Sustainable Aquaculture Through Enzymatic Hydrolysis of Raw Chitin from Crab By-Products: Functional Fish Feeds Targeting Fish Health with Implications for Human Health

Abstract

Valorisation of crab by-products by enzymatic hydrolysis (EH) is proving to be a promising strategy to promote sustainable aquaculture and support a circular economy for crustaceans. Crab processing generates significant amounts of by-products that, if not properly managed, pose an environmental and economic challenge. These by-products are rich in chitin, proteins, and bioactive compounds and offer significant untapped potential for the development of functional feed. This review focuses on the application of enzymatically hydrolysed crab by-products as functional feed additives in aquaculture and their effects on fish growth, health management, and, consequently, human health. Recent studies have shown that EH effectively recovers chitin and bioactive peptides and improves the digestibility and bioavailability of nutrients in aquaculture. The inclusion of crude chitin, along with residual proteins and calcium carbonate, in the diet of farmed fish has been associated with increased growth, improved immune responses, and greater disease resistance, emphasising their critical role in fish health management. In addition, these functional additives contribute to the development of innovative aquafeeds with high added value and improved nutritional quality, while reducing environmental waste. Overall, the utilisation of crustacean by-products through enzymatic hydrolysis represents a valuable tool for the sustainable development of crustacean aquaculture, promotes the circular economy, and supports the development of innovative functional feeds while improving the growth and health of farmed fish, which has a positive impact on human health through their consumption.

1. Introduction

The seafood processing industry generates millions of tonnes of waste from crabs, shrimps and lobsters every year, with the inedible parts accounting for about 50–70% of the total biomass [1,2,3]. Usually, these by-products are disposed of in landfills, incinerated, or composted, leading to environmental pollution and waste of valuable resources [4,5,6]. The chemical composition of the shells contains valuable components such as chitin, chitosan, proteins, calcium carbonate, and astaxanthin [7,8]. These by-products are rich in nutrients, making them suitable for the production of high-quality animal feed. Chitin and its derivatives are used in various fields, including agriculture, aquaculture, the food industry, pharmaceuticals, and cosmetics [8,9]. Chitin, which is abundant in shrimp, crab, and lobster shells, consists of N-acetylglucosamine units linked by β-(1→4)-glycosidic bonds [10,11]. Deacetylation of chitin produces chitosan, while further chemical or EH yields chitooligosaccharides (COS), such as N,N′-diacetylchitobiose (GlcNAc)₂, which have antimicrobial, antioxidant, anti-inflammatory, and immunostimulatory properties [12]. The utilisation of chitin from crab shells is considered strategically important to reduce waste and promote environmental sustainability [4,5]. Chitin isolation can be carried out using traditional chemical methods (acids/bases) or “greener” approaches, such as EH with proteolytic enzymes, as well as more advanced technologies such as microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), the use of ionic liquids (ILs), or electrochemical methods [11,13,14]. The extraction of high-purity chitin requires processes of deproteinisation, demineralisation, decolourisation and deacetylation [15]. Similarly, chitin obtained by EH may contain residual proteins and calcium carbonate but is still suitable as a feed additive in aquaculture, and promotes sustainable waste utilisation [10,14]. Chitosan, which is soluble in water and many organic solvents, has bacteriostatic, antioxidant, and immunomodulatory effects [12]. Low concentrations of chitin (<0.1–1%) in feed have been shown to enhance immune responses and disease resistance in various fish species, including rainbow trout Oncorhynchus mykiss [16,17], Nile tilapia Oreochromis niloticus [18,19], olive flounder Paralichthys olivaceus [20], and Asian seabass Lates calcarifer [21]. In contrast, tilapia hybrids Oreochromis niloticus exhibited reduced growth at higher concentrations of chitin or chitosan [22]. Chitosan, derived from partial deacetylation of chitin, is also used in fish feeds and appears to have similar immunostimulatory and antimicrobial effects. Due to its higher solubility and bioavailability, effective concentrations may be slightly lower than those of chitin. Additionally, the anti-inflammatory, hypocholesterolaemic, and antimicrobial properties of both chitin and chitosan contribute to improved gut health, promote microbial balance, and reduce pathogenic microorganisms [23]. Consuming seafood rich in chitin and chitosan may provide benefits to humans, such as regulating glucose, insulin, cholesterol, and triglycerides, improving lipid metabolism, boosting immune defences and reducing oxidative stress and inflammation. COS offer higher bioavailability and a faster effect. However, their production remains costly and complex, which limits their compatibility with the principles of circular economy and sustainable development [24,25]. This review focuses on the EH of crab by-products, the techniques used to process the waste, the conditions of use and the products obtained, with an emphasis on the utilisation of chitin. The use of crude chitin with residual proteins and calcium carbonate as a feed enrichment ingredient is also discussed, which may improve fish growth, survival, and overall health. The bioactive components can be assimilated by humans through diet, potentially offering health benefits.

2. Methodology

The data used in this literature review were selected according to strict scientific criteria, focussing on the enzymatic hydrolysis of crab by-products to obtain bioactive compounds, with particular emphasis on chitin. The review also examines the application of chitin in fish farming through feeds enriched with this biopolymer, as well as its effects on the growth, health, and physiology of farmed species, with indirect effects on human health through diet. A total of 100 scientific articles were evaluated, of which 96 met the specified criteria and were included in the analysis. Articles were excluded because they combined EH with other processing technologies that fell outside the scope of this study. Data searches were conducted in Elsevier, Springer, Google Scholar, and Scopus databases, using combinations of keywords: Crab by-products, enzymatic hydrolysis, chitin utilisation, experimental chitin diets, benefits to fish, benefits to humans. The included studies cover the period 1997–2025 and thus offer a comprehensive time frame that reflects the development and progress of scientific research in this field. The selection of this time period allows for an in-depth evaluation of the enzymatic hydrolysis method using different proteolytic enzymes, as well as an evaluation of studies on different fish species enriched with different amounts of chitin over different feeding periods, to achieve specific benefits. This approach contributes to a comprehensive understanding of the technological developments, innovations, and trends that have shaped the crustacean processing industry, with a particular focus on the utilisation of crab biomass, thus providing an up-to-date overview of the field.

