Antioxidant Properties of Hemp Proteins: From Functional Food to Phytotherapy and Beyond
1Department of Food, Nutrition, Dietetics and Health, Kansas State University, Manhattan, KS 66506, USA
2John C. Pair Horticultural Center, Department of Horticulture & Natural Resources, Kansas State University, Haysville, KS 67060, USA
3Department of Grain Science and Industry, Kansas State University, Manhattan, KS 66506, USA
4Department of Biological and Agricultural Engineering, Kansas State University, Manhattan, KS 66506, USA
*Correspondence: dwang@ksu.edu (D.W.); wwang@ksu.edu (W.W.)Abstract
As one of the oldest plants cultivated by humans, hemp used to be banned in the United States but returned as a legal crop in 2018. Since then, the United States has become the leading hemp producer in the world. Currently, hemp attracts increasing attention from consumers and scientists as hemp products provide a wide spectrum of potential functions. Particularly, bioactive peptides derived from hemp proteins have been proven to be strong antioxidants, which is an extremely hot research topic in recent years. However, some controversial disputes and unknown issues are still underway to be explored and verified in the aspects of technique, methodology, characteristic, mechanism, application, caution, etc. Therefore, this review focusing on the antioxidant properties of hemp proteins is necessary to discuss the multiple critical issues, including in vitro structure-modifying techniques and antioxidant assays, structure-activity relationships of antioxidant peptides, pre-clinical studies on hemp proteins and pathogenesis-related molecular mechanisms, usage and potential hazard, and novel advanced techniques involving bioinformatics methodology (QSAR, PPI, GO, KEGG), proteomic analysis, and genomics analysis, etc. Taken together, the antioxidant potential of hemp proteins may provide both functional food benefits and phytotherapy efficacy to human health.
1. Introduction
Excessive oxidation is one of the primary causes of mitochondrial damage, DNA damage, cell pyroptosis, carcinogenesis, and chronic diseases [1,2,3,4]. Thus, the biological system needs antioxidant defenses to protect the body against oxidative attacks [3]. In general, antioxidants can be classified into three categories: (1) Hydrophilic and lipophilic antioxidants [5]; (2) Endogenous and exogenous antioxidants [6,7,8]; and (3) Synthetic and natural antioxidants. In recent decades, some antioxidants are being developed as therapeutic agents, nutrition supplements, and food additives to enhance human health [9,10,11,12,13,14]. However, the safety of antioxidants has been questioned. In a typical example, butylated hydroxytoluene at high doses could induce the non-apoptotic cell death of thymocytes in rat models [15,16]. Therefore, natural exogenous antioxidants such as dietary hemp protein have attracted more attention over the past few years.
Throughout history, hemp is one of the oldest plants cultivated by humans [17]. It was found around 800 B.C. Hemp used to be a legal crop before the nineteenth century in the USA. However, the prohibition of hemp cultivation was imposed on the hemp industry in 1970, because hemp contains two neurotoxic ingredients, cannabidiol and delta-9-tetrahydro-cannabinol, which were unable to be legally described [18]. Hemp returned to the USA. as a completely legal crop till the Agricultural Improvement of 2018 was signed by the USA president. As a result, it has stimulated an increasing number of researchers to conduct various novel studies on hemp [19,20,21].
While bioactive peptides derived from hemp proteins have been proven to be strong antioxidants, some controversial disputes and unknown issues are still underway to be explored and verified in the aspects of technique, methodology, characteristic, mechanism, application, caution, etc. Therefore, this review focusing on the antioxidant properties of hemp proteins aims to discuss the multiple critical issues, including in vitro structure-modifying techniques and antioxidant assays, structure-activity relationships of antioxidant peptides, pre-clinical studies on hemp proteins and pathogenesis-related molecular mechanisms, usage and potential hazard, and novel advanced techniques. The antioxidant potential of hemp proteins may provide both functional food benefits and phytotherapy efficacy to human health.
3. Assessments of the Antioxidant Ability
A series of mature techniques such as reactive oxygen species (ROS) scavenging ability assay, metal ion chelating capacity assay, and assessments of antioxidant enzymatic and non-enzymatic activities have been constantly developed and widely applied to evaluate the antioxidant activity of various natural proteins. Through these techniques, a large amount of research work has made considerable contributions to the identification of bioactive antioxidant peptides [32].
3.1. Reactive Oxygen Species Scavenging Ability
ROS including superoxide, hydroxyl, and peroxy radicals are characterized as a cluster of oxidants or free radicals [3]. Free radical scavenging ability is one of the essential functions of antioxidants, which is associated with hydrogen atom transfer (HAT) and electron transfer (ET) [3,32].
