Exploration of rice protein hydrolysates and peptides with special reference to antioxidant potential: Computational derived approaches for bio-activity determination

Accepted Manuscript Exploration of rice protein hydrolysates and peptides with special reference to antioxidant potential: Computational derived approaches for bio-activity determination Sapna Rani, Km Pooja, Gaurav Kumar Pal PII:

S0924-2244(17)30728-8

DOI:

10.1016/j.tifs.2018.07.013

Reference:

TIFS 2274

To appear in:

Trends in Food Science & Technology

Received Date: 12 November 2017 Revised Date:

6 May 2018

Accepted Date: 12 July 2018

Please cite this article as: Rani, S., Pooja, K., Pal, G.K., Exploration of rice protein hydrolysates and peptides with special reference to antioxidant potential: Computational derived approaches for bioactivity determination, Trends in Food Science & Technology (2018), doi: 10.1016/j.tifs.2018.07.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Abstract

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Background Rice processing by-products derived proteins have been well acknowledged as rich

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sources of structurally diverse compounds (especially proteins) possess various health-related

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benefits along with a great therapeutic potential for the treatment and prevention of various

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diseases.

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Scope and approach

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In this paper, we have reviewed and explored the possibilities for adapting the

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sustainable valorisation of rice processing by-products to generate bioactive hydrolysates and

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peptides for food and biotechnological industries. The role of computational derived

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approaches for the production and applications of bioactive hydrolysates and peptides from

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the parent protein has also been explored.

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Key findings and conclusions

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Based on the emerging evidence of potential health benefits, the antioxidant potential

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of rice protein hydrolysate and peptides has been reviewed. The present review mainly

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highlights the recent research on rice proteins derived bioactive compounds for food and

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biotechnological applications using computational derived approaches with special reference

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to antioxidant activity. The safety, bioavailability and technological problems (towards the

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incorporation into food products) to deliver the bioactive peptides on the specific target has

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been discussed. The major opportunities and challenges are discussed for inspiring

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researchers/industries to investigate the critical problems that are responsible for preventing

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the utilization of these approaches for the development of functional food and nutraceutical

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products.

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Keywords: Rice processing by-products; protein hydrolysates; computational approaches;

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bioactive rice peptides; functional food products

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Exploration of rice protein hydrolysates and peptides with special reference to

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antioxidant potential: Computational derived approaches for bio-activity determination

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Sapna Rani1, Km Pooja2,*, Gaurav Kumar Pal3,*

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Dairy Microbiology Division, ICAR-National Dairy Research Institute, Karnal-132001, Haryana, India

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Department of Botany, Chaudhary Charan Singh University, Meerut-250004, Uttar Pradesh, India 3

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Department of Microbiology, Meerut Institute of Engineering and Technology, Meerut250005, Uttar Pradesh, India

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Email-

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[email protected] (Sapna Rani)

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[email protected] (Km Pooja)

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[email protected]; [email protected] (Gaurav Kumar Pal)

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* = corresponding authors

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Corresponding Address:

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Department of Microbiology,

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Meerut Institute of Engineering and Technology,

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Meerut-250005, Uttar Pradesh, India

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Mobile: 91+ 9482382610

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1. Introduction Agricultural processing by-products derived bioactive molecules are considered as an

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emerging field in the present biotechnological era among the researchers. From many years,

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agricultural processing by-products especially rice by-products are considered as undervalued

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substrates due to the complicated treatment and disposal issues (Galanakis, 2012). Nowadays,

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the demands for sustainability in food and agricultural sectors led to their valorisation as a

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source of functional and bioactive compounds. The utilisation of bioactive compounds

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became more popular and acceptable worldwide with increasing scientific knowledge, social

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awareness, along with a broad spectrum of biotechnological validation. In recent years,

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naturally occurring bioactive compounds are being preferred due to their easy accessibility

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and rarer side effect for the development of nutraceuticals and functional food products

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(Galanakis, 2012, 2013).

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Rice (Oryza sativa L.) is a leading staple food crop worldwide. It is a major food

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source for approximately more than one-half of the world’s population, particularly in Asian

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countries (Pooja & Rani, 2017). The global annual production of the rice estimated

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approximately 610 million metric tons (milled rice basis) (Sereewatthanawut et al., 2008;

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Wang, Chen, Fu, Li, & Wei, 2017). It is cultivated in every zone of more than hundred

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countries, except Antarctica (Amagliani, O’Regan, Kelly, & O’Mahony, 2017a, 2017b). It is

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a second most grown dietary staple cereal crop after wheat, globally (Fabian & Ju, 2011). As

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a staple food, rice could be able to fulfill the requirement of essential and unique

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micronutrients such as vitamins, minerals, and phenolic compounds that have potent

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antioxidant activity. Rice is rich in a specific group of flavonoids and other unique

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compounds that may have significant free radical scavenging activities (Zhou, Canning, &

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Sun, 2013).

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ACCEPTED MANUSCRIPT Due to enormous production of the rice, it (rice milling processing industries)

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produces vast quantities of rice by-products (Sereewatthanawut et al., 2008). In most of the

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developing countries, rice by-products are yet not to be efficiently utilized for human

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consumption. Usually, rice by-products regularly used as an animal feed ingredient or

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discarded as waste (Fabian & Ju, 2011). It can be used to provide a considerable proportion

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of the protein intake for millions of people.

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Rice bran is the outer component of raw rice that is obtained as a by-product during

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the rice milling process and has 12-15% protein content. It is a vital underutilised by-product

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of rice milling with a global potential of 29.3 million tons annually (Sharif, Butt, Anjum, &

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Khan, 2014; Sohail, Rakha, Butt, Iqbal, & Rashid, 2016). The protein digestibility and

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biological value of rice have been reported to be higher than those of the other major cereals

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(such as wheat, corn, etc.). Rice bran derived proteins usually have a high-quality and

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generally regarded as a hypoallergenic protein that may be useful in infant food formulations.

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It is also well reported that these proteins have various biological activities (Wang et al.,

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2017). A lot of research studies suggesting that rice proteins and its derived hydrolysates and

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specific peptide fractions have strong biological and functional activities such as anti-

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oxidative, anti-hypertensive, anti-obesity and so on (Cheetangdee & Benjakul, 2015; Fang et

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al., 2017; Wang et al., 2017; Yan, Huang, Sun, Jiang, & Wu, 2015; Zhao, Xiong, et al., 2012;

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Zhou et al., 2013). Therefore, rice can be utilized as a fascinating, cost-effective, and

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potential source of proteins for the development of protein-enriched ingredients for the

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formulation of nutritional enriched food or drink products.