3. Chitin Extraction Technologies from Crustacean By-Products

Several studies focus on the potential recovery of chitin from properly processed biomass of crustacean by-products such as crabs, shrimps and lobsters, as shown in Table 1. The method of EH is based on the action of proteolytic enzymes that cause deproteinisation of the biomass. After completion of the process, the utilisable products are the sediment, which consists of crude chitin with residual protein and calcium carbonate, and the supernatant, which contains hydrolysed proteins [26]. Similarly, the chemical method for chitin recovery is based on the use of dilute acids that remove the calcium carbonate from the chitin through a demineralisation process. The process is completed by deproteinising the chitin by thermal alkaline treatment, which removes the proteins [27]. The concentration of the alkaline solution, typically NaOH, should be indicated, e.g., 1–5 M, to ensure reproducibility [25,27]. The biological method is used in combination with chemical or EH for chitin production and aims to deproteinise the biomass. Fermentation then takes place with lactic acid bacteria, that produce lactic acid, typically using whey, lignocellulose or starch as a nutrient substrate. The drop in pH during fermentation inhibits microbial spoilage by other microorganisms. The lactic acid dissolves the sediment of the processed crustacean biomass and reacts with calcium carbonate, producing calcium lactate that can be easily removed [28]. Chitin extraction by ultrasound-assisted extraction (UAE) is based on the application of ultrasonic waves that induce cavitation phenomena. This method is used in combination with acidic or alkaline solvents that enhance the deproteinisation of crustacean biomass and significantly improve the removal of proteins and inorganic elements [14,29,30]. The particle size of the biomass prior to UAE can influence efficiency, with typical target ranges of 0.5–2 mm after milling [29,30]. The HOW-CA process for chitin extraction combines thermal treatment with supercritical carbon dioxide scCO₂ pressure. First, the material is heated in hot water, which causes partial protein degradation. Then, the application of scCO₂ under pressure (typically 100–300 bar) creates carbonic acid (H₂CO₃), which dissolves inorganic salts, mainly calcium carbonate (CaCO₃). Upon pressure release, calcium carbonate precipitates and is removed by washing and filtration, resulting in the isolation of pure chitin [14,31]. The use of ILs for chitin extraction is based on heating the ILs, which allows chitin dissolution without destroying its molecular structure. Chitin is isolated by adding a catalyst or solvent, followed by precipitation, filtration, and washing with distilled water [32,33]. The electrochemical method combines the use of an electrolytic solution of acid or base. Through electrolytic removal, effective elimination of proteins and inorganic elements is achieved, while preserving the molecular structure of chitin [34,35]. Chitin extraction with scCO₂ is carried out in a high-pressure chamber, utilizing CO₂ in a supercritical state (temperature > 31 °C and pressure > 73.8 bar) [36,37,38,39]. This method achieves effective dissolution of desired compounds. Upon pressure reduction, CO₂ returns to the gaseous phase, facilitating separation and recovery of the extract [36,37,38,39]. EH outperforms other methods of chitin isolation, as it offers a milder and more environmentally friendly approach without the need for aggressive chemicals or complex and costly equipment [14]. In contrast, other techniques such as microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), the use of ILs or electrochemical methods require specialised and expensive equipment, complex handling and often high energy consumption, which limits their wide industrial application [14]. Chitin obtained by EH may contain residual proteins and calcium carbonate. However, these do not appear to hinder incorporation into aquafeed, where they may even provide additional nutritional value. At the same time, further studies are needed to investigate the effects of these residues on fish digestibility and health so that the use of chitin as a component of aquafeed can be optimised [14]. Further studies should investigate the effects of particle size and residual content on fish digestibility and health [14,26,40,41]. Chitin produced through enzymatic hydrolysis (EH), along with its residual proteins and calcium carbonate content, presents advantages for fish feed production [26,40,41,42]. Unlike chitin purified by other methods, the crude form combines nutritional and functional benefits [1,2,43], making it more suitable for incorporation into fish diets [44,45].

Table 1. Comparison of Chitin Extraction Methods.

Method	Chitin Yield	Energy Consumption	Advantages	Disadvantages	References
EH	Satisfactory	Low	Environmentally friendly, mild process	Time-consuming, lower yield	[14,26]
UAE	Moderate to High	Moderate	Increased yield, reduced solvent usage	Specialized equipment required	[29,30]
MAE	Low	Very low	Fast, lower energy requirement	May affect chitin structure	[14,31]
scCO2	High	Moderate	Environmentally friendly, high yield	Expensive equipment	[36,37,38,39]
ILs	High	Moderate	Solvent recycling, high yield	Expensive production	[32,33]

Table 1. Comparison of Chitin Extraction Methods.

Method	Chitin Yield	Energy Consumption	Advantages	Disadvantages	References
EH	Satisfactory	Low	Environmentally friendly, mild process	Time-consuming, lower yield	[14,26]
UAE	Moderate to High	Moderate	Increased yield, reduced solvent usage	Specialized equipment required	[29,30]
MAE	Low	Very low	Fast, lower energy requirement	May affect chitin structure	[14,31]
scCO2	High	Moderate	Environmentally friendly, high yield	Expensive equipment	[36,37,38,39]
ILs	High	Moderate	Solvent recycling, high yield	Expensive production	[32,33]

3.1. Preparation of Crab By-Product Samples

The crab species used in EH studies are summarized in Table 2, while the various sample preparation methods are illustrated in Figure 1. This information originates from a systematic review of the relevant literature. Most studies focus on processing crab by-products to recover bioactive compounds through EH.

These by-products, also referred to as residues or processing waste, include various anatomical parts such as the exoskeleton, shells, carapace, cephalothorax, gills, viscera, hepatopancreas, as well as muscle-containing parts such as legs, claws, frame meat, and muscle remnants. The species reported in these studies include Callinectes sapidus, Portunus trituberculatus, Chionoecetes opilio, Paralithodes camtschaticus, Chionoecetes japonicus, Carcinus maenas, Cancer irroratus, species of the genus Scylla, Portunus pelagicus, Callinectes bellicosus, Portunus segnis and Eriocheir sinensis. In some cases, the crab species were not specified [43,46]. Crab by-products originate from different countries and facility types, reflecting geographic diversity and varied technological approaches. In Brazil, by-products mainly derive from small-scale crab meat processing industries, although the species are not explicitly stated [40]. In Canada, sources include pilot-scale processing lines, large factories, and commercial companies, with species such as Cancer irroratus and Chionoecetes opilio used [41,44,47]. In China, major contributors are Portunus trituberculatus and Eriocheir sinensis, originating from seafood companies, bioproduct factories, and crab processing units, while some studies do not specify species [43,48,49]. In Korea, by-products primarily come from crab processing units involving Chionoecetes japonicus [50,51]. In Malaysia, local fish markets and suppliers provide Portunus pelagicus and Scylla spp. [52,53]. In Mexico, specialized processing companies handle Callinectes bellicosus [54]. In Russia, by-products come from industrial processing facilities and crabs harvested from the Barents Sea, mainly Paralithodes camtschaticus [42,55,56]. In Tunisia, processing plants and fish markets supply Portunus segnis [57]. In the United States, by-products are collected from natural areas such as the Back River [58]. Pre-treatment of raw material is a critical step for effective EH application on crab biomass. Proper handling increases the availability of proteins and other bioactive compounds and maintains the quality and safety of the final product. Typically, crab raw material is initially washed with running or tap water to remove impurities and microbial load. In some protocols, additional parts such as viscera and shells are removed to isolate the meat or desired portions. Moisture removal is achieved either by drying in an air-circulated oven at approximately 60 °C for 8 h [40] or by freezing at low temperatures −20 °C to −40 °C to preserve the raw material until processing [48,58,59]. Alternatively, lyophilization (freeze-drying) can be used to better maintain the bioactivity of the compounds [50,55]. Mechanical grinding is a key stage, using a mortar, Wiley grinder, or electric mill, to convert the raw material into a fine or pulverized form [40,42,49]. Homogenization with food processors can also be applied to produce a uniform paste suitable for hydrolysis [52,53]. In some protocols, biomass is stored under freezing conditions −20 °C to −40 °C in airtight containers or plastic bags to prevent oxidation and spoilage until hydrolysis [41,49]. Other procedures include salt treatment, e.g., soaking in sodium chloride (NaCl) solution, which assists in lipid removal and improves stability [55]. Thermal treatment such as steaming for a few min, e.g., 3–4 min, is used for pasteurization and facilitates meat removal from the shell while simultaneously preparing the biomass for hydrolysis [52,53]. Overall, the choice of an appropriate pre-treatment method depends on biomass type, desired final product, and processing conditions, always aiming for maximum EH efficiency and preservation of the quality of the produced peptides and other bioactive compounds.

Table 2. Pre-treatment Conditions of Crab Raw Materials for Biotechnological Valorization through Enzymatic Hydrolysis.

