http://orcid.org/0000-0002-5898-4108
ABSTRACT
Rice bran is an important underutilized by-product of rice-processing industries, and a huge amount of bran is released during rice processing. It can be used as a prime source of bioactive peptides towards the development of functional food ingredients. The aim of the study was to evaluate the potential of rice bran proteins such as glutelin, globulin, and prolamin as a precursor of bioactive peptides using various in silico approaches (BLAST, BIOPEP, PeptideRanker, PepDraw, Pepcalc, and ToxinPred). Rice bran proteins majorly release angiotensin-I-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. Papain protease can cleave the rice bran proteins effectively compared with other proteases to release ACE-inhibitory peptides. Physicochemical studies have shown that ACE-inhibitory peptides have low molecular weight profiles. These peptides have been classified as bitter peptides with a non-toxic profile. The result of this study demonstrates the usefulness of in silico approaches and provides a theoretical basis for the development of rice bran proteins as a source of bioactive peptides. The predicted prominent ACE-inhibitory peptides could be used for the development of functional food products or nutraceuticals.
Introduction
Rice (Oryza sativa L.) is one of the staple food sources for more than half of the world’s population, especially in Asian countries. It is the second most grown dietary staple cereal crop after wheat, globally.[Citation1] Rice-milling-processing industries produce huge quantities of by-products. In developing countries, most of the rice-processing by-products are not efficiently utilized for human consumption, and instead they are regularly used as an animal feed ingredient or discarded as waste.[Citation2,Citation3] Rice bran is an important underutilized by-product of rice milling, with a global potential of 29.3 million tons annually.[Citation4] Although it is rich in dietary fibres, lipids, proteins, minerals (K, P, Ca, Mg, Mn, and Fe), and vitamins, but usually it is not consumed as food due to its high fibre content. The rice bran contains ~10–15% highly nutritional proteins.[Citation5,Citation6] Most of the proteins found in rice grain are usually present in the bran. These rice bran proteins are mostly storage proteins (glutelin, globulin, and prolamin).[Citation2] Among the agro-industrial by-products, proteins found in rice bran are an exclusive source of bioactive components that find potential applications in food, pharmacy, and cosmetic industries.[Citation3] The essential amino acid such as lysine (a limiting amino acid in cereal grains) are abundantly present in rice bran proteins. Therefore, balanced amino acid profile and the hypoallergenic nature of rice bran-derived peptides and hydrolysates make it a suitable ingredient for infant food formulations.[Citation4]
Nowadays, trends of global research are mainly focused on the valorization of various plant-protein-enriched by-products to improve the functional and nutritional values of food products in a cost-effective manner. The utilization of plant proteins to supplement food products with desirable functional attributes is less expensive than that of animal-source proteins. Rice bran is gaining considerable attention of researchers due to its high nutrient profile, easy availability, potential functional activities, and apparent hypoallergenic nature compared with other cereal proteins. Rice bran protein profile has also been recognized as nutritionally superior to that of other protein sources.[Citation3] Rice bran proteins are highly digestible and well recognized as effective functional food ingredients and nutritional supplements.[Citation3] At present, rice bran protein supplements are not available commercially.[Citation2] Rice bran-derived albumin protein has chelating ability and can be used as an anti-oxidative in food products. The development of new value-added functional food or nutraceutical products using rice bran protein requires more information about protein properties and characteristics.[Citation2,Citation5]
Bioactive hydrolysates and peptides are commonly released by enzymatic hydrolysis from their parent protein using one or more exogenous and/or endogenous enzymes.[Citation7,Citation8] Furthermore, the functional characteristics of rice bran protein hydrolysates and peptides make them suitable for application in an array of food products like nutritional supplements, functional ingredients, flavour enhancers, coffee whiteners, cosmetics, personal care products, confectionery, toppings, beverages, meat, bakery products, and in the fortification of soft drinks and juices, soups, sauces, gravies, etc.[Citation3,Citation5] Rice bran protein hydrolysates have been incorporated in food and drink products without compromising their final quality.[Citation3]
There has been growing interest in the utilization of food-derived bioactive hydrolysates and peptides as an agent to control chronic diseases for reducing the risk of synthetic drugs’ side effects. Currently, hypertension and cardiovascular diseases are considered as the most serious chronic illnesses.[Citation7,Citation8] Angiotensin-I-converting enzyme (ACE, EC 3.4.15.1.) is a multifunctional zinc-containing enzyme that plays a key role in hypertension control, type-2 diabetes, and other metabolic syndromes.[Citation9–Citation11] Inhibition of enzymes involved in various stages of the renin-angiotensin-aldosterone system (RAAS) can play a vital role in combating hypertension and other related diseases.[Citation7] In addition, the screening of ACE-inhibitory peptides from novel substrates using conventional methods is an expensive and time-consuming process compared with in silico approaches. In the past decades, this process was simplified using in silico approaches such as BLAST, BIOPEP, PeptideRanker, Pepdrew, Pepcalc, and ToxinPred.[Citation8,Citation10,Citation12–Citation16] Therefore, the objective of this investigation was to identify and screen the potential ACE-inhibitory peptides from rice bran proteins using in silico approaches. Moreover, the physicochemical characteristics, primary structure, toxicity, and allergenicity of most potent ACE-inhibitory peptides were also evaluated.
