan et al., 2014). Modern approaches such as ATPS and membrane filtration enhance yield while maintaining catalytic activity (Abraha et al., 2018). Immobilisation and microencapsulation further improve enzyme stability and reusability, supporting sustainable bioprocessing strategies (Batista et al., 2022).

Proteases derived from fish viscera play a pivotal role in the hydrolysis of structural and myofibrillar proteins, generating peptides with antioxidant, antihypertensive, antimicrobial, and immunomodulatory properties (Klomklao et al., 2007; Camargo et al., 2021). Lipases are similarly employed in lipid bioconversion reactions, including the synthesis of structured lipids and biodiesel (Sayari et al., 2016). This integration directly facilitates FPH production.

2.3 Fish protein hydrolysate and bioactive peptides

Fish processing generates multiple by-product fractions, including viscera, frames, heads, skin, bones, and trimmings, which represent substantial reservoirs of recoverable proteins and lipids. These fractions can be converted into high-value compounds through integrated extraction and purification strategies, supporting circular bioeconomy principles. The major processing routes and resulting products are summarised in Figure 1.

FPH represents one of the most strategically important pathways for the valorisation of fish-processing residues. Protein-rich fractions such as viscera, frames, heads, and skin contain substantial recoverable protein that can be converted into soluble peptides through controlled hydrolysis (Chalamaiah et al., 2012; He et al., 2013). In addition to serving as substrates, viscera contain endogenous enzymes that may facilitate autolytic hydrolysis, reinforcing integration between enzyme recovery and protein conversion (Bougatef et al., 2008).

FPH production can be achieved through acid, alkaline, or enzymatic hydrolysis (Pasupuleti and Braun, 2010; Tavano, 2013). Among these approaches, enzymatic hydrolysis is widely applied because it allows controlled production of peptides while maintaining nutritional quality. A more detailed discussion of enzymatic hydrolysis processes and enzyme systems is presented in Section 3.

As a result of this process, FPH exhibits desirable solubility, emulsifying capacity, foaming capacity, and gelling properties in food systems (Elavarasan, 2018). More importantly, derived peptides demonstrate antioxidant, antihypertensive, antimicrobial, and emerging metabolic regulatory effects (Balti et al., 2010; Bougatef et al., 2008; Batista et al., 2022).

3 Fish protein hydrolysate

3.1 Overview, nutritional composition, functional properties, and applications of fish protein hydrolysates

FPH can be defined as proteins derived from fish that have been chemically or enzymatically broken down into smaller amino acids and peptides, enhancing their functional and nutritional properties. FPHs are recognised for their high nutritional value, bioactive peptides, and applications in food and aquaculture industries (Gupta et al., 2025). Beyond nutritional benefits, FPH production can also contribute to circular bioeconomy strategies by valorising fishery by-products, reducing waste streams, and supporting sustainable development goals (AESAN, 2019; Coppola et al., 2021). The properties of FPHs depend on several factors, such as types of raw material used, enzymes used, degree of hydrolysis (DH), as well as MW of peptides that need to be addressed in a holistic approach (Zhao et al., 2023; Kristinsson and Rasco, 2000).

Nutritionally, fish is generally low in calories and a rich source of protein, typically ranging from 10% to 25% (FAO, 2022). FPHs are particularly known for high levels of protein that usually range from 60% to 90% (Bui et al., 2021). This high protein content is due to protein solubilisation that occurs during hydrolysis, removal of insoluble matter through centrifugation, and moisture reduction techniques such as spray-drying or freeze-drying (Herranz, 2014). In addition to that, fish is rich in essential amino acids such as tryptophan, lysine, methionine, and threonine that enhance digestibility (Borges et al., 2023). Studies revealed that FPHs may retain larger amounts of amino acids compared to raw fish with glutamic acid, aspartic acid, and arginine usually being the predominant (Khan et al., 2020). The total content of essential amino acids in FPHs is estimated to be nearly 50% (Bui et al., 2021). Recent comparative studies also show that FPHs derived from salmon and tuna by-products provide amino acid profiles comparable to casein hydrolysates, supporting their use in clinical nutrition (Vázquez et al., 2024). Although present in very minimal amounts, polyunsaturated fatty acids (PUFAs) such as EPA, DHA, and docosapentaenoic acid (DPA) are essential constituents of fish with a high concentration in areas such as the head, gill plates, and intestine that deliver cardiovascular health (Rana et al., 2023; Prihanto et al., 2019).

FPHs possess a range of bioactive properties that contribute to human health. They exhibit significant antioxidant activity by reducing hydroperoxides, scavenging free radicals, and chelating metal ions (Araujo et al., 2021). This activity is influenced by enzymatic hydrolysis conditions like pH, temperature, and time (Idowu et al., 2021). Lower-MW peptides, especially those below 3 kDa, are more effective at donating electrons or hydrogen atoms and reacting with free radicals (Hassan et al., 2019). The presence of hydrophobic and aromatic amino acids such as phenylalanine, tryptophan, and tyrosine also enhances the antioxidant capacity of the hydrolysates (Idowu et al., 2019). Additionally, FPHs, particularly those derived from fish by-products, have demonstrated antihypertensive activity through angiotensin-converting enzyme (ACE) inhibition (Pinela et al., 2022). This activity is dependent on the MW, amino acid sequence, and hydrophobicity of the peptides, with lower-MW peptides generally being more effective at inhibiting ACEs (AZTI, 2018; CECOPESCA, 2012). Meta-analyses of clinical trials suggest modest but consistent reductions in systolic blood pressure following supplementation with fish-derived peptides, though larger studies are needed to confirm efficacy (Desai et al., 2023).

