Purification, identification and characterization of two novel antioxidant peptides from finger millet (Eleusine coracana) protein hydrolysate

Accepted Manuscript Purification, identification and characterization of two novel antioxidant peptides from finger millet (Eleusine coracana) protein hydrolysate

Himani Agrawal, Robin Joshi, Mahesh Gupta PII: DOI: Reference:

S0963-9969(18)30913-X https://doi.org/10.1016/j.foodres.2018.11.028 FRIN 8091

To appear in:

Food Research International

Received date: Revised date: Accepted date:

23 August 2018 8 October 2018 15 November 2018

Please cite this article as: Himani Agrawal, Robin Joshi, Mahesh Gupta , Purification, identification and characterization of two novel antioxidant peptides from finger millet (Eleusine coracana) protein hydrolysate. Frin (2018), https://doi.org/10.1016/ j.foodres.2018.11.028

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ACCEPTED MANUSCRIPT Purification, identification and characterization of two novel antioxidant peptides from finger millet (Eleusine coracana) protein hydrolysate Himani Agrawal1 , Robin Joshi* and Mahesh Gupta* 1

Academy of Scientific and Innovative Research (AcSIR)

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CSIR-Institute of Himalayan Bioresource Technology, Palampur-176061, Himachal Pradesh,

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India

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Corresponding authors: *Dr. Mahesh Gupta

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CSIR-Institute of Himalayan Bioresource Technology Palampur, Himachal Pradesh

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India, 176061

*Dr. Robin Joshi

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[email protected]

CSIR-Institute of Himalayan Bioresource Technology

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Palampur, Himachal Pradesh India, 176061

[email protected]

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ABSTRACT Antioxidant peptides were successfully identified from a finger millet protein hydrolysate. The trypsin hydrolysate showed a higher degree of hydrolysis (17.47 ± 0.63%) than the

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pepsin hydrolysate, was further fractionated by ultrafiltration membrane [<3kDa (UF3), 310kDa (UF2 ) and >10kDa (UF1 )]. UF3 with higher antioxidant activity (48.41± 0.21%) was

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further fractionated into five fractions (GF A, GFB, GFC, GFD and GFE) using gel filtration.

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Fraction GFB possessed the highest antioxidant activity (61.79 ± 0.08%) and was purified by reverse-phase ultra- flow liquid chromatography. Two potential antioxidant peptides were

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identified as TSSSLNMAVRGGLTR and STTVGLGISMRSASVR. Synthetic peptides with

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the same sequence were synthesized and characterized for their antioxidant activity. Molecular docking studies revealed that potential antioxidant activity from both peptides resulted from the interaction of serine and threonine residues with free radicals. The current

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study suggested that natural peptides identified from finger millet have potent antioxidant

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activity and regarded as a promising source for a functional food ingredient. Keywords:

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Finger millet peptides, RP-UFLC, MALDI-TOF/TOF-MS/MS, Amino acid analysis, Scanning electron microscopy, Molecular docking Abbreviations:

Finger millet (FM), finger millet flour (FMF), finger millet protein isolate (FMPI), finger millet protein hydrolysate (FMPH), purified peptide (PP), TSSSLNMAVRGGLTR (PP1), STTVGLGISMRSASVR (PP2), scanning electron microscopy (SEM), ultrafiltration (UF), gel filtration (GF) reverse phase - ultra fast liquid chromatography (RP-UFLC), matrixassisted laser desorption ionization-time-of- flight- mass spectrometry (MALDI- TOF/TOF-

ACCEPTED MANUSCRIPT MS/MS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2’-azinobis-(3-ethyl-benzothiazoline-6-

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sulphonate) (ABTS)

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1. Introduction In human diets, cereal grains such as wheat, rice, barley, rye, oat, millet and corn have been used as a staple food since ancient times. Cereals are the most important food crop for several

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populations around the world and serve as a good source of natural antioxidants. An abundance of scientific evidence suggests that consuming whole grains cereals prevents

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chronic diseases such as cancer, cardiovascular disease and diabetes (Kaur, Jha, Sabikhi, &

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Singh, 2014). Among other cereals, millets are placed sixth, accounted for 1.3% of entire cereal production (Devi, Vijayabharathi, Sathyabama, Malleshi, & Priyadarisini, 2014).

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Millets are nutrient-dense food and gluten-free among cereals; therefore, it can be used for

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the production of gluten-free foods and beverages for patients with gluten sensitivity (Amadou, Gbadamosi, & Le, 2011). Earlier, it has been reported that millets are not only rich sources of phenolic compounds but it also contains beneficial proteins (Kamara, Amadou, &

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Zhou, 2012). Protein from finger millet contains an adequate amount of essential amino acids

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required by adult humans but moderately deficient in lysine (Tumkur, Ramachandra, & Nagaraju, 1975). Finger millet is particularly precious as it contains the amino acid

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methionine, which is deficient in the diets of hundreds of millions of the poor who live on starchy staples for example cassava, plantain, polished rice or maize meal (Kumar et al., 2016). Protein structure of the cereals mainly contributes to its functional and nutritional properties. Other than cereals, dietary protein from different food sources have been studied to identify bioactive peptides. Bioactive peptides found in legumes and dairy products play a significant role in chronic disease prevention; although there is very limited research which connects cereal grains with potential bioactive peptides (Cavazos & Mejia, 2013). Bioactive peptides range from 2-20 amino acids, are encrypted within the parental protein and released by the action of food processing (by fermentation or enzymatic hydrolysis) or gastrointestinal