Methods
BLAST analysis of rice bran protein sequences
The primary protein sequences of nine representative rice bran proteins were used in the present study (). The protein sequences of rice bran-derived proteins and their accession number were obtained from UniProtKB database (http://www.uniprot.org/) in the FASTA format at the ExPASy bioinformatics resource portal (https://www.expasy.org/). Glutelin, globulin, and prolamin are the predominant rice storage proteins.[Citation1–Citation4] Hence, the major contributors of the rice bran-derived proteins were selected for this study. The selected protein sequences were used for homology analysis using align two or more sequences option in BLAST tool (http://blast.ncbi.nlm.nih.gov/Blast.cgi).[Citation12,Citation17] BLAST was used to calculate the significant areas of commonly present amino acid sequences.
Table 1. The nine representative rice bran-derived protein sequences and their accession numbers from UniProtKB database.
In silico analysis of the amino acid composition
The amino acid compositions of the selected rice bran protein sequences were determined using ProtParam tool (http://web.expasy.org/protparam/). ProtParam is an in silico analysis program that computes the physicochemical properties of a protein or peptide from its amino acid sequences.[Citation18] The total numbers of amino acids, molecular weight, and theoretical isoelectric point (pI) of the selected rice bran proteins were also evaluated.
In silico assessment of rice bran proteins using BIOPEP tool
Potential biological activity profile
The profiles of potential biological activity of the selected rice bran proteins were carried out using the “profiles of potential biological activity” option in BIOPEP tool (http://www.uwm.edu.pl/biochemia/index.php/en/biopep).[Citation14]
Assessment of potential ACE-inhibitory peptides and their occurrence frequency
The potential of rice bran proteins to release the ACE-inhibitory peptides and their occurrence frequency in the selected rice bran protein sequences were evaluated using the following equation:[Citation14]
where A is the occurrence frequency of bioactive peptides, a is the number of bioactive peptides, and N is the total number of amino acid residues in the selected protein sequences. The potential of ACE-inhibitory activity in the selected rice bran proteins (B) is also predicted. The calculation of the B value majorly depends on the EC50 value of the selected ACE-inhibitory peptide sequences. However, the repetitions of the ACE-inhibitory fragment in the protein sequence, number of different fragments with ACE-inhibitory activity, and the total number of amino acid residues in a protein are also required to calculate the B values. If the EC50 value is not known, then the B value calculation cannot be performed.[Citation14,Citation19]
In silico proteolysis
The sequences of rice bran proteins were subjected to in silico proteolysis using the pepsin pH 2.0, proteinase K, ficain, papain, and bromelain enzymes for the prediction of theoretically released peptides by the enzymatic action program available in BIOPEP tool.[Citation14] In this study, the selected enzymes were chosen based on the previous reports and evidences from published databases. Furthermore, the theoretically released peptides obtained using the selected proteases were submitted to the “search for active fragments” option in BIOPEP tool. The ACE-inhibitory peptides were screened from the list of potential bioactive peptides released from the given proteins. The peptides with ACE-inhibitory activity were selected for further analysis. The possibilities for releasing the ACE-inhibitory peptides by the given proteases were also evaluated. The following equations were used to calculate the release frequency of fragments with ACE-inhibitory activity by the selected protease (AE) and the relative frequency of fragments with ACE-inhibitory activity by the selected enzymes (W):[Citation19]
where d is the number of fragments released by enzymes with ACE-inhibitory activity from a given protein sequence and N is the number of amino acid residues in the given protein.
