Review
Benefits, Challenges, and an Alternative Model of Fish Oil Enrichment in Animal-Based Food Products:A Review
2025 Volume 74 Issue 9 Pages 739-755
Details
2025 Volume 74 Issue 9 Pages 739-755
Abstract: Fish oil (FO) has garnered attention in recent decades because of its omega-3 fatty acid (n-3 FAs) content, which is essential for healthy functions. However, the broad application of FO in food products has pros and cons because n-3 FAs are highly prone to oxidative deterioration, leading to product rejection. Enriching food products with FO is an effective strategy to boost the accumulation of n-3 FAs in the body. The n-3 FAs are considered essential lipids, and their consumption helps maintain normal triacylglycerol and cholesterol levels in the blood, decreases the risk of cancer and cardiac disorders, and augments brain function. The n-3 FAs obtained from FO can be added to animal diets or food products as free FO or protected FO. In this review, we focus on elucidating the benefits and challenges of adding FO to several animal-based foods, such as meat-, egg-, and milk-based products. In addition, we discuss the preparation of edible film/coating-forming emulsions and the design of FO-enriched double-layered edible films/coatings.
The health benefits of fish oil (FO) consumption have contributed to many aspects of healthy human life over the past few decades. The consumption of FO is considered relatively safer than that of fish, as it undergoes a refining process to eliminate harmful toxins and pollutants that may be present in fish. The omega-3 fatty acids (n-3 FAs) present in FO are considered to be health promoters. Reviews by Goel et al. and Liao et al. have elaborated evidence regarding the important role of FO as a potential cardioprotective agent1) ,2) . Similarly, information regarding the role of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) , the n-3 FAs present in FO, in brain development has been summarized in reviews by Dighriri et al. and Dinicolantonio and O'keefe3) ,4) . Regarding the cancer-preventive effects of marine- and plant-derived n-3 FAs, the n-3 FAs present in FO exhibit a remarkable potential in limiting colorectal and breast cancer risk compared with that of n-3 FAs present in olive oil and safflower and flaxseed oils, respectively5) ,6) . Plant-derived oils rich in n-3 FAs principally contain α-linolenic acid (ALA) , and only a small amount of ALA can be converted into essential lipids, such as DHA at the conversion rate of 0.5-5% and EPA at the conversion rate of 5-8%7) .The bioavailability of FO makes it one of the best sources of essential FAs.
Many researchers have made innovative efforts to enrich market-oriented food products such as cookies, bread, granola bars, yogurt, chocolate, and sausages, with FO in different ways8) ,9) ,10) ,11) ,12) ,13) 14) . This is because the n-3 FAs are considered essential lipids and cannot be completely synthesized in the human body because they have a carbon-carbon double bond in the n-3 position, and the enzymes available in the human body can only catalyze double bond formation beyond carbon 915) . Although the direct incorporation of FO into food products is the easiest method, the susceptibility of FO to oxidation, attributed to the double bonds of n-3 FAs, must not be overlooked. Awada et al. reported that the ingestion of a diet containing oxidized n-3 FAs leads to a 5-fold increase in the accumulation of 4-hydroxy-2-hexenal in mice16) . They further noted that the oxidized end-product of n-3 FAs causes an inflammatory process that triggers oxidative stress in the duodenum. Moreover, numerous factors related to the solubility, volatility, and sensory acceptance of FO also limit its direct incorporation into foods.
Consequently, there has been an increasing interest in the development of appropriate techniques to protect FO from oxidation and off-odors to successfully avail the health benefits of including FO in diets. Encapsulation techniques have emerged as a common and mostly used means to tackle the above-mentioned vulnerabilities of FO. It is noteworthy that the carrier/wall material used for entrapping the FO can also be compromised during extensive food processing, leading to a decrease in its functional properties. As a result, the oxidative instability of FO can be induced by intense interactions with food components. To broaden the application field, a potential means of supplementing food products with FO is through its incorporation in the form of an edible film/coating. There has been a large number of scientific review publications regarding the encapsulation of FO in the last few years. We are not attempting to put one more similar review, but providing a clear comprehension of the development of FO-loaded edible films/coatings. A few pioneering researchers have developed edible films impregnated with FO with or without plant-based antioxidants17) ,18) ,19) ,20) ,21) ,22) . The impregnation of FO in edible films markedly improved their physical and mechanical performance. In practical applications, the oxidative stability of FO in edible films/coatings can also be preserved since the edible film/coating lay separately on the food surface and can be applied after the food has been cooked. In this review, we propose an alternative technique for improving the functionality of FO-loaded edible films/coatings. In addition, we highlight an enrichment model for increasing the FO content in animal-based food products and outline the disadvantages associated with its use.
Both in vitro and in vivo experiments have elucidated the beneficial effects of FO supplementation on specific tissues or organs. However, FO supplements may not always be acceptable, as some people avoid fishy breath and burps, an undesirable sweat odor, and unwanted aftertaste after their ingestion. Food can be used as a vehicle for FO, which is relatively more organoleptically acceptable to consumers compared with FO supplements, as they can cause a loss of appetite23) . Therefore, extensive efforts have been made for the development of FO-enriched foods to increase the consumption of n-3 FAs. Figure 1 illustrates various methods used for the enrichment of food products with n-3 FAs obtained from FO. It can be achieved through animal diet modification or direct mixing of FO with food formulations, through pre-emulsification or encapsulation or both before being added to food formulations, or through the preparation of FO-enriched edible films/coatings. Meat, eggs, milk, and their processed products, as well as formula milk, are commonly enriched with FO, as shown in Table 1.