FPHs also contain antimicrobial peptides, typically amino acid chains smaller than 10 kDa and 50 amino acids, which are often amphipathic, rich in cysteine, and positively charged (Rodríguez-Hernández et al., 2021). These peptides can induce bacterial cell death by forming pores or blocking membrane ion gradients, thereby disrupting cell integrity and respiration. Longer hydrolysis times, leading to higher-DH and lower-MW peptides, have been shown to enhance their antimicrobial effectiveness. In addition to these, FPHs demonstrate anticancer properties with research showing cytotoxicity on cancer cell lines especially from lower-MW peptides below 3 kDa and those with high hydrophobic content at the N-terminal position (Baco et al., 2022; Nguyen et al., 2022). Moreover, FPHs can contribute antidiabetic properties through inhibitory action on glucose regulatory enzymes such as α-amylase, α-glucosidase, and dipeptidyl peptidase-4 (DPP-IV) (Pezeshk et al., 2019; Phadke et al., 2021). The inhibitory ability depends on the MW as well as on the composition of the constituent’s amino acids in the FPHs (Guo et al., 2019). Emerging evidence also suggests neuroprotective effects of FPH peptides, with potential applications in delaying cognitive decline (Jimenez-Champi et al., 2024).

Technologically, FPHs possess several functional properties in food systems. Solubility is a significant marker of FPH functionality that is dependent on pH, DH, composition of amino acids, and MW (Pereira et al., 2020). In general, increased pH and DH enhance solubility through increased ionic as well as hydrogen bonding with water and increased polar as well as ionisable groups (Gao et al., 2021). FPHs also exhibit foaming capacity that can be attributed to the unfolding of peptides as well as the orientation of hydrophobic and hydrophilic groups at gas-liquid interfaces (Siddik et al., 2021). However, in certain cases, high DH may lower foaming properties since short peptides may struggle to sustain well-ordered interfacial orientation (Wu and Chen, 2022). Their emulsification capacity is another predominant characteristic since FPHs are surfactant materials exhibiting oil-in-water emulsions due to water solubility and possession of hydrophilic and hydrophobic groups (Saidi et al., 2018). Emulsifying activity index (EAI) and emulsion stability index (ESI) are dependent on FPH concentration and pH (Karoud et al., 2019). Finally, FPHs exhibit water-holding capacity (WHC) through hydrophilic polar groups that can increase cooking yield (Alahmad et al., 2023). Oil-holding capacity (OHC) relates to hydrophobicity and contributes to sensory attributes (Gan et al., 2022). Although lower-MW peptides may possess lower capacity to retain oil, OHC is dependent on enzyme-substrate specificity and DH (Sinthusamran et al., 2020). These functional properties make FPHs versatile ingredients in food formulations, improving texture, stability, and sensory quality (Rivero-Pino et al., 2020).

Owing to their many properties, FPHs are becoming popular within the food industry. They are added to a variety of foodstuffs like mayonnaise, yogurt, pasta, bread, and crackers, contributing to increased protein content and increased nutritional content (Unnikrishnan et al., 2021; Rivero-Pino et al., 2020; Lima et al., 2021; Witono et al., 2019). Besides that, FPHs can be employed as taste enhancers (Gao et al., 2020) as well as food preservatives owing to bioactive peptides (Honrado et al., 2024). Beyond food applications, FPHs are incorporated into animal feed, pet food, as well as cosmetic formulations, with future potential in protein supplements, sports nutrition, as well as infant formulas (Nisov et al., 2022). The FPHs marketplace is predicted to attain a value of USD 372.9 million in 2031 with a 4.4% compound annual growth rate from 2023’s value of USD 267.9 million (Fu et al., 2019). Industry reports also highlight growing demand in Asia-Pacific markets, driven by consumer interest in functional foods and sustainable protein sources (Pires et al., 2024).

In summary, FPHs are a critical and multipurpose ingredient with a broad nutritional profile as well as a variety of bioactive and technologically relevant properties. Their ability to upcycle fish by-products as a source of high-value-added commodities makes them central to sustainability in the fishing industry. Future research should focus on clinical validation of health claims, optimisation of large-scale production methods, and exploration of novel applications in pharmaceuticals and biomaterials.

3.2 Production of FPH via fermentation and enzymatic hydrolysis

3.2.1 Fermentation (microbiological hydrolysis)

Microbial fermentation involves the utilisation of proteolytic microorganisms to hydrolyse fish proteins (Abd El-Rady et al., 2023).

• Advantages: Reduced dependence on commercial enzymes, which lowers production costs while imparting distinctive flavours and functional attributes (Zhou et al., 2012).
• Limitations: Lower protein recovery and DH, together with higher microbial load that requires downstream processing such as pasteurisation or drying (Lee et al., 2022).
• Process steps: Adjustment of pH and solids content, inoculation with starter cultures or reliance on indigenous microbiota, and regulation of temperature, time, and aeration (Abd El-Rady et al., 2023).
• Challenges: Limited standardisation due to strain specificity and poorly reported fermentation conditions, which complicates reproducibility (Zhou et al., 2012).

3.2.2 Enzymatic hydrolysis

Enzymatic hydrolysis is recognised as the predominant method for producing high-quality FPHs, owing to precise control over hydrolysis and product characteristics (Sapatinha et al., 2024).