Sensory characteristics prediction of the ACE-inhibitory peptides
Peptides and amino acids possess the capacity to alter the taste of food commodities and products.[Citation20,Citation21] Therefore, the sensory characteristics of the rice bran-derived ACE-inhibitory peptides were predicted. The various sensory characteristics (such as astringent, bitter, bitterness suppressing, salt enhancer, salty, sour, sweet, umami, and umami enhancing) of the ACE-inhibitory peptides were predicted.[Citation21]
Peptide ranking
The potential of the selected rice bran-derived ACE-inhibitory peptides were predicted by PeptideRanker (http://bioware.ucd.ie/∼compass/biowareweb/) tool.[Citation15] It is a web-based server to predict the probability of biological activity of a given peptide sequence. PeptideRanker tool provides the peptide score in the range of 0–1. The maximum score represents the most active and the least score denotes the least active peptides. The selected most potent rice bran-derived ACE-inhibitory peptides were subjected to further analysis.
Physicochemical characteristics and structure of rice bran-derived ACE-inhibitory peptides
The physicochemical features of rice bran-derived ACE-inhibitory peptides were evaluated using online peptide calculators.[Citation14] BIOPEP tool can also be used to calculate the molecular weight profile and location of the peptides released from the protein sequences. The theoretical molecular weight, isoelectric point, peptide charge at pH 7, estimated solubility, and extinction coefficient of the screened rice bran-derived ACE-inhibitory peptides were estimated by online Pepcalc software (http://pepcalc.com/). Furthermore, the primary structure of the ACE-inhibitory peptides was drawn using PepDraw (http://pepdraw.com/) tool.
Toxicity prediction of rice bran-derived ACE-inhibitory peptides
The toxicity of ACE-inhibitory peptides is one of the major hurdles towards the sustainable utilization of rice bran proteins for the development of nutraceuticals/functional food ingredients. Therefore, in silico toxicity prediction of rice bran-derived ACE-inhibitory peptides was investigated using ToxinPred online tool (http://www.imtech.res.in/raghava/toxinpred/index.html).[Citation13] The support vector machine (SVM)-based prediction method with a threshold value of 0.0 was chosen. The threshold value (0.0) was used to separate toxic and non-toxic peptides.[Citation13]
Allergenicity prediction of rice bran-derived ACE-inhibitory peptides
The rice bran-derived theoretically released ACE-inhibitory peptides were assessed for allergenicity using AllerTOP tool (http://www.pharmfac.net/allertop/).[Citation22] Additionally, Allergen FP v.1.0 tool (http://www.ddg-pharmfac.net/AllergenFP) was also used to predict the allergenicity of rice bran-derived ACE-inhibitory peptides.
Result and discussion
Homology of the selected rice bran proteins
The selected rice bran protein sequences were aligned against each other for homology analysis based on their molecular characteristics, amino acid composition, and length (). BLAST method can be used to compare the homology between different protein sequences and calculate the significant similar regions of amino acid sequences in a given protein.[Citation23–Citation25] The BLAST analysis results were obtained in the form of identities, positives, gaps, and bit scores. The percentage of matched amino acids within the full length of the aligned protein sequences can be represented as identities. In other words, a high value of the identities showed more homology between the aligned sequences. Positive value represents the conservative substitution of amino acids that share similar molecular characteristics.[Citation12,Citation23] The gap (-) represents the numbers of gaps found within the total length of the aligned protein sequences. The gap was applied to successfully align the given protein sequences.[Citation12,Citation17] The score or bit score is a value that is calculated on the basis of the number of gaps and substitutions associated with the aligned protein sequences. Higher bit score indicates reliability of alignment between the given protein sequences.[Citation12,Citation17]
Table 2. Summary of the selected rice bran protein sequence analysis using BLAST.