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Bioactive peptides are specific and small protein fragments that are inactive within the

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sequence of their parent protein. Enzymatic hydrolysis commonly releases bioactive

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hydrolysates and peptides. These peptides are 2-20 amino acids in size and typically possess

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specific amino acid sequences, mainly comprised of hydrophobic groups in addition to

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proline, arginine, and lysine (Pal & Suresh, 2016, 2017). However, there is scanty

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information about the antioxidant activities of rice bran protein and its hydrolysates. For a

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better understanding of antioxidant activities of hydrolysate from rice bran protein, the

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peptides contributing to those activities need to be examined (Wang et al., 2017). There has been growing interest in the use of computational derived approaches for

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screening of bioactive peptides from the novel substrates. The screening for biological

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activity containing peptides or hydrolysates from novel substrates using conventional

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methods is an expensive and time-consuming process as compared with in silico analysis

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(Pooja & Rani, 2017; Rani & Pooja, 2018; Rani, Pooja, & Kumar, 2017). However, this

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process can be simplified using computational approaches such as BLAST, BIOPEP,

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PeptideRanker, Pepdrew, Pepcalc, and ToxinPred, etc. (Altschul et al., 2005; Dimitrov,

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Bangov, Flower, & Doytchinova, 2014; Gupta et al., 2013; Piotr Minkiewicz, Dziuba,

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Iwaniak, Dziuba, & Darewicz, 2008; Mooney, Haslam, Holton, Pollastri, & Shields, 2013;

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Mooney, Haslam, Pollastri, & Shields, 2012).

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The antioxidants play a vital role to reduce oxidative processes in the food

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commodities and in human body. In food commodities, antioxidant can retards the protein

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oxidation, peroxidation of lipid, secondary product formation during lipid peroxidation, and

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also helps to maintain the flavour, texture, and colour of the food product during storage. The

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natural antioxidants (such as vitamin C, tocopherols, rosemary, tea extracts) have been well

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known as alternatives to synthetic antioxidants (Sila & Bougatef, 2016). The potential

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antioxidant activity of the protein hydrolysates and peptides derived from various animals,

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plants and other sources (such as milk, soy, egg, fish, etc.) have also been demonstrated

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(Samaranayaka & Li-Chan, 2011; Pal & Suresh, 2016).

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Therefore, this article emphasized the current knowledge about rice protein derived

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bioactive hydrolysates and peptides with a particular accent on the exploration of bioactive

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peptides using computational approaches and their possible use as natural antioxidants in

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food products.

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2. Distribution of proteins in rice The yield of utilisable protein is higher for rice as compared to wheat, due to the

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superior quality of rice proteins. Most of the protein found in rice grain is usually present in

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the bran. These are the high value nutritionally rich protein that contains ~10-15% of the total

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protein content of the rice (Chandi & Sogi, 2007). These rice bran proteins are mostly storage

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proteins (Glutelin, globulin, and prolamin) (Fabian & Ju, 2011). On the basis of the solubility

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characteristics, the rice protein can be categorized into four main groups. The four main rice

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proteins are albumin (water soluble), globulin (salt soluble), glutelin (alkali/acid soluble), and

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prolamin (alcohol soluble) (Fig 1). Rice proteins are mainly found in the form of storage

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organelles (Amagliani et al., 2017b). These rice protein components have unique gelling and

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emulsifying properties. There is limited research on the preparation of rice protein derived

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bioactive hydrolysates/peptides and their functional properties (Arsa & Theerakulkait, 2018;

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Phongthai, D’Amico, Schoenlechner, Homthawornchoo, & Rawdkuen, 2018; Pooja & Rani,

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2017; Pooja, Rani, & Prakash, 2017; Senaphan et al., 2018; Wang, Chen, Fu, Li, & Wei,

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2017; Zhou et al., 2013). The protein content of rice is influenced by factors such as

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management and cultural practices, climate and genotype. The proper handling and

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processing of the rice processing by-products may provide high nutritional value and could

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find a potential application in the functional food industries (Amagliani et al., 2017a).

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3. Development of the rice bran derived hydrolysate and peptides

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Rice processing by-products has gained much attention as potential biologically active

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hydrolysate and peptides due to the high availability of rice processing by-products (Pooja &

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Rani, 2017; Sharif et al., 2014; Udenigwe, 2016). Rice processing by-product derived

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hydrolysates/peptides are specific protein fragments that play a significant role in preventing

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inactive within the parent sequences and become active after release from the parent

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sequences. Peptides usually contain 2-20 amino acids, and their biological and functional

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activities are based on their amino acid position and composition (Pal & Suresh, 2016, 2017).

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The bioactivity of released hydrolysates and peptides also depends on the primary sequence

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of protein and specificity of the enzymes used. It is also well known that structural feature of

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peptides mostly influences their biological and functional activities profiles. The presence of

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tyrosine, phenylalanine, tryptophan, proline, valine, leucine, lysine, isoleucine, and arginine

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in peptides strongly influences the binding of peptides with angiotensin converting enzyme

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(ACE) (Harnedy & FitzGerald, 2012). The activity of antimicrobial peptides is associated

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with positively charged residues. The radical scavenging activity is associated with histidine,

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leucine, tyrosine, methionine, and cysteine amino acid residues (Pal and Suresh 2017).

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Hydrophobic amino acids (Proline and hydroxyproline) appear to play a role in the inhibition

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of lipid peroxidation (Harnedy & FitzGerald, 2012). The most common methods used for the

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development of hydrolysates and peptides are discussed (Fig 2).

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3.1 Chemical methods

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The bioactive hydrolysates and peptides can be generated by chemical hydrolysis.

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This process was classified into two types: acid hydrolysis and alkali hydrolysis process.

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These hydrolysis methods are cost-effective, simple operative and required short hydrolysis

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time. Acid hydrolysis process is commonly carried out with 6 M HCl under high-temperature

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conditions (110-120°C). Alkaline hydrolysis process carried out with a strong alkali (sodium

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hydroxide or potassium hydroxide) in water at high temperatures (130-180°C). Moreover,

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these methods are usually not acceptable for preparing the bioactive peptides intended for the

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application in the development of functional food ingredients (Anal, Noomhorm, &

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Vongsawasdi, 2013; Pal & Suresh, 2016).