Fish oil enrichment models in food products.
Methods of FO enrichment and several key discoveries.
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Efforts have been made to enrich several animal meats with FO to obtain naturally high levels of n-3 FAs in these meats through the supplementation of FO in the feed formulations of these animals. Bahnamiri et al. observed significant differences in the concentrations of n-3 FAs and the ratio of n-6/n-3 FA concentrations between longissimus dorsi muscles of Holstein bulls fed alfalfa hay diets supplemented with 0, 1, and 2% FO on a dry matter basis24) . The higher the FO inclusion, the higher the levels of n-3 FAs in meat, and the lower the ratio of n-6/n-3 FA concentrations. The inclusion of FO in combination with flaxseed in chicken diet also tremendously improves the proportion of n-3 FAs (up to 15-fold) and the ratio of n-6/n-3 FA concentrations (up to 15-fold) in breast and thigh meat, compared with that in chickens fed with diets not containing FO. Approximately 33% of EPA and 15.5% of DHA can be obtained by consuming 100 g of this FO-enriched chicken meat25) . Moreover, Khiaosa-ard et al. developed processed meat products, such as bacon, Vienna sausage, and Chinese sausage from the meat of pork raised with and without FO supplementation26) . They concluded that FO-enriched pork meat exhibits significantly increased concentrations of n-3 FAs and a decreased ratio of n-6/n-3 concentrations in the final products.
Another approach for FO enrichment, other than the manipulation of animal diets, is the introduction of FO as a food ingredient. When the ingredients of Dutch fermented sausage were modified using FO and flaxseed oil, EPA content was only recorded in sausages containing 15% FO; however, ALA content (1%) was considerably lower in these sausages compared with that in other sausage formulations containing 15-20% flaxseed oil (10-13% ALA) 27) . This could be attributed to the different sources and amounts of n-3 FAs in FO and plant-derived oils.
Recent studies have used encapsulated FO to fortify meat-based products. Aquilani et al. used microencapsulated and bulk FO forms to fortify fresh and cooked Cinta Senese burgers28) . They prepared spray-dried FO microcapsules in a multi-layered system enhanced by soy lecithin, chitosan, and maltodextrin for each layer. The microcapsules had a low moisture content of 4% and an EPA+DHA content of 9.63 mg/g. They further reported that the addition of FO to both fresh and cooked Cinta Senese burgers significantly improves the concentrations of n-3 FAs and the ratio of n-6/n-3 FA concentrations in these burgers, irrespective of the FO form used. Another study indicated a similar trend that fish sausages containing either bulk or emulsified FO had a higher proportion of n-3 FAs, at 4.14-4.27% and 4.48-4.66%, respectively than in fish sausages not enriched with FO29) . The amount of n-3 FAs in fish sausages not enriched with FO was only 2.34-2.53%. The ratios of n-6/n-3 FA concentrations in sausages enriched with bulk FO and emulsified FO and those without FO enrichment were 11.26-11.72, 10.27-10.70, and 20.65-22.20, respectively. The better characteristic of fish sausage enriched with emulsified FO can be attributed to the entrapment ability of soy isolate protein and carrageenan, which protects FO during the cooking process of the sausage. These two reports also revealed that processed meat products enriched with protected FO, i.e., encapsulated or emulsified FO, were relatively more stable and were preferred by the panelists. Thus, the encapsulation or emulsification of FO seems to protect FO from interactions with other food components and external factors; thus, slowing down the oxidation rate of FO in foods.
In a clinical study, the consumption of sausages enriched with n-3 FAs, both EPA and DHA, was reported to successfully increase the concentrations of n-3 FAs in the erythrocytes of 22 healthy individuals from 6.78% at day 0 to 8.56% at week 8, indicating that the accumulation of n-3 FAs in the human body can be gradually increased by the regular consumption of n-3 FA-enriched foods30) . Specifically, the consumption of sausages rich in DHA and EPA obtained from FO in the morning has been shown to considerably reduce the levels of triacylglycerols and total saturated FAs in healthy 20-60-year-old Japanese adults31) .
2.2 Egg and egg productsThe manipulation of chicken diets with FO has been extensively used to produce eggs rich in n-3 FAs. The production and consumption of eggs labeled 〝omega-3 enriched eggs〟 have been increasing because of their health benefits. Khan et al. fortified chicken diets with 1% FO (14.2% DHA and 7.5% EPA) and 8% flaxseed (52% ALA) and reported a significantly higher proportion of n-3 FAs in eggs produced by chickens fed the diet supplemented with FO and flaxseed than that in eggs obtained from chickens fed a diet not supplemented with FO and flaxseed32) . The proportion of n-3 FAs in the former group rose to 5.63-5.73%, whereas that in the latter group reached only 0.42%. Kralik et al. attempted to enhance the proportion of n-3 FAs in chicken eggs produced by hens fed with diets supplemented with FO and microalgae in different proportions33) . They reported that a diet containing 0.75% FO and 0.75% microalgae results in the highest proportion of n-3 FAs (14.65%) in eggs. However, a diet containing 0.5% of both FO and microalgae results in 13.60% n-3 FA concentrations in eggs, with the lowest proportion of n-3 FAs (4.53%) observed in standard commercial eggs. The accumulation of n-3 FAs in chicken eggs is greater during the period when chickens are being fed the FO-supplemented diet until 180 days34) .