In , the glutelin type A1, A2, and A3 showed 82–95% identities and 88–97% positives. Glutelin type B1, B2, and C also showed 72–93% identities and 84–96% positives. However, globulin 1 and 2, prolamin, and glutelin type-C do not share any homology or identities (). The result of this study indicates that glutelin proteins might share ~72–97% sequence homology to each other. The higher percentage of positives showed that these proteins share similar molecular characteristics. Rice bran proteins glutelin type A1, A2, and A3 have almost similar molecular weight profiles and physicochemical and molecular characteristics. It was observed from previous studies that homologous proteins can deliberate the almost similar bioactive peptides/fragments.[Citation23,Citation26] The results of this study are in agreement with similar findings from the literature.[Citation10,Citation27] Therefore, BLAST analysis results indicated that rice bran-derived glutelin type A1, A2, A3, B1, and B2 might share the similar biological potentials. Hence, based on BLAST analysis, rice bran proteins glutelin type A1, B1, C, globulin (1 and 2), and prolamin (13 kDa) were selected for further analysis.
Amino acid composition analysis of the selected rice bran proteins
shows the amino acid profiles of the selected rice bran proteins evaluated using ProtParam online tool. The composition of 22 amino acids was evaluated. The result of this study showed that glutamine is the most abundantly present amino acid in glutelin A1, B1, C, and prolamin proteins. These proteins are also rich in serine, arginine, leucine, valine, glycine, alanine, and asparagine amino acids. However, alanine, arginine, glycine, and leucine are the frequently present amino acids in globulin 1 and 2 proteins. The molecular weights of glutelin A1, B1, and C were observed at ~ 56, 56, and 50 kDa, respectively. Globulin 1, globulin 2, and prolamin had a low molecular weight of ~ 18, 15, and 17 kDa, respectively. Globulin and prolamin proteins possess low molecular weight compared with glutelin proteins due to the short length of proteins. The pI values of glutelin, globulin, and prolamin proteins are predicted at alkali pH except for globulin 2 at a slightly acidic pH (). The results of this study are in agreement with the findings of rice bran-derived globulin proteins reported earlier.[Citation27]
Table 3. In silico amino acid (AA) compositions, molecular weight, and theoretical pI prediction of the selected rice bran proteins.
Biological activity profiles of rice bran proteins
The several potential biological activities of rice bran proteins are reported in the online supplementary information (SI) S1. Traditionally, classical methods were used for the exploration of biological activities of protein hydrolysates/peptides.[Citation10,Citation28] Furthermore, the most potential bioactive peptides are subjected to identification of their sequences and chemical synthesis for validating their biological activity. The classical approaches require much time to conduct the experiments.[Citation10,Citation28] These methods can also lead to a lower yield of isolated bioactive peptides and loss of their potential bioactivities.[Citation16] Therefore, to avoid these drawbacks, the cost-effective and time-saving computer simulated methods or in silico methods can be used to predict the theoretically released bioactive peptides derived from food proteins such as rice bran proteins.[Citation8] Therefore, we have evaluated the potential biological activity profiles of rice bran proteins. The theoretically released peptides from rice bran proteins have several potential biological activities (SI S1). The results of this study are in accordance with the finding of Udenigwe[Citation29], who reported several biological activities in specific rice bran proteins. The results of this study are also in agreement with the study of Thamnarathip et al.[Citation30,Citation31] and Wattanasiritham et al.[Citation32], who reported the in vitro antioxidant activities of rice bran proteins hydrolysates and peptides.
