Review on preparation methods, mechanisms and applications for antioxidant peptides in oil

2.1. Preparation of antioxidant peptides by traditional hydrolysis

Hydrolyzing protein is the most common method for preparing antioxidant peptides. Acid-base hydrolysis uses acid or alkali to hydrolyze protein. Some researchers hydrolyzed ostrich egg white protein with 0.25 mol/L NaOH at 40 ºC for eight hours. They proved that the antioxidant activity of the protein was significantly enhanced after hydrolysis (Khueychai et al., 2019Khueychai S, Jangpromma N, Daduang S, Klaynongsruang S. 2019. Effects of alkaline hydrolysis and storage conditions on the biological activity of ostrich egg white. J. Food Process. Preserv. 43 (4), e13921. http://dx.doi.org/10.1111/jfpp.13921 ). This method is cheap and time-saving. Nonetheless it is difficult to control the degree of hydrolysis and product quality, because the violent reaction conditions involved are destructive to amino acids (Hall and Ahmad, 1997Hall GM, Ahmad NH. 1997. Surimi and fish-mince products. Fish Process. Technol. 74-92. https://doi.org/10.1007/978-1-4613-1113-3_3 ).

Microbial fermentation can produce enzymes and enzymatic hydrolysis simultaneously. This method also can directly isolate and purify antioxidant peptides (Chai et al., 2020Chai KF, Voo AYH, Chen WN. 2020. Bioactive peptides from food fermentation: a comprehensive review of their sources, bioactivities, applications, and future development. Compr. Rev. Food Sci. Food Saf. 19 (6), 3825-3885. http://dx.doi.org/10.1111/1541-4337.12651 ). After fenugreek protein was fermented with Lactococcus lactis for 24 hours, the free radical scavenging activities of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS) increased by 42.9 and 40%, respectively (Setti et al., 2017Setti K, Kachouri F, Hamdi M. 2017. Improvement of the antioxidant activity of fenugreek protein isolates by Lactococcus lactis fermentation. Int. J. Pept. Res. Ther. 24 (4), 499-509. http://dx.doi.org/10.1007/s10989-017-9636-y ). However, the process of screening microorganisms is cumbersome and the safety of microbial enzyme production is uncontrollable (He et al., 2012He R, Ju XR, Yuan J, Wang LF, Girgih AT. 2012. Antioxidant activities of rapeseed peptides produced by solid state fermentation. Food Res. Int. 49 (1), 432-438. https://doi.org/10.1016/j.foodres.2012.08.023 ).

Enzymatic hydrolysis uses proteolytic enzymes to hydrolyze protein into small molecular polypeptides. It is characterized by mild conditions, strong operability, strong specificity and fewer by-products. The same protein hydrolyzed by different enzymes produces different antioxidant peptides (da Rosa et al., 2018da Rosa LOL, Santana MC, Avezedo TL, Santa Brigida AI, Godoy R, Pacheco S, Mellinger-Silva C, Cabral LMC. 2018. A comparison of dual-functional whey hydrolysates by the use of commercial proteases. Food Sci. Technol. 38, 31-36. https://doi.org/10.1590/fst.08417 ). A study has measured the antioxidant activity of camel colostrum protein hydrolyzed by pepsin, trypsin, pancreatin and another two enzymes. The results showed that the proteolytic peptide hydrolyzed by pancreatin had the highest antioxidant activity (Oussaief et al., 2019Oussaief O, Jrad Z, Adt I, Dbara M, Khorchani T, El-Hatmi H. 2019. Antioxidant activities of enzymatic-hydrolysed proteins of dromedary (Camelus dromedarius) colostrum. Int. J. Dairy Technol. 73 (2), 333-340. http://dx.doi.org/10.1111/1471-0307.12668 ). Hydrolysis conditions are also critical. Some researchers have optimized the hydrolysis conditions from egg white with chymotrypsin and pepsin through response surface methodology, and the antioxidant activity of the peptide was improved (Yuan et al., 2020Yuan J, Zheng Y, Wu Y, Chen HB, Tong P, Gao JY. 2020. Double enzyme hydrolysis for producing antioxidant peptide from egg white: optimization, evaluation, and potential allergenicity. J. Food Biochem. 44 (2), e13113. https://doi.org/10.1111/jfbc.13113 ). Table 1 summarizes experiments related to the preparation of antioxidant peptides by enzymatic hydrolysis.

