To exert their biological activity, hydrolysates or peptides must be evaluated for digestibility and subsequent release of bioactive peptides in relevant in vitro intestinal models and the in vivo GI tract lumen. In vitro methods using cultures such as monolayers of human intestinal Caco-2 epithelial cells and in vivo models to determine permeability can aid in the prediction of oral bioavailability. The selective permeability of the intestinal barrier to candidate peptides is based on an understanding of the structural and chemical properties of the active compounds, the interactions in the gastrointestinal tract, and knowledge of the physiology of the GI tract ^[17][164]. As previously mentioned, it has long been known that hydrolysates with many short peptides, especially dipeptides and tripeptides, can lead to their better absorption and are more efficient than free amino acids and larger precursor peptide molecules ^[18][165]. If the MW of the molecule increases above 500 Da, the oral bioavailability decreases dramatically ^[15][162]. For example, the bioavailability of fractions of casein-derived peptides less than 500 Da was 16.23%, compared to 9.54% for fractions in the molecular weight range of 500–1000 Da ^[19][166]. Also of interest is that the length of the peptide chain provides a clue as to whether a transdermal transporter is involved. Specifically, dipeptides and tripeptides have been described as substrates for PepT1 binding and transport, which is a peptide transporter with a transmembrane electrochemical protein gradient located on the apical membrane of enterocytes ^[20][167]. In contrast, the TJs-mediated paracellular pathway is responsible for the translocation of oligopeptides containing four to nine amino acid residues ^[21][22][168,169]. This conclusion is corroborated by the work of the group of Xu et al. ^[23][42]. In an assay evaluating the uptake mechanism and bioavailability of rapeseed protein-extracted peptide WDHHAPQLR (RAP) in Caco-2 cell monolayers, they found that RAP is degraded by brush border peptidases, and then longer fragments of RAP, DHHAPQLR and WDHHAP are transported via the paracellular pathway, while tripeptide QLR is taken up via PepT1. In addition, through pharmacokinetic tests, they found that the absolute bioavailability of RAP (100 mg/kg BW) could reach 3.56% in rats after oral gavage, although the effective permeation rate of the basal side of Caco-2 monolayer measured in the preliminary screening test was only 1% at most, which was not sufficient to exert antioxidant effects. These results suggest that RAP may be developed as an efficient radical scavenging peptide. An earlier study by the same group showed that 65.57% of YWDHNNPQIR was resistant to hydrolysis by brush border peptidase and could be absorbed by the intestinal epithelium intact. More importantly, the absorbed peptides could still exhibit favorable antioxidant activity ^[24][170]. Wang et al. ^[25][82] reported an interesting work to screen and identify antioxidant peptides with digestive stability and bioaccessibility in muscle hydrolysates of silver carp. Two digestion products, i.e., viniferase and papain-induced hydrolysate fractions with molecular weight less than 1 kDa after SGID, showed 9.21% and 11.45% permeability by trans-cortical transport analysis of the Caco-2 monolayer. Then further identified by LC-MS/MS, the fragmented peptide LVPVAVF in the permeate showed the strongest DPPH^• cleavage (EC₅₀ 0.65 mg/mL) and ROS quenching activity (27.23% at 50 µg/mL). Similarly, Feng and Betti ^[26][171] reported that digestion products of bovine collagen hydrolysate could reach up to 21.4% transport efficiency across Caco-2 cell monolayers. These studies support the idea that protein digests screened by in vitro permeability assays to obtain highly permeable fractions have greater potential as natural resistance components in food and drug systems than single or purified peptides. However, further studies focusing on the relationship between intestinal absorption of antioxidant peptides and subsequent in vivo metabolism are needed.

In conclusion, given the properties of antioxidant peptides, such as molecular weight, charge, hydrogen bond potential and hydrophobicity sensitivity to peptidase and intestinal transport, and the correlation between intestinal epithelial transport of peptides and peptidase catalysis, most oligopeptides exhibiting in vitro antioxidant activity rarely maintain their integrity or activity after transmembrane transport and subsequently affect their bioavailability, even though small amounts of target peptides may be detected in plasma. The presence of small amounts of the target peptide may be detected in plasma. As recently novel antioxidant peptides LHSMK ^[27][90], YFCLT and GLLLPH ^[22][169], WDHHAPQLR ^[23][42], GNPDIEHPE, SVIKPPTDE and VIKPPTDE ^[28][172] were reported as such. Therefore, to maximize health benefits, future work needs to shift more towards the development of promotion strategies for the stability and bioavailability of bioactive peptides.