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3.2. Biological methods In biological methods, the bioactive hydrolysates and peptides are majorly released

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from rice processing by-products using exogenous enzymes, endogenous enzymes,

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fermentation, and gastrointestinal digestion process (Chen et al., 2013). However, enzymatic

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hydrolysis is the most widely used method to improve the functional and nutritional

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characteristics of protein hydrolysates and peptides. Enzymatic hydrolysis is the leading

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process for the production of bioactive hydrolysates and peptides from rice proteins. The rice

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proteins are gradually degraded into the low molecular weight peptides in the range of 0.2-4.0

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kDa during the enzymatic hydrolysis. The molecular weight of hydrolysates and peptides is

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one of the most critical parameter to produce bioactive peptides. It is well known and

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extensively accepted among the researchers that low molecular weight peptides can be easily

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absorbed in the gastrointestinal tract and cardiovascular circulation system and finally exhibit

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physiological-regulating properties (Pooja, Rani & Prakash, 2017). The commercial available

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food grade and non-food grade proteolytic enzymes from microbes such as alcalase,

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flavourzyme, protamex, proteinase K, metalloproteases, serine-protease; from plants papain,

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bromelain, ficin; from animal α-chymotrypsin, neutrase, trypsin, etc. have been widely used

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for the preparation of rice protein derived hydrolysates and peptides with functional

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activities. Exogenous enzymes have been preferred due to better control of hydrolysis with an

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optimum degree of hydrolysis and time to obtain the consistent molecular weight profiles of

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hydrolysates and peptides (Samaranayaka & Li-Chan, 2011). However, the choice of

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enzyme, and enzyme reaction conditions (pH, temperature, time, and enzyme concentration)

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are the crucial factors to prepare rice protein derived hydrolysates and peptides with desirable

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functional characteristics (Kim & Wijesekara, 2010; Samaranayaka & Li-Chan, 2011). It is

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also well known that only few peptides have the potential health promoting activity among a

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group of various peptides released after hydrolysis. Therefore, it is essential to highlight that

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ACCEPTED MANUSCRIPT biological and functional activity of the peptides evaluated using in vitro analysis are not

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enough and need to confirm by in vivo methods to observe a real health benefit. The bioactive

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peptides should be resistant to the gastrointestinal digestive enzymes. It must be absorbed

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through enterocytes to the serum and further reach to the target site for exhibiting its

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biological activity. The length of amino acid chain and its composition are of key factors that

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play a crucial role in absorption and resistance to degradation by gastrointestinal digestive

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enzymes (López-Barrios, Gutiérrez-Uribe, & Serna-Saldívar, 2014).

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3.3 Enzymes used in the hydrolysis process

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Rice proteins and their by-product proteins can be hydrolysed with a wide range of

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commercial enzymes, which is derived from the plant, animal, and microbial sources. The

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plant derived enzymes (ficin, bromelain, and papain) and animal derived enzymes (pepsin

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and trypsin) have been widely used for enzymatic hydrolysis process. Alcalase®,

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flavourzyme®, neutrase®, collagenase, and proteinase K are derived from the microbial

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sources and most widely used microbial enzyme for hydrolysis process (P. Minkiewicz,

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Dziuba, & Michalska, 2011; Piotr Minkiewicz et al., 2008). However, the cost of enzyme is a

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rate limiting step for the successful preparation of bioactive hydrolysates and peptides.

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Hence, it is recommended to use the cheap sources of proteinases those derived from waste or

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by-products and microorganisms (Agyei & Danquah, 2011). Neutrase®, subtilisin, orientase,

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alcalase®, flavoursyme®, and proteases from lactic acid bacteria are the most suitable to use

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for the enzymatic hydrolysis due to their cheap cost as compared to others enzymes. Most of

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these cost-effective enzymes derived from the microorganisms or microbial sources

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(Zambrowicz, Timmer, Polanowski, Lubec, & Trziszka, 2013). Bioactive peptides can also

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be generated during the microbial fermentation process and can be influenced by growth

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parameters such as the inoculum conditions and peptide content in the medium (Agyei &

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Danquah, 2011).

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4. Approaches for exploring biological activity of hydrolysates and peptides In the last years, various researchers have been focused their research studies on the

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identification, characterisation, and purification of bioactive hydrolysates and peptides from

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rice processing by-products. Classical or traditional approaches, in silico or computational

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approaches, and integrated approaches are majorly used to explore the potential of bioactive

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peptides.

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4.1 Classical or traditional approaches

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In traditional approaches, various steps are involved in the development of bioactive

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rice protein derived peptides. The classical method involves firstly the selection of particular

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protein sources and their enzymatic hydrolysis using the food-grade/non-food grade enzymes

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for the preparation of the hydrolysates and peptides. Subsequently, prepared protein

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hydrolysates subjected to the fractionation and purification based on their particular

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biological and functional activity (Udenigwe, 2014; Udenigwe & Aluko, 2012). The most

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potential bioactive hydrolysates/peptide sequences are subjected to identification using mass

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spectrometry methods. Further, biological and functional activities of released peptides are

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validated by chemically synthesized peptides. This approach has several drawbacks.

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It is a time-consuming process.

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This approach may lead to the lower yields of isolated bioactive peptides with desired

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Loss of some potential bioactive peptides may occur during the purification process.

4.2 In silico or computational approaches

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The computational simulated approaches can be used for exploring the bioactive

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hydrolysates and peptides (Pooja & Rani, 2017). In silico analysis is a suitable method for

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predicting the release of potential bioactive peptides from the known parental protein

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sequences. It is a suitable and emerging approach for exploring the novel and unexplored

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ACCEPTED MANUSCRIPT proteins which has not been previously studied as sources of bioactive peptides (Lafarga,

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O’Connor, & Hayes, 2014; Pal & Suresh, 2017; Pooja & Rani, 2017). Several popular

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bioinformatics tools, such as basic local alignment search tool (Altschul et al., 1997, 2005),

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BIOPEP database tool (Piotr Minkiewicz et al., 2008), PeptideDB, CAMP, APD2 or

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PepBank, and QSAR (quantitative structure-activity relationship) (Pripp, Isaksson, Stepaniak,

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Sørhaug, & Ardö, 2005; Wu, Aluko, & Nakai, 2006) have been well employed to predict and

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design the potential bioactive peptides from the plant, animal, and food-derived proteins.