Yolk color intensifies when chicken diets are supplemented with up to 2.5% FO compared to those added with 2.5% refined palm oil35) . This can be attributed to the yellow pale hue of the refined palm oil. At this supplementation level (2.5% FO) , there is an improvement in the taste of boiled and fried yolks; however, the odor deteriorates36) . Hydrocarbons, as one of the secondary oxidation products of FO, are responsible for the undesirable odor of heated (65-85°C) eggs rich in n-3 FAs, as disclosed in Chen et al.37) . The deleterious effects of scrambling and hard-boiling FO-enriched eggs at 80 and 100°C, respectively, on the levels of thiobarbituric acid reactive substances (TBARS; 2-fold increase) and total n-3 FAs (5% reduction) have been documented by Cortinas et al.38) . This finding is in agreement with that reported by Douny et al., who found a 4-10% reduction in the levels of n-3 FAs in cooked eggs39) . The cooking process can alter the levels of n-3 FAs in eggs and egg products, particularly when cooking at high temperatures of up to 100°C for a prolonged time.
In a clinical study, Bovet et al. reported that participants who consumed FO-enriched eggs had lower levels of triacylglycerols and high-density lipoproteins (HDLs) at 18 and 1.6%, respectively40) . Further investigation revealed a significant increase in the DHA content of erythrocyte membranes in healthy volunteers from 2.72 to 3.15% after consuming six n-3 FAs-enriched eggs per week, which is higher than the DHA content of erythrocyte membranes in healthy volunteers consuming standard eggs41) . The DHA content of erythrocyte membranes in the latter group decreased by approximately 2.79% at the baseline value and 2.90% at the end value than that of the erythrocyte membranes in the former group. The FA composition of eggs plays a major role in this change in DHA content, as the accumulation of n-3 FAs in the human body can be successfully increased through treatment with an FO-enriched diet. The presence of n-3 FAs in diets helps regulate the levels of various hormones and FA receptors and the structure of cell membranes in the body, thereby resulting in increased accumulation in the blood42) .
2.3 Milk and milk productsAnimal milk, an essential nutrient source, can also be made naturally rich in n-3 FAs through the enrichment of animal feed with FO. Nelson and Martini observed the n-3 FA content and the ratio of n-6/n-3 FA concentrations in milk produced by cows fed a diet containing protected FO and calcium salts43) . They reported that milk produced by cows fed diets containing 71 or 43% protected FO showed no significant changes in both parameters; however, treatments with FO resulted in a slight improvement in the levels of n-3 FAs (0.76-0.82%) in milk produced by cows fed protected FO-enriched diets than that (0.52%) in milk produced by cows fed diets not supplemented with protected FO. The ratio of n-6/n-3 FA concentrations in milk produced by cows fed diets not supplemented with protected FO was the highest between the treatments, at approximately 5.75. These findings are similar to those reported by Bernard et al., who found a significant difference in the n-3 FA content of goat milk obtained from goats fed a diet supplemented with FO and that obtained from goats fed a diet containing sunflower seed oil (5.4 vs. 3.1%, respectively) 44) .
Enrichment through the modification of animal diets or the direct use of FO in food formulations has also been reported in several milk products. The enrichment of strawberry yogurt through the addition of 2% microencapsulated FO significantly impacted its n-3 FA content and the ratio of n-6/n-3 FA concentrations after 4 weeks of storage45) . Encapsulation of FO in the gum arabic (GA) /maltodextrin blend-based wall system was conducted under an ultrasonic processor, before being subjected to a spray-dryer. Moisture content of 2.44%, total oxidation of 13.36, and encapsulation efficiency of 75.49% were observed in the FO microcapsules. The initial n-3 FA content of FO-enriched yogurt was 7.50%, which significantly reduced to approximately 5.82% after storage for 4 weeks in the refrigerator. However, the initial n-3 FA content of yogurt without FO enrichment was only 1.76%, which significantly reduced to 1.47% after 4 weeks. The ratio of n-6/n-3 FA concentrations in FO-enriched yogurt (0.53-0.68) was lower than that in non-enriched yogurt (2.45-3.02) . However, both samples showed a slight increase in the ratio of n-6/n-3 FA concentrations after storage. Dal Bello et al. prepared yogurts enriched with fruit or vegetable oils obtained from raspberry, flaxseed, Camelina sativa, Echium plantagineum, and blackcurrant46) . These fruit- or vegetable-oil-enriched yogurts contained only 0.19-0.74% n-3 FAs. These findings suggest that FO is a promising candidate for n-3 FA enrichment in yogurts and other food products. Nevertheless, the use of FO alone in yogurt formulations has serious consequences on the sensory acceptance of the enriched products, as reported by Murage et al.11) . They observed that FO-enriched yogurt was the most unacceptable sample, whereas yogurt samples enriched with FO in combination with strawberry, passion fruit, mango, and banana flavors were preferred by the panelists. Therefore, it is apparent that the fruity flavors can be used in the development of FO-enriched milk-based products.