However, enzymatic hydrolysis usually obtains crude peptides, so it needs to be separated and purified. Usually, ultrafiltration or gel chromatography can be carried out according to the size of antioxidant peptides. Ion exchange chromatography and reverse chromatography can also be used (based on the charge and hydrophobicity of the antioxidant peptide, respectively) (Vanvi and Tsopmo, 2016Vanvi A, Tsopmo A. 2016. Pepsin digested oat bran proteins: separation, antioxidant activity, and identification of new peptides. J. Chem. 2016, 8216378. http://dx.doi.org/10.1155/2016/8216378 ). After that, tandem mass spectrometry is applied to identify the antioxidant peptides. One study extracted antioxidant peptide from duck plasma hydrolysate (DPH) and then separated and purified the antioxidant peptide by ultrafiltration, dimensional exclusion chromatography and reversed-phase high performance liquid chromatography. The researchers also identified the DPH fraction with the highest antioxidant activity by nanoscale liquid chromatography-tandem mass spectrometry (Yang et al., 2020Yang J, Huang JC, Dong XL, Zhang YL, Zhou XH, Huang M, Zhou GH. 2020. Purification and identification of antioxidant peptides from duck plasma proteins. Food Chem. 319, 126534. https://doi.org/10.1016/j.foodchem.2020.126534 ). In fact, DPPH, ABTS free-radical scavenging and oxygen-radical absorbance capacity (ORAC) tests are generally used to evaluate the effect of antioxidant peptides (Kim et al., 2018Kim SS, Ahn CB, Moon SW, Je JY. 2018. Purification and antioxidant activities of peptides from sea squirt (Halocynthia roretzi) protein hydrolysates using pepsin hydrolysis. Food Biosci. 25, 128-133. https://doi.org/10.1016/j.fbio.2018.08.010 ). Other researchers have pre-treated the protein with ultrasound, microwave and irradiation to improve the antioxidant activity of the peptide (Wang et al., 2019Wang L, Li T, Sun DL, Tang MQ, Sun Z, Chen LF, Luo XH, Li YF, Wang R, Li YA, Li J, Chen ZX. 2019. Effect of electron beam irradiation on the functional properties and antioxidant activity of wheat germ protein hydrolysates. Innov. Food Sci. Emerg. Technol. 54, 192-199. https://doi.org/10.1016/j.ifset.2018.09.003 ; Zhang et al., 2019Zhang M, Huang TS, Mu TH. 2019. Improvement of thermal, microwave and ultrasonication pretreatment on the production of antioxidant peptides from sweet potato protein via in vitro gastrointestinal digestion. Int. J. Food Sci. Technol. 54 (7), 2338-2345. https://doi.org/10.1111/ijfs.14095 ). However, the single use of enzymatic hydrolysis is time consuming when selecting hydrolytic enzymes. The process of identifying antioxidant peptides is complicated and the target antioxidant peptides may not be obtained (Wen et al., 2020Wen CT, Zhang JX, Zhang HH, Duan YQ, Ma HL. 2020. Plant protein-derived antioxidant peptides: isolation, identification, mechanism of action and application in food systems: a review. Trends Food Sci. Technol. 105, 308-322. https://doi.org/10.1016/j.tifs.2020.09.019 ). Therefore, bioinformatics has attracted people’s attention.

2.2. Bioinformatics guides the preparation of antioxidant peptides by enzymatic hydrolysis

Bioinformatics based on computer technology can guide the selection of precursor proteins and simulate enzymatic hydrolysis (Zhou et al., 2019Zhou TY, Gao XC, Dang YL, Pan DD, Cao JX. 2019. Research progress of bioactive peptides based on bioinformatics. Sci. Technol. Food Ind. 40 (12), 335-340. http://dx.doi.org/10.13386/j.issn1002-0306.2019.12.054 ). Moreover, the function of the enzymatic hydrolysis products can be predicted and identified based on the sequence, structure and other parameters of proteins and peptides by bioinformatics. It is widely used in the research of hydrolyzing protein to obtain antioxidant peptides (Tejano et al., 2019Tejano LA, Peralta JP, Yap EES, Panjaitan FCA, Chang YW. 2019. Prediction of bioactive peptides from Chlorella sorokiniana proteins using proteomic techniques in combination with bioinformatics analyses. Int. J. Mol. Sci. 20 (7), 1786. http://dx.doi.org/10.3390/ijms20071786 ). This method is often used with databases such as Peptide Ranker, BIOPEP-UWM and UniProtKB (Chen et al., 2017Chen C, Huang H, Wu CH. 2017. Protein bioinformatics databases and resources. Methods Mol. Biol. 1558, 3-39. https://doi.org/10.1007/978-1-4939-6783-4_1 ; Pearman et al., 2020Pearman NA, Ronander E, Smith AM, Morris GA. 2020. The identification and characterisation of novel bioactive peptides derived from porcine liver. Curr. Res. Food Sci. 3, 314-321. https://doi.org/10.1016/j.crfs.2020.11.002 ). Some commonly used bioinformatic databases are listed in Table 2. These databases contain the amino acid sequences of various proteins and peptides to improve the efficiency of obtaining target peptides at a low cost (Tadesse and Emire, 2020Tadesse SA, Emire SA. 2020. Production and processing of antioxidant bioactive peptides: a driving force for the functional food market. Heliyon 6 (8), e04765. https://doi.org/10.1016/j.heliyon.2020.e04765 ). Darewicz et al. (2016)Darewicz M, Borawska J, Pliszka M. 2016. Carp proteins as a source of bioactive peptides-an in silico approach. Czech J. Food Sci. 34 (2), 111-117. http://dx.doi.org/10.17221/300/2015-cjfs retrieved the amino acid sequence, molecular weight and chain length of Cyprinus carpio protein from UniProtKB. After using the ClustalW2‒Multiple Sequence Alignment program to eliminate the same sequence, they selected 33 carp protein amino acid sequences with less than 90% identity for further analysis. The number of amino acid residues in the analyzed carp protein amino acid sequence ranged from 62 (light myosin, Q90335) to 1938 (myosin heavy chain, Q2HX56).