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PeptideCutter, EnzymePredictor, PeptideRanker or PeptideLocator tools can be used to

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predict possible cleavage sites of the given protein sequences (Mooney et al., 2013, 2012).

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Recently, the computational approaches (in silico analysis) has been used to identify the

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potential sources of various bioactive and functional peptides (such as antioxidant, ACE-I

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and DPP-IV inhibiting peptides) from various sources (egg, milk, pea, oat, barley, meat, fish

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and others). Various researchers reported the potential theoretically released bioactive

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peptides from various plant, milk, and meat sources (Chang & Alli, 2012; Fu, Young, et al.,

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2016; Fu, Wu, Zhu, & Xiao, 2016; Lafarga et al., 2014; P. Minkiewicz et al., 2011;

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Nongonierma & FitzGerald, 2014; Nongonierma, Mooney, Shields, & FitzGerald, 2014; Pal

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& Suresh, 2017; Pooja & Rani, 2017; Udenigwe, 2016; Udenigwe, Gong, & Wu, 2013),

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however, till date the bioactivities of predicted bioactive peptides have not been confirmed.

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The

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(http://pops.csse.monash.edu.au) can be used for in silico proteolysis of given/selected

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protein sequences. Usually, the in silico approach involves computational mining of protein

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sequence information available in the database, followed by in silico hydrolysis of the protein

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based on known enzyme cleavage sites. The in silico approaches have several advantages

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over traditional experimental approaches towards the exploration of potential bioactive

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hydrolysates and peptides.

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online

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2. It is a more economical process for prediction of biologically active peptides.

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3. Computer simulated approaches can also be used to investigate bioactive peptides

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derived from various food-source proteins such as plant proteins, milk proteins, fish

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proteins, muscle proteins and others protein from various sources (Minkiewicz et al.,

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2011).

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4. The utilisation of computational approach can reduce the number of experiments. However, sometimes the peptides released from computational derived approaches

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may not be generated experimentally due to the complex interaction between selected

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enzymes and proteins as well as their post-translational modifications (Mohan, Rajendran,

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He, Bazinet, & Udenigwe, 2015). Hence, following a successful in silico digestion of proteins

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by computational methods to produce the bioactive sequences need to be evaluated by real

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laboratory synthesis under optimal temperature, and pH conditions and their bioactivity also

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need to validate by in vitro and in vivo methods (Rani & Pooja, 2018). QSAR method can

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also help to search the information which relates the chemical structure to biological

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activities using the computer simulated analysis. In the research area of bioactive

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hydrolysates and peptide studies, the utilisation of QSAR and other methods has increased

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which is crucial prior to in vitro and in vivo experiments (Iwaniak, Minkiewicz, Darewicz,

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Protasiewicz, & Mogut, 2015; Pripp et al., 2005). In QSAR methods, the bioactivities of

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selected peptides are closely related to their structural variations (Hellberg, Sjoestroem,

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Skagerberg, & Wold, 1987). Furthermore, QSAR models can also be employed for the

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activity prediction of synthetic/natural peptides, and to identify the novel functional and

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biological activity containing peptides.

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4.3 Integrated approaches

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an integrated approach is projected for the exploration of potential bioactive peptides and

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hydrolysates. Enzymatic hydrolysis using one or more proteases/enzymes is an efficient way

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of releasing bioactive peptides from their parental protein sources (Korhonen & Pihlanto,

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2006). This proposed approach involves the selection of the optimum protease/enzyme

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according to the computer simulated approaches or in silico approaches. The computational

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based analysis and tools such as BIOPEP and Expasy Peptide Cutter can be simplified the

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enzyme/protease selection process. Further, the selected protein needs to be subjected to in

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vitro digestion by selected enzymes to generate the protein hydrolysates and peptides. The in

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vitro released hydrolysates and peptides can be characterized by tandem mass spectroscopy

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(Harnedy & FitzGerald, 2012). Thereafter, the identified hydrolysates and peptide profiles are

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subjected to the prediction of activity by in silico methods. In integrated approaches, the

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experimental analyses play a crucial role in the exploration of bioactive hydrolysates and

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peptides. The details of the various in silico tools utilised for the exploration of bioactive

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peptides and hydrolysates are given below.

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4.3.1 ProtParam tool

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The amino acid compositions of the selected protein sequences can be predicted using

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ProtParam tool. ProtParam is an in silico analysis program that computes the physico-

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chemical properties of a protein or peptide from its amino acid sequences (Gasteiger et al.,

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2005). The total number of the amino acids, molecular weight, and theoretical pI of the

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selected protein sequences can also be predicted using this tool.

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4.3.2 PeptideRanker tool

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PeptideRanker is a web-based server to predict the probability of bio-activity of a

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given peptides. PeptideRanker tool can rank the peptide sets and assign the peptide score in

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the range of 0-1, on the basis of structure-function patterns. The maximum score represents

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the most potent active peptides, and least score denotes the least active peptides (Mooney et

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al., 2012).

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4.3.3 BIOPEP tool

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Profile of potential biological activity of selected protein sequences The potential biological activity profile of any chosen protein sequences can be

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predicted by BIOPEP tool (Piotr Minkiewicz et al., 2008). This tool provides the complete

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profile of selected protein for the presence of possibly released bioactive peptides. The

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occurrence frequency of potential bioactive fragments in the selected protein sequences can

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also be calculated using the BIOPEP tool. The occurrence frequency of bioactive fragments

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or peptides derived from the selected proteins has been computed using the following

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equation

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A = a/N

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Where, A = occurrence frequency of the bioactive fragments or peptides, a = number of

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bioactive peptides, and N = total number of amino acid residues in the selected protein

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sequences.