ACCEPTED MANUSCRIPT proteolysis to achieve their specific “bioactive” roles (Harnedy & FitzGerald, 2012). Enzymatic hydrolysis is one of the safest, fastest and most controllable processes to generate antioxidant peptides using the proteolytic enzymes such as trypsin, pepsin, pancreatin and alcalase. At present, several antioxidant peptides had been extracted and isolated from different cereal protein sources, such as wheat protein (Chen, Lin, Gao, Cao, & Shen, 2017),

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rice endosperm protein (Wang, Chen, Fu, Li, & Wei, 2017), peanut kernels (Hwang, Shyu,

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Wang, & Hsu, 2010), soy protein (Park, Lee, Baek, & Lee, 2010), buckwheat protein (Ma,

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Xiong, Zhai, Zhu, & Dziubla, 2010), pearl millet (Agrawal, Joshi, & Gupta, 2016), foxtail millet (Amadou, Le, Amza, Sun, & Shi, 2013), green sorghum (Agrawal, Joshi, & Gupta,

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2017) and corn gluten meal (Zhuang, Tang, & Yuan, 2013) but peptides from finger millet are still not identified and are unexplored. Earlier studies have been done to explore its

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nutrient and anti-nutrients, phenolic acid and phytochemical compound in the different variety of finger millet (Thippeswamy, Junna, & Shinde, 2016). The present study deals with

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the use of a chromatography technique used to fractionate, purify and characterise the peptides. Moreover, peptides were synthesised with the same mas to confirm the antioxidant

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activity. The molecular interaction mechanism between identified peptides and DPPH and ABTS free radical was also proposed according to results analysed from molecular docking

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using structure-activity relationship. 2. Materials and methods 2.1.Materials

Finger millet seeds were procured from a local market of Palampur, Himachal Pradesh, India. Trypsin from porcine pancreas (E.C. 3.4.21.4), pepsin (E.C. 3.4.23.1), 1,1-diphenyl-2picrylhydrazyl (DPPH), 2, 2’-azinobis-(3-ethyl-benzothiazoline-6-sulphonate) (ABTS), bradford reagent, o-phthaldialdehyde (OPA), sephadex G-25, ἀ-cyano-4-hydroxycinnamic acid, trifluoroacetic acid and trichloroacetic acid were purchased from Sigma-Aldrich India.

ACCEPTED MANUSCRIPT Methanol, hydrochloric acid, ethanol, hexane, formic acid and acetonitrile (HPLC grade) were purchased from Merck India Pvt Ltd. Ferrozine, hydrogen peroxide, ferrous chloride and ferric chloride were purchased from Himedia. The synthetic peptides Thr-Ser-Ser-SerLeu-Asn-Met-Ala-Val-Arg-Gly-Gly- Leu-Thr-Arg

and

Ser-Thr-Thr-Val-Gly- Leu-Gly-Ile-

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Ser-Met-Arg-Ser-Ala-Ser-Val-Arg were also obtained from Sigma-Aldrich India.

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2.2.Preparation of finger millet protein isolate and hydrolysates

Initially, finger millet flour (FMF) was obtained by milling the whole seeds in a

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laboratory scale cutting mill (Retsch, SM-100), screened by 0.250 mm mesh and defatted by

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hexane extraction at the ratio of 1: 2 (w/v) for 4 h at room temperature. Finger millet protein isolate (FMPI) was prepared according to the method reported by Najafian & Babji, 2014.

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Defatted FMF was soaked in phosphate buffer (0.02 mol/L, pH 6.5) at the ratio of 1:100 (w/v) and homogenized (IKA T10 basic) for 1 h at room temperature. Afterwards, the

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solution was centrifuged (Eppendorf) at 13,000 rpm for 20 min at 4°C and the supernatant was collected followed by filtration using a filter (0.22 µm) (Merck Millipore, India). FMPI

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was then lyophilized and kept at -20°C for further analysis. FMPI was digested by two enzymes separately using trypsin (from porcine pancreas) and

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pepsin (from porcine gastric mucosa). Lyophilized FMPI was dispersed in Milli-Q water at a proportion of 1: 25 (w/v) and placed into the water bath for 10 min at 80°C to denature the protein. After cooling to room temperature the reaction mixture was adjusted to optimum conditions (based on the manufacturer recommendations) (Fan, He, Zhuang, & Sun, 2012) for the respective enzymes used (Trypsin- pH 8.5, 37°C, 3h, E: S 1:100 (15U/mg protein) and Pepsin- pH 2.0, 37°C, 6h, E: S 1:100 (2.5U/mg protein)). Enzymes were added with continuous stirring to initiate the hydrolysis. Enzyme inactivation was performed by heating the mixture at 80 °C for 10 min in boiling water. The supernatant was collected by

ACCEPTED MANUSCRIPT centrifugation at 4000 rpm for 20 min followed by lyophilization and stored at -20°C for further use. 2.3.Degree of hydrolysis (DH) The degree of hydrolysis was determined using the previously described method of Chi,

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Wang, Wang, Deng, & Ma, 2014. Hydrolysed protein (50 μL), phosphate buffer (500 μL, 0.2

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M, pH 8.2) and TNBS reagent (500 μL, 0.05 %) was mixed properly. TNBS was freshly prepared by diluting with deionized water before use. After incubating the mixture for 1 h at

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50 °C in a circulating water bath (VELP Scientifica GDE), 0.1 M HCl (1 mL) was added to

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the reaction mixture to stop the reaction. Again mixture was incubated for 30 min at room temperature and absorbance was recorded at 420 nm using a Kinetic Bio spectrometer

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Eppendorf. FMPH was completely hydrolyzed with 6M HCl in a sample to acid ratio of 1: 100 at 120 °C for 24 h to determine the total amino acid content. L- Leucine was used as a

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standard and DH (%) was calculated as Degree of hydrolysis (%) = At – A0 / Amax – A0 x 100. Where At was the amount of α-amino acids released at time t, A0 was the amount of α-amino

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acids in the supernatant at 0 h, and Amax was the total amount of α-amino acids obtained after acid hydrolysis at 120 °C for 24 h.

