Evaluation of head and broken rice of long grain Indica rice cultivars: Evidence for the role of starch and protein composition to head rice recovery

Journal Pre-proof Evaluation of head and broken rice of long grain Indica rice cultivars: Evidence for the role of starch and protein composition to head rice recovery

Amardeep Singh Virdi, Narpinder Singh, Priyanka Pal, Parmeet Kaur, Amritpal Kaur PII:

S0963-9969(19)30561-7

DOI:

https://doi.org/10.1016/j.foodres.2019.108675

Reference:

FRIN 108675

To appear in:

Food Research International

Received date:

22 May 2019

Revised date:

26 August 2019

Accepted date:

11 September 2019

Please cite this article as: A.S. Virdi, N. Singh, P. Pal, et al., Evaluation of head and broken rice of long grain Indica rice cultivars: Evidence for the role of starch and protein composition to head rice recovery, Food Research International (2019), https://doi.org/ 10.1016/j.foodres.2019.108675

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© 2019 Published by Elsevier.

Journal Pre-proof Evaluation of head and broken rice of long grain Indica rice cultivars: Evidence for the role of starch and protein composition to head rice recovery Amardeep Singh Virdi1, Narpinder Singh1*, Priyanka Pal1, Parmeet Kaur1, Amritpal Kaur1 1

Department of Food Science & Technology, Guru Nanak Dev University, Amritsar, Punjab-

143005, India *

Author for correspondence Email: [email protected] (N. Singh).

Highlights

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1) Head rice yield decreased with extended degree of milling (DoM).

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2) Extended DoM caused decrease in protein content, fat content in both HR and BR. 3) Peak viscosity and final viscosity of HR and BR was increased with extended DoM.

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4) MALDI-ToF/MS analysis revealed the absence or least accumulation of glutelin type-D 1 protein in BR.

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5) The differential accumulation of 60S ribosomal protein L10a, glutelin type-D 1

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(GLUD1_ORYSJ), pathogenesis-related protein 1 (Oryza sativa Japonica Group), and Oryza sativa 1-Cys peroxiredoxin A was also observed in HR and BR different Indica

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rice cultivars.

Journal Pre-proof Evaluation of head and broken rice of long grain Indica rice cultivars: Evidence for the role of starch and protein composition to head rice recovery Amardeep Singh Virdi1, Narpinder Singh1*, Priyanka Pal1, Parmeet Kaur1, Amritpal Kaur1 1

Department of Food Science & Technology, Guru Nanak Dev University, Amritsar, Punjab-

143005, India *

Author for correspondence Email: [email protected] (N. Singh).

Abstract Brown rice of different long-grain Indica cultivars was polished to variable degree of

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milling (DoM) to see the difference in proteins and starches characteristics in head (HR) and

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broken rice (BR). Study revealed differential accumulation of starch, fat and proteins in both HR and BR. Extended DoM of brown rice resulted in a progressive decrease in HR yield and

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increase in BR yield. The extended DoM caused a decrease in protein and fat content in both HR and BR, whereas; an increase in peak viscosity and final viscosity was observed. On the

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contrary, the setback viscosity of HR and BR of different rice cultivars was influenced by

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cultivars and extended DoM. Milled rice from different cultivars milled to 6% DoM showed higher levels of 59 kDa, 54 kDa, 51 kDa, 32 kDa, 31 kDa, 30 kDa, 28 kDa, 24 kDa, 23 kDa, 15 kDa, 13 and 12 kDa PPs, while 28 kDa, 24 kDa, 23 kDa, 15 kDa, 13 kDa and 12 kDa PPs was

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the least or not observed in BR. The major quantitative changes were observed in 28 kDa, 24 kDa, and 23 kDa PPs. MALDI-ToF/MS analysis revealed the identity of 28 kDa PP as 60S

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ribosomal protein L10a and glutelin type-D 1 proteins. Whereas, the identity of 24 and 23 kDa

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PP, respectively was established as pathogenesis-related protein 1 (Oryza sativa Japonica Group) and Oryza sativa 1-Cys peroxiredoxin A. HR showed the presence of highly condensed packaged starch granules with smooth edges, which were tightly imbibed in the proteins matrix. However, the inter-cultivar differences in the starch structure and packaging were also observed. On the contrary, BR revealed lesser accumulation of starch particles with abnormal protein filling and several fissures and cracks were also observed in the starch granules of BR of different cultivars. Keywords: Head rice, broken rice, RVA, protein, field-emission scanning electron microscopy, MALDI-ToF/MS.