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In silico proteolysis

Eq. (1)

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The selected protein sequences can be subjected for in silico proteolysis to predict the

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theoretically released peptide sequences using enzymatic action program available in the

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BIOPEP tool. This tool allows the user to predict the potential of various substrates (known

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protein sequences) to generate theoretical bioactive peptides using enzymes with known

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cleavage specificities. In this tool, more than 25 enzymes can be applied alone or in

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combination to release the theoretical bioactive peptides and hydrolysates. Further,

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theoretically released fragments and peptides can be used to search the bioactive fragments or

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peptides using search for active fragments option of BIOPEP tool. The possibilities for the

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release of bioactive peptides by using selected proteases can also be predicted using the

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BIOPEP tool (P. Minkiewicz et al., 2011; Piotr Minkiewicz et al., 2008). The following

325

equation is used to calculate the release frequency of bioactive fragments by selected protease

326

(AE) and the relative frequency for the release of bioactive fragments by selected enzymes

327

(W). AE = d/N

Eq. (2)

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Where, d = the number of bioactive fragments released by enzymes in the protein sequence,

330

N = the number of amino acid residues in the protein chain. W = AE/A

Eq. (3)

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The values of these parameters were calculated based on Equations (1) and (2), respectively.

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Sensory characteristics profile

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The sensory characteristics prediction of the peptides released from selected protein

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can be predicted using the BIOPEP tool. The various sensory characteristics such as

336

astringent, bitter, bitterness suppressing, salt enhancer, salty, sour, sweet, umami, and umami

337

enhancing can be predicted by BIOPEP tool (Iwaniak, Minkiewicz, Darewicz, &

338

Hrynkiewicz, 2016; Iwaniak, Minkiewicz, Darewicz, Sieniawski, & Starowicz, 2016).

339

Peptides and amino acids have the capacity to altering the taste of food commodities and

340

products. The overall sensory profiles of the protein hydrolysates can also be predicted using

341

BIOPEP tool.

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4.3.4 Physico-chemical characteristics

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The physico-chemical features of the potential bioactive peptides can be evaluated

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using the online peptide calculators. The BIOPEP tool can also be used to calculate the

345

molecular weight profile, location of the peptides released from selected protein sequences.

346

The theoretical molecular weight, isoelectric point, the peptide charge at pH 7, estimated

347

solubility and extinction coefficient of the peptides can be estimated using online Pepcalc

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ACCEPTED MANUSCRIPT 348

software. Additionally, theoretical MW and isoelectric point of the peptides can be calculated

349

using the ExPASy Compute PL/MW Tool (Gasteiger et al., 2005).

350

4.3.5 Primary structure The primary structure of the selected food derived bioactive peptides can be draw

352

using the PepDraw tool. The web link for drawing the primary structure has been given in

353

Table 1.

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4.3.5 Toxicity prediction

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The toxicity of the bioactive peptides can be predicted by using ToxinPred online

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tool. The SVM (support vector machine) based prediction method with a threshold value of

357

0.0 can be chosen for the toxicity prediction of peptides released from food proteins. The

358

threshold value (0.0) was usually used to separate toxic and non-toxic peptides (Gupta et al.,

359

2013).

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4.3.6 Allergenicity prediction

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The potentially bioactive peptides and hydrolysates released from the selected

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proteins were assessed for their potential allergenicity using AllerTOP tool. However,

363

Allergen FP v.1.0-Another in silico tool can also be used to predict the allergenicity of the

364

protein hydrolysates and peptides (Dimitrov et al., 2014). These tools use a variety of amino

365

acid principal properties, such as hydrophobicity and β-strand forming propensities for

366

allergenicity prediction.

367

5. Purification and characterisation of rice bran derived peptides

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After the hydrolysis of protein, it is frequently essential to fractionate and purify the

369

mixture of peptides. Ultrafiltration, reversed-phase chromatography, ion exchange

370

chromatography, gel filtration chromatography and size exclusion chromatography are the

371

most common methods used for the purification of peptides. Reversed-phase (RP)-high-

372

performance liquid chromatography (HPLC) is one of the extensively used methods for the

15

ACCEPTED MANUSCRIPT purification and characterisation of peptides. However, nowadays liquid chromatography

374

(LC)-mass spectrometry (MS) is one of the preferred methods for the characterisation of

375

bioactive peptides which is separated by HPLC (Pal & Suresh, 2016). MS is an extremely

376

useful tool for the identification and characterisation of peptides and proteins due to its ability

377

to calculate the accurate molecular weight of the molecule. Electrospray ionisation (ESI) and

378

matrix-assisted laser desorption/ionisation (MALDI) MS have also been used for peptide and

379

protein identification. However, these methods cannot be economically viable due to their

380

higher cost. In order to reduce the cost, the use of immobilized enzymes and membrane

381

separation techniques were recommended for large-scale processes (Agyei & Danquah, 2011;

382

Korhonen & Pihlanto, 2006). Immobilized enzymes allow the user to reuse the enzymes and

383

also allows for more organized working conditions (Agyei & Danquah, 2011).

384

6. Biological activity of hydrolysates/peptides from rice processing by-products

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The hydrolysates and peptides released from the rice processing by-products have a

386

wide range of the applications in food and other allied sectors. In food industries, rice bran

387

derived protein hydrolysates can be used as functional and nutraceutical ingredients towards

388

the development of novel functional foods. In the last decades, the demand for food enriched

389

with plant proteins derived functional hydrolysates is growing due to the improvement in the

390

nutritive and functional properties of the food commodities. During last five years, various

391

research findings indicate the potential biological activities of rice bran derived

392

hydrolysates/peptides (Fig. 3). They have several biological activities such as antioxidative,

393

antihypertensive, immunomodulatory, antimicrobial, and other biological activities. Here, we

394

have discussed the antioxidative activity of rice bran derived bioactive hydrolysates and

395

peptides.

396

6.1. Antioxidant activity

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ACCEPTED MANUSCRIPT In vertebrates and humans, oxidation is a vital process that leads to the formation of

398

free radicals. The oxygen radicals can react with every cellular component and create the

399

functional and morphological disturbances in cells. Antioxidants are extensively used to

400

check the oxidation reaction (Najafian & Babji, 2012; Pal & Suresh, 2016). The deterioration

401

of the food takes place due to the lipid oxidation and formation of various undesirable

402

secondary lipid peroxidation products such as peroxides, aldehydes, and ketones

403

(Cheetangdee & Benjakul, 2015). Synthetic antioxidants are widely used in food products to

404

reduce the deterioration. Butylated hydroxyanisole, butylated hydroxytoluene, tert-butyl

405

hydroquinone, and propyl gallate are the commonly used antioxidants in the food and allied

406

sectors. Recently, there has been a growing interest to replace the synthetic antioxidants with

407

natural antioxidants owing to the concerns of health issues associated with synthetic

408

antioxidants (Cheetangdee & Benjakul, 2015; Najafian & Babji, 2012).