• Process overview:
• Preparation of raw fish by-products: cleaning and thermal treatment to remove non-protein debris and inactivate endogenous enzymes (Five, 2013).
• Mixing of substrates with selected proteolytic enzymes under controlled pH and temperature (Sapatinha et al., 2024).
• Termination of hydrolysis at the desired DH through heat inactivation (75–100 °C, approximately 30 min) (Abd El-Rady et al., 2023).
• Advantages:
• High protein yield with reproducible peptide size distribution (Geirsdóttir et al., 2011).
• Hydrolysates tailored for nutritional or functional applications (Nirmal et al., 2022).
• Sequential or combined enzyme strategies (endo- and exopeptidases) that improve peptide yield and bioactivity (Sapatinha et al., 2024).
• Limitations:
• High cost of commercial enzymes (Five, 2013).
• Risk of bitterness due to excessive hydrolysis (Abd El-Rady et al., 2023).
• Increased energy demand associated with thermal inactivation (Kristinsson and Rasco, 2000).

Representative studies on enzymatic hydrolysis of fish proteins are summarised in Table 1.

Table 1. Enzymes used for FPH production.

3.3 Enzymatic hydrolysis: overview of commercial enzymes

Enzymatic hydrolysis relies on:

• Endogenous enzymes naturally present in fish tissues (Five, 2013).
• Commercial proteases, such as Alcalase^®, Flavourzyme^®, Protamex^®, papain, and trypsin (Geirsdóttir et al., 2011).

Key process features:

• Hydrolysis under controlled pH, temperature, enzyme-to-substrate ratio, and time (Toma et al., 2022).
• DH affects peptide size, bioactivity (antioxidant, ACE-inhibitory), and functional properties such as emulsification and solubility (Lapeña et al., 2018).
• Sequential or combined enzyme strategies (endo- and exopeptidases) enhance peptide yield and bioactivity (Coscueta et al., 2024).
• Standardised use of commercial enzymes produces reproducible hydrolysates scalable for industry (Hayes et al., 2024).

Applications:

• Food, feed, and nutraceutical formulations (Liaset et al., 2003).
• In vitro studies show antioxidant and ACE-inhibitory activities, though clinical validation remains limited (Hayes et al., 2024; Lapeña et al., 2018).

3.4 Hydrolysis mechanisms of Alcalase^®, Flavourzyme^® Neutrase^®, and Protamex^®

The hydrolysis mechanisms of major commercial enzymes differ in cleavage specificity, peptide size, and resulting functional properties, shaping the composition and bioactivity of FPH (Kristinsson and Rasco, 2000; Je et al., 2007; Merz et al., 2015; Liaset et al., 2003). The enzymatic hydrolysis mechanisms of major commercial proteases are illustrated in Figure 2.

Key enzymes and mechanisms:

• Alcalase^® (serine endopeptidase, Bacillus licheniformis)
• • Operates via a catalytic triad of serine, histidine, and aspartate residues (Rao et al., 1998).
• • Cleaves peptide bonds at the carboxyl side of hydrophobic residues such as leucine, tyrosine, and phenylalanine (Je et al., 2007).
• • Produces low-MW peptides (<5–10 kDa) enriched in hydrophobic amino acids, contributing to antioxidant and emulsifying properties (Kristinsson and Rasco, 2000).
• • Optimal conditions: pH 8–10, 50–60 °C.
• • Often used first in sequential hydrolysis to release bioactive peptides (Hoyle and Merritt, 1994).
• Flavourzyme^® (endo- and exopeptidase complex, Aspergillus oryzae)
• • Combines endopeptidase activity (internal cleavage) with exopeptidase activity (terminal amino acid removal), producing short peptides and free amino acids (Merz et al., 2015).
• • Prefers hydrophobic amino acids such as leucine, phenylalanine, and tyrosine, altering flavour characteristics (Wu et al., 2003).
• • Operating conditions: pH 6–7, 45–55 °C, enzyme-to-substrate ratio 0.5–1%, hydrolysis time 2–4 h (Alahmad et al., 2023).
• • Produces peptides <1 kDa, improves digestibility, reduces bitterness, and enhances umami taste (Cui et al., 2021).
• Neutrase^® (neutral metalloprotease, Bacillus amyloliquefaciens)
• • Functions at neutral pH (6.5–7.5), with a Zn²⁺ ion in its catalytic centre (Mótyán et al., 2013).
• • Targets hydrophobic residues, producing shorter peptides with reduced bitterness compared to Alcalase^®.
• • Effective against collagen and connective tissue proteins, generating peptides with antioxidant potential and possible benefits for skin and joint health (Jongjareonrak et al., 2006).
• • Optimal conditions: 40–45 °C, pH 7, enzyme-to-substrate ratio 0.8% (Shen et al., 2012).
• Protamex^® (broad-spectrum protease complex, Bacillus spp.)
• • Exhibits both endopeptidase and exopeptidase activity, hydrolysing sarcoplasmic and connective tissue proteins (Liaset et al., 2003).
• • Cleaves near hydrophobic and aromatic residues, producing small- to medium-sized peptides with balanced nutritional and sensory properties (Thiansilakul et al., 2007).
• • Optimal conditions: pH 6.5–7.5, 50–55 °C, hydrolysis time 1–3 h.
• • Often used in combination with Alcalase^® or Flavourzyme^® to enhance hydrolysis efficiency while maintaining palatability (Di Filippo et al., 2024).