Journal Pre-proof 1.0 Introduction The milling of rough results in two types of rice namely head rice (HR) and broken rice (BR). HR is widely consumed by peoples globally and HR yield is an important sign of rice quality. As per USDA (1983), the unbroken rice and at least three-fourths of an unbroken endosperm of rice are considered as HR. The production of rice raised 750 Mt paddy, however, the

annual

yield

of

milled

rice

(HR+BR)

remains

490

Mt

(http://faostat.fao.org/site/339/default.aspx) thus indicating a huge loss of HR during milling. The demand for quality rice is directly associated with the economic status of the peoples of the

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developing countries and it greatly influences the global market of rice. HR is directly associated with brown rice and milled rice yield. The moisture sorption during drying imposes stress and

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strains on rice endosperm, which results in the formation of fissures and cracks in the mature

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endosperm of rice (Buggenhout, Brijs, Celus, & Delcour, 2013). Whereas, the chalkiness is due to the presence of air pockets, fissures and cracks in the rice endosperm and is also associated

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with poor starch and protein synthesis. Rice of high chalkiness, large number of fissures and cracks showed lower HR yield (Chun, Song, Kim, & Lee 2009). Studies have shown that starch

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and protein composition, pre- and post-harvesting conditions greatly influence the HR yield (Siebenmorgen, Grigg, & Lanning, 2013; Sreenivasulu et al., 2015; Siebenmorgen, Counce, Lu,

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& Kocher, 1992; Siebenmorgen, Nehus, & Archer, 1998). Studies of Patindol and Wang, (2003) revealed that the chalky rice endosperm starch contained less amylose (more amylopectin) and

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more short branch-chain amylopectin (less long branch-chain amylopectin), as compared to starch from the translucent kernel of milled rice. Albumin and globulin stored at the outer

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regions while glutelins distributed towards the center of the rice endosperm. Conversely, prolamins are evenly distributed in the entire rice endosperm. The levels of globulins and albumins, but not the proportion of prolamins and glutelins, affect the grain hardness (Houstan et al., 1968). However, Balindong et al. (2018) reported that globulin, glutelins and prolamin positively correlated with head rice yield (HRY). Therefore, the levels and distribution of seedstorage proteins in rice endosperm not only affect the pasting properties but the grain harness also. The lower accumulation of starch, proteins, majority of 17 amino acids and contents of Mn, K and Mg in white core chalky grains of rice was also observed (Xi et al., 2016). More than 600 quantitative trait loci (QTLs) related to yield and grain quality have been reported (Song, Huang, Shi, Zhu, & Lin, 2007; Pinson, Jia, & Gibbons, 2013). Studies have shown that heat stress

Journal Pre-proof imposed a profound impact on the seed development, milling quality and sensory attributes of raw and cooked rice (Siebenmorgen, Grigg, & Lanning, 2013; Sreenivasulu et al., 2015). Relative humidity, the moisture content in endosperm and temperature of air used during drying process also influences the breakage of milled rice (Siebenmorgen, Counce, Lu, & Kocher, 1992; Siebenmorgen, Nehus, & Archer, 1998). Indian long-grain rice or basmati has a huge demand in the world market and rice-breeding program resulted in the development of several popular long rice grain cultivars with a strong aroma and high yield. Conversely, some of the cultivars were highly successful and accepted by a wide range of consumer worldwide. However, other

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cultivars were rejected by rice miller and consumers because of low HR yield, lack of golden

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color after parboiling and poor sensory attributes (Pal, Singh, Kaur, & Kaur, 2018). The objective of the present study was to identify possible reasons that led to more breakage during

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milling of brown rice through the evaluation of differences in the proximate composition, physicochemical, textural, protein characteristics of HR and BR of different long-grain rice

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cultivars.

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2.0 Materials and Methods 2.1 Materials

Indica rice cultivars PB1, PS44, PB1509, PB1121, and PS5 were procured from the

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Indian Agricultural Research Institute, Karnal. These cultivars were grown on loamy sand soil and harvested in the year of 2014.