These bioinformatic methods can also simulate protease hydrolysis. In 2018, researchers used Peptide Cutter to simulate 100 tripeptides from the myosin of Mizuhopecten yessoensis by pepsin and trypsin (Yu et al., 2018Yu ZP, Wu SJ, Zhao WZ, Ding L, Shiuan D, Chen F, Li JR, Liu JB. 2018. Identification and the molecular mechanism of a novel myosin-derived ACE inhibitory peptide. Food Funct. 9 (1), 364-370. https://doi.org/10.1039/c7fo01558e ). For example, the BIOPEP-UWM database can not only simulate hydrolysis, but also predict the bioactive peptides released from protein sequences and their bioactivity and physicochemical properties (Yang et al., 2017Yang RW, Li XF, Lin SY, Zhang ZM, Chen F. 2017. Identification of novel peptides from 3 to 10 kDa pine nut (Pinus koraiensis) meal protein, with an exploration of the relationship between their antioxidant activities and secondary structure. Food Chem. 219, 311-320. http://dx.doi.org/10.1016/j.foodchem.2016.09.163 ). Researchers estimated the solubility of antioxidant peptides from flaxseed proteins in water by using Innovagen Peptide Solubility Calculator Proteomic. They also predicted the release of antioxidant peptides by opening the ‘search for active fragments tab of BIOPEP. Thereafter, they estimated several properties and indices of the silico-derived antioxidant peptides by using the application of the ‘Peptides’ package in R or ProtParam of ExPASy. These properties included amino acid composition, amino acid length, molecular weight (MW), isoelectric point (pI), Boman index, net charge and hydrophobicity index (Ji et al., 2019Ji DW, Udenigwe CC, Agyei D. 2019. Antioxidant peptides encrypted in flaxseed proteome: an in silico assessment. Food Sci. Hum. Well. 8 (3), 306-314. https://doi.org/10.1016/j.fshw.2019.08.002 ). In another research, Salim and Gan (2020)Salim MASM, Gan CY. 2020. Dual-function peptides derived from egg white ovalbumin: bioinformatics identification with validation using in vitro assay. J. Funct. Foods 64, 103618. https://doi.org/10.1016/j.jff.2019.103618 used Peptide Cutter to simulate the digestion of egg white ovalbumin, then Peptide Ranker was used to screen the obtained peptide sequences. After that, they predicted the peptide binding sites by Pepsite2 and finally used an in vitro assay to verify the bioactivity of the peptide. The AnOxPePred, a method for predicting the free-radical scavenging ability and chelating properties of antioxidant peptides was developed. When constructing the predictive variable, a standard database consisting of peptides and their free-radical scavenging capabilities and chelating properties was created (Olsen et al., 2020Olsen TH, Yesiltas B, Marin FI, Pertseva M, Garcia-Moreno PJ, Gregersen S, Overgaard MT, Jacobsen C, Lund O, Hansen EB, Marcatili P. 2020. AnOxPePred: using deep learning for the prediction of antioxidative properties of peptides. Sci. Rep. 10, 21471 . https://doi.org/10.1038/s41598-020-78319-w ).

Although bioinformatics is an effective method to study and predict the antioxidant peptides released by proteins and their potential activities, there are some limitations to this approach. For example, there is a lack of research on the protein sequence in simulated hydrolysis and its influence on enzymatic hydrolysis (Zhou et al., 2019Zhou TY, Gao XC, Dang YL, Pan DD, Cao JX. 2019. Research progress of bioactive peptides based on bioinformatics. Sci. Technol. Food Ind. 40 (12), 335-340. http://dx.doi.org/10.13386/j.issn1002-0306.2019.12.054 ). Bioinformatics simulates the hydrolysis of the protein sequence completely, but it is often necessary to consider the secondary and tertiary structures of protein and other factors such as inhibitors, pH and temperature when verifying in vitro (Bechaux et al., 2020Bechaux J, Ferraro V, Sayd T, Chambon C, Page JFL, Drillet Y, Gatellier P, Sante-Lhoutellier V. 2020. Workflow towards the generation of bioactive hydrolysates from porcine products by combining in silico and in vitro approaches. Food Res. Int. 132, 109-123. https://doi.org/10.1016/j.foodres.2020.109123 ). Some scientists showed that in the peptides obtained by computer simulation of a porcine actin sequence, only a few could be verified through laboratory experiments (Keska and Stadnik, 2016Keska P, Stadnik J. 2016. Porcine myofibrillar proteins as potential precursors of bioactive peptides-an in silico study. Food Funct. 7 (6), 2878-2885. https://doi.org/10.1039/C5FO01631b ). This proved that bioinformatics was an auxiliary method for selecting protein families and optimal hydrolyses. Bioinformatics was still theoretical, so it needed to be verified by in vitro experiments.