With the regard to the antioxidant ability assays caused by HAT-based reactions, the Oxygen Radical Absorbance Capacity (ORAC) method is a common method to assess antioxidant activities [3,33,34]. The mechanism of the ORAC assay is as follows. One 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH) molecule loses the dinitrogen (R-N=N-R) to produce two radicals (R·). Under an O2-saturated condition, the radical abstracts an O2 to generate a peroxyl radical (ROO·). ROO· reacts with a hydrogen atom (XOH) to form hydroperoxide (ROOH) and an antioxidant radical (XO·) (R-N=N-R→2R· + N2; R·+ O2 →ROO·; ROO· + XOH→ROOH + XO·) [35,36,37,38].
Similarly, other standard antioxidant ability assays are usually employed to analyze the antioxidant ability triggered by ET-based reactions, such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, Ferric Reduction Antioxidant Power (FRAP) assay, and Trolox Equivalent Antioxidant Capacity (TEAC) [3,33,34].
In a study of hemp antioxidant ability [39], Lu et al. (2010) reported that hemp protein hydrolyzed by alcalase for 1 h, had good DPPH radical scavenging ability (3.75 ± 0.09 mg/mL), superoxide radical scavenging ability (3.5 ± 0.1 mg/mL), hydroxyl radical scavenging ability (7.25 ± 0.3 mg/mL), and FRAP ability (0.21 ± 0.03 mg/mL) [39]. Likewise, the results of Frassinetti et al. displayed that hemp seed extracts had high ORAC activity (127 ± 5 µmol TE/g), and DPPH radical scavenging activity (40 ± 2%) [40]. The likely explanations for these results were that hemp antioxidant peptides contained the donors of electrons and hydrogens, which could interrupt the radical-chain reactions, so they could convert free radicals to stable species [39,40].
The free radical scavenging ability assays described above had already been extended in the hemp application. For instance, a novel gluten-free cracker fortified with hemp seed oil pressed-cake and decaffeinated green tea leaves was developed. The antioxidant property of crackers was tested by DPPH (30.3 μmol TE/g d.w.). The overall score of 8.9 indicated it had good antioxidant properties. Moreover, the researchers further interpreted that the crackers still had antioxidant properties even without green tea. They implied that hemp seed as a nutritional food additive had promising applications in snack marking [41].
3.3. Assessments of Antioxidant Enzymatic and Non-Enzymatic Activities
The human body has its own endogenous antioxidants to defend against the damage of ROS. These antioxidant-related mechanisms possess two arms, enzymatic antioxidants and non-enzymatic antioxidants.
Enzymatic antioxidants including glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD) can prevent ROS damage by converting superoxide radicals to stable compounds. Due to this mechanism, they are favorable biological indicators of antioxidant activity. Take SOD as a good example. The superoxide is formed from an oxygen atom and an electron (O2 + electron→O2·). It is a long-lived molecule that can diffuse readily. The transition from superoxide to hydrogenous oxide is catalyzed by SOD. Therefore, SOD activity can reflect the state of superoxide under the actions of antioxidants [46,47]. In a cell culture experiment, Hong et al. (2015) illustrated that hemp seed extracted by ethanol and supercritical fluid could enhance the gene expression of antioxidant enzymes (SOD, CAT, and GPx) in HepG2 cells [48]. Another in vivo experiment conducted by Girgih, Abraham T. et al. demonstrated that the plasma SOD and CAT activities in spontaneously hypertensive rats (SHRs) consuming the hemp diet were significantly higher than those in the control group [46,49].
Non-enzymatic antioxidants including glutathione (GSH), bilirubin, α-tocopherol, and ascorbic acid can also provide defense against ROS, clear up free radicals, and maintain the balance of redox [46,47]. A relatively recent study by Kubiliene et al. (2021) indicated that hemp extract could significantly reduce the level of GSH (26.81%) in the brain and liver of mice [50]. Similarly, a decreasing concentration of GSH was detected in the splenocyte cells treated with hemp derivatives [51]. These results supported that hemp could work well as an antioxidant.
So far, we also completed a pilot experiment on the antioxidant ability of hemp protein. In our pilot experiment, the hemp protein and soybean (control) were hydrolyzed by neutrase and the hydrolyzates were further assessed by the ORAC method modified from Ou et al. [52] and the metal iron chelating activity assay modified from Elias et al. [53]. The results in Table 1 showed that the antioxidant activities of hemp protein hydrolysate were significantly higher than that of soybean protein hydrolysate (Table 1). This pilot experiment paves the way for future in vitro and in vivo studies involving cell and small animal models to explore more biological significance of hemp protein.