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2.4.Determination of peptide content Peptide content was determined using the method of Zhu et al., 2013 with some modification. Briefly, sample (50 µL) was mixed with o-phthaldialdehyde (2 mL) [50 mL of OPA mixture containing 100 mM sodium tetraborate (25 mL), 20% w/w sodium dodecyl sulphate (2.5 mL), OPA (40 mg) dissolved in methanol (1 mL), β- mercaptoethanol (100 µL) and distilled water (21.4 mL)]. Sample mixture was incubated for 20 min at room temperature and absorbance was read at 340 nm. A standard curve constructed by using L-

ACCEPTED MANUSCRIPT glutathione to calculate the peptide content and calculated as Peptide content (%) = (peptide contents / protein content) x 100 2.5. Purification of the antioxidant peptides from FMPH FMPH was purified using ultrafiltration (UF), gel filtration (GF) followed by reverse

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phase- ultra fast liquid chromatography (RP-UFLC) as previously reported (Agrawal, Joshi, & Gupta, 2016). The first step of peptide purification after hydrolysis is ultrafiltration. The

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lyophilized FMPH was dissolved in distilled water and fractionated by the ultrafiltration

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membrane system (Amicon ultra centrifugal filter units, Merck Millipore) with the molecular weight cut off (MWCO) 3 kDa and 10 kDa. The FMPH was first passed through the 10 kDa

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membrane to generate a retentate with MW>10 kDa (UF1). Afterwards, the 10 kDa permeate

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was passed through the 3 kDa membrane and two fractions of 3kDa retentate with MW 3-10 kDa (UF2), and 3 kDa permeate with MW<3 k Da (UF3) were collected. All fractions were

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lyophilized for further analysis and purification.

After ultrafiltration, the fraction with the highest antioxidant activity was re-dissolved in

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distilled water and further purified using a gel filtration chromatography. A sample with the concentration of 50 mg /mL, loaded on to the column (2 x 30 cm dimension) packed with

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Sephadex G-25. Elution was carried out with 0.01 N hydrochloric acid with constant flow rate. Each fraction was collected based and monitored at 280 nm and lyophilized. Reverse phase ultra- flow liquid chromatography (RP-UFLC) (Shimadzu Prominence UFLC system, Shimadzu Technologies Co. Ltd., Japan) with a Betabasic-18 (100 x 0.32 mm, 5 µm) column, UV/VIS detector (SPD-20A), autosampler (SIL-20AC), pump (LC-20AD), column oven (CTO-20AC) was used for further purification. Elution was carried out with a linear gradient followed in Agrawal, Joshi and Gupta, (2016). The elution peaks were monitored at a UV wavelength of 280 nm. The active fraction was collected and lyophilized for further determination of amino acid sequence and molecular mass of purified peptides.

ACCEPTED MANUSCRIPT 2.6.Identification of peptides sequence by MALDI-TOF/TOF-MS/MS The amino acid sequence of the purified peptides with the highest antioxidant activity was determined using a MALDI-TOF/TOF-MS/MS (Bruker, Ultraflextrame, USA) as previously reported (Agrawal, Joshi, & Gupta, 2016). Fractions with the higher antioxidant

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activity, collected from RP-UFLC was loaded on an Anchor Chip ATP plate. MALDI matrix α-cyano-4-hydroxycinnamic acid (0.5 µL) (20 mg/mL in 0.1 % trifluoroacetic / 30 %

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acetonitrile (1: 2)) was mixed with the each loaded fraction and dried at room temperature.

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MALDI- ToF Proteomics Analyzer (Ultrafle Xtreme TM mass spectrometer; Bruker Daltonics Inc. Germany) was used for the mass spectrometric analysis of the peptides. A

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standard tryptic BSA digest (Bruker DaltonicsInc, Germany) and mass standard starter kit

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(Bruker DaltonicsInc, Germany) was used to calibrate the system. BioTools 3.0 software (Bruker DaltonicsInc, Germany) was used to perform LIFT-MS/MS and a combined MS. Positive ion reflector mode with a mass range from 700 to 3500 Da was used to record the

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TOF spectra. For each spectrum, five hundred shots were collected. To determine the peptide

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sequence, fragmentation analysis was applied for the two most abundant peptide ions. All peptide masses were assumed monoisotopic and [M+H]+. Peptide identification was

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performed by a database driven approach using Matrix Science (Mascot software, Sequences: 70861097, Residues: 25660401164). 2.7.Synthesis of purified peptides and their antioxidant activity In order to validate the antioxidant activity of the purified peptides, synthetic peptides with the same sequence were chemically synthesized and tested. The purified peptides were synthesized by Sigma-Aldrich, India with the final purity of ≥95.4 %. The purity was determined by Sigma-Aldrich using HPLC and LC-ESI-MS. The antioxidant activity of both synthesized peptides was also determined.