Scientific Names of Species	A: Country B: Company	Crab Biomass	Required Pre-Treatment of Crab Raw Material for the Application of Enzymatic Hydrolysis	References
Callinectes sapidus	A: Brazil B: Small processing crab meat industries	Carapaces, Legs	Biomass pre-treatment procedure: Washed in running water. Dried in air-circulated oven for 8 h at 60 °C. Ground in a Willey grinder. Conditioned in plastic bags. Stored at −20 °C until the processing.	[40]
Portunus trituberculatus	A: China B: Seafood Company	Processing by-products	Biomass pre-treatment procedure: Minced in a meat grinder Stored at −20 °C until use and/or used immediately	[48]
Portunus trituberculatus	A: Not mentioned B: pilot plant transformation line	Legs, Claw, Cephalothorax, Shells	Biomass pre-treatment procedure: Ground. Stored at −20 °C until use.	[59]
Chionoecetes opilio	A: Canada B: pilot plant transformation line	By-products	Biomass pre-treatment procedure: Stored at 0–−4 °C until use	[41]
Not mentioned	A: Not mentioned B: Local supermarkets	Surimi	Biomass pre-treatment procedure: the product was homogenized Stored at −20 °C, until use.	[46]
Paralithodes camtschaticus	A: Not mentioned B: ship-derived	Processing waste	Biomass pre-treatment procedure: Disjointed. Frozen at −18 °C. Ground to a particle size of 3 mm.	[42]
Chionoecetes japonicus	A: Republic of Korea B: Crab processing plant	Shell	Biomass pre-treatment procedure: Lyophilized Ground into powder Stored in a freezer at −20 °C until use.	[50]
Carcinus maenas	A: USA B: harvested at the back river	Carapace	Biomass pre-treatment procedure: Washed with tap water Βlast-frozen at −30 °C for 1 h Stored at −20 °C until use.	[58]
Cancer irroratus	A: Canada B: Factory	Legs, Claws, Cephalothorax,	Biomass pre-treatment procedure: Ground used immediately.	[44]
Scylla sp.	A: Malaysia B: Supplier	Meat	Biomass pre-treatment procedure: Cut in half and cleaned (bellies and gills removed). Washed and steamed for 4 min. Homogenized using a food processor. Stored in a freezer (−20 °C) until further use.	[53]
Portunus pelagicus	A: Malaysia B: Fish market	Meat	Biomass pre-treatment procedure: Cut in half and washed to remove contaminants. Steamed for 4 min, and the meat was separated from the shell. Homogenized using a food processor for 5 min until it formed a paste.	[52]
Callinectes bellicosus	A: Mexico B: Processing company	Exoskeletons	Biomass pre-treatment procedure: Washed Dried Ground in a mortar Stored refrigerated at 4 °C until use.	[54]
Chionoecetes japonicus	A: Republic of Korea B: Crab processing factory	Shells, Frame Meats	Biomass pre-treatment procedure: Used immediately for enzyme extraction.	[51]
Portunus segnis	A: Tunisia B: Processing plant and Fishery market	Viscera, Shells	Biomass pre-treatment procedure: Washed with tap water Viscera and shells were removed and thoroughly rinsed with cold distilled water. Used immediately for enzyme extraction.	[57]
Paralithodes camtschaticus	A: Russia B: industrial processing	Gills, Carapace	Biomass pre-treatment procedure: Frozen at −20 °C, until use.	[56]
Chionoecetes opilio	A: Canada B: Company	By-products	Biomass pre-treatment procedure: Ground, Solid and liquid phases were separated by decanting. Liquid phase was centrifuged (11,000× g) at 0–4 °C. The supernatant stored in polypropylene bags at −40 °C until use.	[47]
Not mentioned	A: China B: Biological Products Factory	Shell	Biomass pre-treatment procedure: Used as raw material without prior processing until further use	[43]
Paralithodes camtschaticus	A: Russia B: Caught in the Barents Sea	Hepatopancreas	Biomass pre-treatment procedure: Dissected in situ Frozen in liquid nitrogen Thawed in 5% NaCl with periodic stirring to allow complete autolysis. NaHCO3 and chitosan ascorbate solutions were added to cluster lipids for removal. Filtered Dried using a freeze dryer until use	[55]
Eriocheir sinensis	A: China B: Crab products factory	Muscle residuals	Biomass pre-treatment procedure: Steamed Frozen at −40 °C. Dried. Ground using a high-speed electric grinder. Sieved to separate particles by size (60 mesh, 0.3 mm). Stored in airtight foil Ziplock bags to prevent moisture and oxidation. Kept frozen at −40 °C until use.	[49]

Table 2. Pre-treatment Conditions of Crab Raw Materials for Biotechnological Valorization through Enzymatic Hydrolysis.

Scientific Names of Species	A: Country B: Company	Crab Biomass	Required Pre-Treatment of Crab Raw Material for the Application of Enzymatic Hydrolysis	References
Callinectes sapidus	A: Brazil B: Small processing crab meat industries	Carapaces, Legs	Biomass pre-treatment procedure: Washed in running water. Dried in air-circulated oven for 8 h at 60 °C. Ground in a Willey grinder. Conditioned in plastic bags. Stored at −20 °C until the processing.	[40]
Portunus trituberculatus	A: China B: Seafood Company	Processing by-products	Biomass pre-treatment procedure: Minced in a meat grinder Stored at −20 °C until use and/or used immediately	[48]
Portunus trituberculatus	A: Not mentioned B: pilot plant transformation line	Legs, Claw, Cephalothorax, Shells	Biomass pre-treatment procedure: Ground. Stored at −20 °C until use.	[59]
Chionoecetes opilio	A: Canada B: pilot plant transformation line	By-products	Biomass pre-treatment procedure: Stored at 0–−4 °C until use	[41]
Not mentioned	A: Not mentioned B: Local supermarkets	Surimi	Biomass pre-treatment procedure: the product was homogenized Stored at −20 °C, until use.	[46]
Paralithodes camtschaticus	A: Not mentioned B: ship-derived	Processing waste	Biomass pre-treatment procedure: Disjointed. Frozen at −18 °C. Ground to a particle size of 3 mm.	[42]
Chionoecetes japonicus	A: Republic of Korea B: Crab processing plant	Shell	Biomass pre-treatment procedure: Lyophilized Ground into powder Stored in a freezer at −20 °C until use.	[50]
Carcinus maenas	A: USA B: harvested at the back river	Carapace	Biomass pre-treatment procedure: Washed with tap water Βlast-frozen at −30 °C for 1 h Stored at −20 °C until use.	[58]
Cancer irroratus	A: Canada B: Factory	Legs, Claws, Cephalothorax,	Biomass pre-treatment procedure: Ground used immediately.	[44]
Scylla sp.	A: Malaysia B: Supplier	Meat	Biomass pre-treatment procedure: Cut in half and cleaned (bellies and gills removed). Washed and steamed for 4 min. Homogenized using a food processor. Stored in a freezer (−20 °C) until further use.	[53]
Portunus pelagicus	A: Malaysia B: Fish market	Meat	Biomass pre-treatment procedure: Cut in half and washed to remove contaminants. Steamed for 4 min, and the meat was separated from the shell. Homogenized using a food processor for 5 min until it formed a paste.	[52]
Callinectes bellicosus	A: Mexico B: Processing company	Exoskeletons	Biomass pre-treatment procedure: Washed Dried Ground in a mortar Stored refrigerated at 4 °C until use.	[54]
Chionoecetes japonicus	A: Republic of Korea B: Crab processing factory	Shells, Frame Meats	Biomass pre-treatment procedure: Used immediately for enzyme extraction.	[51]
Portunus segnis	A: Tunisia B: Processing plant and Fishery market	Viscera, Shells	Biomass pre-treatment procedure: Washed with tap water Viscera and shells were removed and thoroughly rinsed with cold distilled water. Used immediately for enzyme extraction.	[57]
Paralithodes camtschaticus	A: Russia B: industrial processing	Gills, Carapace	Biomass pre-treatment procedure: Frozen at −20 °C, until use.	[56]
Chionoecetes opilio	A: Canada B: Company	By-products	Biomass pre-treatment procedure: Ground, Solid and liquid phases were separated by decanting. Liquid phase was centrifuged (11,000× g) at 0–4 °C. The supernatant stored in polypropylene bags at −40 °C until use.	[47]
Not mentioned	A: China B: Biological Products Factory	Shell	Biomass pre-treatment procedure: Used as raw material without prior processing until further use	[43]
Paralithodes camtschaticus	A: Russia B: Caught in the Barents Sea	Hepatopancreas	Biomass pre-treatment procedure: Dissected in situ Frozen in liquid nitrogen Thawed in 5% NaCl with periodic stirring to allow complete autolysis. NaHCO3 and chitosan ascorbate solutions were added to cluster lipids for removal. Filtered Dried using a freeze dryer until use	[55]
Eriocheir sinensis	A: China B: Crab products factory	Muscle residuals	Biomass pre-treatment procedure: Steamed Frozen at −40 °C. Dried. Ground using a high-speed electric grinder. Sieved to separate particles by size (60 mesh, 0.3 mm). Stored in airtight foil Ziplock bags to prevent moisture and oxidation. Kept frozen at −40 °C until use.	[49]

3.2. Description of the Enzymatic Hydrolysis Process

The production of bioactive substances from crab by-products requires as a crucial step the deproteinisation of the biomass, as demonstrated by several studies (Table 3, Figure 2).