4. Structure-Activity Relationship of Antioxidant Peptides
Considering that the antioxidant activities of bioactive peptides are mainly associated with the molecular weight (MW) of peptides, the compositions of amino acids, and the sequences of amino acid groups [54,55,56], the structure-activity relationship of antioxidant peptides can be studied by the two techniques below.
The first technique includes three traditional methods, membrane ultrafiltration (MU), mass spectrometry (MS), and reverse-phase high-performance liquid chromatography (RP-HPLC). They are extensively used to detect the relationship between the peptide structure and antioxidant activity through the isolation and identification of peptides. Membrane ultrafiltration separates the small MW of peptides from the large MW of peptides by membranes with specific pore sizes [54]. MS analyzes the compositions of amino acids using the optics principles [57,58]. RP-HPLC isolates bioactive peptides based on the hydrophobic characteristics of peptide [30,59]. In practice, the hemp hydrolysates can be filtered by MU first. Subsequently, the low MW of hemp hydrolysate can be tested through in vitro and in vivo assays to detect its antioxidant abilities. Finally, the compositions and structures of antioxidant peptides are verified by RP-HPLC and MS. This experimental flow chart of antioxidant peptides is shown in Figure 1 [32].
The second technique is a novel bioinformatics method, quantitative structure-activity relationship (QSAR) statistical model, which can predict the biological efficacy of peptides. Although there are a lot of existing studies on the QSAR of anti-hypertensive peptides, the number of studies on the QSAR of antioxidant peptides is far less than that of anti-hypertensive peptides. This may be because antioxidant peptides have many dependent, independent, and control variables, making the statistical analysis of antioxidant peptides extremely complicated [60,61,62,63,64,65].
4.1. Molecular Weight
Generally, the short-chain peptides derived from food protein have several physiological benefits such as promoting the gastrointestinal digestion process, being more absorbed into the blood circulation to reach target organs rapidly and preventing oxidative damage [66,67,68,69]. Hence, Girgih et al. (2011) suspected that peptides with appropriately low MW had strong antioxidant abilities. In their study, the hemp seed protein was hydrolyzed by alcalase and separated by MU to obtain the low MW of peptide fractions, e.g., >5, 3–5, 1–3, and <1 kDa MW of peptides. The <1 kDa fractions demonstrated the highest DPPH radical scavenging ability (24.2%) [44].
4.2. Composition of Amino Acids
Notably, the composition of amino acids is a determining factor in the antioxidant activities of peptides. Amino acids can be divided into hydrophobic amino acids (Ala, Leu, Met, Ile, Pro, Phe, Trp, Val) and hydrophilic amino acids. The hydrophilic amino acids can be subclassified into neutral amino acids (Gly, Thr, Tyr Ser, Cys, Gln, Asn), acidic amino acids (Asp, Glu), and alkaline amino acids (Lys, Arg, His) [70,71,72].
The hydrophobic amino acids serve as great hydroxyl radical scavengers owing to their indole structure and the benzene ring, which can donate hydrogen in lipid peroxidation reactions (RH + OH· →R· + H2O; R· + O2→ROO·; ROO· + RH→ROOH + R·) [3,27,54]. They deter lipid peroxidation reactions by converting hydrogen from the indole structure and benzene ring to free radicals. That may be the reason why the hydrophobic amino acids have a strong antioxidant ability [70,71,72].
For example, the peptides from tuna muscle exhibited strong antioxidant ability due to their high proportion of hydrophobic amino acids [73]. Likewise, Chi et al. (2015) analyzed bluefin protein and verified that peptides (Trp-Glu-Gly-Pro-Lys, Gly-Pro-Pro, Gly-Val-Pro-Leu-Thr) containing high hydrophobic amino acid content could work well as natural antioxidants [74].
On the contrary, other scholars do not agree with above-mentioned perspective. They think that hydrophilic amino acids, especially neutral amino and alkaline amino acids may have good performance in antioxidant activities. For instance, Chen et al. (1996) discovered that the bioactive peptide chains containing His were credited with good metal ion chelating ability due to the presence of α-amido, carboxyl, and imidazole structures [66]. Moreover, Tian et al. (2005) predicted Cys was one of the most active antioxidant amino acids by a QSAR model. It might be associated with the number of strong disulfide bonds in a precursor molecule [75].