ACCEPTED MANUSCRIPT 2.8.Determination of free amino acid composition using ultra performance liquid chromatography (UPLC) The free amino acid analysis was performed on Waters Acquity UPLC-H class system equipped with eʎ photodiode array detector (PDA), an autosampler, 600 controller™pump

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with online degasser, column heater and a binary solvent manager. Water BEH C18 column (2.1 x 100 mm, 1.7 µm particle size) was used fitted with a suitable guard column. Mobile

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phase A was sodium acetate (0.14 mol/L) containing triethylamine adjusted to pH 6.7 with

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glacial acetic acid and methanol (90:10). Mobile phase B was 60% acetonitrile. Gradient program was 0 min A 100%, 0.5 min A 95%, 6.5 min A 47% and 12 min A 100% at a flow

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rate of 0.2 mL min-1 . The injection volume was 1 µL. Before injection, samples were

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derivatized with O- phthalaldehyde and passed through a 0.45 µm Millipore membrane syringe filter. Free amino acids in each sample were monitored at 254 nm and quantified with

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calibration curve using amino acid mix (Sigma Aldrich, India). 2.9.Determination of the antioxidant activities

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Antioxidant activities were determined using ABTS, DPPH, metal-chelating, and hydroxyl radical scavenging activity as previously reported by Agrawal, Joshi, & Gupta,

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2016. Percent inhibition of each free radical was calculated for the FMPI, FMPH, UF, GF and RP-UFLC fractions. Expression of percent inhibition of DPPH radical by synthetic peptides was also determined. 2.10.

Scanning electron microscopy (SEM)

Morphological characteristic of the FMPI, FMPH (Trypsin and Pepsin), PP1 (TSSSLNMAVRGGLTR) and PP2 (STTVGLGISMRSASVR) was determined by using a scanning electron microscope (S-3400N, Hitachi, Japan). Briefly, samples were fixed on the aluminium stub containing a double-sided adhesive carbon tape. A sputter (E1010 ion sputter

ACCEPTED MANUSCRIPT Hitachi, Japan) was used to make all samples conductive by gold coating at 10 Pa vacuum for 20 sec. The electron microscope was operated using the following conditions: accelerating voltage, 15 kV; sample distance, 18-23 mm; tilt angle, 0°; and objective aperture, 10 µm. Samples were examined at 500× magnification. Molecular docking of antioxidant peptides on ABTS and DPPH binding site

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2.11.

The 3D structure of the ligand molecules (DPPH and ABTS) was retrieved from

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PubChem (https://pubchem.ncbi.nlm.nih.gov). The PubChem CID of ABTS is 5360881 and

was

constructed

using

I-TASSER

software

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STTVGLGISMRSASVR)

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for DPPH is 2735032. The structure of the purified peptides (TSSSLNMAVRGGLTR and

(https://zhanglab.ccmb.med.umich.edu/I-TASSER/). Peptides and ligands were prepared

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using the AutoDockTools software (http://autodock.scripps.edu/) by the addition of atom types, partial charges, and polar hydrogen atoms. The ligand rigid root was generated

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automatically and all possible rotatable bonds and torsions were defined as active. Grid maps were generated with 0.375 Å spacing and dimensions of 60 x 60 x 60 points by the AutoGrid

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program (Morris et al., 1998). AutoGrid (version 2.2, Autodock, Autogrid) has been used for making gridbox, where peptide was docked with ligand ABTS and DPPH, respectively. To

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study the binding site between them, the AutoDock 4.2 program was used and the Lamarckian genetic algorithm (LGA) was applied for minimization using default parameters. Hundred different conformers were generated for each docking simulation. The best pose was selected based on lowest binding energy among peptide and ligand docking and also concern the major cluster to select. Pymol software was used to visualize the docked confirmation and to locate the binding interaction between peptides and ligands (W.L. Delano, PyMOL software, version 0.99, USA, San Carlos, 2004). 2.12.

Statistical analysis

ACCEPTED MANUSCRIPT All analytical values were conducted in three independent determinations. Data were represented as the mean ± standard deviation. One-way analysis of variance (ANOVA) followed by Duncan’s multiple range test was performed to analyze data using the SPSS 19.0 statistical software program. Differences between the means of parameters were co nsidered significant at p < 0.05.

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3. Results and discussion

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Previous studies on millet reported that foxtail millet, pearl millet, Barnyard millet and

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proso millet contain potential antimicrobial, antioxidant and antibacterial peptides (Amadou, Le, Amza, Sun, & Shi, 2013; Agrawal, Joshi, & Gupta, 2016; Bisht, Thapliyal, & Singh,

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2016). Finger millet has been reported for its high nutritional value. It has been also identified as a rich source of amino acids such as phenylalanine, leucine, isoleucine and methio nine

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(Chandra, Chandra, Pallavi, & Sharma, 2016). In the current study, for the first time finger millet used to discover natural products in the form of antioxidant peptides. Trypsin and

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pepsin hydrolysate of the crude protein isolate fro m finger millet was prepared in order to generate antioxidant peptides. Furthermore, the hydrolysate with the higher degree of

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hydrolysis and antioxidant activity was fractionated using the different chromatographic route.

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3.1.Antioxidant activity of FMPI, FMPH, and hydrolysate with different molecular weight distribution

To obtain protein hydrolysate with desirable bioactive and functional properties, selection of suitable enzyme and digestion conditions are essential for the best action of the enzyme, including temperature and pH, enzyme-to-substrate ratio and hydrolysis time (Samaranayaka & Li-Chen, 2011). For the production of peptides with antioxidant activity, FMPI was separately digested by the sequential action of the digestive enzymes such as trypsin and pepsin. The degree of hydrolysis (DH) assay was used to estimate the extent of protein

ACCEPTED MANUSCRIPT degradation by proteolytic enzymes. There was a significant difference found in the degree of hydrolysis of both peptides as 17.47 ± 0.23 % and 13.73 ± 0.18 % for trypsin and pepsin, respectively (P < 0.05). An earlier study reported that squid muscle hydrolysed with αchymotrypsin, trypsin and pepsin. Tryptic hydrolysate showed the highest degree of hydrolysis and inhibition of linoleic acid oxidation (Rajapakse, Mendis, Byun and Kim,

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2005). The peptide content of FMPH (Trypsin) and FMPH (pepsin) was also measured to