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The antioxidant potential of the rice bran derived protein hydrolysates and peptides

410

have been widely demonstrated in various oxidative systems such as free radical scavenging

411

activity, reducing power and metal ion-chelating capacity. In the low molecular weight of

412

protein hydrolysates and peptides, the presence of some specific amino acids could enhance

413

antioxidant activities (Cheetangdee & Benjakul, 2015; Liu, Wang, Li, Liang, & Yang, 2016).

414

Free radical-scavenging activities response of rice protein in an in vitro digestive system has

415

been highlighted to suppress the oxidative damage (Liu et al., 2016). It was also reported that

416

the antioxidant potential of heat stable defatted rice bran derived protein hydrolysates

417

associated with its reduced molecular weight profile, amino acid composition and

418

hydrophobicity (Zhang, Wang, Zhang, & Zhang, 2014). Incorporation of the rice bran

419

derived protein hydrolysates could improve the oxidative stability of bulk oil and emulsion

420

(Cheetangdee & Benjakul, 2015). The commercially available rice dreg protein hydrolysates

421

(RDPH) (released by trypsin hydrolysis) have been studied as natural antioxidants in a

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variety of emulsion-type food systems. RDPH containing corn oil-in-water emulsion was

423

more stable when used in combination with Tween 20 as compared to the emulsions prepared

424

by either RDPH or Tween-20 alone (Zhao, Selomulya, et al., 2012). Furthermore, selenium-enriched rice protein hydrolysates exhibit the potential

426

antioxidant activity evaluated by cellular antioxidant activity test. Additionally, a positive

427

correlation has been found between the antioxidant activity and immunomodulatory activity

428

of selenium-enriched rice protein hydrolysates (Fang et al., 2017). The protein hydrolysates

429

released from rice protein isolate showed the potent antioxidant activity. These protein

430

hydrolysates demonstrated as effective natural antioxidants to prevent the lipid oxidation for

431

improving the shelf-life of meat products (Zhou et al., 2013).

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The rice bran derived peptide sequence Tyr-Ser-Lys has been released after digestion

433

with trypsin enzyme. This peptide exhibited the strong scavenging activities on DPPH,

434

reducing power assay (Wang et al., 2017). Hence, rice protein hydrolysates could help to

435

enhance the stability of food emulsions, as well as act as a natural antioxidant. Numbers of

436

studies have shown in vitro and in vivo that rice bran derived protein hydrolysate/peptides

437

could be a potential natural antioxidant (Table 2).

438

7. Safety and bioavailability profile of the rice bran derived antioxidant peptides

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The rice bran protein derived hydrolysates and peptides must be safe for the

440

consumption. The rice bran proteins are usually safe for human consumption. If the bioactive

441

peptides are produced using digestive enzymes and food-grade enzymes, then there has been

442

a little concern about the safety of bioactive hydrolysates and peptides (Agyei, Tsopmo, &

443

Udenigwe, 2018). However, the released hydrolysates and peptides must be subjected to

444

analyse their toxicity and allergenicity profile. The rice bran derived bioactive hydrolysates

445

and peptides must have the acceptable level of toxicological profiles. Peptides (derived from

446

plant and animal proteins) released using the enzyme (obtained from plant or animal sources)

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ACCEPTED MANUSCRIPT which are frequently employed in several food-processing industries are reported to be safe

448

without any health risk (Pooja, Rani & Prakash, 2017). Peptides with low molecular weight

449

profile are non-toxic and known to be less allergenic compared with their native proteins

450

(Lafarga, O’Connor, & Hayes, 2014; Pooja, Rani & Prakash, 2017). Val, Thr, Arg, Gln, Met,

451

Leu, Lys, Ile, Phe, and Ala are the primary components of non-toxic peptides. Sometimes, the

452

nature of the food products (in which hydrolysates/peptides incorporated) also affects the

453

safety and toxicological profiles of the selected bioactive hydrolysates and peptides. Mass

454

spectrometry techniques are widely used to evaluate the possible formation of toxic

455

compounds when peptides interact with the food product matrix. Mass spectrometry and

456

computational biology approaches could also be used to evaluate the quality, and safety

457

(included toxicity, allergenicity etc.). Hence, it is required to evaluate the safety and

458

toxicological

459

peptides/hydrolysates following the international authority’s guidelines.

characteristics

of

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the

food

products

incorporated

with

these

Bioavailability of the bioactive hydrolysates/peptides can be characterized as the

461

proportion of a given hydrolysates/peptides/nutrient in a selected food product that is actually

462

utilized by the body across the gastrointestinal tract. These hydrolysates/peptides may be

463

resistant or can be absorbed during gastrointestinal digestion (Guo et al., 2014). Several

464

products are under development by various food industries to explore the potential of food-

465

derived bioactive hydrolysates and peptides. One of the most important task that needs for

466

delivering the functional/biological activity of bioactive hydrolysate/peptides is the

467

bioavailability. The bioavailability of hydrolysate/peptides is one of the major concerns to

468

develop the novel functional food or nutraceutical products. The optimisation of healthy and

469

functional food products through its nutrition value by using hydrolysates or peptides is a

470

major scientific and technological challenge (Agyei et al., 2018). The food processing

471

variables such as pH, temperature, and time should be appropriately controlled and must be

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ACCEPTED MANUSCRIPT chosen very carefully to preserve the activity of the incorporated hydrolysates and peptides in

473

the food products. As well as, the behaviour of the bioactive peptides in a given food product,

474

deterioration and shelf life of the food products must be considered for finalisation of the

475

food product (Carrasco-Castilla, Hernández-Álvarez, Jiménez-Martínez, Gutiérrez-López, &

476

Dávila-Ortiz, 2012). The in vitro and in vivo methods (Cell culture, animal models, and

477

human studies) have been widely used to study the bioavailability of selected hydrolysates

478

and peptides.

479

8. Major challenges and the future research directions

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The assessment of error rates in the prediction of bioactive hydrolysates and peptides

481

is an important issue. The faster speed of the computational approaches may be lead to the

482

false prediction of the bioactive peptides from the selected proteins. The identification of

483

false-positive bioactive peptides and hydrolysates are particularly dangerous for

484

biologists/scientist interested in studying the function of these selected hydrolysates and

485

peptides. Hence, the most popular approach for searching the protein from the various

486

biological databases should be practiced with great caution (Carrasco-Castilla et al., 2012).