Additionally, the amino acid contents of hemp protein can be modified by physical and chemical techniques. Table 2 exhibits that the amino acid contents of defatted hemp seed, hemp protein hydrolysate, hemp protein isolations, and hemp protein fractions are different, which verifies this viewpoint. Although the underlying mechanism is less clear, the results of hemp protein studies listed in Table 2 can provide scholars with useful information for reference. For example, the Arg content of hemp protein was the highest by enzymatic hydrolysis treatment (pepsin and pancreatin) and the lowest by pH-shift treatment (acid extraction). If the Arg content of hemp is studied, acid extraction might be avoided.
4.3. Sequence of Amino Acids
Importantly, some scientists found that the amino acids of the same composition did not have similar bioactive effects. They deemed that the sequence of the amino acids played a significant role in antioxidant ability [55,56]. Despite the controversy, the following studies are still good examples to interpret how the sequence of amino acids affects the antioxidant activities of specific peptides. Chen et al. (1996) discovered that the metal ion chelating capacity of His-Leu was higher than that of Leu-His, the same as His-His-Pro > Pro-His-His, His-His-Leu-Pro > Pro-Leu-His-His, and His-His-Pro-Leu-Leu > Leu-Leu-Pro-His-His. Apparently, His at the N-terminus of antioxidant peptides had better metal ion chelating capacity [66]. In contrast, other scholars consider that the peptides containing hydrophobic amino acids at the N-terminal and hydrophilic amino acids at the C-terminal have high free radical scavenging ability. Bougatef and his coworkers studied seven peptides from ardinella, Pro-His-Tyr-Leu, Gly-Ala-His, Gly-Ala-Leu-Ala-Ala-His, Leu-His-Tyr, Gly-Gly-Glu, Leu-Ala-Arg-Leu, and Gly-Ala-Trp-Ala. They stated that the Leu-His-Tyr had the highest antioxidant ability (DPPH 63 ± 1.57%) [78].
Specially, Xu et al. (2004) studied the antioxidant peptide, Leu-Asp-Tyr-Glu from corn. In their opinion, the reason why this peptide had a strong antioxidant ability was that Tyr was adjacent to Asp and Glu, so the hydrogen donating capacity of Tyr could be enhanced. The carboxylate of Asp and Glu could attract electrons, and then the density of the electron cloud in phenolic hydroxyl of Tyr was decreased. These reactions made it easier for hydrogen of Tyr to be released [79]. Similar results had been observed by Bamdad et al. (2005) that among the six peptides from barley protein, Pro-Tyr-Pro, Gln-Pro-Tyr-Pro-Gln, Gln-Gln-Pro-Tyr-Pro-Gln, Gln-Pro-Gln-Pro-Tyr-Pro-Gln, Thr-Gln-Gln-Pro-Tyr-Pro-Gln, and Glu-Pro-Tyr-Pro-Glu, Gln-Pro-Tyr-Pro-Gln had the strongest antioxidant ability since it contained symmetrically repeated sequences. The Gln and Pro near Tyr could help release the hydrogen in the phenolic hydroxyl of Tyr, boosting the antioxidant ability of peptides [80].
Surprisingly, in alignment with these results, a recent QSAR study also observed that Tyr, Trp, or Cys located at the C-terminal position played a more dominant role in antioxidant activity than other C-terminal residues. Based on the predicted antioxidant values of 7870 unknown tripeptides, Du et al. compared 14 popular machine learning methods for the QSAR models and then screened tripeptides to identify critical amino acid features that determine the antioxidant activity. Later, they found that potentially high antioxidant activity tripeptides all have a Tyr, Trp, or Cys residue at the C-terminal position [65].
Supplementarily, Table 3 shows the amino acid sequence of nutritious food with antioxidant ability, which can also support the above viewpoints. However, the structure-activity relationship of antioxidant peptides has not been completely understood. More studies on the advanced structure-modifying techniques such as glycosylation and high-intensity ultrasound, the structure identification of antioxidant peptides, as well as the correlation between antioxidant protein and other antioxidant phytochemicals that can fortify protein function such as polyphenols are required to expand the utilization of the hemp protein as a functional food [81,82,83,84,85].