Thanks to the natural properties and generally high biological activity of antioxidant protein hydrolysates or peptides, the use of these high value-added products as functional and nutritional fortification ingredients in specialty formulations is an area of increasing interest. Currently, the application of these active compounds is focused on four main areas: (1) as matrix enhancers, preservatives, or nutritional supplements in food systems; (2) as therapeutic agents to be incorporated into pharmaceutical systems; (3) as feed additives to improve animal immunity; and (4) development of anti-aging and photoprotective pharmaceuticals. Unlike living organisms, the quality features of food products suffer from irreversible decay from the date of production, which can be postponed from a few hours to months or even years if appropriate strategies are adopted. In this case, it turned out to be a common trend for the food industry to prioritize the use of synthetic antioxidants such as BHA (butylated hydroxyanisole), BHT (dibutylated hydroxytoluene), and TBHQ (tert-butylated hydroquinone) to promote food stability and extend shelf life ^[29][11]. However, given the potential health hazards of synthetic antioxidants, the current consumer trend is a dramatic increase in demand for ‘clean label’ foods and functional foods enriched with natural active ingredients. This trend has reinforced the demand of the food industry and researchers to obtain and apply food additives of natural origin that also exert bioactivity to prevent the development of NCDs. In this respect, antioxidant peptides have acted, at the laboratory level, as potential food additives. Few studies have evaluated the effects of antioxidant peptides in real food matrices, which could support their potential use as additives (Table 1). Meat products are a more studied food system as they are susceptible to lipid oxidation and require exogenous antioxidants to scavenge the active substances. For example, Shen et al. ^[30][173] reported that the addition of silver carp protein hydrolysate (2 and 4%, w/w) to surimi attenuated the formation of myofibrillar protein carbonyls, inhibited the reduction of free sulfhydryl content, and slowed down the formation of peroxidized lipid MDA and the rate of change of flavor compounds. Similarly, Lin et al. ^[31][174] proved that the incorporation of bighead carp gills hydrolysate (1 and 2%, w/w) treated with neutral protease to surimi increased the concentration of sulfhydryl and salt-soluble proteins, enhanced Ca²⁺-ATPase activity, reduced disulfide bonds, carbonyls, and hydrophobicity, and improved gel strength and texture. Nowadays, rarely commercial foods containing antioxidant active peptides exist on the market, despite the considerable literature on them. The possible reason is the lack of sufficient evidence for the biological effectiveness, processing and matrix stability, and toxicological safety of most antioxidant peptides. The stability of peptides during food processing and storage is critical for their application as functional ingredients, as peptides are vulnerable to chemical modifications of the backbone or side chains. These chemical reactions involve disulfide bond formation, dehydration, glycation, and aromatic ring oxidation, inducing changes in the structure and bioactivity of peptides ^[32][33][99,175]. The interaction between peptides and food matrix components such as proteins, lipids, and polysaccharides during food processing and storage could trigger a number of physicochemical reactions, such as hydrophobic interactions, disulfide interactions, and Maillard reaction, thereby favorably or adversely impacting the biological activity, solubility, sensory profiles, and color and texture parameters of peptide-based functional foods ^[32][34][99,176]. Over the decades, remarkable progress has been made in exploring the role of peptides in textural, sensory, and health aspects. Previous studies have focused on how peptides shape textural and technical functional properties, such as how mackerel gelatine hydrolysate affects flavor, color, emulsion activity and stability, and foaming properties and stability of carbonated beverage ^[35][86]. However, understanding how and to what extent peptides affect the functional properties of foods requires a comprehensive consideration of multiple complexities (e.g., peptide amphipathicity, solubility, and gelation capacity, food composition and ingredient distribution, and food processing and storage conditions) for better tailoring the type of hydrolysate in the formulated product to obtain the desired functional properties. Thus, further studies are requested to assess the impact of antioxidant bioactive peptides on the technical properties and consumer acceptance of final products, even though promising outcomes have been recorded in literatures (especially for minced meat and beverages).