Rice bran-derived ACE-inhibitory peptides and their occurrence frequency
The theoretically released rice bran-derived bioactive peptides with ACE-inhibitory activity are screened and subjected to further investigation. The predicted rice bran-derived peptides with potential ACE-inhibitory activities are summarized in . The rice bran protein glutelin type A1, B1, and C majorly released the ACE-inhibitory and DPP-IV-inhibitory peptides. The glutelin type A1, B1, and C theoretically released 87, 97, and 133 ACE-inhibitory peptides, respectively. The rice bran-derived globulin 1, 2, and prolamin released 51, 41, and 38 fragments with ACE-inhibitory activity, respectively. The occurrence frequencies of DPP-IV- and ACE-inhibitory peptides released from rice bran-derived glutelin type A1, B1, and C sequences are 0.5983, 0.5928, 0.6123 and 0.3138, 0.3179, 0.3188, respectively. The occurrence frequencies values of ACE-inhibitory (0.3197, 0.3058) and DPP-IV-inhibitory (0.5164, 0.4175) peptides in globulin 1 and 2 proteins are also predicted. However, the prolamin protein sequence resulted a low value for the occurrence frequency of ACE-inhibitory peptides (0.2140) compared with other rice bran proteins. The predicted occurrence frequencies of the selected glutelin and globulin proteins have similar value to release ACE-inhibitory peptides, which also indicates the consequence of homology against each other.[Citation27] The calculated value B is the second discriminant of the potential biological activity of the protein. The higher B value found in the glutelin type-B1 protein sequence indicates the presence of the most potent ACE-inhibitory activity containing peptides (SI S2). The predicted peptides sequences with ACE-inhibitory activity are matched by BIOPEP tool from the published literature of various plant-, animal-, and other food product-derived peptides.[Citation14] The total number of ACE-inhibitory dipeptides is higher compared with that of tri-peptides (). It is well known and extensively accepted among the scientific community that dipeptides and tri-peptides can be easily absorbed in the gastrointestinal tract then in the cardiovascular circulation system and then finally exhibit physiological-regulating properties.[Citation26,Citation33–Citation35]
Table 4. The predicted rice bran protein-derived dipeptides and tri-peptides with ACE-inhibitory activity.
ACE-inhibitory peptides released after in silico proteolysis
The theoretically released ACE-inhibitory peptides using in silico proteolysis are summarized and presented in . The ACE-inhibitory peptides are released from selected rice bran proteins using specific proteases (pepsin pH 2.0, proteinase K, ficain, papain, and bromelain). In silico proteolysis demonstrates the ability of given enzymes to release the ACE-inhibitory peptides from rice bran proteins. Papain theoretically released the highest number of ACE-inhibitory peptides compared with all other enzymes used in this study. On the contrary, bromelain and ficain enzymes comparatively released smaller numbers of ACE-inhibitory peptides from the selected rice bran proteins. The ACE-inhibitory dipeptides are the most abundantly released from rice bran proteins, whereas few tri-peptides and tetra-peptides sequences were also observed (). It was also reported that hydrophobic amino acids (alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, valine, and proline) present in the ACE-inhibitory peptides can increase the inhibition of ACE, specifically when these amino acids are located at the C-terminal.[Citation23,Citation36–Citation39] These amino acid residues containing peptides had been reported with stronger ACE-inhibitory activity.[Citation37] Chang and Alli[Citation23] reported that ficain and proteinase K proteases might release a large number of hydrophobic amino acids at C-terminal positions. Udenigwe and Aluko[Citation8] and Aluko[Citation38] reported that application of the exogenous proteases with comprehensive cleavage sites is crucial to release the ACE-inhibitory peptides from food-derived proteins. The maximum AE (for the release frequency of bioactive ACE-inhibitory peptides) and W (relative frequency to release the fragments with ACE-inhibitory activity using proteases) values are obtained using pepsin and papain enzymes. AE and W are the two most important parameters that can be used to determine the possible release of bioactive peptides using proteases from the protein sequences.[Citation19]
Table 5. In silico proteolysis of rice bran proteins to release ACE-inhibitory peptides using BIOPEP enzyme action tool.