In order to efficiently and economically prepare antioxidant peptides, more and more researchers combine enzymatic hydrolysis and bioinformatics. The amino acid sequence of the target protein can be searched in the database to facilitate the selection of hydrolases, and it is estimated that there are more than 8 million amino acid sequences of proteins in the database (Agyei et al., 2018Agyei D, Tsopmo A, Udenigwe CC. 2018. Bioinformatics and peptidomics approaches to the discovery and analysis of food-derived bioactive peptides. Anal. Bioanal. Chem. 410 (15), 3463-3472. https://doi.org/10.1007/s00216-018-0974-1 ). Some researchers used the amino acid sequence of the 11S coffee globulin (the entry Q9ZNY2_COFAR and accession number NCBI 13443) deposited in the UniProtKB database. Subsequently, they selected enzymes (pepsin (EC 3.4.23.1), trypsin (EC 3.4.23.4) and chymotrypsin (EC 3.4.21.1)) to simulate gastrointestinal digestion. They carried out the prediction of peptides generated through simulation by using the BIOPEP-UWM “enzyme action”. Based on the results for the prediction of bioactivity of the 11S globulin, a series of in vitro studies was carried out, including protein extraction, enzymatic hydrolysis, protein isolation and determination of its antioxidant capacity. Finally, they proved that the hydrolyzed peptides of roasted coffee beans have higher antioxidant activity (Ribeiro et al., 2021Ribeiro E, Rocha TD, Prudencio SH. 2021. Potential of green and roasted coffee beans and spent coffee grounds to provide bioactive peptides. Food Chem. 348, 129061. https://doi.org/10.1016/j.foodchem.2021.129061 ). Computer simulation of hydrolysis can also greatly improve the screening efficiency of hydrolytic enzymes. Jie et al. (2020)Jie Y, Hu JL, Zhao HF, Zhang BL. 2020. Optimization of the preparation of antioxidant peptides from Caragana korshinskii seed protein based on bioinformatics. Food Sci. 41 (20), 278-284. http://dx.doi.org/10.7506/spkx1002-6630-20190822-224 used gluten as the bioinformatics template of Caragana korshinskii seed protein, then carried out virtual enzymatic hydrolysis with the assistance of the BIOPEP-UWM database. They determined that papain was the main protease to hydrolyze the Caragana korshinskii seed protein, which improved preparation efficiency.

After simulated hydrolysis, the computer generates an amino acid sequence. By comparing the sequence with the antioxidant peptide sequences reported in databases such as Swiss-Prot, Peptide Ranker or in the literature, the activity of the antioxidant peptide is predicted, evaluated and the target peptide is selected (Tu et al., 2018Tu ML, Cheng SZ, Lu WH, Du M. 2018. Advancement and prospects of bioinformatics analysis for studying bioactive peptides from food-derived protein: sequence, structure, and functions. TrAC Trends Anal. Chem. 105, 7-17. http://dx.doi.org/10.1016/j.trac.2018.04.005 ). For example, Sun et al. (2021)Sun XH, Wang K, Gao S, Hong H, Zhang LT, Liu HG, Feng LG, Luo YK. 2021. Purification and characterization of antioxidant peptides from yak (Bos grunniens) bone hydrolysates and evaluation of cellular antioxidant activity. J. Food Sci. Technol. 58, 3106-3119 https://doi.org/10.1007/s13197-020-04814-7 first prepared defatted collagen hydrolysates from yak bone, and then separated and purified the hydrolysate by ultrafiltration and reversed-phase high-performance liquid chromatography. After that, they determined the antioxidant activities of different hydrolysates and screened the best components for identification by mass spectrometry. Mass spectrometry was used to identify components with better antioxidant activity- Swiss-Prot was used to determine the amino acid sequence of the target peptide. Finally, they selected peptides with molecular weight of less than 1 kDa for synthesis according to the possible structural features of antioxidant peptides and the number of enzyme-cutting sites contained in the peptides. Other researchers isolated and purified antioxidant peptides from Asparagus by-products by reverse-phase chromatography, and then identified the peptides by tandem mass spectrometry. Peptide identification was carried out by using the Swiss-Prot database. PeptideRanker was used to predict the probability of the antioxidant activities of these peptides. This study only considered peptides with probability scores higher than 0.95. Therefore, only five peptides were retained and five novel peptide sequences were synthesized, and their antioxidant activity was finally evaluated in vitro (Montone et al., 2019Montone CM, Chiozzi RZ, Marchetti N, Cerrato A, Antonelli M, Capriotti AL, Cavaliere C, Piovesana S, Lagana A. 2019. Peptidomic approach for the identification of peptides with potential antioxidant and anti-hyperthensive effects derived from Asparagus by-products. Molecules 24 (19), 3627. https://doi.org/10.3390/molecules24193627 ). It is also necessary to predict the toxicity of antioxidant peptides. Harvian et al. (2019)Harvian ZA, Ningrum A, Anggrahini S, Setyaningsih W. 2019. In silico approach in evaluation of Jack bean (Canavalia ensiformis) canavalin protein as precursors of bioactive peptides with dual antioxidant and angiotensin I-converting enzyme inhibitor. Mater. Sci. Forum 948, 85-94. https://doi.org/10.4028/www.scientific.net/MSF.948.85 used ToxinPred to analyze the antioxidant peptide obtained from jack bean (Canavalia ensiformis) canavalin protein. ToxinPred is a in silico database for the prediction of the toxicity of selected peptides. The support vector machine (SVM)-based prediction method and SVM threshold value (0.0) were used to separate toxic from non-toxic peptides. Their results showed that the antioxidant peptide was non-toxic. Of course, in vivo or in vitro tests are required to further determine the physiological activity of antioxidant peptides eventually.

In summary, the bioinformatics method can simulate protein hydrolysis, determine the most suitable hydrolase and screen out peptides with good antioxidant effects by comparing the amino acid sequences of peptides gained through experiments with those in the database for in vitro verification. The combination of the two methods will simplify the cumbersome experimental process of traditional enzymatic hydrolysis, save cost, improve preparation efficiency and provide a promising method to improve not only the availability but also the high-value utilization of antioxidant peptides (Ji et al., 2019Ji DW, Udenigwe CC, Agyei D. 2019. Antioxidant peptides encrypted in flaxseed proteome: an in silico assessment. Food Sci. Hum. Well. 8 (3), 306-314. https://doi.org/10.1016/j.fshw.2019.08.002 ).