For the recovery of these bioactive compounds, an initial stage of deproteinisation and, in some cases, defatting of the biomass is essential [54,57]. Specifically, for the deproteinisation of crab biomass, the use of various proteolytic enzymes is required, applied under controlled conditions such as pH, temperature, incubation time, and enzyme inhibition method [40,41,52,53]. EH of crab by-products is usually performed by mixing the suitably prepared biomass with water at a ratio of 1:1–1:3 (w/v) [53]. The hydrolysis process is carried out under controlled physicochemical parameters, such as pH, temperature, and incubation time, which vary depending on the enzyme and the desired product [41,53]. The most commonly used proteolytic enzymes for EH of crab biomass are alcalase, flavourzyme, papain, neutrase, and trypsin, with incubation pH ranging from 6.0–9.0, temperatures between 50–65 °C, and hydrolysis times from 1–7 h [40,48,50,52,53,58]. In contrast, enzymes such as protamex and bromelain are used less frequently but have proven equally effective at pH 6.0–8.0 and temperatures up to 53 °C [40,53,59]. EH depends significantly on pH: for example, pepsin works optimally in an acidic environment at pH 3.0 [59], while alcalase acts in an alkaline environment at pH 7.0–8.5 [51,53]. Pancreatin can act at an incubation temperature of about 37 °C, while flavourzyme can operate at temperatures up to 65.3 °C [46,49], with incubation times usually ranging from 5 min to 12 h [40,46]. EH inhibition is achieved by thermal treatment at temperatures between 85–95 °C for 5–20 min [40,41,51,57]. At the end of EH, recovered bioactive substances with high added value are produced, such as protein hydrolysates and carotenoids, proteins and peptides, lipids, chitin and minerals, free amino acids, antioxidant compounds, and bioactive peptides, as well as special products such as crab flavouring rich in sweet–bitter free amino acids and astaxanthin-enriched hydrolysates. These can be used for various industrial applications, such as in aquafeed, functional foods, and pharmaceutical products [40,41,42,49,52,53]. This review focuses on the processing of crab by-products through EH to recover crude chitin and its further use as dietary supplements in farmed fish, which are kept in captivity, conferring benefits to the health, growth, and survival of these fish [60]. EH of crab biomass using the enzyme Neutrase is conducted in a neutral to slightly acidic environment at pH between 6.5–7.0, which is the ideal condition for optimal enzyme activity. The incubation temperature is set at 50 °C to ensure high enzymatic activity without enzyme denaturation. Hydrolysis time ranges from 3–6 h, sufficient for the breakdown of proteins surrounding the chitin and its release. EH is completed with thermal enzyme inhibition, where the mixture is heated at 90–95 °C for 20 min to limit further enzymatic degradation of the substrate [55,57], During hydrolysis, the hydrolysate separates into two phases: the supernatant liquid and the sediment. The supernatant contains soluble proteins, peptides, and other bioactive compounds, products of protein and organic compound hydrolysis. In contrast, the sediment mainly consists of insoluble compounds such as crude chitin and calcium [55,57,61,62]. For chitin recovery, the sediment is collected and undergoes further processing, which usually includes demineralisation (removal of inorganic salts, mainly calcium) and alkaline deproteinisation (removal of residual proteins). Through this process, chitin is isolated in a purer form and can be used for further conversion to chitosan or other industrial applications [27,40,41,63]. Therefore, the enzymatic hydrolysis process constitutes a simple and environmentally friendly method for the utilisation of crab by-products, providing valuable bioactive components such as crude chitin, which is expected to be applied in various industrial sectors of aquaculture.

Table 3. Description of the enzymatic hydrolysis process with the use of proteolytic enzymes for the recovery of bioactive components from crab by-products.

Enzymes	Incubation (pH)	Incubation Temperature	Incubation Time	Enzyme Inhibition Temp./Time	Recovered Extracts	References
Alcalase Βromelain	Single pH 9.0 6.0	Single temp 53 °C 53 °C	Different periods: 5, 15, 30, 45, 60, 90, 120, 180, 240 min.	90 °C for 5 min.	Protein hydrolysate, Carotenoid	[40]
Neutrase Flavorase Papain	Single pH 6.0 6.0 6.0	Single temp 55 °C 55 °C 55 °C	Single time 7 h 7 h 7 h	95 °C for 10 min.	Protein hydrolysate	[48]
Trypsin Neutrase Bromelin Protamex Pepsin	Single pH 7.0 7.0 8.0 7.0 3.0	Single temp 55 °C 55 °C 45 °C 50 °C 35 °C	Single time 1 h 1 h 1 h 1 h 1 h	95 °C for 15 min.	Proteins	[59]
Protamex	Single pH 8.0	Single temp 40 °C	Single time 60 min	85 °C for 10 min.	Proteins, Lipids, Chitin, Minerals	[41]
Pancreatin Lipase	Single pH 7.4 7.4	Single temp 37 °C 37 °C	Single time 12 h	-	TiO2 nanoparticle	[46]
Proteinase preparation	Single pH 6.0–9.5	Single temp 50.0 ± 0.5 °C	-	-	Free Amino Acids, Protein hydrolysates, Chitin, Antioxidant compounds	[42]
Alcalase	Single pH 7.0	Single temp 50 °C	Single time 25 h	100 °C	Protein hydrolysates	[50]
Alcalase Protamex Flavourzyme Papain	Single pH pH 8.0 pH 7.0 pH 7.0 pH 6.0	Single temp 50 °C 50 °C 50 °C 65 °C	Single time 1 h 1 h 1 h 1 h	85–90 °C for 10 min.	Protein hydrolysates	[58]
Protamex	Single pH pH 9.0	Single temp 40 °C	Single time 90 min.	85 °C for 10 min.	Protein hydrolysates	[44]
Alcalase Protamex Neutrase Papain	Single pH 8.5 6.5 7.0 6.0	Single temp 55 °C 50 °C 55 °C 50 °C	Different periods: 1, 2, 3, 4 h	85 °C	Protein hydrolysates, Bioactive peptides, free amino acids	[53]
Alcalase Protamex Neutrase Papain	Single pH 8.0 6.5 6.5 6.0	Single temp 55 °C 55 °C 50 °C 60 °C	Different periods: 2–4 h	85 °C for 20 min.	Bioactive peptides, Protein hydrolysates	[52]
Pectinase Lipase Hemicellulase	Single pH 7.0	Single temp 40 °C ± 5 °C	Single time 1 h	-	Chitin, Lipids	[54]
Flavourzyme Neutrase alcalase protamex	Single pH 7.0 7.0 7.0 7.0	Single temp 60 °C 60 °C 60 °C 60 °C	Single time 5 h 5 h 5 h 5 h	95 °C for 5 min	Protein, Protein Hydrolysate, Free Amino Acids	[51]
Neutrase	Single pH 7.0	Single temp 50 °C	Single time 3 h	90 °C, 20 min	Chitin	[57]
Neutrase	Single pH 7.0	Single temp 50 °C	-	-	Protein hydrolysates, free amino acids	[56]
Enzyme	Single pH 6.5–7.0	Single temp 50 °C	Single time 6 h	95 °C	Chitin	[55]
Flavourzyme	Single pH 6.5	Single temp 65.3 °C	-	-	Crab flavoring rich in sweet-taste free amino acids	[49]
Alcalase Bromelain	Single pH 8.0 6.0–7.0	Single temp	120 min		Protein hydrolysates, chitin, Astaxanthin-enriched	[40]

Table 3. Description of the enzymatic hydrolysis process with the use of proteolytic enzymes for the recovery of bioactive components from crab by-products.