6. Hemp Seed Protein Product and Usage
Compared with other plant foods, hemp seed is a superior nutraceutical resource, since hemp seed protein has a balanced amino acid profile and contains a great amount of essential amino acids such as His, Arg, Met and Leu. Hemp seed can provide sufficient amino acids to meet the daily requirements of children according to the recommendations of the Food and Agriculture Organization (FAO), World Health Organization (WHO), and United Nations University (UNU) (Table 4) [125,126,127]. Therefore, hemp protein products can be recommended for people with low-protein intakes, vegans and vegetarians, lactose intolerant people, patients with osteoporosis, athletes, dieters, children, etc. In recent years, hemp protein has been directly added to daily diets through advanced food science technologies, such as bakery products, milk, and meats for health benefits [128,129,130,131]. For instance, hemp flour could be blended into gluten-free bread. It limited the recrystallization of amylopectin and the hardening of crumb (the main symptom of bread staling) during bread storage [132]. Meanwhile, gluten-free bread fortified with hemp provided a higher intake of macroelements and microelements, especially proteins and iron. 10% of hemp derivatives could keep the balance between nutraceutical value and bread quality [130]. Additionally, hemp derivatives were applied to meat products. The quality of pork loaves added with hemp ingredients was evaluated by Zajac et al. (2019). The contents of fiber, magnesium, manganese, iron, copper, and polyunsaturated fatty acids in meat products were elevated, but the microbial growth was not affected. It can be generalized that hemp-treated meat products are healthy foods [131]. Specifically, hemp as a vegetable protein might be a good way to improve nutritional level of yogurt. In reviewing a recent literature, the authors studied that the fortification of plant protein-mixed yogurt. The effects of 5 vegetable proteins (hemp, pea, soy, wheat, and pumpkin) as additives were determined by rheological, physicochemical, and sensory properties of yogurt before pasteurization [129]. Hemp protein had the lowest syneresis value and the high viscosity. Furthermore, the yogurt fortified with hemp proteins also displayed an increase in acidity. These results revealed that the addition of hemp had good performance in dairy products [129,133,134].
7. Potential Hazard
However, hemp is a mild allergen for special populations because it can directly trigger side effects such as wheal, pruritus, and severe symptoms caused by type I hypersensitivity [141,142]. A case report showed that a 30-year-old man who worked in hemp harvesting suffered from contact urticaria. It demonstrated that hemp might be a resource of occupational allergen [141,143]. Additionally, hemp is an indirect allergen. When consumed with other specific plant allergens, hemp would magnify the adverse reactions, which are called “cannabis-fruit” or “cannabis-vegetable” syndromes [142,144]. To improve beneficial functions and alleviate side effects, the experiments on hemp seed proteome have emerged in recent years. Scientists identified the proteomic profile of hemp seed by gel electrophoresis, MS. Followed by bioinformatics methods such as protein–protein interactomics (PPI) network analysis, Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways analysis, the correlation between the proteomic characteristics of hemp seed and its related physiological functions involving energy regulation, metabolism, stress response, cytoskeleton, and binding, protein synthesis was studied (Figure 5) [145,146]. Fortunately, two major hemp allergens, thaumatin-like protein and lipid transfer protein had been found and they could be purged by the food processing techniques [147]. The more negative properties of hemp seed protein can be discovered and eliminated, the more beneficial hemp seed protein will be.
Finally, protein is one of the most essential macronutrients for human beings. Since the excessive intake of animal protein is associated with a high risk of chronic diseases, hemp protein products gradually become a good alternative to animal protein. However, excessive protein may bring about side effects because the body dose not store proteins. Long-term and high-protein intake may increase the renal glomerular filtration rate and impose a significant metabolic burden on kidneys, thus leading to kidney damage, especially for those who already have chronic kidney diseases, such as diabetic nephropathy and nephrotic syndrome. Therefore, regular clinical follow-ups with healthcare professionals are essential for individuals consuming protein products. On the one hand, it is important to discuss the dosages of hemp protein intake with healthcare professionals, who can identify the best dietary intervention course for consumers. On the other hand, it is necessary to take into account the anti-nutritional compounds.
8. Conclusions and Future
Hemp seed is a novel protein-rich functional food with promising phytotherapy efficacy. The research and applications on hemp are relatively few compared with other plant foods. Thus, hemp intrigues the growing interest of scientists. This review focuses on the antioxidant ability of hemp seed protein and summarizes the characteristics of antioxidant peptides. It found that the structure-activity relationship of antioxidant peptides is impacted by multiple factors involving the molecular size, the compositions of amino acids and the sequences of amino acids. Considering the positive pre-clinical properties of hemp protein, many scholars offered a broad spectrum of potential pharmacology on various diseases such as hypertension, inflammatory diseases, cancer. Since hemp seed is still an underexploited food, more high-quality experiments such as proteomic analysis, genomics analysis, and the clinical trials of large sample size are warranted for more scientific evidence. Despite the current limitations of hemp protein products, we believe they will be better applied to human health worldwide with the development of food, agriculture, biology, and medicine technologies. We are looking forward to seeing a brilliant future of hemp protein.