Notes

• Enzyme selection controls peptide size, DH, and bioactivity (Lapeña et al., 2018; Coscueta et al., 2024).
• Operational challenges include enzyme cost, pH/temperature control, risk of bitterness, and potential salt increase at alkaline conditions (Kristinsson and Rasco, 2000).

Detailed characteristics, optimum pH, temperature, main products, and bioactivities are summarised in Table 2.

Table 2. Characteristics and functional properties of major commercial enzymes used in FPH production.

4 Endogenous protease from fish source

4.1 Background of endogenous proteases from fish by-products

Historically, scientists have depended on various commercial enzyme mixtures, including Alcalase^®, Flavourzyme^®, Neutrase^®, and Protamex^®, as substrates for making industrial FPH because they provided reliable and predictable enzymatic activity. The endogenous or autolytic proteases constitute these natural enzymes that are present within various tissues and organs within a fish because they are primarily responsible for the process of digestion and protein turnover (Ghosh et al., 2024).

The appeal of endogenous proteases for FPH production primarily relates to cost and sustainability. They offer a cost-effective and sustainable alternative by reducing or eliminating reliance on commercial enzymes, while simultaneously valorising fish waste into useful products. This approach aligns with circular economy principles and zero-waste strategies. However, autolysis produces hydrolysates with different functionalities compared to those generated by commercial enzymes and typically results in lower DH (Domínguez et al., 2024).

Fish and other marine organisms provide a wide range of endogenous enzymes, including proteases, lipases, carbohydrases, chitinases, and transglutaminases (Khiari, 2024; Shahidi and Kamil, 2001). These enzymes are distributed across tissues such as stomach, viscera, pancreas, and muscle, where they contribute to natural metabolic processes. Representative examples and their functions are summarised in Table 3.

Table 3. Enzymes extracted from fish waste.

4.2 Classification of fish protease

Fish proteases can be categorised into four main mechanistic classes based on their mechanisms and active site residues, with unique sets of biochemical properties influencing their applications in production processes for FPH. Aspartyl proteases are the primary gastric digestive enzymes of fish stomachs, and pepsin is the most prominent among them. They prefer very low and highly acidic conditions, with optimal activity at pH 2–3, as it occurs in a fish stomach (Peña-Marín et al., 2021; Córdova-Murueta et al., 2024). Their activity can be inhibited by pepstatin A, and they prefer to cleave peptide bonds before hydrophobic amino acids. Pepsin-like enzyme activity with an optimal pH at an extremely low value and sensitivity to pepstatin A has been detected in Pacific whiting and Pacific red snapper stomachs using zymography (Peña-Marín et al., 2021; Córdova-Murueta et al., 2024). The optimal temperatures for these proteases lie within 30–45 °C, but they differ based on thermal ecology.

Serine proteases, particularly trypsin and chymotrypsin, are recognised as prominent enzymatic components in the fish intestine and associated tissues. Unlike the case with the acidic proteases, serine proteases are more active in an alkaline medium but typically at pH 6–10 (Córdova-Murueta et al., 2024; Jesus-Ramirez et al., 2017; Kulsum and Nugrahapraja, 2023). Their activity can be inhibited by serine protease inhibitors like phenylmethylsulfonyl fluoride. Trypsin acts on the carboxyl side of basic residues lysine and arginine, and chymotrypsin prefers an aromatic residue. Fish species such as Merluccius productus, Fat Snook (Centropomus parallelus), and eels (Anguilla bicolor) have multiple forms of trypsins and chymotrypsins with slightly varied preferences for pH and temperature (Córdova-Murueta et al., 2024; Jesus-Ramirez et al., 2017). These varied characteristics denote adaptabilities within the fish digestive system for optimal processing of varied protein molecules.

Cysteine proteases, and more specifically cathepsins, play a prominent role in muscle cell-protein breakdown and muscle proteolysis. These enzymes normally act at near-neutral to slightly low pH and are characterised by the presence of a cysteine residue at their active site. Cathepsins play an active role in muscle texture development and changes that occur because of storage and processing (Ghosh et al., 2024). Although they are less frequently involved in FPH production compared to digestive proteases, they still have an important role in autolytic hydrolysis. Their role contributes to understanding changes that occur because of traditional processing, for instance, fermentation involved in fish sauce production.

Metalloproteases rely on metal co-factors, usually Zn²⁺, as a catalyst and can be inhibited by metalloprotease inhibitors like EDTA. These enzymes take part in extracellular matrix turnover and general protein breakdown (Ghosh et al., 2024; Peña-Marín et al., 2021). While they have not been as widely characterised within fish digestive systems as other classes, metalloproteases have been identified in Pacific red snapper intestinal extracts using inhibitor methods (Peña-Marín et al., 2021). Their specific contribution within FPH production has yet to be completely understood but would appear to play supporting roles within protein hydrolysis. Their specific contribution within FPH production has yet to be completely understood but would appear to play supporting roles within protein hydrolysis.

A summary of the major protease classes derived from fish by-products, together with their representative enzymes, anatomical sources, and functional properties, is presented in Table 4 (Khiari, 2024; Mótyán et al., 2013; Shahidi and Kamil, 2001; Sriket, 2014).

Table 4. Classification of major protease enzymes from fish by-products, their sources, and functional properties.

Adapted from Khiari (2024), Mótyán et al. (2013), Shahidi & Kamil (2001), and Sriket (2014).

4.3 Anatomical sources of endogenous proteases

Proteolytic enzymes in a fish are not evenly distributed within its body. Instead, they are classified according to tissue location, reflecting their roles in digestion and metabolism. The anatomical distribution of major endogenous proteases in fish tissues is schematically illustrated in Figure 3.