3.1. Scavenging free radicals

Free radicals exhibit strong reactivity, so they are initiators in oxidation reactions (Tomycz et al., 2011Tomycz ND, Richardson RM, Friedlander RM. 2011. Seizures and free radicals: the antioxidant may become the next anticonvulsant. Neurosurgery 69 (6), N12-N13. https://doi.org/10.1227/01.neu.0000407917.06492.a8 ). At present, it is generally recognized that an important cause of oil automatic oxidation is the chain reaction initiated by free radicals, such as hydroxyl radicals, peroxy free radicals and alkoxy free radicals (Sonklin et al., 2018Sonklin C, Laohakunjit N, Kerdchoechuen O. 2018. Assessment of antioxidant properties of membrane ultrafiltration peptides from mungbean meal protein hydrolysates. PeerJ. 6, e5337. https://doi.org/10.7717/peerj.5337 ). Hydroxyl radicals can remove the hydrogen atoms in the adjacent fat chain and induce oxidation chain reactions. High-energy peroxy free radicals and alkoxy free radicals can combine hydrogen atoms from adjacent fatty chains to destroy fatty acids and deteriorate oil. Therefore, it is necessary to combine free radical scavengers with free radicals to slow down the rate of chain reactions. Many antioxidant peptides are free radical scavengers. As shown in Scheme 2, together with amino acids, they act as hydrogen donors or electron donors to terminate the chain reactions of free radicals by converting them into stable products (Yarnpakdee et al., 2015Yarnpakdee S, Benjakul S, Kristinsson HG, Kishimura H. 2015. Antioxidant and sensory properties of protein hydrolysate derived from Nile tilapia (Oreochromis niloticus) by one-and two-step hydrolysis. J. Food Sci. Technol. 52 (6), 3336-3349. http://dx.doi.org/10.1007/s13197-014-1394-7 ).

The antioxidant capacity of peptides and their amino acid composition are closely related, and the mechanism of amino acids depends on the functional groups of their side chains (Elias et al., 2008Elias RJ, Kellerby SS, Decker EA. 2008. Antioxidant activity of proteins and peptides. Crit. Rev. Food Sci. Nutr. 48 (5), 430-441. http://dx.doi.org/10.1080/10408390701425615 ). For example, peptide sequences with hydrophobic amino acids, aromatic amino acids and sulfur-containing amino acids usually exhibit strong antioxidant activity (Wu et al., 2015aWu RB, Wu CL, Liu D, Yang XH, Huang JF, Zhang J, Liao BQ, He HL, Li H. 2015a. Overview of antioxidant peptides derived from marine resources: the sources, characteristic, purification, and evaluation methods. Appl. Biochem. Biotechnol. 176 (7), 1815-833. http://dx.doi.org/10.1007/s12010-015-1689-9 ). Antioxidant peptides containing hydrophobic amino acids such as alanine, valine and leucine have strong antioxidant capacity. The non-polar aliphatic hydrocarbon side chains of these amino acids can enhance the interaction between fatty acids and peptides to prevent hydrogen atoms from being attacked (as shown in Figure 1A) (Rajapakse et al., 2005Rajapakse N, Mendis E, Jung WK, Je JY, Kim SK. 2005. Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties. Food Res. Int. 38 (2), 175-182. https://doi.org/10.1016/j.foodres.2004.10.002 ). Their presence can also increase the solubility of antioxidant peptides in oil, thus promoting interactions with free radicals and exhibiting higher antioxidant activity (Aguilar-Toalá and Liceaga, 2020Aguilar-Toalá JE, Liceaga AM. 2020. Cellular antioxidant effect of bioactive peptides and molecular mechanisms underlying: beyond chemical properties. Int. J. Food. Sci. Tech. 56 (5), 2193-2204. http://dx.doi.org/10.1111/ijfs.14855 ). Some scientists proposed that hydrophobic amino acids such as valine and aspartic acid present in the peptide sequence could contribute to improving the antioxidant activity of peptides (Najafian and Babji, 2014Najafian L, Babji AS. 2014. Production of bioactive peptides using enzymatic hydrolysis and identification antioxidative peptides from patin (Pangasius sutchi) sarcoplasmic protein hydolysate. J. Funct. Foods 9, 280-289. https://doi.org/10.1016/j.jff.2014.05.003 ).

Antioxidant peptides with aromatic amino acids such as tyrosine, tryptophan and phenylalanine in the sequence also have strong antioxidant properties. Aromatic amino acids can not only enhance the solubility of antioxidant peptides in oil like hydrophobic amino acids, but can also scavenge free radicals produced by oxidation by removing a H· from the phenolic hydroxyl or indole group in these amino acids. These amino acids are converted into relatively stable free radicals to terminate the chain reaction and inhibit the automatic oxidation of oil (as shown in Figure 1B) (Wang et al., 2014Wang B, Gong YD, Li ZR, Yu D, Chi CF, Ma JY. 2014. Isolation and characterisation of five novel antioxidant peptides from ethanol-soluble proteins hydrolysate of spotless smoothhound (Mustelus griseus) muscle. J. Funct. Foods 6, 176-185. https://doi.org/10.1016/j.jff.2013.10.004 ). One study isolated two peptides containing tryptophan and tyrosine from the hydrolysate of Sardinella aurita, both of which have high DPPH scavenging activity (Bougatef et al., 2010Bougatef A, Nedjar-Arroume N, Manni L, Ravallec R, Barkia A, Guillochon D, Nasri M. 2010. Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinella aurita) by-products proteins. Food Chem. 118 (3), 559-565. https://doi.org/10.1016/j.foodchem.2009.05.021 ). A related study also showed that the antioxidant activity of the dipeptides Tyr-Leu and Phe-Tyr derived from Perilla is related to the presence of tyrosine (Yang et al., 2018Yang JJ, Hu L, Cai TT, Chen QL, Ma Q, Yang J, Meng C, Hong J. 2018. Purification and identification of two novel antioxidant peptides from perilla (Perilla frutescens L. Britton) seed protein hydrolysates. PLoS One 13 (7), e0200021. https://doi.org/10.1371/journal.pone.0200021 ).