A clinical study evaluating the effects of the intake of FO-enriched milk on the development of cardiovascular biomarkers in healthy children aged 8-14 years reported that children receiving FO-enriched milk for 5 months had higher levels of HDL and DHA and lower levels of low-density lipoprotein (LDL) and cholesterol than in those consuming standard commercial milk47) . In contrast, Molinari et al. reported that intervention with FO-enriched milk in healthy people aged 30-65 years for 8 weeks led to a reduction in their HDL levels even though their n-3 FA content increased48) . This does not necessarily mean that the reduction of HDL levels is an adverse effect, since LDL levels were also found to be lower after an 8-week intervention. Lifestyle parameters are important factors that need to be assessed because children and adults have different activities in their lives that can affect the efficacy of their daily consumption of FO-enriched foods.
2.4 Formula milkBreast milk is an ideal food for infants, as it possesses complete nutrients in the right amounts depending on the maternal diet. However, the availability of breast milk is occasionally insufficient for the daily nutritional needs of infants; therefore, infants are not exclusively breastfed. To carefully support the daily growth of infants, formula milk can be provided to fulfill their nutritional needs. Notably, FO is commonly used as an ingredient to enrich many types of commercial bovine milk-based formula milk, such as starter infant formula, infant formula for premature and low-weight newborns, extensively hydrolyzed whey protein infant formula, and partially hydrolyzed whey protein infant formula with n-3 FAs49) . Stimming et al. reported that the n-3 FA content of formula milk, enriched with FO and fungal oil, accounted for approximately 2.49%, whereas breast milk and non-FO-enriched formula milk contained lower concentrations of n-3 FAs, at approximately 1.64 and 2.12%, respectively50) . The formula milk without FO enrichment had the least DHA content (<0.01%) among the three milk types. The DHA content (0.23%) of FO-enriched formula milk was recorded to be comparable to that of breast milk. This finding confirms the effectiveness of FO as an n-3 FA-enriching agent in food products.
A clinical study showed that FO-enriched formula milk significantly increased the DHA content in the red blood cell (RBC) phospholipids of infants after 4 months of intervention51) . The DHA content was reported to be 6.02-9.22% at different intervals of intervention, higher than the DHA contents of infants receiving breast milk and standard formula milk, accounting for 3.51-6.39% and 2.78-5.89%, respectively. Regarding the difference in the DHA content based on the source of n-3 FAs, the findings of this study revealed a higher DHA content following the use of FO-enriched milk than that reported in a recent study by Gianni et al., who used vegetable oils derived from rapeseed, palm, and sunflower in formula milk52) . The supplementation with vegetable oils helped infants achieve a DHA content of 3.39-4.34% after 4 months of intervention. The conversion of ALA obtained from plant-derived oils to EPA and DHA seems to be the main limiting step affecting the DHA content in the RBC phospholipids of infants.
Lipid oxidation is a crucial problem affecting the efficacy of FO enrichment in food formulations. This problem may lead consumers to avoid FO-enriched products. Once the lipid in the food matrix is oxidized, it leads to multiple issues associated with the safety (health-related aspects) and quality (flavor, taste, and shelf-life) of food products. Intensive exposure to heat, air, or light can lead to the rapid oxidation of unsaturated FAs present in FO. Initiation, propagation, and termination are step-by-step oxidation processes wherein unsaturated FAs are attacked by free radicals (OH･, O2-, and H2O2) to produce various oxidation products. For example, aldehydes, alkanes, alcohols, and ketones are the products of the decomposition of the unstable lipid hydroperoxides, a primary product of lipid oxidation. These oxidation products are responsible for oxidative stress-induced diseases. Mayyas et al. reported that the serum and cardiac levels of TBARS significantly increased in male hyperthyroid rats after treatment with a FO-enriched diet for 8 weeks53) . The elevation of the levels of reactive oxygen species is attributed to hyperthyroidism, a condition that facilitates the oxidation of unsaturated FAs in FO, thereby increasing the levels of oxidative stress biomarkers. The adverse effects of oxidative stress have also been reported in the mouse duodenum caused by the high accumulation of the toxic aldehyde 4-hydroxy-2-hexenal. A diet containing oxidized n-3 FAs initiates the formation of injurious compounds16) . Regardless of the health condition of the subject, the oxidized n-3 FAs of FO can promote the formation of toxic and reactive compounds that cause cell death.
In the food system, the incorporation of bulk FO quickly increased TBARS levels in chicken nuggets before cold storage, which slowly went up to reach just below 2.5 mg malondialdehyde (MDA) /kg chicken nuggets after 3 months of cold storage. However, the TBARS levels in chicken nuggets enriched with microencapsulated FO stored for 3 months were significantly lower than those in chicken nuggets without FO, at approximately 1.0 and 1.9 mg MDA/kg chicken nuggets, respectively54) . The multilayer encapsulation of unsaturated FAs prevents their rapid degradation under heat exposure during the frying of chicken nuggets. However, the oxidation of lipids in chicken nuggets continues to occur rapidly, even after cold storage. This process may be induced by the formation of free radicals at low water activity levels. Oxygen readily diffuses into the relatively more porous matrix of frozen nuggets to hasten oxidation. Similar studies have demonstrated that the protection of FO via encapsulation or emulsification retarded the production of TBARS in Cinta Senese burgers and fish sausages, respectively28) ,29) . Microencapsulated or emulsified FO effectively suppressed the production of TBARS (2- to 3-fold reduction) from day 0 to day 30 in storage. Interestingly, Aquilani et al. and Jiménez-Martín et al. utilized similar ingredients (soy lecithin, chitosan, and maltodextrin) to fabricate the wall system of the FO microcapsules, and they demonstrated positive results in their studies28) ,54) . Encapsulation technology can be exploited to protect FO against oxidation in the food matrix and to restrain its release during storage.