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determine the generated peptides during the hydrolysis process. Trypsin hydrolysate

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possessed significantly higher peptide content (2.14% ± 0.15) as compared to pepsin hydrolysate (1.53% ± 0.03) (P < 0.05). Hence, the trypsin hydrolysate with higher peptide

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content was selected for further purification. Antioxidant activities were measured using DPPH, ABTS, hydroxyl radical scavenging activity and metal-chelating assay. The free

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radical scavenging activity of FMPI, FMPH (Trypsin and pepsin) and different molecular weight distribution (UF1, UF2, and UF3) were compared (Figure 1A). Antioxidant activity

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of FMPI was significantly increased after digestion. This is because the hydrolysis process enhances the capacity of peptides to scavenge free radicals. Furthermore, it has been reported

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that a partially hydrolyzed protein chain could act as a hydrogen donor due to the higher exposure of amino acid residue (Sbroggio, Montilha, Figueiredo, Georgetti, & Kurozawa,

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2016). Trypsin digested hydrolysate showed significantly higher DPPH (38.41 ± 0.02 %), ABTS (40.15 ± 0.19 %) and metal chelating (35.11 ± 0.05%) activity as compared to pepsin digested hydrolysate and FMPI (P < 0.05). There was no significant difference found in the hydroxyl radical scavenging activity of trypsin hydrolysate (33.01 ± 0.12%) and pepsin hydrolysate (30.18 ± 0.04%) (P > 0.05) but it was found to be significantly higher as compared to FMPI (P < 0.05) (Figure 1A). Subsequently, small peptides from FMPH (trypsin) were separated using an ultrafiltration process to obtain hydrolysate with better antioxidant activity. Results demonstrated that fraction with MW <3 kDa (UF3) at the

ACCEPTED MANUSCRIPT concentration of 1 mg/mL possess significantly higher DPPH radical scavenging activity with 48.41 ± 0.02 % of inhibition as compared to the FMPI, FMPH and other UF fractions (P < 0.05). ABTS radical scavenging activity was also increased after separation and UF3 showed the higher ABTS radical scavenging activity with 42.17 ± 0.12 % of inhibition at the same concentration as compared to other fractions (P < 0.05). Similar activity was also reported by

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Zhuang, Tang, & Yuan, 2013 that peptides with MW < 3 kDa possess higher free radical

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scavenging capacity as compare to other fractions. At a concentration of 1mg/mL metal-

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chelating ability of UF3 was also found to be significantly higher with 51.2 ± 0.11 % as compared to FMPI, FMPH, UF1 and UF2 (P < 0.05). This could be due to the peptide

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cleavage resulted in binding of Fe2+ with amino and carboxyl groups in their side chains (Zhang, Xiao, Himali, Lee, & Ahn, 2010). There was no significant difference found in the

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metal-chelating ability of UF1 and UF2 fractions at 1 mg/mL concentration but was found to be significantly higher than FMPI and FMPH. Hydroxyl radical is the one of the most diverse

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ROS and destructive one as it reacts with biological macromolecules such as DNA and protein. At 1 mg/mL concentration, OH radical scavenging activity of UF2 and UF3 was

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found to be significantly higher with 42.22 ± 0.05 % and 43.17 ± 0.18 % inhibition observed respectively as compared to FMPI, FMPH and UF1 (P < 0.05). Results are supported by the

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previous study which showed that 1-3 kDa fractions possessed highest free radical scavenging activity against different free radical such as hydroxyl, DPPH, superoxide and peroxyl (Wang, Ding, Wang, Zhang, & Liu, 2015). Antioxidant activity of these peptides is increased upon digestion and separation into low MW peptide fractions because low molecular weight peptides can easily react with free radical and terminate the radical chain reaction (Lobo, Patil, Phatak, & Chandra, 2010). 3.2.Antioxidant activity of peptide separated by gel-filtration chromatography

ACCEPTED MANUSCRIPT Gel filtration chromatography is a most widely used technique to separate components in a mixture by their size and remove other salts and impurities from the mixture (Chi, Wang, Wang, Deng, & Ma, 2014). As shown in Figure 1B UF3 (<3 kDa) fraction with the higher antioxidant activity was subsequently purified into five sub- fractions (GFA, GFB, GFC, GFD, and GFE) using column packed with Sephadex G-25. Among the five fractions, GFB fraction

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exhibited significantly higher DPPH radical scavenging activity (61.79 ± 0.06 %), ABTS

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radical scavenging activity (78.61 ± 0.28 %) and metal-chelating ability (51.20 ± 0.19 %) as

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compared to other fractions at concentration of 1.0 mg/mL (P < 0.05) (Figure 1B). There was no significant difference found between hydroxyl radical scavenging ability of GFA (64.76 ±

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0.21%), GFB (66.66 ± 0.27 %) and GFC (63.80 ± 0.04) at concentration of 1 mg/mL (P > 0.05) (Figure 1B) but was found to be higher as compared to other fractions GF D and GFE at

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the same concentration (P < 0.05) (Figure 1B). Earlier, it has been reported that the antioxidant ability of the peptide was closely related to the MW distribution (Onuh, Girgih,

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Aluko, & Aliani, 2014). Peptide with low molecular weight could interact more effectively

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with the radical in the oxidizing process (He, Girgih, Malomo, Ju, & Aluko, 2013). 3.3.Antioxidant activity of peptide separated by RP-UFLC