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2.2 Dehusking

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The different paddy samples were subjected to a McGill sample sheller (Rapsco, Brookshire, TX, U.S.A.) for dehusking followed by milling in a McGill number 2 mill (Rapsco) to remove 6%, 8%, and 10% bran, as described by Singh et al. (2000). 2.3 Protein profiling Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of seed storage proteins was carried out according to modified method of Laemmli (1970). The extraction of total seed storage proteins for SDS-PAGE was done according to Kawakatsu, Yamamoto, Hirose, Yano, and Takaiwa, (2008). Briefly, 100 mg of rice flour was weighed in pre-sterile eppendorff tubes (ET) and 1.0 mL of extraction buffer contains [50 mM Tris buffer (pH 6.8), 4% sodium dodecyl sulfate, 8M urea, 5% β-mercaptoethanol, 20% glycerol, and 15 µl/mL protease inhibitor cocktail (Sigma-Aldrich, USA)] was added. ETs were placed in

Journal Pre-proof standing position on orbital shaker (200 rpm) for overnight at 25 °C followed by centrifugation of ETs for 20 min at 15,000 rpm and 25 C to separate supernatant from undissolved materials. The 40 µl supernatant was transferred on to fresh ETs and equal volume of 2x Laemmli buffer [100 mM Tris buffer (pH 6.8); 4% SDS; 2% β-mercaptoethanol; 20% glycerol; 0.04% bromophenol blue] was mixed together and loaded on to the wells. The electrophoresis was carried out at 35 mA constant current and gels were stained with coomassie brilliant blue R250 (CBB-R250) dye (50% methanol; 10% glacial acetic acid; 0.2% w/v CBB-R250). Stained gels were destained by using destaining solution (20% methanol and 12 % glacial acetic acid) and

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scanned with HP Scanjet 4010 scanner at 600 dots per inch resolution. AlphaEaseFC ® v6.0.0

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was used for the determination of molecular weight of different polypeptide bands. 2.4 Mass spectrometric analysis of gel isolated proteins

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Tryptic digestion of the polypeptide bands, excised from the gels, and sample preparation were done as described by Koistinen et al. (2002). Briefly, polypeptide bands were excised

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aseptically from CBBR-250 stained SDS-PAGE gel, chopped into small pieces and destained

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with repeated washing with 40 mM ammonium bicarbonate (100 µl) and 500 µl acetonitrile (ACN), each with 10 min at room temperature (RT). The polypeptides in destained gel pieces

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were reduced in situ at 56 °C for 30 min using 10 mM DTT in 100 mM ammonium bicarbonate (ABC). Gel pieces were thoroughly washed with acetonitrile (ACN) and soaked in 55 mM

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iodoacetamide, prepared in 100 mM ABC solution, for 20 min in dark at RT. Gel pieces were washed twice with ABC and dehydrated with ACN. Tryptic digestion of the polypeptides was

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done by soaking gel pieces in 20 µl trypsin solution (20 ng/µl, Promega, Medison, WI, USA) in buffer (40 mM ABC solution and 10% ACN) for 1 h at 4 °C, followed by 37 °C for overnight. The in-gel digests were vortexed, briefly centrifuged and the supernatant was collected and poured into sterile ETs. The gel pieces containing in-gel digests were re-extracted two more time with 20 µl of ACN and pooled with previous supernatants. Supernatant was lyophilized and rehydrated with 0.5 ml of matrix solution (5 mg/mL a-Cyano-4-hydroxycinnamic acid in 50% ACN containing 0.1% TFA) and spotted on stainless steel 384 well target plate. The peptidematrix spots were allowed to air dry at RT and were then subjected to mass spectrometric analysis using Ultra fleXtreme TM 4 MALDI TOF/TOF system (Bruker Daltonics, Germany), which was calibrated with a mass standard starter kit (Bruker, Germany) and a standard tryptic BSA digest (Bruker, Germany). Mass data was recorded in positive ionization mode and protein

Journal Pre-proof identification was performed by searching acquired MS and MS/MS data in non-redundant protein sequence database (Swissprot) using MASCOT search engine version 3.5 (Matrix science: www.matrixscience.com), with the following parameters: parent ion mass tolerance and MS/MS mass tolerance at 100 ppm, peptide mass tolerance ±1.2 Da, mass values-monoisotopic, peptide charge state-1+, max missed cleavages-1, variable modifications-oxidation of methionine (M) and carbamidomethylation of cysteine (C) was considered as fixed modification, enzyme used as trypsin, taxonomic group was Oryza sativa and the significance threshold was set to p <0.05. Proteins with mascot score greater than 58 were considered as significant (p<0.05).