Enzymes	Incubation (pH)	Incubation Temperature	Incubation Time	Enzyme Inhibition Temp./Time	Recovered Extracts	References
Alcalase Βromelain	Single pH 9.0 6.0	Single temp 53 °C 53 °C	Different periods: 5, 15, 30, 45, 60, 90, 120, 180, 240 min.	90 °C for 5 min.	Protein hydrolysate, Carotenoid	[40]
Neutrase Flavorase Papain	Single pH 6.0 6.0 6.0	Single temp 55 °C 55 °C 55 °C	Single time 7 h 7 h 7 h	95 °C for 10 min.	Protein hydrolysate	[48]
Trypsin Neutrase Bromelin Protamex Pepsin	Single pH 7.0 7.0 8.0 7.0 3.0	Single temp 55 °C 55 °C 45 °C 50 °C 35 °C	Single time 1 h 1 h 1 h 1 h 1 h	95 °C for 15 min.	Proteins	[59]
Protamex	Single pH 8.0	Single temp 40 °C	Single time 60 min	85 °C for 10 min.	Proteins, Lipids, Chitin, Minerals	[41]
Pancreatin Lipase	Single pH 7.4 7.4	Single temp 37 °C 37 °C	Single time 12 h	-	TiO2 nanoparticle	[46]
Proteinase preparation	Single pH 6.0–9.5	Single temp 50.0 ± 0.5 °C	-	-	Free Amino Acids, Protein hydrolysates, Chitin, Antioxidant compounds	[42]
Alcalase	Single pH 7.0	Single temp 50 °C	Single time 25 h	100 °C	Protein hydrolysates	[50]
Alcalase Protamex Flavourzyme Papain	Single pH pH 8.0 pH 7.0 pH 7.0 pH 6.0	Single temp 50 °C 50 °C 50 °C 65 °C	Single time 1 h 1 h 1 h 1 h	85–90 °C for 10 min.	Protein hydrolysates	[58]
Protamex	Single pH pH 9.0	Single temp 40 °C	Single time 90 min.	85 °C for 10 min.	Protein hydrolysates	[44]
Alcalase Protamex Neutrase Papain	Single pH 8.5 6.5 7.0 6.0	Single temp 55 °C 50 °C 55 °C 50 °C	Different periods: 1, 2, 3, 4 h	85 °C	Protein hydrolysates, Bioactive peptides, free amino acids	[53]
Alcalase Protamex Neutrase Papain	Single pH 8.0 6.5 6.5 6.0	Single temp 55 °C 55 °C 50 °C 60 °C	Different periods: 2–4 h	85 °C for 20 min.	Bioactive peptides, Protein hydrolysates	[52]
Pectinase Lipase Hemicellulase	Single pH 7.0	Single temp 40 °C ± 5 °C	Single time 1 h	-	Chitin, Lipids	[54]
Flavourzyme Neutrase alcalase protamex	Single pH 7.0 7.0 7.0 7.0	Single temp 60 °C 60 °C 60 °C 60 °C	Single time 5 h 5 h 5 h 5 h	95 °C for 5 min	Protein, Protein Hydrolysate, Free Amino Acids	[51]
Neutrase	Single pH 7.0	Single temp 50 °C	Single time 3 h	90 °C, 20 min	Chitin	[57]
Neutrase	Single pH 7.0	Single temp 50 °C	-	-	Protein hydrolysates, free amino acids	[56]
Enzyme	Single pH 6.5–7.0	Single temp 50 °C	Single time 6 h	95 °C	Chitin	[55]
Flavourzyme	Single pH 6.5	Single temp 65.3 °C	-	-	Crab flavoring rich in sweet-taste free amino acids	[49]
Alcalase Bromelain	Single pH 8.0 6.0–7.0	Single temp	120 min		Protein hydrolysates, chitin, Astaxanthin-enriched	[40]

4. Description of the Production of Aquafeed with Chitin and Its Benefit to Farmed Fish

The rapid development of the aquaculture sector has led to a corresponding increase in the production of fishery products. However, intensive fish farming, especially at high stocking densities, creates favorable conditions for predictable outbreaks of diseases. To address these challenges, bioactive substances such as chitin and chitosan are incorporated into aquafeed at regular intervals and/or seasonal periods, offering various benefits [64]. Even when there are clear signs of disease onset, chitin- and chitosan-enriched aquafeed are administered preventively to farmed fish, acting as immunostimulant substances [19,65,66]. Several studies perform experimental disease challenge trials under controlled conditions, which constitute a key tool for studying pathogenesis and immune responses, with the aim of evaluating the effectiveness of administered bioactive compounds. Bacterial infections affecting fish include pathogens such as Aeromonas hydrophila [19,60], Vibrio anguillarum [21], Aeromonas veronii-like, Cellulomonas hominis-like, Bacillus oceanisediminis-like [67], as well as Streptococcus agalactiae [68]. These infections cause septicemia, skin lesions, encephalitis, and high mortality. In contrast, fungal infection by Aphanomyces invadans [17,69] is characterized by skin ulcers and systemic infection. Therefore, chitin and chitosan contribute to the prevention and treatment of such diseases [17,19,21,60,67,68,69]. Moreover, fish kept in captivity under high stocking densities exhibit increased stress [66,70,71,72], which negatively affects their health and reduces aquaculture productivity. Chitin and chitosan possess antioxidant properties [18,71,72], which are effective in combating stress [66,70]. Additionally, the enhancement of innate immunity [18,21,65,69,73] and the increased resilience to stressors in farmed fish have been documented in numerous studies, which emphasize the important role of bioactive compounds such as chitin and chitosan in improving the natural defense mechanisms of aquaculture species, developing stress-coping mechanisms [66,70,71,72], as well as their strong antioxidant activity, which protects cells from oxidative damage [18,71,72]. Chitin and chitosan are not used solely as immunostimulant and antioxidant agents or for combating pathogenic microorganisms. A large body of research [17,18,60,70,72,73,74,75,76] demonstrates that their use significantly contributes to improved growth and survival of farmed fish. In particular, studies such as [60] highlight the dual role of chitin and chitosan, not only in promoting the immune response but also in optimizing growth and survival parameters [77]. Chitin and chitosan play an important role in regulating increased protein synthesis [70], as well as reducing lipid levels [22,71,72]. The regulation of these parameters supports the smooth functioning of metabolism, creating a favorable and stable growth environment. Studies have investigated (Table 4 and Figure 3) the benefits of chitin and chitosan as dietary supplements in farmed fish. Factors such as fish species, inclusion level of chitin and chitosan in the diet, initial weight at the start of the trial, and feeding duration are critical in evaluating the effect of these compounds on the growth, survival, and health of farmed fish. The absorption and benefits of crude chitin may vary depending on the fish species, a topic that will be analysed in detail in Section 6 [18,21,65,71]. Despite the technical advantages of enzymatic hydrolysis, industrial-scale application of raw chitin in aquafeeds faces several challenges. These include the high cost of proteolytic enzymes [14,40], the difficulty in scaling up hydrolysis reactors to industrial volumes [40,42], and the uncertainty regarding market acceptance of feeds containing raw chitin [14,40]. Addressing these practical issues is crucial to ensure the commercial success of this sustainable approach. Nevertheless, the incorporation of crude chitin in Aquafeeds remains an environmentally friendly and cost-effective solution compared to chemically processed chitin derivatives [14,40,42].