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Currently, RP-UFLC has become the most common technique for the separation of peptides owing to its high resolution, high speed, high sensitivity and good reproducibility and considered as the final step in the peptide purification (He, Girgih, Malomo, Ju, & Aluko, 2013). RP-UFLC is used to separate compounds based on hydrophilic and hydrophobic character, the hydrophilic or large polar peptides eluted first then the hydrophobic or nonpolar peptides followed (Zhang, Mu, & Sun, 2014). Fraction GFB with the highest free radical scavenging activity was further separated by RP-UFLC on a C18 column, of which four peaks (GFB1 , GFB2 , GFB3 , GFB4 ) were obtained (Figure 1C) and determined for its DPPH

ACCEPTED MANUSCRIPT radical scavenging activity. As shown in Figure 1C, among all fractions, fraction GFB3 possessed significantly higher DPPH radical scavenging activity (73.27 ± 0.37%) as compared to fraction GFB1 (56.58 ± 0.21%), GFB2 (49.54 ± 0.17) and GFB4 (67.27 ± 0.11) at 1.0 mg/mL concentration (P < 0.05). Results also demonstrated that DPPH radical scavenging activity of GFB3 was significantly higher as compared to FMPI, FMPH (trypsin

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and pepsin) UF fractions and GF fractions (P < 0.05). In our previous study Agrawal, Joshi,

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& Gupta, 2016, we also reported that antioxidant peptide fraction from pearl millet purified

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on the C18 column, eluted last and possess higher antioxidant activity than other fractions. Above mentioned results demonstrated that free radical scavenging activity of peptide was

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not only related to the size but also to the hydrophobic nature of the peptides.

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3.4. Free amino acid composition of FMPH fractions

FMPH digested with trypsin with a higher degree of hydrolysis was further separated

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based on their molecular weight and analysed for their free amino acid composition. The major amino acid was Ser in all fractions <3kDa (236.88mg/g), 3-10kDa (226.47 mg/g) and

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>10kDa (271.95 mg/g) (Table 1). The ratio of hydrophobic amino acid was found to be higher in fraction FMPH <3kDa and 3-10kDa compared to >10kDa (103.44, 103.41 and

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84.43 mg/g, respectively). These fractions are rich in hydrophobic amino acids (Val, Leu, and Trp) which contribute to its potential antioxidant activity (Wiriyaphan, Chitsomboon, & Yongsawadigul, 2012). Among aromatic amino acid, only Tyr is found in all fractions. It was significantly higher in fraction <3kDa (52.04 mg/g) as compared to 3-10kDa (34.96 mg/g) and >10kDa (30.51 mg/g) (p<0.05). According to Rajapakse, Mendis, Jung, Je, & Kim, 2005 aromatic amino acid convert free radical into the stable molecule by transferring their electrons. In the present study, trypsin hydrolysate possesses higher hydrophobic amino acids possibly due to the degradation of the peptide bond of hydrophobic or aromatic amino acids. Earlier, it has been suggested that low molecular weight peptides react easily with free radical

ACCEPTED MANUSCRIPT and convert it into the stable product by terminating the chain reaction (Li et al., 2013). Therefore, peptides with low molecular weight are the main contributor to the antioxidant activity of FMPH. 3.5.Determination of amino acid sequence of the antioxidant peptide by MALDI-

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TOF/TOF-MS/MS Antioxidant peptides are significantly influenced by molecular structure and mass (Zou,

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He, Li, Tang, & Xia, 2016). Sarmadi & Ismail, 2010 reported that peptides with low

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molecular weight possess free radical scavenging capacity have always been more effective in vitro. The amino acid sequence and molecular mass of the antioxidant peptides purified

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from RP-UFLC (fraction GFB3 and GFB4 ) were determined by MALDI-TOF/TOF-MS/MS.

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Table 2 represented the characteristic features of the two novel peptides, including their calculated mass/observed mass, net charge, hydrophobic ratio, protein accession number

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from Mascot database, species and the antioxidant capacity of the synthesized peptide with same sequences. Figure 2 (A & B) demonstrate the mass spectra and MS/MS analysis of the

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two purified peptides. The amino acid sequence of fraction GFB3 was identified as Thr-SerSer-Ser-Leu-Asn-Met-Ala-Val-Arg-Gly-Gly-Leu- Thr-Arg

(TSSSLNMAVRGGLTR)

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(MASCOT protein accession number = gi|170522269, UDP-glucuronic acid 4-epimerase) with calculated mass 1564.7991 Da, which was closely related to the observed mass with 1566.1993 Da (M+H+ ). Similarly, fraction GFB4 was identified as Ser-Thr-Thr-Val-Gly- LeuGly-Ile-Ser-Met-Arg-Ser-Ala-Ser-Val-Arg (STTVGLGISMRSAS VR) (MASCOT protein accession number = gi|460385567, PREDICTED: uncharacterized protein LOC101247521) with calculated mass 1636.8567 Da and observed mass 1638.2910 Da (M+H+). The deviation between the calculated mass and observed mass of both sequences was deemed acceptable. Peptides with specific bioactivity contain 2-20 amino acid residue and can cross the intestinal barrier to exert their effect at the tissue level (Sarmadi & Ismail, 2010). The hydrophobic

ACCEPTED MANUSCRIPT ratio of the peptides TSSSLNMAVRGGLTR and STTVGLGISMRSASVR were found at 46.66% and 50%, respectively. It is reported that hydrophobicity increases the solubility of the peptides in lipid medium and support the accessibility to hydrophobic radical species. Lipid inhibitory activity also enhanced by the interaction of peptide and free radical species (Siow & Gan, 2013).

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Furthermore, two identified peptides were synthesized with same sequences to confirm

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their antioxidant activity, for which the DPPH radical scavenging activity was 80.55 ± 0.93%

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and 75.11 ± 2.08 % at 1.0 mg/mL for TSSSLNMAVRGGLTR and STTVGLGISMRSASVR, respectively suggesting that TSSSLNMAVRGGLTR possessed the highest radical

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scavenging activity. Antioxidant activity is associated with the sequences and presence of specific amino acids. The structural action bond of antioxidant peptides up till now has been

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completely recognized and may be incorporated into functional foods as original antioxidant ingredients or as finger millet protein based value-added products.