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2.5 Proximate Composition

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2.5.1 Amino acid determination

The amino acid composition from head rice (HR) and broken rice (BR) was carried out as

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described earlier (Pal et al., 2016). Briefly, rice flour of varied DoM was hydrolyzed with 6M HCl for overnight followed by derivatization of amino acid residues with a mixture of acid,

o-phthalaldehyde,

and

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mercaptopropionic

9-fluorenylmethyl

chloroformate.

The

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derivatized amino acids of different rice flours were loaded on the amino acid analyzer (LC-30 AD, Shimadzu, Kyoto, Japan) equipped with a C18 column (pore size, 5 μm; length, 150 × 4.6 mm) and sepration of amino acid was done as per the manufacturers’ instructions. The column

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was pre-conditioned with mobile phase A and B of 20 mM phosphate buffer and acetonitrile/methanol/water (45/40/15 v/v/v), respectively, and the oven temperature was

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(5.54SP 5, Shimadzu).

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maintained at 40 °C. The chromatograms were generated by using LAB solutions software 2.5.2 Blue value and λmax

The Iodine absorption spectrum of different rice flour samples was done as described by Takeda, Takeda, and Hizukuri, (1983) with minor modifications. Briefly, 100 mg of milled rice flour was poured into sterile Oakridge tubes and 1.0 mL of ethanol and 9.0 mL of 1.0M sodium hydroxide was poured into tubes followed by heating in a boiling water bath for 10 min with intermittent mixing to completely dissolve the flour. The pH of the suspension was adjusted to 6.5 with 1.0M HCl. The volume of the suspension was raised up to 100 ml with distilled water and 5 mL of diluted sample was mixed with 1 mL of 0.2% iodine solution. The final volume of the starch suspension was raised to 100 mL with distilled water and the mixture was kept at room temperature for 15 min. The minimum and maximum absorbance spectra of each the suspension

Journal Pre-proof was recorded from 450 to 800 nm with a spectrophotometer (Lambda Bio 35, Perkin Elmer, Norwalk, CT, U.S.A.) to determine λmax. The blue values of iodine–starch complexes at 680 nm were determined from absorbance maxima spectrum manually. 2.5.3 Pasting properties An Anton Paar Rheo Plus/32 model MCR-301 rheometer and the method described by Kaur et al. (2013) were used for the evaluation of the pasting properties of different rice flours. Briefly, rice flour suspensions consists of 3.5 g of flour and 24.5 g of de-ionized water, were heated from 50 to 95°C at 6°C/min (after an equilibration of 1 min at 50°C). The temperature of

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the suspension was kept on hold for 5 min at 95°C and then allowed to cool at the rate of

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6°C/min until it was not reached at 50°C. Pasting properties (pasting temperature: PT; peak viscosity: PV; final viscosity: FV; breakdown viscosity: BDV; and setback viscosity: SBV) were

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calculated by using software provided by the manufacturer. 2.6 Field emission scanning electron microscopy

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The morphology of HR and BR of different rice cultivars was done by using field

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emission scanning electron microscopy (FESEM) (Carl Ziess, SUPRA 55) facility provided by Emerging Life Sciences Centre of Guru Nanak Dev University, as per the manufacturer’s

between 1 µM to 100 µM. 2.7 Statistical Analysis

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instructions. The accelerating voltage was ranged from 2-10 keV and resolution was kept

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The data were subjected to two way analysis of variance (ANOVA) and Duncan's test (p

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≤ 0.05) using Minitab Statistical Software (MINITAB® v 14.12.0, State College, PA). 3.0 Results and Discussion