487

The computational approaches used for the preparation and development of the bioactive

488

hydrolysate and peptides are the comprehensive, cost-effective and time saving process. After

489

the successful development and production of bioactive hydrolysates and peptides, the

490

incorporation of these hydrolysates/peptides into a food matrix is considered as a potential

491

challenge for the scientific community. Bioactive peptides must be delivered to the cellular

492

sites of action without affecting its functional characteristics. These hydrolysates/peptides can

493

be administered using different vehicles such as beverages, gummy paste, chewing gum etc.

494

Microencapsulation method can also be used to enhance their stability and absorption

495

(Carrasco-Castilla et al., 2012). The generation of peptides and hydrolysates from the parent

496

protein and assessment of their biological activity have been performed using computational

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ACCEPTED MANUSCRIPT methods and published in various biological databases. The use of computational biology

498

approaches may speed up the screening of high-potential peptides from different sources.

499

Computational methods may also be helpful to optimize controlled hydrolysis of the parent

500

protein for the production of peptides with potential biological activity. It is also well known

501

that computer simulated results are not always replicated in laboratory analysis. Hence, there

502

is a need to establish a more predictive accuracy for the generation of bioactive peptides

503

using different in silico tools and methods (Agyei et al., 2018). However, the results of these

504

studies need to be verified by using the in vitro and in vivo analysis. These approaches do not

505

replace the need for further experimental verification and analysis. As well as, in the area of

506

food science and technology, computational approaches (in silico simulation) analysis can be

507

used as a fast tool for the initial screening of high potential sources of bioactive hydrolysates

508

and peptides. Computational approaches can be used to study the following characteristics of

509

the bioactive hydrolysates and peptides derived from the food sources: Searching for

510

potential precursors of bioactive hydrolysates and peptides, computational derived structure

511

simulations, protein/protease selection for hydrolysis, prediction of sensory characteristics,

512

structure-function relationships, screening and prediction of biological activity, simulated

513

proteolysis, secondary structure prediction, toxicity prediction, allergenicity prediction,

514

evaluation of physico-chemical properties and structure-activity relationship analysis etc.

515

(Agyei et al., 2018; Carrasco-Castilla et al., 2012; Rani & Pooja, 2018). Still, it is important

516

to consider that mathematical model used for the optimisation cannot be applied in all

517

conditions (such as temperature, pH and processing time) and sometimes may result in the

518

false positive information to the scientific community. Further work is also needed to

519

improve the predictive capability of computational tools for the development and analysis of

520

functional bioactive peptides present in food and other complex biological matrices (Agyei et

521

al., 2018).

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9. Sensory characteristics of rice bran derived antioxidant peptides The bioactive hydrolysates and peptides generation has gained the attention of the

524

scientific community to design novel nutraceutical and functional food products. Several

525

factors need to be looked after optimisation of bioactive peptides production. The sensory

526

characteristic is one of the major concerns when incorporated into the desired food products

527

(Agyei et al., 2018). Taste is one of the critical factor responsible for determining the quality

528

of any food commodities and responsible for differentiating among food products. It is well

529

reported that sweet, bitter, and umami are superior taste attributes of peptides and

530

hydrolysates released after enzymatic hydrolysis. The presence of bitter peptides was

531

dominant compared with that of the other tastes. Pooja, Rani & Prakash, (2017) reported the

532

ACE-inhibitory peptides released from the rice proteins with bitter taste, bitterness-

533

suppressing taste and sweet taste using the in silico analysis. Peptides that are composed of

534

up to eight amino acid residues have more bitterness. Phenylalanine, tyrosine, and glycine are

535

major amino acids that have an impact on the bitterness of peptides (Rani, Pooja, & Kumar,

536

2017). Encapsulation approach can be used to improve the bioavailability and sensory

537

characteristics of bitter peptides and hydrolysates.

538

10. Conclusion

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The rice processing industry generates a huge amount of by-products, which should

540

be adequately managed not only to attend to environmental concerns but also to generate the

541

added valued food products. The enzymatic process can achieve the conversion of rice

542

protein into bioactive hydrolysates and peptides. The utilisation of the computational and

543

integrated approaches can be efficiently used to explore the bioactive peptides and

544

hydrolysates from novel substrates. Computational approaches will be useful for the food

545

manufacturers to predict the possible bioactivity of the peptide in a cost-effective and less

546

labour intensive manner. The combination of clinical trials related to bioactive peptides in a

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ACCEPTED MANUSCRIPT food matrix and computational analysis will make easier confirmation of their health-

548

promoting activities as well as recognized by international agencies (such as Food and Drug

549

Administration and European Food Safety Authority). Rice-derived bioactive hydrolysates

550

and peptides with antioxidative properties may be a potential substitute for synthetic

551

antioxidants towards the development of functional foods. Moreover, additional research is

552

necessary to evaluate the safety profile of peptide-based products prior to commercialisation.

553

In addition, the rice protein hydrolysates/peptides incorporation as free or encapsulated

554

ingredients is also an exciting area for future research.

555

Acknowledgement & Conflict of interest

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The authors would like to thank anonymous reviewers for their valuable comments to

557

improve the manuscript. This research did not receive any specific grant from funding

558

agencies in the public, commercial, or not-for-profit sectors. The authors declare that we have

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no conflict of interests.