Peptide ranker analysis
The ACE-inhibitory peptides derived from selected rice bran proteins using in silico proteolysis were subjected to peptide ranker tool for the prediction of feasible activity. PeptideRanker server can rank the peptide sets according to their structure–function patterns.[Citation15] lists the most potential ACE-inhibitory peptides released from the selected proteins. The maximum PeptideRanker scores (0.99) showed by FR, FG and RF dipeptides. The ACE-inhibitory peptides generated from globulin 1 proteins (RP-0.82, AP-0.63) demonstrated a low PeptideRanker score compared with other selected protein sequences (). Amino acid profile of small peptides is responsible for their biological activity.[Citation15] The activity of the peptide sequence depends on two major determinants – the position of amino acid in the peptide sequence and its composition. It is well documented that short peptides containing phenylalanine are more likely to be predicted as bioactive. Despite these facts, the number of glycine in the peptide sequence has paramount correlation with its PeptideRanker score.[Citation15,Citation24,Citation37] The maximum peptide rank score of rice bran-derived ACE-inhibitory peptides was observed in the peptides that contains phenylalanine and glycine in their sequences (). It is well known that PeptideRanker cannot exactly predict the potential and biological activities of the peptides. Therefore, the unreported biological activities of the predicted peptides using in silico tool need to be confirmed with in vitro and in vivo analyses of synthetic peptides.[Citation40] Hence, this tool analysis will be helpful to advance the goal for designing the desired ACE-inhibitory bioactive peptides from the selected rice bran proteins with improved efficiency.
Table 6. Peptide ranking of ACE-inhibitory peptides derived from selected rice bran proteins after in silico proteolysis and their sensory prediction.
Sensory analysis of rice bran proteins-derived ACE-inhibitory peptides
Taste is the major factor that is responsible to differentiate among different food products. Taste is the key factor responsible for determining the quality of any food commodities. Humans most commonly recognize five basic taste sensations – bitter, salty, sour, sweet, and umami. The taste of food is affected by some molecules with a specific chemical nature such as peptides derived from food proteins.[Citation20,Citation21] In this study, a bitter taste of ACE-inhibitory peptides released from the in silico proteolysis has been predicted. The dipeptides FR released from the glutelin type-A1, B1, and C showed bitter and bitterness-suppressing taste. Moreover, PG, GM, MG, and AP dipeptides showed sweet taste ().
In the literature, it is well reported that sweet, bitter, and umami are superior taste attributes of peptides. The presence of bitter peptides was dominant compared with that of the other tastes.[Citation41] Several biological activities such as inhibition of enzymes that regulate the body functions are also associated with the peptide taste.[Citation20] Kim and Li-Chan[Citation42] reported that peptides composed of up to eight amino acid residues have more bitterness. Phenylalanine, tyrosine, and glycine are major amino acids that have a major impact on the bitterness of peptides.[Citation42] Various methods have been successfully used to remove/reduce the bitterness of bioactive peptides. Bitterness can be most effectively masked through the addition of other substances like sugars, skim milk, soybean casein, casein hydrolysate, sugar alcohols, and sodium salts of organic acids.[Citation20,Citation28,Citation43] Isoelectric precipitation, extraction with food-grade solvents, adsorption on chromatographic resins or activated carbon, silica gel chromatography, dialysis, and enzymes (carboxypeptidase) can also be used to remove the bitterness of peptides.[Citation44] Encapsulation (using liposomes, double emulsions, and nanoparticles) is a new approach that can be used to improve the bioavailability and sensory characteristics of bitter peptides as nutraceuticals.[Citation41] The de-bitter peptides may be successfully incorporated as an ingredient for the development of functional foods. The major concerns about the biological activity of bitter peptides should remain after the de-bittering process.[Citation20] Therefore, this study provided a technical feasibility for the application of in silico approaches to predict and release ACE-inhibitory peptides from selected protein sequences. Based on the PeptideRanker score and sensory analysis prediction, the most potential ACE-inhibitory peptides have been screened and subjected to further analysis.
Physicochemical characteristics and primary structure of rice bran-derived ACE-inhibitory peptides
The rice bran protein-derived ACE-inhibitory peptides were studied to predict the various physicochemical characteristics and their primary structure. The molecular weight of rice bran protein-derived ACE-inhibitory peptides was found to be ~ 0.1–0.3 kDa. The ACE-inhibitory peptides containing molecular weight ~ 300 Da showed the alkali isoelectric point. However, peptides of molecular weight ~ 200 Da showed the acidic isoelectric point (). The results of this study indicate that most of ACE-inhibitory peptides have low molecular weight. The isoelectric points of the predicted ACE-inhibitory peptides were found to be in the pH range of 3.28–10.6. The alkali isoelectric point containing ACE-inhibitory peptides showed good water solubility, whereas the acidic isoelectric point containing peptides showed poor water solubility (). The primary structure of the selected ACE-inhibitory peptides is shown in SI S3. The results of the study are in accordance with Pooja et al. [Citation27], who reported physicochemical characteristics of DPP-IV-inhibitory peptides released from rice bran-derived globulin proteins. Pal and Suresh [Citation28] also reported that most of the bioactive peptides have low molecular weight profiles.