3.1 SDS-PAGE analysis of seed storage proteins of rice HR and BR from brown rice from different cultivars milled to 6% DoM was subjected to SDS-PAGE analysis which revealed the presence of 27 major polypeptide (PP) bands ranged from 12.0-116.0 kDa (± 2.0 kDa) (Fig. 1). These PPs were categorized into four classes i) acidic subunits (GAS) (33 and 31kDa), (ii) globulins (24kDa), (iii) glutelin basic subunits (GBS) (24 and 22 kDa) and (iv) prolamins (15 kDa, 16 kDa and 18 kDa) by Kawakatsu, Yamamoto, Hirose, Yano, and Takaiwa, (2008). Analysis of gels revealed that PB1, PS44, PB1509, PB1121, and PS5 contained identical banding pattern for all major PPs. However, the accumulation of all major PPs was differential for HR and BR of different cultivars. The higher levels of 59 kDa, 54

Journal Pre-proof kDa, 51 kDa, 32 kDa, 31 kDa, 30 kDa, 28 kDa, 24 kDa, 24 kDa, 23 kDa, 15 kDa, 13 kDa and 12 kDa PPs was observed in HR of different rice cultivars. Whereas, the storage of 28 kDa, 24 kDa, 23 kDa, 15 kDa, 13 kDa and 12 kDa PPs was lesser or not observed in BR of all the cultivars (Fig. 1). These results thus demonstrated that the quantity of PPs in BR was differential and cultivar-dependent. DoM also influenced the levels of these PPs, as depicted in SDS-PAGE analysis (Fig 1Sa and 1Sb). The major quantitative changes were observed in 28 kDa, 24 kDa, and 23 kDa PPs, therefore, identification of these PPs was carried out by MALDI-ToF MS/MS. 3.2 MALDI-ToF/MS analysis of PPs

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The MALDI-ToF MS/MS analysis of selected rice proteins was carried out. It revealed

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that the identity of 28 kDa PP was matched to 60S ribosomal protein L10a (R10A_ORYSI) and glutelin type-D 1 (GLUD1_ORYSJ) of rice. The mascot score of 39 and e-value of 0.6 for 60S

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ribosomal protein L10a was observed (Table 1). Total 8 peptides were matched with known database along with the sequence coverage of 36% and theoretical molecular weight and pI of

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24467 Da and 9.83, respectively for 60S ribosomal protein L10a were observed (Table 1).

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Whereas, glutelin type-D 1 (GLUD1_ORYSJ) also showed a high mascot score of 86 and evalue of 1.1e-05 (Table 1). Total 15 peptides of 28 kDa PP were matched with glutelin type-D 1 with the sequence coverage of 20%. The theoretical molecular weight and pI of 54827 Da and

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9.34, respectively for glutelin type-D 1 protein were observed (Table 1). The identities of 24 and 23 kDa PP were established as pathogenesis-related protein 1 [Oryza sativa Japonica Group

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(REHYA_ORYSI)] and Oryza sativa 1-Cys peroxiredoxin A, respectively. Total of 6 peptides of

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24 kDa PP were matched with protein sequence of pathogenesis-related protein 1 with 53% sequence coverage. The mascot score and e-values of 119 and 2.2e-07, respectively for 24 kDa PP were observed. While theoretical molecular weight and pI of 17163 Da and 5.85, respectively for the 24 kDa were observed (Table 1). The mascot score and e-values of 182 and 1.1e-13, respectively for 23 kDa PP were observed (Table 1). Total 12 peptides of 23 kDa PP were matched with Oryza sativa 1-Cys peroxiredoxin A protein with a sequence coverage of 66%. The theoretical molecular weight and pI of 24057 Da and 5.97, respectively for the Oryza sativa 1-Cys peroxiredoxin A were depicted (Table 1). The role of these proteins in physico-chemical, textural and sensory attributes of rice, however, was not evaluated. Balindong et al. (2018) also reported the greatest differences in the accumulation of a glutelin between HR and BR using high pressure-liquid chromatography, whereas, the difference between globulin and prolamines

Journal Pre-proof was marginal. Study of Balindong et al. (2018) also revealed a positive correlation among globulin, glutelins and prolamin with HRY. Present study showed that the accumulation of not only glutelins but the levels of prolamines were also different in HR and BR of different rice cultivars. 3.3 Head rice yield and proximate composition HRY of milled rice was differential for different rice cultivars and also affected by extended DoM (Table 2). F values revealed the significant effect of DoM on HRY as compared to cultivars (Table 7). Average HRY of 57%, 51% and 46%, respectively for 6%, 8% and 10%