A wide range of farmed fish have been used for chitin- and chitosan-based dietary supplementation, including Oreochromis niloticus, Paralichthys olivaceus, Misgurnus anguillicaudatus, Sparus aurata, Oncorhynchus mykiss, Cirrhinus mrigala, Lates calcarifer, Clarias gariepinus, Dicentrarchus labrax, Carassius auratus gibelio, and Larimichthys crocea. The minimum mean individual weight ranges from 0.18 g for Misgurnus anguillicaudatus [71], while the maximum mean individual weight reaches 125 g in Sparus aurata [65]. The supplementation levels of chitin and chitosan vary from as low as 0.025 g/kg [65]) to as high as 20 g/kg [21]. Chitin is administered in various forms, such as chitosan, nano-chitosan, recovered chitosan from crab and shrimp shells [69,74], chitosan-based coating solution [20], and COS [20]. Feeding duration in different studies ranged from as little as 1 week to up to 90 days [18,21,71,74], indicating flexibility in the administration period of chitin-based bioactive substances. Based on the available data in Table 4 and Figure 3, the maximum utilization of bioactive compounds derived from chitin in farmed fish can be achieved under specific parameters. In particular, the use of nano-chitosan recovered from shrimp shell chitin appears to offer the most comprehensive benefits, especially in Nile tilapia Oreochromis niloticus. Optimal effectiveness is observed when administered at levels of 1–5 g/kg feed for a duration of 45–70 days, leading to significant improvement in growth, enhanced immune response, increased antioxidant activity, reduced intestinal lipid levels, and greater resistance to pathogenic microorganisms. The initial mean weight of fish ranges between 5 and 20 g, which seems to facilitate the effective absorption and action of these compounds. This approach offers a flexible and effective strategy for improving fish health and aquaculture productivity, making chitin, chitosan, and nano-chitosan derived from crab shells a highly promising solution for enhancing the sustainability and efficiency of the sector.

Table 4. Dietary Supplementation of Farmed Fish with Chitin and Chitosan and Its Benefits.

Scientific Name	Chitosan Addition (g/kg)	Feeding Trial at Initial Weight (g)	Feeding Duration	Benefits	References
Carassius auratus gibelio	Different chitosan levels: 1.8, 4, 7.5, 10, 20	Average Initial weight: 4.80 ± 0.01 g	75 days	Resistance against Aeromonas veronii-like, improved Cellulomonas hominis-like, Bacillus oceanisediminis-like	[67]
Cirrhinus mrigala	Recovered chitin and chitosan from exoskeleton of Giant freshwater prawn: 0.05, 0.5, 5	Average Initial weight: 25.6 ± 1.7 g	1–4 weeks	Enhancement of innate immunity. Resistance against Aphanomyces invadans	[69]
Clarias gariepinus	Recovered chitosan nanoparticles from crab shell: 5	Average Initial weight: 2.79 ± 0.05 g	90 Days	Growth and survival improvement	[74]
Dicentrarchus labrax	Different chitosan levels: 5, 10, 20, 30, 40	Average Initial weight: 0.21 ± 0.01 g	75 days	Growth improvement	[75]
Larimichthys crocea	Different chitosan levels: 3, 6, 9	Average Initial weight: 3.81 ± 0.20 mg	30 days	Growth and survival improvement. Enhancement of digestive enzyme activity and intestinal development. Antioxidant activity. Immunostimulant agent.	[76]
Lates calcarifer	Different chitosan levels: 5, 10, 20	Average Initial weight: 15 ± 2 g	15–60 Days	Enhancement of innate immunity. Resistance against Vibrio anguillarum	[21]
Misgurnus anguillicadatus	Different chitosan levels: 1, 5, 10	Average Initial weight: 3.14 ± 0.05 g	10 weeks	Growth and survival improvement. Resistance against Aeromonas hydrophila	[60]
Misgurnus anguillicaudatus	Different chitosan levels: 5, 10, 20, 50	Average Initial weight: 0.18 g	50 days	Antioxidant activity. Reduction in intestinal lipid content. Stress resistance	[71]
Oncorhynchus mykiss	Different nano-chitosan levels: 0.05, 0.5, 5	Average Initial weight: 27.75 ± 0.34 g	70 Days	Growth improvement. Resistance against Aphanomyces invadans	[17]
Oncorhynchus mykiss	Different chitosan levels: 2.5, 5, 10	Average Initial weight: 25 ± 0.1 g	8 weeks	Immunostimulant agent. Stress resistance	[66]
Oreochromis nilotica	Chitosan nanoparticles: 2.5, 5, 10, 20	Average Initial weight: 19.8 ± 0.59 g	45 Days	Growth improvement. Antioxidant activity. Enhancement of innate immunity.	[18]
Oreochromis nilotica	Recovered chitin from shrimp shells: 5, 10, 20	Average Initial weight: 40.12 ± 4.25 g	4 weeks	Immunostimulant agent. Resistance against Aeromonas hydrophila.	[19]
Oreochromis nilotica	Recovered chitin from shrimp shells: 5	Average Initial weight: 23.56 ± 1.23 g	60 days	Growth and survival improvement.	[77]
Oreochromis nilotica	Recovered chitosan from shrimp shells: 20, 40, 60, 80	Average Initial weight: 50.13 ± 4.13 g	56 days	Growth improvement. Increased protein levels. Stress resistance.	[70]
Oreochromis nilotica	Different chitosan nanoparticle levels: 1, 3, 5	Average Initial weight: 5.66 ± 0.02 g	70 days	Growth improvement. Enhancement of innate immunity.	[73]
Oreochromis nilotica	Different chitin and chitosan levels: 20, 50, 100	Average Initial weight: 0.99 ± 0.01 g	8 weeks	Reduction in lipid content	[22]
Oreochromis nilotica	Different chitosan levels: 30, 50	Average Initial weight: 39.3 ± 0.3 g	-	Resistance against Streptococcus agalactiae	[68]
Oreochromis nilotica	Different chitosan levels: 50, 100	Average Initial weight: 17.32 ± 2.2 g	60 days	Growth improvement. Antioxidant activity. Stress resistance. Reduction in lipid content.	[72]
Paralichthys olivaceus	Chitosan-coating solution: 10	Average Initial weight: 80 g	12 weeks	Immunostimulant agent. Reduction of COD and suspended solids.	[20]
Sparus aurata L.	Different chitin levels: 0.025, 0.05, 0.1	Average Initial weight: 125 ± 13 g	2–6 weeks	Immunostimulant agent. Enhancement of innate immunity.	[65]

Table 4. Dietary Supplementation of Farmed Fish with Chitin and Chitosan and Its Benefits.