Volatile oxidation products are also related to the state of the FO, whether it is in free form or protected form. Jiménez-Martín et al. screened 13 of 88 volatile compounds that correlated strongly with the oxidation of FO-enriched chicken nuggets54) . Chicken nuggets supplemented with bulk FO had relatively higher amounts of these 13 volatile compounds (2,4-decadienal, nonanal, octanal, 2-pentylfuran, 2,4-hexadienal, heptanal, hexanal, pentanal, hexane, butanal, dimethyl disulfide, and 2- and 3-methylpropanal) during the 3 months of storage than in the samples treated with microencapsulated FO and those in the control group. The levels of oxidation products, namely, ketones and alcohols, were further quantified to be the highest in the chicken nugget samples enriched with bulk FO, which led to a reduction in the panelists' preference for these samples. In the other samples, acids, aldehydes, terpenes, and esters were the most abundant volatile compounds detected in dry-cured sausages enriched with FO. In contrast, aldehydes, cyclic hydrocarbons, aliphatic hydrocarbons, and alcohols were the most abundant in FO-containing sausages cooked by heating at 85°C55) . Different FO-enriched meat products contain different main volatile compounds. However, the use of encapsulated FO resulted in a lower proportion of volatile compounds than when the bulk FO was used. Notably, the aldehyde levels of FO-enriched sausages prepared using a heating process were higher than those in FO-enriched sausages prepared by dry curing below 9°C, which could be attributed to the oils used for cooking56) . In addition to the cooking medium, the levels of secondary oxidation products generated depend on the cooking method, including cooking time and temperature. These secondary oxidation products are responsible for the off-flavors of FO-enriched food products.
Thus far, the role of volatile oxidation products, such as aldehydes, in determining the odor and taste perception of FO and FO-containing food products remains hypothetical. Regardless of whether trained or untrained panelists were used, FO-enriched food products, such as cupcakes, chocolate, chicken nuggets, sausage, yogurt, and granola bars, scored relatively low on the attributes of odor and taste10) ,11) ,12) ,13) ,57) . Panelists occasionally experience an odor in a food that has been previously smelled, or they have observed that others may perceive similar odors, and the odor reminds the panelists of their previous experience. Hence, for FO odors that they do not like, the panelists' responses will be the same, regardless of the techniques applied to mask the FO odor. However, although the findings may vary, the utilization of encapsulation techniques and fishy odor-masking agents is likely to result in improved odor and taste preferences.
In the previous section, we primarily elaborated on FO enrichment through food formulation and its welcome and unwelcome effects; however, another possible breakthrough needs to be discussed. In addition to the direct incorporation of FO into food dough, an edible film/coating can be used to protect FO from the oxidative activities of other reactive molecules or species during food processing. Notably, this alternative enrichment is specifically intended for solid foods. Figure 1 illustrates that FO can be emulsified inside a wall system/material and immediately cast onto a Petri dish to make an edible film. In addition, solid foods can be immersed in or sprayed with edible coating solutions. A detailed discussion on multilayer films for general purposes can be found in a previous review58) . In this review, we propose an alternative technique for FO enrichment in the form of a double-layer edible film or coating using our methodological approach. This alternative approach is expected to improve the oxidative stability and functionality of edible films rich in FO. The information presented in this review can pave the way for wide implementation of this technique in the future.
We underscore three main factors for the successful production of the FO-containing edible films/coatings in the double layer model, including biopolymer selection, bioactive selection, and drying conditions. They can determine the functionality and stabilization of the edible film/coating. According to a systematic review by Khoshnoudi-Nia et al., protein/polysaccharide blend-based wall materials are the most appropriate and stable complexes for coating FO59) . When protein is used alone as a wall material, its solubilization is majorly influenced by the pH, which can create different interaction types between protein, solvent, surfactant, and other molecules at different pH values. Ruiz-Álvarez et al. reported that whey protein hydrolysate (WPH) and blue whiting (Micromesistius poutassou) protein hydrolysate (BPH) stabilized the FO-in-water emulsion in different ways as impacted by pH values60) . The charges on the peptides in both these hydrolysates determined the physical stability of the FO emulsion. At alkaline pH, WPH significantly retained smaller FO emulsion droplets (0.375-0.730 µm) , while emulsions prepared with BPH showed immediate breakage of their droplets after homogenization. At an acidic pH, BPH was incapable of retaining small oil droplets after 7 days of storage at pH 2 because of the separated serum layer, while WPH was found to maintain the droplet size of the emulsion at 1.7 µm after 7-day storage time. However, BPH exhibited better interfacial elasticity and viscosity at the same measurement interval. Moreover, at pH 8, WPH lowered the interfacial tension, as the concentration of WPH increased to 10 mg/mL in the aqueous phase because WPH exhibited a better balance of aspartic acid and glutamic acid contents, which helped improve its solubility and eventually prevented aggregation. García-Moreno et al. reported that higher adsorption of fish protein hydrolysates at the interface of the FO-in-water emulsion resulted in a lower peroxide value while stabilizing the droplet size of the emulsion61) . On the other hand, previous studies have also shown the limitation of using polysaccharides alone for the encapsulation of FO62) ,63) ,64) . Maltodextrin, sodium alginate, sugar beet pectin, and GA were relatively ineffective in entrapping FO due to a low loading capacity, resulting in the encapsulation efficiency of less than 70%. It seems reasonable that their binding affinity impairs the entrapment process. Hydrophilic bioactives are likely to mix relatively more readily and evenly in a hydrophilic biopolymer-based liquid system, whereas lipophilic bioactives dissolve readily in a lipophilic biopolymer-based liquid system. Therefore, protein/polysaccharide blends are the most common wall materials applied in the encapsulation technique.