Acknowledgments
Contribution No. 21-173-J from the Kansas Agricultural Experiment Station.
Conflicts of Interest
The authors declare no conflict of interest.
| Soybean Hydrolysate | Hemp Hydrolysate | p Value | |
|---|---|---|---|
| ORAC (μM trolox/g sample) | 38.78 ± 0.362 | 0.539 ± 0.124 | <0.001 |
| Metal iron chelating activity (%) | 42.71 ± 0.247 | 0.591 ± 0.152 | <0.05 |
| Amino | Defatted | Hemp Protein | Hemp Protein Isolate | Hemp Protein Fraction | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| His | 3.07 ± 0.01 | 2.78 ± 0.03 | 1.09 ± 0.33 | 2.23 ± 0.20 | 2.85 ± 0.05 | 2.81 ± 0.47 | 2.61 ± 0.06 | 2.47 ± 0.01 | 2.47 ± 0.03 | 2.49 ± 0.08 |
| Arg | 12.68 ± 0.21 | 14.07 ± 0.40 | 6.05 ± 0.56 | 10.64 ± 0.92 | 13.44 ± 0.03 | 9.91 ± 0.91 | 13.87 ± 0.11 | 12.96 ± 0.13 | 13.6 ± 0.25 | 13.31 ± 0.24 |
| Thr | 3.79 ± 0.08 | 3.68 ± 0.29 | 2.06 ± 0.18 | 3.36 ± 0.37 | 3.79 ± 0.10 | 4.57 ± 0.35 | 3.60 ± 0.04 | 3.77 ± 0.35 | 4.01 ± 0.19 | 4.00 ± 0.19 |
| Val | 5.27 ± 0.11 | 4.66 ± 0.02 | 2.51 ± 0.09 | 3.90 ± 0.12 | 3.77 ± 0.40 | 4.98 ± 0.13 | 5.67 ± 0.14 | 5.26 ± 0.11 | 4.45 ± 0.19 | 4.24 ± 0.14 |
| Met | 2.14 ± 0.11 | 1.81 ± 0.39 | 1.83 ± 0.15 | 1.63 ± 0.05 | 3.79 ± 0.10 | 1.39 ± 0.06 | 1.94 ± 0.04 | 2.03 ± 0.06 | 1.85 ± 0.14 | 1.80 ± 0.15 |
| Lys | 3.78 ± 0.09 | 2.97 ± 0.06 | 1.20 ± 0.23 | 1.97 ± 0.18 | 0.89 ± 0.34 | 4.16 ± 0.87 | 3.19 ± 0.11 | 2.94 ± 0.14 | 2.56 ± 0.03 | 2.51 ± 0.07 |
| Ile | 4.23 ± 0.08 | 3.84 ± 0.07 | 1.93 ± 0.02 | 3.12 ± 0.10 | 3.02 ± 0.40 | 3.99 ± 0.08 | 4.15 ± 0.13 | 4.16 ± 0.11 | 3.98 ± 0.05 | 3.90 ± 0.04 |
| Leu | 7.12 ± 0.05 | 6.75 ± 0.04 | 3.13 ± 0.08 | 4.90 ± 0.19 | 7.13 ± 0.11 | 6.63 ± 0.23 | 9.91 ± 0.06 | 7.26 ± 0.10 | 5.15 ± 0.04 | 4.82 ± 0.43 |
| Phe | 4.76 ± 0.09 | 4.60 ± 0.04 | 2.34 ± 0.19 | 3.86 ± 0.42 | 5.12 ± 0.02 | 4.57 ± 0.11 | 7.68 ± 0.05 | 5.01 ± 0.14 | 3.21 ± 0.02 | 2.85 ± 0.40 |
| Non-essential | ||||||||||
| Trp | 0.76 ± 0.05 | 1.23 ± 0.16 | 0.04 ± 0.00 | 0.02 ± 0.00 | 0.55 ± 0.02 | NA | 1.58 ± 0.01 | 1.44 ± 0.15 | 1.16 ± 0.12 | 1.11 ± 0.02 |
| Asp | 11.34 ± 0.37 | 11.39 ± 0.03 | 4.06 ± 0.40 | 6.55 ± 0.48 | 12.35 ± 0.34 | 9.41 ± 0.39 | 9.49 ± 0.06 | 11.70 ± 0.29 | 12.79 ± 0.47 | 12.70 ± 0.11 |
| Ser | 5.57 ± 0.11 | 4.63 ± 0.09 | 2.37 ± 0.23 | 3.90 ± 0.31 | 6.31 ± 0.21 | 5.18 ± 0.02 | 4.73 ± 0.21 | 4.79 ± 0.19 | 4.69 ± 0.07 | 4.47 ± 0.38 |