The stomach is the primary site of aspartyl proteases, mainly pepsins, which begin the process of decomposing proteins at an acidic pH (Peña-Marín et al., 2021; Córdova-Murueta et al., 2024). The stomach tissues in carnivorous and omnivorous species have large concentrations of acid proteases adapted to a pH 2–3. The stomach-based organs have been shown to display maximum acid protease activity among all enzymatic contents within different species based on diet (Peña-Marín et al., 2021; Jesus-Ramirez et al., 2017).

The intestine and associated structures, such as pyloric caeca have been recognised as the most prominent sources of high-alkaline serine proteases, including trypsin and chymotrypsin (Peña-Marín et al., 2021; Córdova-Murueta et al., 2024; Jesus-Ramirez et al., 2017). These organs are primarily associated with the process of protein digestion in fish and have shown multiple proteolytic activities at an alkaline pH range, indicating the presence of multiple forms of trypsin and chymotrypsin. During autolytic FPH production at an alkaline pH, organs like the intestine and hepatopancreas are highly preferred. Additionally, as indicated from rainbow trout viscera research, these tissues with high enzyme activity possess excellent scope for valorisation via autolysis at an operating pH of 8 and a temperature of 40 °C (Domínguez et al., 2024).

Viscera, as well as hepatopancreas and associated digestive glands, accumulate both digestive and metabolic proteases, thus identifying these as outstanding sources for enzyme-assisted processing. Studies on fish processing side streams have constantly listed viscera at the top among sources fit for autolytic hydrolysis due to high endogenous protease levels (Domínguez et al., 2024; Siddiqui et al., 2023). This practice has triggered an ever-increasing interest in making something out of viscera as FPH, as an approach meant to reduce waste and make use of sources that constitute as much as 30–40% of the whole weight of a whole fish.

Muscle tissue, while not being as rich in enzymes as digestive organs, still retains significant levels of cathepsins and other proteases (Ghosh et al., 2024). Muscle-associated proteolytic enzymes are implicated in softening and flavour enhancements that occur during storage. Although muscle proteolytic enzyme activity remains lower compared with viscera, it becomes very significant when working with whole by-products and with prolonged autolysis times. An understanding of regions with muscle-associated proteases would enable control and prediction of autolytic procedures using mixed feedstocks.

5 Recovery and purification of endogenous proteases

5.1 Conventional methods for recovery and purification

5.1.1 Overview of conventional purification approaches

The conventional method refers to traditional, well-established techniques commonly used to recover and purify enzymes, such as proteases, from biological sources like fish. These approaches exploit physicochemical properties such as solubility, charge, molecular size, and affinity. Although originally developed for studying enzyme mechanisms and protein structures, they remain widely applied for preparing biocatalysts. A key measure of purification efficiency is the specific activity of enzyme preparation, known as units of activity per mg protein. Improvements in specific activity after each purification step indicate higher enzyme purity (Westphal and van Berkel, 2021).

Conventional purification typically follows a multistep scheme including cell disruption, clarification of crude extracts, and successive separation processes such as selective precipitation and chromatography (Westphal and van Berkel, 2021). These methods often involve steps such as precipitation by using the ammonium sulphate precipitation method to concentrate enzymes by reducing their solubility, centrifugation to separate solid from liquid phases, dialysis or ultrafiltration to remove small molecules and concentrate the enzyme, and chromatographic techniques such as IEC and SEC for further purification in some cases (Ketnawa et al., 2013).

Although effective, these methods are usually time-consuming, costly, and require multiple steps, which may lead to loss of enzyme activity and reduced yields. In addition, large chemical volumes raise concerns about scalability and environmental impact (Ketnawa et al., 2013; Westphal and van Berkel, 2021). For example, ammonium sulphate fractionation often enhances recovery but may only achieve modest purification folds unless combined with other steps such as IEC or SEC (Westphal and van Berkel, 2021).

5.1.2 Precipitation techniques

Precipitation is widely used in protease purification, with ammonium sulphate precipitation being the most common. This technique is based on the principle of differential solubility, where proteins precipitate out of solution when the ionic strength is increased by adding salts such as ammonium sulphate. The precipitated proteins are collected by centrifugation and resuspended in buffer for further purification. This method is simple, cost-effective, and suitable for large volumes, but often yields low purification folds, requiring subsequent steps (Klomklao et al., 2007). Several studies have successfully employed conventional methods to isolate proteases from fish viscera and by-products. Ammonium sulphate fractionation followed by chromatography has yielded proteases with enhanced purity and specific activity, though overall recovery remains low, limiting industrial application.

5.1.3 Chromatographic techniques

Chromatography is another important technique used in protease purification, and various chromatographic methods have been developed to separate proteins based on their physicochemical properties. IEC separates proteins based on their net charge, where proteins bind to charged resins and are eluted by changing the ionic strength or pH of the buffer. SEC separates proteins based on their molecular size, where larger proteins elute first and smaller proteins elute later (Bougatef et al., 2008). Affinity chromatography is a highly specific technique that separates proteins based on their biological function or chemical structure, where proteins bind to specific ligands immobilised on a solid support and are eluted by changing the buffer conditions (Bougatef et al., 2008). Hydrophobic interaction chromatography (HIC) separates proteins based on their hydrophobicity, where proteins bind to hydrophobic resins at high salt concentrations and are eluted by decreasing salt concentration. These chromatographic techniques are often used in combination to achieve high purification folds and enzyme purity (Bougatef et al., 2008).