This is also important to note that the use of protein/polysaccharide blends is pivotal for the preparation of emulsion-based edible film/coating. Protein/polysaccharide blends are expected to eliminate the shortcomings of using individual biopolymers, either protein or polysaccharide source. Lower moisture resistance and mechanical strength are the main undesirable properties in this case. Even if lipidic materials are incorporated into the film/coating-forming solution, the wall materials must have the ability to control them, so that oil separation and hydrophobic saturation in the film matrix can be avoided. Such phenomena will lead to the formation of cracks, holes, and surface roughness that eventually compromise structural integrity and other functionalities of the films, as proven by the previous studies65) ,66) ,67) . Therefore, the biopolymer selection is the first taken into account because of its vital role in stabilizing the entrapment of FO, either in the emulsion form or in the edible film/coating form.
In this review, we propose using zein to construct the protein part of the wall material, and the polysaccharide part can be constructed using GA. Zein is a corn-derived protein that is water-insoluble due to its richness in nonpolar amino acids, approximately more than 50%, making it reliable in interacting with FO in the internal phase of the oil-in-water (O/W) emulsion system68) . Rahmani-Manglano et al. have noted that zein limited FO oxidation by increasing the encapsulation efficiency (92.4%) of FO nanocapsules69) . Zein alone is also capable of synthesizing stable micro- to nano-capsules containing a diverse range of lipophilic bioactives, including eucalyptus essential oil (EO) , Litsea cubeba EO, cumin EO, algae oil, resveratrol, and lovastatin70) ,71) ,72) ,73) ,74) . With respect to the isoelectric point of zein (pI) of 6.2, some features of the zein, such as electrical charges and functional groups, can be affected when it is prepared at pH ranges close to neutral75) . Hence, blending with supporting biopolymers will promote the good properties of the wall system. On the other hand, an important polysaccharide source is the GA, which is commonly used in food processing due to its favorable thickening, emulsifying, and stabilizing properties. This biopolymer is naturally hydrophilic, comprised of arabinogalactan (80%) , arabinogalactan protein (18%) , and glycoprotein (2%) 76) . These fractions are responsible for such functional properties. Benefiting from its excellent water solubility, GA can demonstrate a more profound function in the external phase of the O/W emulsion system to serve a long-term stability of emulsion droplets. A similar phenomenon was observed when whey protein concentrate-GA mixes were used to encapsulate FO77) . The physical properties of encapsulated FO were also significantly affected by GA. Thus, the polysaccharide components of the wall system are also the key molecules that stabilize encapsulated or emulsified FO or both, which may be attributed to their ability to adsorb the hydrophobic parts of the protein while facilitating the interaction with an aqueous phase. The good performance of the combination of zein and GA has been documented in previous works dealing with the encapsulation of hydrophobic and hydrophilic cores, such as peppermint oil, cinnamon bark oil, medium chain triacylglycerol oil, resveratrol, anthocyanin, and epigallocatechin gallate68) ,78) ,79) ,80) ,81) ,82) . In edible film applications, their combination has been evaluated to improve functional properties of edible films containing oregano EO and trans-cinnamaldehyde83) ,84) .
A schematic representation of FO stabilization using zein-GA complex is shown in Fig. 2. Glycerol and sodium dodecyl sulfate (SDS) are the chosen surfactants to protect the emulsion from destabilization and to generate smaller droplet sizes85) ,86) . They can also improve the miscibility of zein and GA in water and promote associative effects between them to improve the encapsulation efficiency. Acetic acid has been reported to cause partial unfolding of zein polypeptides via electrostatic repulsion because of the acetate and hydrogen ions present in acetic acid87) . Moreover, acetic acid can be removed using a rotary evaporator before being mixed with glycerol. Glycerol reacts with the partially unfolded zein polypeptides via hydrophilic/hydrophobic interactions88) . The addition of SDS induces the electrostatic binding of the SDS head with the positively charged amino acids of zein, whereas the hydrophobic tail of SDS anchors the non-polar amino acids of zein. Glycerol strengthens the electrostatic effect of SDS by reducing the dielectric constant of the medium89) . Arabinogalactan, arabinogalactan protein, and glycoprotein of GA provide electrostatic- or polarity-based interactions or both between SDS, glycerol, and zein to form the zein-GA complex. Finally, FO can be controlled by the hydrophobic amino acids of zein, whereas GA can work cooperatively with SDS and glycerol to maintain the integrity of droplets in water. This mixture can be poured onto a Petri dish as the bottom layer of the film, which is rich in FO. Figure 3 shows the image of the bottom layer prepared with 50 mg of FO per 1 g of layer-forming formulation. It can be seen that this layer has a smooth surface and a rough surface on its bottom (Fig. 3A) and upper (Fig. 3B) sides, respectively. The latter also clearly shows spaces inside the film matrix in some areas due to the formation of water bubbles in heterogeneous sizes during evaporation. Even though the peroxide value (PV) of this layer is still relatively low at approximately 10 meq/kg at 0-day storage time, it should be significantly minimized to guarantee the quality of the wrapped foods during the long-term storage time21) . To prevent rancidity flavor, PV should not be more than 10-20 meq/kg90) . Therefore, a double-layering technique is suggested to tackle this issue.