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DoM was observed for different rice cultivars. On the contrary, 6 to 10% DoM resulted in a

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significant increase in the BR yield (BRY) (Table 2). The average BRY of 43%, 49%, and 54%, respectively, for 6%, 8% and 10% DoM was observed. F value revealed the significant effect of

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DoM and cultivar on HRY and the interaction effect of both was also significant (Table 7). PB1121 milled to 6% DoM produced maximum HRY of 61%, whereas milling to 8% and 10%

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DoM yielded HRY of 55% and 51% respectively, for PB1. PB1509 showed significantly higher

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loss of HRY on extended DoM than PB1121 and this may be associated with less maturation period for PB1509 than PB1121. PB1509 took 120 days for maturation against 140 - 145 days for PB1121 (Singh, Singh, Mohapatra, Gopala, & Ellur, 2018). Study of Reid, Siebenmorgen,

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and Mauromoustakos, (1998) also showed that factors such as over-milling, cultivars and moisture content at the time of milling largely affected the HRY of long-grain rice. The long-

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grain rice showed lower HRY than short grain rice (Reid, Siebenmorgen, Mauromoustakos,

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1998). Several studies have also shown that the loss of HRY at higher temperature attributed to the altered starch metabolism (Fitzgerald, McCouch, & Hall, 2009; Siebenmorgen, Grigg, & Lanning, 2013; Zhao and Fitzgerald, 2013; Sreenivasulu et al., 2015). Abnormal starch synthesis under adverse environmental conditions resulted in loosely packing of rice endosperm with fissure and air gaps (Buggenhout, Brijs, Celus, & Delcour, 2013), which resulted in higher BRY during milling. The effect of agro-climatic, genotypic and date of sowing on HRY of PB1509 need more detailed investigations. It was, therefore, likely that the effect on DoM, short duration as well as intercultivar changes may have played a key role in HRY. The protein, starch and fat content in HR and BR from different rice cultivars were also affected by DoM. The milling of brown rice resulted in a decrease in protein and fat content in HR and BR. Average protein content (PC) of 8.90% for brown rice of different cultivars was

Journal Pre-proof observed. Average PC of 8.51%, 6.47% and 4.39%, respectively for HR obtained from 6%, 8% and 10% DoM of different cultivars was observed (Table 3). While the average PC in BR was decreased with extended DoM. Average PC of 7.47%, 5.53%, and 3.30%, respectively for BR of 6%, 8% and 10% DoM of different rice cultivars was observed (Table 3). SDS-PAGE analysis also showed the lower accumulation of 28 kDa, 24 kDa, 23 kDa and different prolamines in BR than HR of different cultivars (Fig 1). F values revealed that the effect of DoM on PC of HR and BR was significantly higher than cultivars, while the interaction effect of both was also significant (Table 7). Lyon et al. (1999) also reported that a decrease in PC for deep-milled rice,

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as compared to regular-milled rice of different cultivars. PC of 8.6%, 8.1%, and 8.7%,

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respectively for regular-milled rice of Bengal, Koshihikari and M-401 grown in Arkansas was observed. On the contrary, PC of 8.1%, 7.3%, and 8.1%, respectively for deep-milled rice of

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Bengal, Koshihikari and M-401, grown in Arkansas was reported (Lyon et al., 1999). Balindong et al. (2018) also reported lower accumulation of glutelins in BR than HR. In present study, F

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values also revealed the greater and highly significant effect of DoM on PC than that of cultivars.

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F values further demonstrated a significant interaction effect of cultivars and DoM on PC of HR (Table 6).