IEC has been widely used to purify proteases from fish viscera due to its high resolution and capacity to separate proteins with different charges. For example, Khantaphant and Benjakul (2008) purified a protease from the hepatopancreas of Pacific white shrimp using DEAE-Sepharose IEC and achieved a purification fold of 15.6 with a recovery of 23.4%. Similarly, Bougatef et al. (2008) purified a protease from the viscera of sardine using Q-Sepharose IEC and achieved a purification fold of 12.3 with a recovery of 18.7%. These studies demonstrate that IEC is an effective technique for purifying proteases from fish viscera, although the recovery may be relatively low due to protein loss during the purification process (Khantaphant and Benjakul, 2008; Bougatef et al., 2008).

SEC has also been used to purify proteases from fish by-products, particularly for separating proteins based on their MW. For instance, Klomklao et al. (2007) purified a trypsin-like protease from the pyloric caeca of walleye pollock using Sephacryl S-200 SEC and achieved a purification fold of 8.4 with a recovery of 31.2%. This technique effectively removes high- and low-MW contaminants from enzyme preparations and is often used as a polishing step after IEC. However, SEC has a relatively low resolution compared to other chromatographic techniques and may not be suitable for separating proteins with similar-MW (Klomklao et al., 2007). Representative studies reporting the recovery and purification of endogenous proteases from fish waste and by-products using conventional techniques are summarised in Table 5.

Table 5. Recovery and purification of protease enzymes from fish waste and by-products.

5.1.4 Limitations of conventional methods

Despite their effectiveness, conventional methods have several limitations. They are time-consuming, require multiple steps, and often lead to enzyme denaturation and activity loss. Large volumes of chemicals and buffers increase costs and environmental concerns. Furthermore, low recovery and yields make them economically unfeasible for large-scale applications, especially for low-value enzymes from fish waste (Ketnawa et al., 2013). These drawbacks highlight the need for alternative purification methods that are more efficient, cost-effective, and environmentally sustainable.

5.2 Alternative methods: aqueous two-phase systems

5.2.1 Principles and advantages of ATPS

To overcome the limitations of conventional purification methods, alternative techniques such as ATPS and TPP offer several advantages for fish protease recovery. ATPS provides a gentle, non-denaturing environment that preserves enzyme activity and minimises loss. This is achieved by using biocompatible solvents such as PEG and salts such as magnesium sulphate, which create a mild phase separation without exposing enzymes to high temperatures (Kuepethkaew et al., 2017).

ATPS is scalable and cost-effective, making it suitable for large-scale applications. High yield and purity can be achieved in a single step by separating biomolecules based on physicochemical properties. Compared to conventional methods, ATPS is more efficient, requires fewer purification steps, and reduces environmental impact by using biodegradable and recyclable components (Ketnawa et al., 2013). It is based on liquid–liquid partitioning, where two immiscible aqueous phases are formed by mixing polymers or a polymer and a salt. Proteins partition between phases according to their physicochemical properties, enabling selective purification (Ketnawa et al., 2013). The overall workflow and key differences between ATPS and TPP for fish protease purification are summarised in Figure 4.

5.2.2 Applications of ATPS in fish protease recovery

Several studies have demonstrated the effectiveness of ATPS for purifying proteases from fish viscera and by-products. For example, Ketnawa et al. (2013) used a PEG/MgSO₄ ATPS to purify proteases from hybrid catfish viscera, achieving a 6.8-fold purification with 89.3% recovery in a single step. Similarly, Klomklao et al. (2007) purified a serine protease from tuna viscera using PEG/salt ATPS, achieving a 5.2-fold purification with 85.7% recovery. These findings highlight ATPS as a promising alternative to conventional methods, offering high recovery and purification in one step.

The performance of ATPS can be optimised by adjusting parameters such as polymer type and concentration, salt composition, pH, and temperature. For instance, Kuepethkaew et al. (2017) reported that PEG-4000 combined with MgSO₄ provided the best partitioning coefficient and recovery for proteases from shrimp hepatopancreas. Careful optimisation of ATPS parameters is therefore essential for maximising enzyme recovery.

5.2.3 Mechanism and factors affecting ATPS performance

Protein partitioning in ATPS is influenced by polymer MW and concentration, salt type and concentration, pH, temperature, and protein physicochemical properties. Typically, hydrophobic proteins partition into the PEG-rich phase, while hydrophilic proteins remain in the salt-rich phase (Klomklao et al., 2007). By manipulating these factors, it is possible to selectively partition the target protein to one phase while leaving contaminants in the other phase, thereby achieving purification.

5.3 Alternative methods: three-phase partitioning

5.3.1 Principles and mechanisms of TPP

TPP is another alternative purification technique used for proteases from fish by-products. TPP combines salting-out with organic solvent addition, typically ammonium sulphate and t-butanol, resulting in three distinct phases. The target protein precipitates at the interface between the aqueous and organic phases and can be collected by centrifugation. TPP offers advantages such as simplicity, rapidity, high recovery, and the ability to concentrate and purify proteins in a single step (Devi et al., 2015).