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ACCEPTED MANUSCRIPT Figure captions

814

Fig 1 Graphical presentation of the rice bran derived proteins

815

Fig 2 Outline of (Traditional and computational) process for developing the bioactive

816

peptides from rice processing by-products

817

Fig 3 Biological activities of the rice protein derived hydrolysates and peptides

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34

ACCEPTED MANUSCRIPT Table 1: List of the various computational based tool and their web address to predict the potential biological activity Web address for tool

ProtParam tool

http://web.expasy.org/protparam/

PeptideRanker

http://bioware.ucd.ie/~compass/biowareweb/Server_pages/peptideranker.ph

RI PT

Tool

p

http://www.uwm.edu.pl/biochemia/index.php/pl/biopep

Expasy Peptide http://web.expasy.org/peptide_cutter/ Cutter

Allergen

http://www.pharmfac.net/allertop/

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AllerTOP tool

SC

BIOPEP

FP http://www.ddg-pharmfac.net/AllergenFP

v.1.0 http://pepdraw.com/

ToxinPred

http://www.imtech.res.in/raghava/toxinpred/index.html

Pepcalc

http://pepcalc.com/

ExPASy

AC C

Compute

http://web.expasy.org/compute_pi/

EP

software

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PepDraw

PL/MW Tool

ACCEPTED MANUSCRIPT Table 2: Antioxidative capacity of rice protein derived hydrolysates and peptides Source

Enzyme used Hydrolysates Amino Antioxidant /peptides acid used sequen ce Protease Hydrolysates DPPH

Defatted

scavenging

sativa

Reducing

L.) licheniformis)

bran

Ferrous

Porcine

protein

pepsin

(Oryza

pancreatin

Hydrolysates and

-

power; (2015)

chelating

ABTS

scavenging

M AN U

Rice

activity; Benjakul,

SC

activity

radical Cheetangdee &

RI PT

rice (Oryza (Bacillus

assay References

radical Y. Liu et al.,

activity; (2016)

DPPH

sativa L.)

scavenging

radical activity;

iron chelating activity;

TE D

copper

-

Protamex

activity, and reducing power DPPH scavenging

AC C

rice bran

EP

Heat stable Alcalase, and Hydrolysates

chelating

radical H. J. Zhang et activity; al., (2014)

reducing

power;

ferrous ion chelating activity,

and

lipid

peroxidation inhibition activity Rice

Trypsin,

(Oryza

papain,

Hydrolysates and

-

Cellular activity

antioxidant Fang (2017)

et

al.,

ACCEPTED MANUSCRIPT sativa L.)

pepsin

Rice

Neutral

protein

protease

absorbance

isolate

(Bacillus

ABTS

subtilis),

scavenging

validase

DPPH

(Aspergillus

scavenging activity

-

Oxygen

alkaline

(Bacillus licheniformis)

Hydrolysates

Tyr-

DPPH

and peptides

Ser-

scavenging

TE D

Trypsin

activity;

radical

radical X. Wang et al., activity; (2017)

Reducing power assay

Hydrolysates

RPNY

DPPH

and peptides

TDA,

scavenging

TSQL

ABTS

Protamex,

LSDQ,

scavenging

Pepsin,

TRTG

Ferrous

Papain,

DPFF

activity

Alcalase,

residue

Flavourzyme,

protein

Protamex,

AC C

EP

Rice

Rice

capacity; (2013)

Lys

Trypsin

radical Yan

et

al.,

activity; (2015) radical activity; chelating

and

and

their

NFHP

combinations

Q,

Alcalase,

al.,

radical

M AN U

protease

Rice bran

et

SC

oryzae), and

radical Zhou

RI PT

Hydrolysates

Hydrolysates

-

ABTS

radical Dei Piu et al.,

ACCEPTED MANUSCRIPT proteins

Neutrase,

scavenging activity

(2014)

Flavourzyme, B.

subtilis

(SV27, B.

RI PT

SV20I), pumilus

(AGI) and B.

(AG2) dreg Alcalase,

protein

Hydrolysates

Neutrase,

DPPH

radical Zhao,

scavenging

Flavourzyme,

TE D

Trypsin Rice

Alcalase,

endosperm

Chymotrypsi

protein

n,

Xiong,

activity; et al., (2012)

Reducing power assay

Protamex and

Hydrolysates

FRDE

DPPH

and peptides

HKK,

scavenging

KHNR

Superoxide

radical

GDEF

scavenging

activity;

EP

Neutrase,

and

AC C

Papain,

-

M AN U

Rice

SC

licheniformis

Flavorase

radical J. Zhang et al., activity; (2010)

Hydroxyl

radical

scavenging

activity

and

inhibition

linoleic

acid

of

model

system Defatted

Alcalase,

commercia

Flavourzyme

Hydrolysates

-

ABTS scavenging

radical Thamnarathip, activity; Jangchud,

ACCEPTED MANUSCRIPT l rice bran

and Neutrase

ferric

ion

reducing Nitisinprasert,

antioxidant power

& Vardhanabhuti, (2016)

Porcine

proteins

pepsin

Hydrolysates

-

and

ABTS

scavenging

pancreatin

DPPH

radical Z. Wang, Liu,

RI PT

Rice

radical (2016)

activity;

SC

scavenging

activity; Li, & Yang,

iron chelating activity;

M AN U

copper

chelating

activity, and reducing power

Rice bran

Trypsin

and Hydrolysates and peptides

Oxygen

radical Wattanasiritha

absorbance capacity

TE D

ProteaseMax

-

Theerakulkait, Wickramaseka

AC C

EP

ra, Maier, & Stevens, (2016)

Selenium

Neutrase,

Hydrolysates

SeMet- DPPH

enriched

Alcalase,

and peptides

Pro-

scavenging

brown rice

Flavorase,

Ser

Superoxide

and Papain

m,

scavenging Hydroxyl

radical K. Liu, Zhao, activity; Chen, & Fang, radical (2015) activity; radical

scavenging activity

ACCEPTED MANUSCRIPT Raw

Alcalase

Hydrolysates

-

DPPH

radical Phongthai,

organic

scavenging

activity; Lim,

rice bran

Ferric

reducing Rawdkuen,

Metal activity Bromelain

Hydrolysates

-

ABTS

chelating

and protease and peptides

scavenging

FP51

DPPH

radical Selamassakul,

activity; Laohakunjit,

SC

Brown rice

power; (2016)

RI PT

antioxidant

radical Kerdchoechue

rice flour

pancreatin

and Hydrolysates

-

AC C

EP

TE D

Pepsin

M AN U

scavenging activity

Defatted

&

n,

&

Ratanakhanokc hai, (2016)

ABTS

radical- Z. Wang, Li,

scavenging

activity; Liang,

Superoxide

radical- Yang, (2016)

scavenging

activity;

Hydrogen

peroxide

scavenging

activity;

Nitric oxide radicalscavenging

activity;

iron chelating activity; copper

chelating

activity, and reducing power

&

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

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TE D

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SC

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights By-products of rice processing are major sources of high-value proteins. Rice-derived protein hydrolysates and peptides have important biological activities. The safety and challenges of rice protein derived antioxidative peptides are discussed.

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Bioactive peptides can be predicted using computational approaches.