The fat content (FC) also decreased with extended DoM of different rice cultivars (Table

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3). Average FC of 1.90% for brown rice of different cultivars was observed. The average FC of 1.21%, 1.04% and 0.67%, respectively for HR obtained from 6%, 8% and 10% DoM of different

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cultivars was observed (Table 3). While average FC of 1.06%, 0.82% and 0.53%, respectively

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for BR obtained from 6%, 8% and 10% DoM was observed (Table 3). These results showed that loss of FC in BR was higher than HR. These results also showed that PC and FC in HR of different cultivars was higher and also influenced by extended DoM. Furthermore, F values revealed that the effect of DoM on FC of HR was significantly higher than cultivars. The interaction effect of cultivars and DoM was also highly significant for FC of HR (Table 7). Liu, Zheng, and Chen, (2017) also reported that FC for different rice cultivars decreased with extended DoM. A decrease in FC from 2.43% to 0.6% and 2.86% to 0.60%, respectively for 0 to 14.8% and 0 to 15.5% DoM, respectively for japonica Xinfeng 2 and Indica T-You 15 cultivars was reported by Liu, Zheng, and Chen, (2017). Ye, Hua, Luoa, McClementsb, and Liang (2018) also reported a significant increase in starch digestibility after the removal proteins, lipids, or both from rice of Indica cultivar (Hua et al., 2018). Earlier studies also demonstrated the effect

Journal Pre-proof of caffeic acid and procyanidins addition on thermal and reterogradation properties of starches (Igoumenidisa, Zoumpoulakisb, and Karathanosa, 2018; Takahama, Hirota, and Yanase, 2019). The pericarp and testa of outermost endosperm contain higher levels of protein, fat and considerable amount of antioxidants, vitamins and minerals and deep-milling of brown rice caused loss of these nutraceuticals molecules from polished rice (Dinesh Babu, Subhasree, Bhakyaraj, & Vidhyalakshmi, 2009; Shobana et al., 2011; Somaratne et al., 2017). Therefore, a decline in FC was mainly occurred due the continuous removal of bran layer from endosperms of brown rice.

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3.4 Blue value and λmax

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The blue value (BV) of different milled rice was also affected by DoM and cultivars (Table 3). Extended DoM from 6 to 10% resulted in an increase in BV for HR and BR of

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different cultivars. Average BV of 0.1377 for brown rice of different cultivars was observed. The average BV of 0.1708, 0.2671 and 0.3368, respectively for HR obtained from 6%, 8% and 10%

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DoM of different rice cultivars was observed (Table 3). On the contrary, average BV of 0.2966,

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0.2299 and 0.2747, respectively for BR obtained from 6%, 8% and 10% DoM of different rice cultivars was observed (Table 3). The λmax values were not different between rice cultivars but it was highly influenced by extended DoM. Average λmax value of 599.3 for brown rice of different

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cultivars was observed. The average λmax value of 597.8 nm, 599.5 nm, and 600.2 nm, respectively for HR obtained from 6%, 8% and 10% DoM of different rice cultivars was

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observed (Table 3). Whereas, λmax value of 587.2 nm, 597.1 nm, 598.8 nm, respectively for BR

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obtained from 6%, 8% and 10% DoM of different rice cultivars was observed (Table 3). These results thus demonstrated that BV of HR was increased by extended DoM, while λmax was marginally affected by extended DoM. The average BV and λmax of HR was higher than BV of BR. The lower BV and λmax indicated the presence of lower apparent amylose content in BR than HR and consistent with earlier reports (Chun, Song, Kim, & Lee 2009). Lyon et al. (1999) also reported higher amylose content (AC) for deep-milled rice, than regular-milled rice, grown in Arkansas region. AC of 19.5%, 20.5%, and 20.1%, respectively for regular-milled rice against 20.3%, 21.8%, and 20.8%, respectively for deep-milled rice for Bengal, Koshihikari and M-401 was observed (Lyon et al., 1999). Studies have also shown that rice with Wxa allele showed ~10 fold higher synthesis of granule-bound starch synthase (GBSS) enzyme as well as higher levels of apparent amylose content in endosperms, as compared to Wxb allele (Sano 1984; Zhang et al.,

Journal Pre-proof 2014). Rice grown at low temperature produced kernels with high levels of Wx protein as well as higher levels of apparent amylose content. It was postulated that the promoter of Wx gene was sensitive to cool temperature (18 °C) and a mutation at the 5′ splicing site in the first intron of the Wx gene is coupled to the sensitivity to temperature at seed maturation (Larkin and Park 1999). Therefore the role of Wx genes in differential starch accumulation in different rice genotypes under similar growth conditions may not be over ruled. It was likely that the composition and quantity of seed storage polypeptides as well starch accumulation largely affected HRY in different rice cultivars.