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3.6.Morphological determination by SEM

Changes in microstructure of protein powder after hydrolysis were analyzed using SEM.

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Figure 3 represented the morphology of FMPI, FMPH (trypsin), FMPH (pepsin), PP1 and PP2. Results demonstrated that after enzymatic treatment protein has degraded into small

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fragments. Due to the reduction in particle size, FMPH (trypsin) (Figure 3B) and FMPH (pepsin) (Figure 3C) showed a smoother matrix compared to FMPI (Figure 3A) showing the aggregates of packed flake- like structures under the same parameter (Mag = 500x; AV = 15 kV). Results are also supported by the previous study (Radha, Kumar, & Prakash, 2007) which showed the structural changes in oilseed protein after enzymatic hydrolysis. The difference found in the morphology of the purified peptides due to the purification process. Both peptides resulted in a smooth cotton like structure compared to FMPH trypsin and pepsin (Figure 3D & 3E).

ACCEPTED MANUSCRIPT 3.7. Mechanism of interaction with DPPH and ABTS radical using molecular docking In silico analysis of the interaction between antioxidant peptides and free radicals has not been clearly established yet but it has been reported that peptides effectively interact with free radicals to neutralize them (Hu, Chi, Wang, & Deng, 2015). The study of the interaction mechanism between free radical and antioxidant peptides have many potential applications to

between

antioxidant

peptides

(TSSSLNMAVRGGLTR

and

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simulation

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design and synthesize their derivatives. AutoDock tool was used to study the docking

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STTVGLGISMRSASVR) and the free radicals (DPPH and ABTS). All bound cofactors, ligands and water molecule were removed from the protein, prior to stimulation. Binding free

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energy of a given inhibitor conformation in the macromolecular structure was calcula ted using Auto Dock. It evaluates how small molecule (inhibitor, substrate, drug or drug

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candidate) and the target macromolecule (enzyme, receptor or nucleic acid) fit together which is useful for understanding the nature of binding and developing better drug candidate.

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Therefore, in order to explain the in silico antioxidant study of the peptide, molecular docking study was carried out. Peptide TSSSLNMAVRGGLTR and STTVGLGISMRSASVR were

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identified as the target for the antioxidant compound. The important binding interaction of the actively docked conformations of the ligand with the target molecule (DPPH and ABTS) are

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identified one by one within the average of 2.0 Å of the active site of the target molecule. The binding interactions of identified peptides have shown strong hydrogen bonding and hydrophobic interactions with the target molecule. As shown in Figure 4 single hydrogen bond was formed between TSSSLNMAVRGGLTR and DPPH (Figure 4A1) while three hydrogen bond was formed during interaction with ABTS molecule (Figure 4A2). On the other hand, upon the interaction between STTVGLGISMRSASVR and DPPH (Figure 4B1), two hydrogen bond formed and a single hydrogen bond formed with ABTS interaction (Figure 4B2). It has been reported that inhibitors interact through the force of hydrophobic,

ACCEPTED MANUSCRIPT ven der Waals, hydrogen bonds and electrostatic interaction (Mirzaei, Mirdamadi, Ehsani, & Aminlari, 2017). Serine and threonine residue present in peptide sequence effectively form a hydrogen bond with a target molecule (DPPH and ABTS). Earlier it has been reported that Lserine has antioxidant and cytoprotective effect as it elevated the antioxidant factors such as Nrf2, HO-1 and NO (Movahedian, Haghjooy, & Javanmard, 2012). From the current study, it

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suggested that amino acid residues present in peptides effectively interacted with free radical

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3.8.Structure-activity relationship of antioxidant peptides

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molecules resulted in the contribution to the strong free radical scavenging activity.

The structural analysis of antioxidant peptides helps to evaluate the food derived protein

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as a potential precursor of antioxidant peptides (Sila & Bougatef, 2016). Peptides are highly

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dependent on their composition and amino acid sequence to possess potent antioxidant activity. Earlier, it has been reported that glycine (Gly) residue contain single hydrogen atom

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in the side chain provide high flexibility to the peptide backbone (Nimalaratne, Bandara, & Wu, 2015) and neutralizes active free radical species by donating a proton thus contribute to

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antioxidant activity (Chen, Chi, Zhao, & Xu, 2012). As shown in Figure 5 both peptides contain two glycine residues in their amino acid sequence which might be an important

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contributor to the antioxidant activity of peptides. Hydrophobic amino acids such as leucine (Leu), Alanine (Ala) and Valine (Val) are reported for the potential antioxidant activity of peptides (Sila & Bougatef, 2016). Therefore, the presence of two Leu, one Ala residue in PP1 and two Val, one Ala residue in PP2 should have a positive impact on their antioxidant activity (Figure 5). Acidic (Asp and Glu) and basic (Arg) amino acids contain carboxyl and amino groups in their side chains have been reported to be critical to the metal ion chelating activity of the peptide (Gimenez, Aleman, Montero, & Gomez-Guillen, 2009). PP1 and PP2 contain two residues of Arg in their sequences which might be favourable to their antioxidant activity. The peptide chain was also found to possess repeating amino acid s. For example,

ACCEPTED MANUSCRIPT repeating dipeptides that constitute the same amino acid residuals such as Gly-Gly; Thr-Thr and Ser-Ser-Ser are found as the module in the peptide sequence. According to Siow & Gan, (2013), some di- and tri-peptides have revealed superior biological action in contrast to their constituent amino acids and could be absorbed more quickly than free amino acid.