Schematic representation of the formation of FO emulsion stabilized by zein-GA complex and the model of double-layer edible film. Sodium dodecyl sulfate (SDS) , gum arabic (GA) , fish oil (FO) .
The bottom (A) and upper (B) sides of the bottom layer of film.
The solution for the active/upper layer can then be poured once the bottom layer dries. This layer does not contain FO; however, potential antioxidant or antimicrobial compounds can be incorporated. In the context of edible coating application, the mixture for the bottom layer can be applied to solid food products through dipping, immersion, brushing, or spraying techniques. Once the bottom layer is formed, the solution for the upper layer can then be applied onto the bottom layer. As the upper layer lay in the outer part, where it is highly exposed to environmental factors (chemical, microbiological, and physical) , the selection of the source of the active compound becomes important.
Today, societal trends are strongly shifting toward the use of natural active compounds, which are safer for human intake for long-term use. Natural resources-generated active compounds have also gained significant attention in the development of edible film/coating because of their powerful biological functions and appealing odors, owing to their active compound constituents. Moreover, these compounds can strongly mask fishy odors. Previous studies have utilized the green tea extract, as well as EOs from oregano and rosemary, to prevent oxidation of FO-enriched edible films in a single-layer model17) ,19) ,22) . These natural substances can be mixed with the zein-GA complex to provide active functions in the upper layer of the edible film.
We suggest using a similar biopolymer (zein-GA complex) when constructing the upper layer to prevent the incompatibility of structural properties between the bottom and upper layers. Our recent publication presented the image of FO-loaded edible film double-layered with an upper layer containing extract of guava leaves (GLE) 21) . We used zein-GA complex to prepare the bottom and upper layers. As a result of using different types of bioactives, the characteristics of each layer remain different. FO renders the lipophilic properties of the bottom layer, while GLE forms the upper layer more hydrophilic. The hydrophilicity of the upper layer can restrict the infiltration of FO. At the same time, the lipophilicity of the bottom layer hinders GLE particles from migrating. Terpenoids and flavonoids are key odor-active compounds in guava leaves91) ,92) ,93) . We employed a simple extraction technique using agitation and heating with water solvent to obtain GLE, which is a safe natural substance for consumption with potential active actions. The edible film showed oxidative stability for one month of storage time, having PV constantly below 10 meq/kg. The inhibitory effects of the edible film against Bacillus subtilis were remarkable but relatively weak against Escherichia coli, probably due to phenolic and flavonoid contents were relatively low. The use of newly developed extraction techniques can be suggested to optimize the extraction yield of active compounds derived from guava leaves. It has been proven that the yield of phenolic compounds was enhanced using ultrasound-assisted extraction and microwave-assisted cryoconcentration with water solvent94) ,95) ,96) . It is also possible to combine GLE and other sources of active compounds, such as EOs derived from guava leaves, mint leaves, tea tree, and rosemary. Such combinations provide distinctive bacterial inhibition modes, which are related to their diverse active compounds. GLE enriches phenolics in the edible film matrix, while those EOs enrich terpenes that contribute more intensively to the inhibition of E. coli97) ,98) . Though we suggest considering the GLE-to-EO ratio to maintain the hydrophilicity of the upper layer. Besides, zein and GA enable proper structural formation between GLE and EO in the film matrix by utilizing their functional groups, so that structural, mechanical, and physical properties of the edible film can be maintained.
In a similar model, previous works have successfully developed double-layered edible films embedded with active compounds99) ,100) ,101) ,102) . The bottom layer was prepared based on the blends of furcellaran and soybean hydrolysate or carp skin gelatin hydrolysate. Ethanolic extract of soybean bran and dipeptide Alanine-Tyrosine were used as active agents in the upper layer. Yet, they revealed that the developed films did not significantly improve the oxidative and microbiological stabilities of tofu, butter, and fresh Atlantic mackerel during prolonged storage, compared to those wrapped in single-layer films without active agents or a control group. Moreover, the use of ethanolic extract may also limit the customization for diverse non-alcoholic food products. Their findings lack functional actions that may be due to the poor biological activity of active agents, the interaction between biopolymers and active agents, and the degradation of active agents. The latter phenomenon may occur during the drying process, given that the active agents in their works were stand-alone without a biopolymer. Due to this reason, the upper layer in our method is prepared with biopolymer. Another similar effort was also performed by Hernández-Carranza et al., who developed the whey-based upper layer embedded with probiotics with or without mango pulp103) . Potato starch and glycerol were mixed to make the bottom layer. Nowak et al. demonstrated another alternative to prepare double-layered edible film embedded with plant extracts104) . They employed aqueous extracts of granulated garlic, dried tomato, black pepper fruit, tansy herb, and lingonberry fruit in the bottom layer prepared with furcellaran solution. The upper layer was constructed using a combination of furcellaran and gelatine without plant extracts. It is worth noting that these studies have designed the double-layered edible film with different placements of active compounds depending on the sensitivity of active compounds and specific use on targeted foods.