Table 7. Prediction of the physicochemical properties, toxicity, and allergenicity of the potential ACE-inhibitory peptides (http://pepcalc.com/).
Toxicity prediction of rice bran-derived ACE-inhibitory peptides
Toxicity of ACE-inhibitory peptides may prevent the development of bioactive peptides as functional food ingredients.[Citation13,Citation26,Citation27] In this study, ACE-inhibitory peptides are derived from plant proteins, and the enzymes used to release these peptides are obtained from plant or animal sources which is frequently employed in several food-processing industries without any health risks reported earlier. Peptides with low molecular weight profile are non-toxic and known to be less allergenic compared with their native proteins.[Citation25,Citation45] It was predicted from ToxinPred analysis that the predicted ACE-inhibitory peptides are non-toxic (SVM scores < 0) (). Val, Thr, Arg, Gln, Met, Leu, Lys, Ile, Phe, and Ala are the primary components of non-toxic peptides.[Citation13] In the present study, rice bran-derived ACE-inhibitory peptides contain the amino acids residues that are usually found in non-toxic peptides. Therefore, these peptides can be used as potential functional ingredients.[Citation27] For human consumption, in vitro and in vivo toxicity assessment of food products must be carried out for these peptides following the international authorities’ guidelines that are usually performed in animal models, cell lines, or unicellular microbial species.[Citation25]
Allergenicity prediction of rice bran-derived ACE-inhibitory peptides
The frequency of food allergy is increasing consistently. It affects approximately 8% of children and 1–2% of adults.[Citation46] Most of the allergens are proteins derived from various plant and animal sources. The European food safety authority (EFSA) encourages the use of in silico approaches to predict the potential allergens from food proteins.[Citation25] The hydrolysis of proteins using food-grade enzymes such as pepsin may also lead to the elimination of linear epitopes, which is the major factor of allergenicity.[Citation28,Citation47,Citation48] In this study, the peptides RF, FR, FG, NF, and KF are predicted as probable allergens. The peptide AF is predicted as a non-allergen, whereas peptides GP, MG, IF, GM, and SF peptides are predicted as probable non-allergens, as predicted by AllerTOP tool.[Citation22] The ACE-inhibitory peptides RF, FR, FG, AF, GP, MG, IF, NF, KF GM, and SF are also predicted as probable non-allergens by AllergenFP v.1.0 tool. [Citation22] The predicted allergenicity of the selected rice protein-derived peptides needs to be clarified using in vitro and in vivo methods.[Citation22,Citation25]
In conclusion, BLAST and in silico amino acid composition analysis suggested that rice bran protein glutelin showed amino acid sequence homology to each other and might share similar biological activities containing peptides. BIOPEP analysis showed that rice bran proteins glutelin, globulin, and prolamin could be utilized as precursors for releasing ACE-inhibitory peptides with enzymatic hydrolysis using food-grade enzymes pepsin, papain, ficain, and bromelain. PeptideRanker, pepcalc, BIOPEP, and pepdrew can be used to predict the bioactive potential, physicochemical, sensory characteristics, and primary structure, respectively. ToxinPred, AllerTOP, and AllergenFP v.1.0 were used to predict potential toxicity and allergenicity of selected rice bran-derived ACE-inhibitory peptides. In silico tools provide a theoretical basis for further research to utilize these by-product proteins as nutraceuticals. Furthermore, in vitro and in vivo studies should be carried out in order to confirm the obtained results. Therefore, these findings will open new avenues for the utilization of rice bran by-products for the development of high value-added functional food ingredients/formulation.
LJFP_A_1368552_Supplementary_file.docx
Download MS Word (1.1 MB)Acknowledgement
The authors would like to thank the anonymous reviewers for their valuable comments that improved the manuscript. The authors declare that they have no conflict of interests. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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