In

the

current

study,

two

novel

peptides

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4. Conclusions TSSSLNMAVRGGLTR

and

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STTVGLGISMRSASVR were isolated, for the first time, from finger millet (Eleusine

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coracana) protein hydrolysate by trypsin enzyme using ultrafiltration, gel filtration and RPUFLC chromatographic methods. Antioxidant activities were significantly increased after

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digestion and purification process. These two peptides possessed strong free radical

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scavenging activity due to the presence of aromatic and hydrophobic amino acids in their sequences and low molecular weights. Morphological changes were observed after digestion

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and purification of the peptides. Molecular docking study showed that the peptide effectively interacts with the free radical by forming hydrogen bonds, hydrophobic, and electrostatic

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interactions which responsible for the free radical inhibition. From the above- mentioned results, it can be concluded that these two peptides with high antioxidant activity may be used

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as a natural antioxidant or functional ingredients for the development of food product or prevention of the oxidation of related food. The further studies are required to determine its cellular activity and in vivo effects in the animal model. Acknowledgements Researchers express their deep gratitude to the Director, CSIR-Institute of Himalayan and Bioresource Technology for their encouragement to conduct the study. The authors gratefully acknowledge Dr Vishal Acharya for docking analysis.

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ACCEPTED MANUSCRIPT Zou, T. B., He, T. P., Li, H. B., Tang, H. W., & Xia, E. Q. (2016). The structure-activity

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ACCEPTED MANUSCRIPT Figure 1. Antioxidant activities of (A) Finger millet protein isolate, tryptic hydrolysate, pepsin hydrolysate and ultrafiltration fractions, (B) Gel- filtration chromatography fractions and (C) Reverse phase ultra-flow liquid chromatography fractions. Figure 2. The MS/MS spectra, molecular mass and amino acid sequence of antioxidant peptides from finger millet tryptic hydrolysate. (A) TSSSLNMAVRGGLTR (B)

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STTVGLGISMRSASVR.

hydrolysate,

(D)

Peptide

TSSSLNMAVRGGLTR

and

(E)

Peptide

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Pepsin

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Figure 3. Scanning electron microscopy of (A) Protein isolate, (B) Tryptic hydrolysate, (C)

STTVGLGISMRSASVR from a finger millet. (Mag = 500x; AV = 15 kV)

with

DPPH

and

ABTS.

(A1)

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Figure 4. Molecular docking of antioxidant peptides from finger millet tryptic hydrolysate TSSSLNMAVRGGLTR

with

DPPH,

(A2)

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TSSSLNMAVRGGLTR with ABTS, (B1) STTVGLGISMRSASVR with DPPH and (B2) STTVGLGISMRSASVR with ABTS. Yellow capsules indicate hydrogen bond formation

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(Ball and stick model).

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hydrolysate fraction.

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Figure 5. Structure-antioxidant activity relationship of peptides from a finger millet tryptic

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Table 1. Free amino acid composition of ultrafiltration fractions of a finger millet tryptic hydrolysate. FMPH (<3 kDa) (mg/g)

FMPH (3-10 kDa) (mg/g)

FMPH (>10 kDa) (mg/g)

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Amino acid

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6.24 4.18 6.12 His Ser 236.88 226.47 271.95 Asp 44.35 61.15 53.21 Thr 12.24 17.84 10.88 Tyr 52.04 34.96 30.51 Val 12.78 14.04 10.01 Leu 9.56 22.77 21.25 Trp 81.10 66.60 53.17 HAAa 103.44 103.41 84.43 PCAAb 6.24 4.18 6.12 c NCAA 44.35 61.15 53.21 AAAd 52.04 34.96 30.51 a Combined total of hydrophobic amino acids (HAA) = Valine, leucine and tryptophan b Positively charged amino acids (PCAA) = Histidine c Negatively charged amino acids (NCAA) = Aspartic Acid d Aromatic amino acids (AAA) = Tyrosine

ACCEPTED MANUSCRIPT

Table 2. Determination of molecular mass and amino acid sequence of antioxidant peptides

GFB3

TSSSLNMAVRG GLTR

Calcula ted mass /observe d mass

Net char ge a

Hydr ophob ic ratio b (%) 46.66

Protein accessio nc

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Purified peptides

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Fracti on

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from a finger millet fraction.

Species

DPPH radical scavengi ng activity (%)d 80.55 ± 0.93

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+2 gi|170522 Boehmeri 1564.79 269 a nivea 91/ 1566.19 93 GFB4 STTVGLGISMRS 1636.85 +2 50 gi|460385 Solanum 75.11 ± ASVR 67/ 567 lycopersic 2.08 1638.29 um 10 a Net charge was calculated based on negatively charged amino acid (E and D) and positively charged amino acid (K, R and H) in the peptide sequence. b Calculated the percentage of hydrophobic residues (G, A, V, L, I, P, F, M and W) in the peptide sequence. c Predicted index from MASCOT Database of MALDI. d DPPH radical scavenging activity of the synthesized peptides was tested at 1.0 mg/mL.

ACCEPTED MANUSCRIPT Highlights 

FMPI was enzymatically hydrolyzed and purified using ultrafiltration, gel filtration and RP-UFLC



Amino acid sequences TSSSLNMAVRGGLTR, STTVGLGISMRSASVR identified

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using MALDI-TOF-MS/MS Antioxidant activity of purified peptides and synthetic peptides were evaluated



UPLC analyzed Ser as a major amino acid in all fractions <3kDa, 3-10kDa and

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

Docking studies revealed interactions between free radical and active amino acid

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residues

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

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>10kDa

Figure 1A

Figure 1B

Figure 2

Figure 3

Figure 4

Figure 5