Methionine and cysteine are two kinds of sulfur-containing amino acids with active sulfhydryl in their structures. They can be used as precursors for the synthesis of glutathione. The methionine and cysteine in the antioxidant peptide sequence contribute to scavenging free radicals through the electron transfer pathway (Atmaca, 2004Atmaca G. 2004. Antioxidant effects of sulfur-containing amino acids. Yonsei Med. J. 45 (5), 776-788. https://doi.org/10.3349/ymj.2004.45.5.776 ). The sulfhydryl group loses electrons to form free radical cations. The free radical cations then undergo a proton transfer reaction to generate stable free radicals and hydrogen ions (Scheme 3).

Jiang et al. (2014)Jiang H, Tong T, Sun J, Xu Y, Zhao Z, Liao D. 2014. Purification and characterization of antioxidative peptides from round scad (Decapterus maruadsi) muscle protein hydrolysate. Food Chem. 154, 158-163. https://doi.org/10.1016/j.foodchem.2013.12.074 obtained His-Asp-His-Pro-Val-Cys and His-Glu-Lys-Val-Cys from Decapterus maruadsi. The DPPH scavenging activity of the former was similar to the glutathione (Jiang et al., 2014Jiang H, Tong T, Sun J, Xu Y, Zhao Z, Liao D. 2014. Purification and characterization of antioxidative peptides from round scad (Decapterus maruadsi) muscle protein hydrolysate. Food Chem. 154, 158-163. https://doi.org/10.1016/j.foodchem.2013.12.074 ). In addition, the CFCTKPC synthesized by Huang et al. (2012)Huang GJ, Deng JS, Chen HJ, Huang SS, Liao JC, Hou WC, Lin YH. 2012. Defensin protein from sweet potato (Ipomoea batatas [L.] Lam ‘Tainong 57’) storage roots exhibits antioxidant activities in vitro and ex vivo. Food Chem. 135 (3), 861-867. https://doi.org/10.1016/j.foodchem.2012.05.082 contained three cysteines, and showed strong free radical scavenging ability and inhibited linoleic acid oxidation.

Histidine is one of the most common amino acids in antioxidant peptides. Its imidazole group can act as hydrogen donors to react with free radicals, especially superoxide anion-free radicals (Jie et al., 2019bJie Y, Zhao HF, Zhang B. 2019b. The role of an acidic peptide in controlling the oxidation process of walnut oil. Foods 8 (10), 499. http://dx.doi.org/10.3390/foods8100499 ). Kim et al. (2020)Kim HK, Song HS. 2020. Antioxidant effect of histidine-containing low-molecular-weight peptides seperated from tuna meat. J. Food Hyg. Saf. 35 (5), 513-520. https://doi.org/10.13103/JFHS.2020.35.5.513 obtained histidine-containing low-molecular-weight (LMW) peptides from tuna waste meats. The histidine-containing LMW peptides exhibited a high DPPH radical scavenging effect in a dose-dependent manner.

3.2. Inhibiting the activity of catalytic metal ions

Catalytic metal can accelerate the decomposition of oil oxidation primary products to generate free radicals as shown in Scheme 4 (Muhr et al., 2020Muhr L, Pontvianne S, Selmeczi K, Paris C, Boschi-Muller S, Canabady-Rochelle L. 2020. Chromatographic separation simulation of metal-chelating peptides from surface plasmon resonance binding parameters. J. Sep. Sci. 43 (11), 2031-2041. http://dx.doi.org/10.1002/jssc.201900882 ).

The catalysis of catalytic metal depends on their ability to react with unoxidized oil to form hydroperoxides (Juita et al., 2011Juita, Dlugogorski BZ, Kennedy EM, Mackie JC. 2011. Oxidation reactions and spontaneous ignition of linseed oil. P. Combust. Inst. 33 (2), 2625-2632. https://doi.org/10.1016/j.proci.2010.06.096 ). For example, Fe²⁺ catalyzes hydrogen peroxide to generate hydroxyl radicals with high activity through the Fenton reaction, thus promoting oil oxidation (Walters et al., 2018Walters ME, Esfandi R, Tsopmo A. 2018. Potential of food hydrolyzed proteins and peptides to chelate iron or calcium and enhance their absorption. Foods 7 (10), 172. http://dx.doi.org/10.3390/foods7100172 ). Another theory was that the presence of transition metal would significantly reduce the content of tocopherols, thereby affecting the oxidation stability of the oil (Fomuso et al., 2002Fomuso LB, Corredig M, Akoh CC. 2002. Metal-catalyzed oxidation of a structured lipid model emulsion. J. Agric. Food Chem. 50 (24), 7114-7119. https://doi.org/10.1021/jf020256j ).