Understanding the adequate drying temperature and duration is another crucial step when designing double-layered edible films/coatings. Notably, these parameters influence the mechanical and structural properties of the film. It is difficult to generalize temperature and duration of drying for all developed edible films, as they mainly depend on biopolymers, bioactives, and modifications involved. For instance, Nowak et al. performed the drying process in a lab hood at room temperature for double-layered edible film based on furcellaran alone and furcellaran in combination with gelatine104) . Whereas, Hernández-Carranza et al. conducted drying of potato starch- and whey-based double-layered films at different conditions, 40°C for 2 h and 40°C for 24 h, respectively103) . The differences in the amino acid compositions of whey and gelatin, as well as carbohydrate compositions of furcellaran and potato starch, have contributed to the distinct drying conditions since these components provide holding capacities for the solvent, bioactives, and other molecules. In our method, drying process for the bottom and upper layers takes place in a standard oven at 40°C for 48 h for each layer, and totalling 96 h for the bottom layer. In this sense, the upper layer-forming solution is spread over the dried bottom layer. As a consequence, the drying duration for the bottom layer is extended until the upper layer achieves the desired dryness. In a single-layer model, previous studies prepared FO-containing edible films at lower temperatures (22-30°C) and in a shorter time (20-24 h) 19) ,20) ,22) . When compared to the drying condition of the bottom layer in our method, their studies indicated better efficiency in the drying process. A longer drying duration of our single- and double-layered films can be attributed to the slow rate of water evaporation as caused by the strong binding process of water induced by hydrophilic amino acids of zein, hydrophilic fractions of GA, and surfactant (SDS and glycerol) , as well as the presence of free/unencapsulated oils and GLE particles that lay on the surface area during the drying process of the bottom and upper layer, respectively. Drying duration is one of the primary issues in our methodological approach that can restrict the expansion of this edible film for small- to large-scale industrial applications. Therefore, the opportunity for future study to address this concern is widely opened by tuning the parameters of drying conditions for FO-enriched double-layered edible films/coatings or exploring other types of drying equipment, such as low-pressure superheated steam drying and vacuum drying.
Furthermore, although an intensive drying process has been performed, our double-layer film poses another concern in its thickness (0.87-0.97 mm) , where it is too thick21) . In addition to physical and mechanical properties, this thickness value can also influence the sensory acceptance of the wrapped/coated foods. Consumers may not prefer a thicker film because it is less convenience-oriented when consumers require a direct sensation of food texture, without being obstructed by repeated texture exposure of such a thick film. A typical range of thickness of single-layer edible films is 0.15-0.40 mm, and this thickness may be more convenient for the consumer105) . Even Jamróz et al. produced a thinner (0.14 mm) edible film in a double-layer model101) . They used different volumes for the bottom and upper layers, with the volume of the upper layer at nearly half that of the bottom layer. On the other hand, our method uses a similar volume for each layer, accounting for 50 mL in total in a 9 cm (diameter) Petri plate. Here, by reducing total volume and adjusting the volume for each layer, we expect to optimize the drying efficiency, thereby yielding a film with a proper thickness. In this regard, we offer some possible scenarios; (1) using a similar volume (15 mL) for each layer and dried at 40°C within 12-24 h for each layer, (2) using different volumes, 20 mL and 10 mL for the bottom and upper layer, respectively, and dried at 40°C within 12-24 h for each layer, and (3) using similar or different volumes, but the upper layer solution is slowly poured when the gel consistency of the bottom layer is observed, and continues drying process at 40°C for not more than 48 h.
In summary, it seems reasonable that the oxidation and volatility characteristics of FO hinder the efficacy of the enrichment of animal-based food products with FO, as these characteristics affect the sensory acceptance and the health outcomes associated with the use of these products. Encapsulation and emulsification are promising alternative strategies for protecting FO against undesirable external factors. However, the efficacy of these technologies depends on the mode of enrichment, i.e., whether they are applied upstream (supplementation in animal diets) or downstream (incorporation in food formulation) , such that the n-3 FAs of FO can reach the household table safely and optimally. In addition, film-forming emulsions/mixes can be used to fabricate FO-rich edible films/coatings to address the adverse effects of direct FO enrichment in food formulations. The desired film/coating properties mainly rely on the versatility of biopolymers, as it is involved in the entrapment of bioactives and all interacting molecules, and ultimately influence the efficiency of drying rate.
The below-mentioned points can be considered in future studies to increase the use of FO in the food industry: (1) The choice of compatible and stable biopolymers and their preparation conditions is important in determining the efficacy of FO enrichment in food products. Therefore, the types of proteins and polysaccharides required for FO preservation must be consistently compared in future pilot-scale studies. (2) The development of flavorful active compounds to mask fishy off-flavors is essential for the wide acceptance of FO-enriched foods. (3) The evaluation of the techno-economic aspects of FO enrichment needs to be considered to ensure its sustainability in the food industry. This process will lead to the determination of a suitable process and product based on several essential factors, including economic feasibility, environmental sustainability, and resource availability.
Aji Sukoco is grateful to the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and the Prefectural University of Hiroshima, Japan, for financial assistance as an MEXT-awardee student.