Scientific Name	Chitosan Addition (g/kg)	Feeding Trial at Initial Weight (g)	Feeding Duration	Benefits	References
Carassius auratus gibelio	Different chitosan levels: 1.8, 4, 7.5, 10, 20	Average Initial weight: 4.80 ± 0.01 g	75 days	Resistance against Aeromonas veronii-like, improved Cellulomonas hominis-like, Bacillus oceanisediminis-like	[67]
Cirrhinus mrigala	Recovered chitin and chitosan from exoskeleton of Giant freshwater prawn: 0.05, 0.5, 5	Average Initial weight: 25.6 ± 1.7 g	1–4 weeks	Enhancement of innate immunity. Resistance against Aphanomyces invadans	[69]
Clarias gariepinus	Recovered chitosan nanoparticles from crab shell: 5	Average Initial weight: 2.79 ± 0.05 g	90 Days	Growth and survival improvement	[74]
Dicentrarchus labrax	Different chitosan levels: 5, 10, 20, 30, 40	Average Initial weight: 0.21 ± 0.01 g	75 days	Growth improvement	[75]
Larimichthys crocea	Different chitosan levels: 3, 6, 9	Average Initial weight: 3.81 ± 0.20 mg	30 days	Growth and survival improvement. Enhancement of digestive enzyme activity and intestinal development. Antioxidant activity. Immunostimulant agent.	[76]
Lates calcarifer	Different chitosan levels: 5, 10, 20	Average Initial weight: 15 ± 2 g	15–60 Days	Enhancement of innate immunity. Resistance against Vibrio anguillarum	[21]
Misgurnus anguillicadatus	Different chitosan levels: 1, 5, 10	Average Initial weight: 3.14 ± 0.05 g	10 weeks	Growth and survival improvement. Resistance against Aeromonas hydrophila	[60]
Misgurnus anguillicaudatus	Different chitosan levels: 5, 10, 20, 50	Average Initial weight: 0.18 g	50 days	Antioxidant activity. Reduction in intestinal lipid content. Stress resistance	[71]
Oncorhynchus mykiss	Different nano-chitosan levels: 0.05, 0.5, 5	Average Initial weight: 27.75 ± 0.34 g	70 Days	Growth improvement. Resistance against Aphanomyces invadans	[17]
Oncorhynchus mykiss	Different chitosan levels: 2.5, 5, 10	Average Initial weight: 25 ± 0.1 g	8 weeks	Immunostimulant agent. Stress resistance	[66]
Oreochromis nilotica	Chitosan nanoparticles: 2.5, 5, 10, 20	Average Initial weight: 19.8 ± 0.59 g	45 Days	Growth improvement. Antioxidant activity. Enhancement of innate immunity.	[18]
Oreochromis nilotica	Recovered chitin from shrimp shells: 5, 10, 20	Average Initial weight: 40.12 ± 4.25 g	4 weeks	Immunostimulant agent. Resistance against Aeromonas hydrophila.	[19]
Oreochromis nilotica	Recovered chitin from shrimp shells: 5	Average Initial weight: 23.56 ± 1.23 g	60 days	Growth and survival improvement.	[77]
Oreochromis nilotica	Recovered chitosan from shrimp shells: 20, 40, 60, 80	Average Initial weight: 50.13 ± 4.13 g	56 days	Growth improvement. Increased protein levels. Stress resistance.	[70]
Oreochromis nilotica	Different chitosan nanoparticle levels: 1, 3, 5	Average Initial weight: 5.66 ± 0.02 g	70 days	Growth improvement. Enhancement of innate immunity.	[73]
Oreochromis nilotica	Different chitin and chitosan levels: 20, 50, 100	Average Initial weight: 0.99 ± 0.01 g	8 weeks	Reduction in lipid content	[22]
Oreochromis nilotica	Different chitosan levels: 30, 50	Average Initial weight: 39.3 ± 0.3 g	-	Resistance against Streptococcus agalactiae	[68]
Oreochromis nilotica	Different chitosan levels: 50, 100	Average Initial weight: 17.32 ± 2.2 g	60 days	Growth improvement. Antioxidant activity. Stress resistance. Reduction in lipid content.	[72]
Paralichthys olivaceus	Chitosan-coating solution: 10	Average Initial weight: 80 g	12 weeks	Immunostimulant agent. Reduction of COD and suspended solids.	[20]
Sparus aurata L.	Different chitin levels: 0.025, 0.05, 0.1	Average Initial weight: 125 ± 13 g	2–6 weeks	Immunostimulant agent. Enhancement of innate immunity.	[65]

5. Chitin and Their Impact on Human Health

Several studies have investigated the biological effects of chitin and its derivatives (Table 5 and Figure 4). An adequate and balanced diet is a prerequisite for producing farmed fish of high nutritional value, and the incorporation of bioactive compounds such as chitin derivatives could play a role in both fish health and human nutrition. The farming process plays a crucial role in the sustainability of aquaculture. Nutrition involves a series of biological and physiological processes by which farmed fish absorb and metabolise food. These processes ensure growth, survival, and a strengthened immune system, helping fish reach the desired commercial weight and size [78]. Clinical and in vitro studies suggest that chitin and chitosan contribute to the regulation of glucose and insulin levels [79,80,81] while lowering cholesterol and triglycerides through binding of negatively charged fats and bile acids [80,81,82]. They also improve lipid metabolism by influencing the expression of genes related to fatty acid storage and oxidation [81,83,84]. Their lipid-binding capacity reduces caloric load, supporting their use as dietary supplements for body weight regulation [82]. Chitin positively affects biological functions and the immune system [85,86]. It neutralises free radicals and reduces oxidative stress, which is associated with chronic diseases and ageing [86]. It also modulates inflammation by regulating cytokine production and secretion [86,87]. Consequently, chitin exhibits significant antioxidant and anti-inflammatory properties that create an unfavorable environment for cancer cell growth [88,89]. Additionally, it enhances immune function by inhibiting pathogenic microorganisms and stimulating macrophages and other immune cells [86,87,88]. Chitosan shows strong antimicrobial properties and serves as an effective treatment for bacterial infections, including antibiotic-resistant strains, while also acting as a carrier for targeted delivery of bioactive substances [87]. It is used for antiviral drug delivery, improving treatments for infections such as HIV [87]. In cancer therapy, it contributes to immunotherapy and targeted drug delivery, reducing side effects [89]. Chitosan also promotes immunoregulation and is applied in immunotherapies and vaccines [82,86,87]. It aids in wound and hemorrhage healing due to its hemostatic and antibacterial properties [87,90], and as nanoparticles, it improves ocular disease treatment through localized drug delivery [87]. Building on existing knowledge, research is driving the development of chitin products with enhanced bioactivity, such as COS Chitin, and Chitosan. COS can be produced via enzymatic, chemical, microbial, physical, or combined methods to improve yield and product quality [91]. Due to their lower molecular weight, COS exhibit higher bioavailability than crude chitin, allowing faster absorption and enhanced biological activity [80,81,83,86,89,90,92]. This improved uptake strengthens the immune system via antioxidant and anti-inflammatory effects. COS are efficiently absorbed by both farmed fish and humans. However, their production is expensive and technically challenging. In contrast, crude chitin, although less bioavailable, can be produced via simpler and less energy-intensive processes such as enzymatic hydrolysis (EH) using discarded crustacean by-products (e.g., crabs, shrimps). This reduces ecological impact, and its inclusion in aquafeed offers a simple, economical, and sustainable solution [78,80,81,83,86,89,90,92]. Beyond their nutritional and biomedical potential, the use of raw chitin aligns with circular economy principles, reducing environmental impact while promoting sustainable aquafeed production. Despite extensive evidence demonstrating the bioactive properties of chitin and chitosan [86,88,89], the current literature lacks quantitative data on their absorption in the human body following consumption of fish or marine by-products, and no threshold levels have been established that correlate with their biological effects. Moreover, most studies are based on in vitro experiments or animal models, rendering any extrapolation to humans hypothetical and highlighting the need for further investigation. Despite these advantages, significant questions remain regarding the absorption of raw chitin by farmed fish and its consequent bioavailability to humans. Therefore, future research should clarify interspecies variability, optimal dietary inclusion levels, and long-term health outcomes in both fish and humans, bridging the gap between preclinical evidence and translational applications.

Table 5. Biological Effects and Evidence of Chitin, Chitosan, and COS.

Biological Effect	Compound	Mechanism	Evidence (Study Type)	References
Regulation of glucose & insulin	Chitin, Chitosan	Improves glucose metabolism	Mainly in vitro studies, limited small-scale human clinical trials	[79,80,81]
Lipid metabolism (reduction of cholesterol and triglycerides, enhancement of fatty acid oxidation)	Chitin, Chitosan	Binds fats & bile acids, regulates lipid-related genes	In vitro and animal studies, limited human clinical trials	[80,81,82,83,84]
Antioxidant activity	Chitin, COS	Neutralizes free radicals, reduces oxidative stress	In vitro and animal studies, limited human trials; COS shows higher bioavailability	[80,81,83,86,89,90,92]
Anti-inflammatory	Chitin, COS	Cytokine regulation	In vitro and animal studies, limited human evidence	[86,87]
Antimicrobial & Antiviral	Chitosan	Inhibits bacteria, carrier for antiviral drugs	Mainly in vitro and preclinical studies, limited human applications	[87]
Cancer prevention & therapy	Chitin, Chitosan	Immunostimulation, targeted drug delivery	In vitro and animal studies, few human data	[88,89]
Wound healing	Chitosan	Hemostatic, antibacterial	Strong preclinical support, applied in clinical wound healing	[87,90]
Ocular disease treatment	Chitosan nanoparticles	Local drug delivery	Preclinical studies, limited human applications	[87]
Blood pressure regulation (ACE inhibition)	Chitin, Chitosan	ACE inhibition	In vitro studies only	[90]

Table 5. Biological Effects and Evidence of Chitin, Chitosan, and COS.