Antioxidant peptides can reduce the chemical reactivity of metals by chelating with metal ions to form stable complexes. Antioxidant peptides can also block the interaction between metals and oil in space. For example, changing the physical position of metal ions to reduce the rate of free radical reactions indirectly achieves the antioxidant effect (Feng et al., 2016Feng JH, Cao AL, Cai LY, Li XX, Yu ZP, Zhao W, Li JR. 2016. Research progress of antioxidant peptides in fish by-products and their active mechanism. Sci. Tech. Food Ind. 37 (15), 365-374. http://dx.doi.org/10.13386/j.issn1002-0306.2016.15.063 ). Regarding antioxidant peptides, suitable amino acids and their correct position in the sequence play a crucial role in their chelating activity (Walters et al., 2018Walters ME, Esfandi R, Tsopmo A. 2018. Potential of food hydrolyzed proteins and peptides to chelate iron or calcium and enhance their absorption. Foods 7 (10), 172. http://dx.doi.org/10.3390/foods7100172 ). The imidazole group of histidine in the antioxidant peptide sequence can chelate with metal ions, thus inhibiting the generation of free radicals catalyzed by them (Burkitt, 2001Burkitt MJ. 2001. A critical overview of the chemistry of copper-dependent low density lipoprotein oxidation: roles of lipid hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin. Arch. Biochem. Biophys. 394 (1), 117-135. https://doi.org/10.1006/abbi.2001.2509 ). Basic amino acids such as glutamic acid and aspartic acid (with carboxylic acid groups) can also inhibit the catalytic oxidation of transition metal ions (Sonklin et al., 2018Sonklin C, Laohakunjit N, Kerdchoechuen O. 2018. Assessment of antioxidant properties of membrane ultrafiltration peptides from mungbean meal protein hydrolysates. PeerJ. 6, e5337. https://doi.org/10.7717/peerj.5337 ). Besides, amino acids such as arginine and lysine with amino groups, cysteine with thiol groups and serine and threonine with hydroxyl may form complexes with antioxidant peptides by electrostatic interactions or H-bond coordination (Canabady-Rochelle et al., 2018Canabady-Rochelle LLS, Selmeczi K, Collin S, Pasc A, Muhr L, Boschi-Muller S. 2018. SPR screening of metal chelating peptides in a hydrolysate for their antioxidant properties. Food Chem. 239, 478-485. https://doi.org/10.1016/j.foodchem.2017.06.116 ; Walters et al., 2018Walters ME, Esfandi R, Tsopmo A. 2018. Potential of food hydrolyzed proteins and peptides to chelate iron or calcium and enhance their absorption. Foods 7 (10), 172. http://dx.doi.org/10.3390/foods7100172 ). It has also been reported that casein-hydrolyzed peptides have the ability to inhibit the oxidation of oil. The peptides can oxidize Fe²⁺ into less reactive Fe³⁺ to delay oil oxidation (Diaz et al., 2003Diaz M, Dunn CM, McClements DJ, Decker EA. 2003. Use of caseinophosphopeptides as natural antioxidants in oil-in-water emulsions. J. Agric. Food Chem. 51 (8), 2365-2370. https://doi.org/10.1021/jf025984l ). Some antioxidant peptides with metal chelating properties can act as potential antioxidants because of their charged amino acids in their sequences. The charged amino acids can be electrostatically attracted to metal ions to inhibit their catalytic effect. For example, the positive charge of histidine can attract negative free radicals, which is consistent with the results of Saiga et al. (2013)Saiga AE, Nishimura T. 2013. Antioxidative properties of peptides obtained from porcine myofibrillar proteins by a protease treatment in an Fe(II)-induced aqueous lipid peroxidation system. Biosci. Biotechnol. Biochem. 77 (11), 2201-2204. https://doi.org/10.1271/bbb.130369 . It is worth noting that antioxidant peptides with molecular weight less than 3 kDa have less chelating force on Fe²⁺ than the peptides with the formula weight of 5-10 kDa (He et al., 2013He R, Girgih AT, Malomo SA, Ju XR, Aluko RE. 2013. Antioxidant activities of enzymatic rapeseed protein hydrolysates and the membrane ultrafiltration fractions. J. Funct. Foods 5 (1), 219-227. https://doi.org/10.1016/j.jff.2012.10.008 ). Therefore, antioxidant peptides with large molecular weight have a better effect on inhibiting oil oxidation.

Moreover, it must be noted that even though a peptide chelates metal, that does not mean it inhibits metal-promoted oil oxidation. Some chelators bind iron but do not decrease its reactivity (Durand et al., 2021Durand E, Beaubier S, Fine F, Villeneuve P, Kapel R. 2021. High metal chelating properties from rapeseed meal proteins to counteract lipid oxidation in foods: controlled proteolysis and characterization. Eur. J. Lipid Sci. Technol. 123 (6), 2000380. https://doi.org/10.1002/ejlt.202000380 ). Therefore, additional assays should be conducted to better assess the ability of these peptides to inhibit oil oxidation induced by prooxidant metals.