| Glu | 18.41 ± 0.13 | 20.06 ± 1.34 | 6.92 ± 0.45 | 11.46 ± 0.82 | 18.86 ± 0.35 | 16.14 ± 0.26 | 15.18 ± 0.96 | 19.31 ± 1.04 | 22.71 ± 1.58 | 22.87 ± 1.02 |
| Gly | 4.74 ± 0.07 | 4.29 ± 0.23 | 2.01 ± 0.24 | 3.19 ± 0.29 | 4.79 ± 0.23 | 3.99 ± 0.06 | 3.23 ± 0.06 | 3.93 ± 0.07 | 4.54 ± 0.20 | 4.71 ± 0.44 |
| Ala | 4.89 ± 0.10 | 4.47 ± 0.16 | 1.79 ± 0.04 | 2.73 ± 0.09 | 5.01 ± 0.14 | 4.50 ± 0.36 | 4.91 ± 0.06 | 4.77 ± 0.12 | 4.30 ± 0.19 | 4.12 ± 0.09 |
| Pro | 2.66 ± 0.69 | 4.00 ± 0.07 | 1.73 ± 0.07 | 2.66 ± 0.10 | 3.02 ± 0.00 | 4.53 ± 0.39 | 3.19 ± 0.33 | 4.04 ± 0.56 | 4.23 ± 0.11 | 4.89 ± 0.73 |
| Cys | 1.22 ± 0.02 | 1.32 ± 0.23 | 0.71 ± 0.02 | 0.99 ± 0.06 | 0.89 ± 0.34 | 0.17 ± 0.01 | 0.29 ± 0.13 | 0.66 ± 0.01 | 1.26 ± 0.07 | 1.58 ± 0.28 |
| Tyr | 3.79 ± 0.08 | 3.45 ± 0.00 | 1.51 ± 0.13 | 2.71 ± 0.26 | 3.79 ± 0.10 | 3.67 ± 0.23 | 4.78 ± 0.02 | 3.50 ± 0.02 | 3.06 ± 0.06 | 3.62 ± 0.96 |
| Reference | [76] | [59] | [77] | [77] | [76] | [7] | [59] | [59] | [59] | [59] |
| Protein Resource | Hydrolytic Enzyme | In Vitro Antioxidant Assays | Amino Acid Sequence | Reference |
|---|---|---|---|---|
| Sardine Muscle | Pepsin | Superoxide radical scavenging activity, hydroxyl radical scavenging activity. | Leu-Gln-Pro-Gly-Gln-Gly-Gln-Gln | [86] |
| Casein | Pepsin | Superoxide radical scavenging activity, hydroxyl radical scavenging activity, DPPH radical scavenging activity. | Tyr-Phe-Tyr-Pro-Glu-Leu | [87] |
| Royal Jelly | Protease N | Superoxide radical scavenging activity, hydroxyl radical scavenging activity, hydrogen peroxide scavenging activity, metal chelating activity. | Ala-Leu, PheLys, Phe-Arg, lle-Arg, Lys-Phe, Lys-Leu, Lys-Tyr, Arg-Tyr, Tyr-Asp, Tyr-Tyr, Leu-Asp-Arg, Lys-Asn-Tyr-Pro | [88] |
| Egg White | Pepsin | ORAC, low-density lipoprotein lipid oxidation induced by Cu2+. | Tyr-Ala-Glu-Glu-Arg-Tyr-Pro-Ile-Leu | [89] |
| Porcine Myofibrillar | Actinase E, Papain | DPPH radical scavenging activity, metal ion chelating activity, hydroperoxides in a peroxidation system. | Asp-Ser-Gly-Val-Thr, Ile-Glu-Ala-Glu-Gly-Glu, Asp-Ala-Gln-Glu-Lys-Leu-Glu, Glu-Glu-Leu-Asp-Asn-Ala-Leu-Asn, Val-Pro-Ser-Ile-Asp-Asp-Gln-Glu-Glu-Leu-Met | [90] |
| Sardinelle | Alcalase | DPPH radical-scavenging assay, the lipid peroxidation inhibition activity, reducing power assay. | Leu-Ala-Arg-Leu, Gly-Gly-Glu, Leu-His-Tyr and Gly-Ala-Leu-Ala-Ala-His | [78] |