Biological Effect	Compound	Mechanism	Evidence (Study Type)	References
Regulation of glucose & insulin	Chitin, Chitosan	Improves glucose metabolism	Mainly in vitro studies, limited small-scale human clinical trials	[79,80,81]
Lipid metabolism (reduction of cholesterol and triglycerides, enhancement of fatty acid oxidation)	Chitin, Chitosan	Binds fats & bile acids, regulates lipid-related genes	In vitro and animal studies, limited human clinical trials	[80,81,82,83,84]
Antioxidant activity	Chitin, COS	Neutralizes free radicals, reduces oxidative stress	In vitro and animal studies, limited human trials; COS shows higher bioavailability	[80,81,83,86,89,90,92]
Anti-inflammatory	Chitin, COS	Cytokine regulation	In vitro and animal studies, limited human evidence	[86,87]
Antimicrobial & Antiviral	Chitosan	Inhibits bacteria, carrier for antiviral drugs	Mainly in vitro and preclinical studies, limited human applications	[87]
Cancer prevention & therapy	Chitin, Chitosan	Immunostimulation, targeted drug delivery	In vitro and animal studies, few human data	[88,89]
Wound healing	Chitosan	Hemostatic, antibacterial	Strong preclinical support, applied in clinical wound healing	[87,90]
Ocular disease treatment	Chitosan nanoparticles	Local drug delivery	Preclinical studies, limited human applications	[87]
Blood pressure regulation (ACE inhibition)	Chitin, Chitosan	ACE inhibition	In vitro studies only	[90]

6. Trends and Future Challenges of Crab By-Product Utilization

In recent years, there has been growing interest in the recovery of chitin, chitosan, and COS from crustacean by-products such as crabs, shrimp, and lobsters [93,94]. The extraction of these biomolecules can be carried out using a variety of methods, both traditional and modern. These include chemical, biological, and EH, as well as more advanced techniques such as ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), the HOW-CA method, (scCO₂), and the use of ILs [94,95]. The chemical method is not environmentally friendly, as it relies on strong solvents such as acids and bases, while the biological method is applied over long periods and under low-pH conditions, which can alter the quality of the recovered products [95]. Modern technologies offer high speed and increased extraction yields; however, their industrial application is often limited by high costs and increased energy consumption [94]. In contrast, EH stands out as a milder and more cost-effective approach. The process preserves the molecular structure of chitin, although residual proteins and calcium carbonate are still present [93,96]. Crude chitin obtained via EH, containing residual proteins and calcium carbonate, exhibits significant advantages for aquafeed production [93,94,96]. Unlike purified chitin from other methods, it combines both nutritional and functional benefits, making it suitable for incorporation into fish diets [93,94]. The residual proteins provide nitrogen and bioactive peptides, while calcium carbonate contributes to mineral supplementation [96], thereby enhancing growth, immune response, and overall fish health [45]. Nevertheless, the recovered substances can be utilized as raw material for the enrichment of industrial aquafeeds [93]. Conversely, they are not immediately suitable for pharmaceutical and/or dietary supplement applications due to their limited purity and the presence of residual proteins and calcium carbonate [94]. Similarly, pure chitin also has limitations, as it cannot compete with COS in terms of bioactivity and bioavailability [45]. The incorporation of raw chitin into long-term feeding regimes of farmed fish species that inherently possess high bioavailability capacity may enable the realization of beneficial effects reported in the literature, such as enhanced immune response and improved overall fish health [93]. However, further investigation is required into the actual bioavailability of these compounds in fish and, consequently, in humans. Particularly important is the accurate quantification and determination of the appropriate consumption pattern (daily or weekly intake) to assess whether the consumption of fish fed with raw chitin obtained through EH can provide similar benefits to the human body through targeted consumption. Understanding these parameters is a critical step toward the reliable evaluation of the nutritional and biomedical value of raw chitin. With the industry’s shift toward “greener” and more sustainable production technologies, EH emerges as a strategic method for the sustainable valorisation of crustacean by-products, combining environmental sustainability, economic efficiency, and high product quality [45,94,95].

7. Discussion

EH is a mild, cost-effective, and environmentally friendly method for extracting chitin from crab by-products. It preserves the molecular integrity of the bioactive compounds while avoiding the use of strong chemicals, expensive equipment, and excessive energy consumption. Enzymes such as pepsin, protamex, bromelain, neutrase, alcalase, pancreatin, and Flavourzyme enzymes are generally used to extract chitin from crabs. Hydrolysis usually takes place in a pH range of 3.0–9.0, at temperatures of 37–65 °C, and with incubation times of 5 min to 12 h, while inactivation of the enzymes is achieved by heat treatment at 85–95 °C for 5–20 min. Chitosan, in the form of powder or nanoparticles, usually obtained from crabs or shrimps, is administered in aquafeed at concentrations of 0.5–20 g kg⁻¹ for experimental durations of 1 week to 90 days in fish with an initial weight of 0.18–125 g, most commonly in farmed Nile tilapia Oreochromis niloticus. The hydrolysis product obtained from raw chitin residues, which contains protein residues and calcium carbonate, can also be used to supplement aquafeed. Bioavailable chitin in farmed fish promotes growth, boosts innate immunity, and exhibits immunostimulant, antioxidant, and antimicrobial effects, that increase resistance to pathogens. In addition, dietary chitin can provide humans with beneficial effects such as antioxidant, anti-inflammatory, antimicrobial, and anticancer activities, as well as strengthening the immune system. Despite these positive results, there is still considerable variability in the experimental parameters, including the type of enzyme, hydrolysis conditions, method of enzyme inactivation, origin and pretreatment of biomass, form and dosage of chitin, and duration of the experiments, which limits the generalisability of the results. The utilisation of crab waste by enzymatic hydrolysis for chitin production can contribute to sustainable crustacean aquaculture, promote the transition to a circular economy and the development of innovative functional feed additives, while improving the growth performance and health management of farmed fish, which in turn benefits human health through their consumption.

8. Conclusions

EH is a mild, cost-effective, and environmentally friendly method for extracting chitin from crustacean by-products. It preserves the molecular integrity of the biomolecules and avoids the use of strong chemicals, expensive equipment, and excessive energy consumption.

The enzymes pepsin, protamex, bromelain, neutrase, alcalase, and pancreatin, as well as flavour enzymes, are commonly used for the extraction of chitin from crabs. EH is usually carried out in a pH range of 3.0–9.0, at temperatures of 37–65 °C, and with incubation times of 5 min to 12 h. Inactivation of the enzyme is achieved by heat treatment at 85–95 °C for 5–20 min.

A crude form of chitin is obtained from crabs by EH, which contains residual proteins and calcium carbonate and could potentially be used to enrich industrial aquafeed.

Chitosan, in powder or nanoparticle form, usually obtained from crab or shrimp shells, is used to fortify aquafeed at concentrations of 0.5–20 g kg⁻¹ for experimental durations of 1 week to 90 days in fish with an initial weight of 0.18–125 g, most commonly in farmed Nile tilapia Oreochromis niloticus.

Bioavailable chitin in farmed fish, when administered through dietary supplementation with chitosan or chitin at levels of 0.1–50 g/kg, provides a full range of biological, immunological, and growth-related benefits.

Overall, chitin, chitosan, and their derivatives exhibit a wide spectrum of biological effects, ranging from metabolic regulation to wound healing, with most findings supported by in vitro and preclinical studies, while clinical evidence in humans remains limited.