3.3. Restraining the formation and reactivity of hydroperoxide

Hydroperoxide is the primary product of oil oxidation. It can react with the ingredients in food, and reduce its quality. With the development of oil oxidation, hydroperoxide eventually decomposes into small molecules such as aldehydes and ketones (Chen et al., 2020aChen C, Sun-Waterhouse DX, Zhang Y, Zhao MM, Sun WZ. 2020a. The chemistry behind the antioxidant actions of soy protein isolate hydrolysates in a liposomal system: their performance in aqueous solutions and liposomes. Food Chem. 323, 126789. https://doi.org/10.1016/j.foodchem.2020.126789 ). Hydroperoxides may even decompose into toxic substances such as malonaldehyde (MDA) and 4-hydroxynonenal (HNE). During the chain transfer period, the free radicals generated by the decomposition of the hydroperoxides can react with unoxidized oil, continuously creating new free radicals and hydroperoxides to accelerate the oxidation of oil (Krugovov et al., 2014Krugovov DA, Mengele EA, Kasaikina OT. 2014. Acetylcholine as a catalyst of hydroperoxide decomposition to free radicals. Russ.Chem.Bull. 63 (8), 1837-1842. http://dx.doi.org/10.1007/s11172-014-0673-9 ).

The acidic amino acids in the antioxidant peptide sequence form hydrogen bonds with unsaturated fatty oil to protect the double bonds of fatty acids and decrease the cleavage of C-H bonds, thereby reducing the generation of hydroperoxides (Jie et al., 2019bJie Y, Zhao HF, Zhang B. 2019b. The role of an acidic peptide in controlling the oxidation process of walnut oil. Foods 8 (10), 499. http://dx.doi.org/10.3390/foods8100499 ). A new antioxidant peptide was isolated from the Caragana ambiguas seed protein. The glutamine in the Caragana ambigua seed peptide (CSP) can form hydrogen bonds with unsaturated oil to protect the double bond of fatty acids, thus reducing the generation of hydroperoxides. In addition, CSP can delay the autoxidation of oil by protecting the phenolic compounds (Jie et al., 2019aJie F, Zhao HF, Sun XQ, Lv XR, Zhang Z, Zhang B. 2019a. Isolation of antioxidative peptide from the protein hydrolysate of Caragana ambigua seeds and its mechanism for retarding lipid auto-oxidation. J. Sci. Food Agric. 99 (6), 3078-3085. http://dx.doi.org/10.1002/jsfa.9521 ). Antioxidant peptides can also reduce the reactivity of hydroperoxides by proton or electron transfer, thus inhibiting the chain transfer period of oil oxidation (Li and Yu, 2015Li Y, Yu JM. 2015. Research progress in structure-activity relationship of bioactive peptides. J. Med. Food 18 (2), 147-156. https://doi.org/10.1089/jmf.2014.0028 ; Lacou et al., 2016Lacou L, Leonil J, Gagnaire V. 2016. Functional properties of peptides: from single peptide solutions to a mixture of peptides in food products. Food Hydrocolloids 57, 187-199. https://doi.org/10.1016/j.foodhyd.2016.01.028 ). Some researchers prepared an antioxidant peptide (APHPH) with the molecular weight of 1801 Da. The hydrophobic amino acids in its sequence can provide protons to inhibit the reaction between hydroperoxides and free radicals, and effectively inhibit oil oxidation in the linoleic acid emulsion system (Kim et al., 2007Kim SY, Je JY, Kim SK. 2007. Purification and characterization of antioxidant peptide from hoki (Johnius belengerii) frame protein by gastrointestinal digestion. J. Nutr. Biochem. 18 (1), 31-38. http://dx.doi.org/10.1016/j.jnutbio.2006.02.006 ). An antioxidant peptide was prepared by using Alcalase alkaline protease and ultrafiltration technology from silkworm sericin. Silk sericin peptide has an inhibitory effect on the hydroperoxides induced by 4NQO (an oral carcinogen) in liver tissue (Fan et al., 2016Fan JB, Zhou SZ, Cai QT, Hou Y. 2016. Protective effect of sericin peptide on liver tissue injury induced by 4NQO. J. Chin. Institute of Food Sci. Tech. 16 (2), 44-49. http://dx.doi.org/10.16429/j.1009-7848.2016.02.007 ). Other researchers believe that antioxidant peptides can also reduce hydroperoxides to relatively inactive hydroxides through non-radical reactions. The reaction mechanism is to generate sulfenic acid and sulfoxide derivatives through double electron transfer from the sulfur of cysteine or methionine (Esfandi et al., 2019Esfandi R, Walters ME, Tsopmo A. 2019. Antioxidant properties and potential mechanisms of hydrolyzed proteins and peptides from cereals. Heliyon 5 (4), e01538. http://dx.doi.org/10.1016/j.heliyon.2019.e01538 ).

In summary, antioxidant peptides can inhibit oil oxidation by scavenging free radicals, chelating metal ions that promote oxidation and reducing hydroperoxides generation. In addition to these common mechanisms, it has been proposed that antioxidant peptides may interact with secondary oxidation products of oil, such as aldehydes. These reactions may interfere with oil oxidation and alter the rate and pathway of oil rancidity (Berton-Carabin et al., 2014Berton-Carabin CC, Ropers MH, Genot C. 2014. Lipid oxidation in oil-in-water emulsions: involvement of the interfacial layer. Compr. Rev. Food Sci. Food Saf. 13 (5), 945-977. https://doi.org/10.1111/1541-4337.12097 ). However, since the secondary oxidation products of oil are bound to the antioxidant peptides and not available for determination (e.g., by GC headspace analysis), the mechanism remains to be further explored (McClements and Decker, 2018McClements DJ, Decker E. 2018. Interfacial antioxidants: a review of natural and synthetic emulsifiers and coemulsifiers that can inhibit lipid oxidation. J. Agric. Food Chem. 66 (1), 20-35. https://doi.org/10.1021/acs.jafc.7b05066 ).