| Barley | Alcalase, Flvourzyme and Pepsin | DPPH radical scavenging assay, superoxide radical scavenging assay, hydroxyl radical scavenging assay, metal chelating activity, ORAC. | Pro-Tyr-Pro, Gln-Pro-Tyr-Pro-Gln, Gln-Gln-Pro-Tyr-Pro-Gln, Gln-Pro-Gln-Pro-Tyr-Pro-Gln, Thr-Gln-Gln-Pro-Tyr-Pro-Gln, Glu-Pro-Tyr-Pro-Glu | [80] |
| Corn | Alcalase | Superoxide dismutase activity. | Leu-Asp-Tyr-Glu | [79] |
| Amino Acids | Hemp Seed | Brown Rice | Soybean | Brown Rice | Oat | Pea | Potato | Wheat | Fababean | Corn | Infant (FAO/WHO/UNU) | Child 2–5 Years Old (FAO/WHO/UNU) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Essential | ||||||||||||
| His | 3.07 | 1.8 | 2.3 | 1.8 | 0.9 | 1.6 | 1.4 | 1.7 | 2.7 | 2.2 | 2.6 | 1.90 |
| Arg | 12.68 | 6.3 | 1.8 | 6.3 | 3.1 | 5.9 | 3.3 | 4.1 | 11.2 | 6.3 | NA | NA |
| Thr | 3.79 | 2.9 | 7.9 | 2.9 | 1.5 | 2.5 | 4.1 | 2.3 | 3.1 | 3.4 | 4.3 | 3.40 |
| Val | 5.27 | 4.6 | 5.9 | 4.6 | 2.0 | 2.7 | 3.7 | 3.6 | 6.8 | 7.3 | 5.5 | 3.50 |
| Met | 2.14 | 2.3 | 1.7 | 2.3 | 0.1 | 0.3 | 1.3 | 1.1 | 0.7 | 1.6 | NA | NA |
| Lys | 3.78 | 2.4 | 9.7 | 2.4 | 1.3 | 4.7 | 4.8 | 2.0 | 7.5 | 4.3 | 6.6 | 5.80 |
| Ile | 4.23 | 3.5 | 4.3 | 3.5 | 1.3 | 2.3 | 3.1 | 3.2 | 4.3 | 2.6 | 4.6 | 2.80 |
| Leu | 7.12 | 6.4 | 10.2 | 6.4 | 3.8 | 5.7 | 6.7 | 6.7 | 9.6 | 10.4 | 9.3 | 6.60 |
| Phe | 4.76 | 4.4 | 2.6 | 4.4 | 2.7 | 3.7 | 4.2 | 2.3 | 5.1 | 4.1 | NA | NA |
| Non-essential | ||||||||||||
| Trp | 0.76 | 1.2 | 1.1 | 1.2 | NA | NA | NA | 1.2 | NA | NA | NA | NA |
| Asp | 11.34 | 6.9 | 10.2 | 6.9 | NA | NA | NA | 4.5 | 7.6 | 7.1 | NA | N |
| Ser | 5.57 | 3.9 | 4.6 | 3.9 | 2.2 | 3.6 | 3.4 | 4.2 | 5.6 | 5.5 | NA | NA |
| Glu | 18.41 | 13.9 | 17.5 | 13.9 | 11.0 | 12.9 | 7.1 | 36.1 | 18.1 | 18.7 | NA | NA |
| Gly | 4.74 | 3.5 | 3.6 | 3.5 | 1.7 | 2.8 | 3.2 | 3.3 | 5.3 | 6.6 | NA | NA |
| Ala | 4.89 | 4.5 | 4.8 | 4.5 | 2.2 | 3.2 | 3.3 | 2.8 | 4.7 | 7.6 | NA | NA |
| Pro | 2.66 | 2.9 | 5.7 | 2.9 | 2.5 | 3.1 | 3.3 | 8.4 | 5.4 | 9.0 | NA | NA |
| Cys | 1.22 | 1.7 | 2.1 | 1.7 | 0.4 | 0.2 | 0.3 | 2.0 | 1.0 | 1.9 | NA | NA |
| Tyr | 3.79 | 4.3 | 1.1 | 4.3 | 1.5 | 2.6 | 3.8 | 4.0 | 1.3 | 1.4 | 1.7 | 1.10 |
| Reference | [76] | [135] | [135] | [135] | [136] | [136] | [136] | [137] | [138] | [138] | [139] | [140] |