5.3.2 Applications of TPP in fish protease purification

Several studies have reported on the successful application of TPP for purifying proteases from fish viscera and by-products. For example, Devi et al. (2015) used TPP to purify a protease from the viscera of rohu and achieved a purification fold of 7.2 with a recovery of 82.5% in a single step. These studies suggest that this technique is a promising alternative to conventional methods. The performance of TPP can be optimised by adjusting various parameters, such as the concentration of ammonium sulphate, the ratio of t-butanol to crude extract, pH, temperature, and incubation time. These findings highlight the importance of optimising TPP parameters to achieve maximum enzyme recovery and purification (Devi et al., 2015).

5.3.3 Advantages and limitations of TPP

TPP offers several advantages over conventional purification methods, including simplicity, rapidity, low cost, and the ability to concentrate and purify proteins in a single step (Devi et al., 2015). However, TPP has some limitations, including the use of organic solvents such as t-butanol, which may pose safety and environmental concerns. Despite these limitations, TPP remains a promising alternative purification technique for fish proteases (Devi et al., 2015).

5.4 Comparison of efficiency, sustainability, and yield of purification methods

5.4.1 Recovery and purification efficiency

The recovery and purification efficiency of different methods can be compared using parameters such as purification fold, recovery, specific activity, and processing time. Conventional methods such as ammonium sulphate precipitation followed by chromatography typically achieve high purification folds (10.0–20.0-fold) but relatively low recovery (20–40%) due to protein loss during multiple steps (Ketnawa et al., 2013). In contrast, ATPS and TPP achieve moderate purification folds (5.0–10.0-fold) with high recovery (80–90%) in a single step, making them more efficient for large-scale enzyme production. For example, Ketnawa et al. (2013) reported that ATPS purification of hybrid catfish viscera proteases achieved a 6.8-fold purification with 89.3% recovery, compared to conventional methods that achieved a 12.3-fold purification but only 23.4% recovery. This demonstrates that ATPS offers higher recovery and operational efficiency, although purification folds may be lower.

5.4.2 Sustainability and environmental impact

Sustainability and environmental impact are critical considerations for industrial applications, particularly in the valorisation of fish-processing by-products. Conventional methods require large volumes of chemicals and buffers, generating significant waste and raising environmental concerns. In contrast, ATPS and TPP use fewer chemicals and can incorporate recyclable components, making them more environmentally friendly. For example, PEG and salts in ATPS can be recovered and reused, while ammonium sulphate in TPP can be recycled, reducing waste and cost (Ketnawa et al., 2013). These features position ATPS and TPP as more sustainable alternatives to conventional purification methods.

5.4.3 Economic considerations and scalability

Cost-effectiveness defines the viability of a purification technique for industrial scale-up. Conventional chromatographic systems involve high equipment costs, expensive resins, buffers, and maintenance, which are justified for pharmaceutical proteins but not for enzymes derived from waste streams. ATPS and TPP systems require only basic equipment, which includes mixing tanks, centrifuges, or settling tanks that are relatively low-cost and accessible, particularly in developing countries. The agents used in ATPS and TPP are inexpensive, especially when recovery schemes are applied, making them more economically viable than conventional chromatography (Klomklao et al., 2007).

5.4.4 Processing time and operational simplicity

Processing time is another important factor for industrial applications, as longer procedures increase the risk of enzyme denaturation and activity loss. Conventional methods often require multiple steps over several days, for example, precipitation, dialysis, and chromatography. In contrast, ATPS and TPP can be completed within hours. Phase separation in ATPS occurs spontaneously, while protein precipitation in TPP is rapid. For instance, Ketnawa et al. (2013) reported that ATPS purification of catfish proteases was completed in 2–3 hours, compared to 3–4 days for conventional methods. This highlights the time-saving advantage of alternative methods, which preserve enzyme activity and improve efficiency.

5.4.5 Conclusion

In summary, ATPS and TPP represent valid alternatives to conventional purification methods, offering higher recovery, reduced processing time, lower costs, and improved sustainability. These features make them promising techniques for industrial applications and for the valorisation of fish-processing by-products.

6 Applications of fish proteases

Fish proteases recovered from fish by-products are versatile biocatalysts with broad industrial relevance, including applications in food, detergents, leather, pharmaceuticals, aquafeed, waste management, and research. Their utilisation enhances process efficiency and sustainability while contributing to circular economy strategies by valorising fishery side streams. The required purity grade of proteases is critical, since it directly influences activity, stability, and specificity depending on the intended application (Jimenez-Champi et al., 2024).

In the food industry, fish proteases are widely applied in protein hydrolysis processes that improve texture, flavour, and nutritional quality. They accelerate fermentation in traditional products such as fish sauce and contribute to the production of FPH and bioactive peptides with recognised health benefits, including cardiovascular support and immune modulation (Kim et al., 2002; Tavano et al., 2018). Purity is particularly important in food applications, since contaminants could compromise taste, safety, or nutritional value (Tavano et al., 2018). For large-scale FPH production, partially purified or crude extracts may suffice if safety and quality standards are met.

Beyond food systems, fish proteases play a key role in detergent formulations, where they catalyse the degradation of protein-based stains in laundry and dishwashing products. Their catalytic efficiency at lower temperatures supports energy-saving formulations, while stability under alkaline conditions and in the presence of surfactants makes them environmentally favourable alternatives to harsh chemical agents (Ward, 2011; Tavano et al., 2018). In such mid-grade applications, partial purification through techniques such as ultrafiltration or TPP is generally adequate to ensure performance and stability.

In the leather and textile industries, proteases replace aggressive chemicals in dehairing, bating, wool processing, and silk degumming. This biocatalytic approach improves selectivity, reduces environmental burden, and enhances the quality of final products. However, insufficient purification could damage fibres and compromise texture and durability, making controlled purity levels essential for maintaining process reliability (Kim et al., 2002).

Fish proteases also contribute significantly to the pharmaceutical and nutraceutical sectors. They are employed in the controlled production of bioactive peptides with antihypertensive, antioxidant, antimicrobial, and anti-inflammatory properties, which are incorporated into functional foods and therapeutic formulations (Chen et al., 2024; Jimenez-Champi et al., 2024). In pharmaceutical manufacturing, highly purified proteases are required to ensure safety, reproducibility, and regulatory compliance. Multi-step chromatographic purification is therefore commonly applied for high-grade biomedical uses.

In aquaculture, protease-generated hydrolysates enhance protein digestibility, feed palatability, and nutrient absorption, thereby improving growth performance and overall health of cultured species (Kim et al., 2002). This application represents a sustainable strategy for recycling fish-processing residues into value-added feed ingredients. In such systems, crude or minimally processed enzyme preparations are often sufficient, as complete purification is not economically necessary.

Fish proteases further contribute to waste management and environmental sustainability by facilitating organic waste decomposition and reducing pollutant load. Their integration into valorisation frameworks enables conversion of fish-processing by-products into fertilisers, biodiesel precursors, and other bioactive extracts, strengthening circular bioeconomy models (Jimenez-Champi et al., 2024). These applications typically tolerate lower purity levels, focusing instead on catalytic efficiency and cost-effectiveness.

In research and diagnostics, fish proteases serve as laboratory reagents in proteomics, biochemical assays, and enzyme characterisation studies (Chen et al., 2024). Their unique catalytic properties, including activity at specific temperature and salinity ranges, make them useful tools in specialised analytical workflows. Such applications demand higher purity and well-characterised enzyme fractions to ensure reproducibility and specificity.

The applicability of fish proteases therefore depends strongly on the required level of purification. Low-grade applications, such as FPH production, aquafeed, and waste management, can rely on crude extracts with minimal downstream processing. Mid-grade industrial uses, including food additives, detergents, and textile treatments, generally require partial purification to balance performance and cost. High-grade applications in pharmaceuticals and diagnostics demand highly purified enzymes obtained through multi-step chromatographic techniques to ensure safety, stability, and specificity (Jimenez-Champi et al., 2024).

Collectively, these diverse applications demonstrate how fish proteases function not only as industrial catalysts but also as strategic components of integrated fish by-product valorisation systems, reinforcing the circular framework outlined throughout this review.

7 Concluding remarks and future perspectives

Increased processing of fish globally has resulted in a significant generation of by-products, thereby creating environmental challenges but also offering opportunities for valorisation. It is evident from this review that fish processing waste valorisation represents a viable and sustainable strategy to address environmental concerns associated with waste disposal by converting underutilised by-products into valuable biocompounds. Recovering proteins, enzymes, lipids, and bioactive compounds from fish waste mitigates environmental problems while supporting the development of a global blue bioeconomy (Coppola et al., 2021; Fotodimas et al., 2025; Desai et al., 2023).

Among the valorisation techniques discussed, enzymatic hydrolysis is one of the most effective approaches for converting protein-rich fish by-products into value-added products such as FPH. The use of natural proteases present in fish viscera, together with commercial proteases, has proved effective in the production of bioactive compounds with desirable MW characteristics, making them suitable for functional foods, nutraceuticals, and pharmaceutical products (Borges et al., 2023; Iñarra et al., 2023). At the same time, developments in separation and purification technologies, such as membrane processing and encapsulations, continue to improve the yield and biostability of these bioactive compounds, enhancing their industrial applicability (Li, 2022; Ghalamara et al., 2024).

Despite these promising advancements, several challenges remain before fish waste bioprocessing can be fully adopted at an industrial scale. Future studies must be aimed at optimising and standardising hydrolysis process variables to ensure consistent product quality regardless of starting material. Several variables must be precisely controlled, including enzyme type, enzyme-to-substrate ratio, temperature, and reaction time. These parameters influence peptide profiles and bioactivity (Iñarra et al., 2023; Vázquez et al., 2024). Furthermore, greater emphasis should be placed on the use of stable, naturally derived enzyme preparations isolated from fish viscera, which represent a cheaper and eco-friendly alternative to commercial enzymes and support zero-waste processing (Borges et al., 2023; Manni et al., 2024).

There also needs to be further investigation into the biological efficiency, safety, and consumer acceptance of bioactive peptides derived from fish. Extensive in vivo testing will be required to substantiate health-related claims. In addition, new non-thermal approaches such as subcritical water extraction could serve as alternative processes to enzymatic hydrolysis, potentially reducing processing costs while preserving bioactivity (Pires et al., 2024).

In summary, valorisation of fish-processing by-products is a promising and strategic pathway to enhance sustainability in fisheries and aquaculture. Future innovations, together with collaboration between different stakeholders, including researchers, industry actors, and policymakers, will be essential for achieving a shift from a linear “catch-processing-discard model to a circular bioeconomy (Coppola et al., 2021; Desai et al., 2023).By emphasising the bridge between fish by-products, proteases, and hydrolysates, this review highlights the potential of fish waste valorisation to contribute to environmental protection, food security, and sustainable production of bio-based materials.

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Footnotes

Edited by: Elenice Souza Dos Reis Goes, Federal University of Grande Dourados, Brazil

Reviewed by: Theresia Desy Askitosari, University of Surabaya, Indonesia

Federica Grasso, University of Genoa, Italy