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3.5 Amino acid composition analysis

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Amino acid (AA) analysis of HR and BR of different rice cultivars revealed decrease in levels of all essential amino acids (EAA) with extended DoM. Whereas, the levels of cysteine,

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glutamine and glycine in HR and BR of different cultivars was increased with extended DoM (Table 4a and 4b). The levels of acidic and aromatic AAs in HR and BR of different cultivars

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decreased with extended DoM (Table 4a and 4b). HR of PS44 and PB1509 showed an increase

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in basic AAs, while HR and BR of rest of the rice cultivars showed a decrease in levels of basic amino acids. F values revealed highly significant effect of DoM on glutamine, glycine, threonine, arginine, tyrosine, GABA, methionine, phenylalanine, lysine, acidic and basic AAs of

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HR of different rice cultivars (Table 5). Serine, histidine, glutamine, glycine, threonine, tyrosine, GABA, cysteine, valine, methionine, tryptophan, phenylalanine, lysine, and aromatic AAs

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showed much higher for BR of different cultivars (Table 5). The interaction effect of DoM and

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cultivars was also highly significant for AAs of HR and BR of different rice cultivars. However, these values for glutamine, glycine, isoleucine, and proline were much higher for BR, as compared to HR of different rice cultivars. These results thus demonstrated that extended DoM resulted in loss of nutritional properties of rice. The increase in cysteine and other AAs for rice milled to higher DoM may be due to the increase in glutelins and prolamines, as the distribution of these proteins are concentrated in higher levels in the endosperm (Houstan et al., 1968). The decreased accumulation of proteins along with majority of 17 amino acids and Mn, K and Mg contents was also reported for the white core of chalky rice grains (Xi et al., 2016). SDS-PAGE results also confirmed the lower accumulation of prolamines in BR than HR of different cultivars (Fig. 1). 3.6 Pasting properties

Journal Pre-proof As starch is a major component of rice endosperm therefore, the pasting behaviour of starch determines rice cooking quality and functionality. The cultivars and extended DoM affected the pasting properties of HR and BR (Table 6). The average pasting temperature (PT) of 88 °C, 86 °C, and 85 °C, respectively for HR obtained from 6%, 8% and 10% DoM of different rice cultivars was observed. Whereas, average PT of 82 °C, 81 °C and 81 °C, respectively for BR obtained from 6%, 8% and 10% DoM of different cultivars was observed (Table 6). These results demonstrated that PT of BR was lower from HR and also decreased with deep milling. Lower apparent amylose content, as depicted from BV and λ max values, may be attributed to

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lower PT of BR of different rice cultivars (Table 3). The average peak viscosity (PV) of 1519 cP,

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1543 cP, and 1602 cP, respectively for HR obtained from 6%, 8% and 10% DoM of different cultivars was observed (Table 6) (Fig 2). These results showed that PV of HR of different rice

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cultivars increased with extended DoM (Table 6). However, average PV of 1481 cP, 1494 cP and 1552 cP, respectively for BR obtained from 6%, 8% and 10% DoM of different cultivars was

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observed (Table 6) (Fig 2). These results showed that the PV of BR was also increased with

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extended DoM. However, HR obtained from different cultivars showed lower PV than BR (Table 6). Perdon, Siebenmorgen, Mauromoustakos, Griffin, and Johnson, (2001) also showed an increase in PV for medium-grain and long-grain rice with an increase in DoM. However, the

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rate of changes in PV was higher for medium grain-rice than long-grain rice. These changes in PV were linked to varied levels of apparent amylose content in different rice cultivars. The

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amylase accumulated in bran-layers of endosperm and extended milling led to their removal

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(Perdon, Siebenmorgen, Mauromoustakos, Griffin, & Johnson, 2001). Therefore, an increase in PV in 8% and 10% HR of different cultivars of rice may be associated with lower amylase activity, when compared to HR from 6% milled rice. The average final viscosity (FV) of 3558 cP, 3600 cP, and 3687 cP, respectively for HR obtained from 6%, 8% and 10% DoM of different rice cultivars was observed (Table 6). While average FV of 3515 cP, 3554 cP, and 3610 cP, respectively for BR obtained from 6%, 8% and 10% DoM of different rice cultivars was observed. These results showed higher FV for HR than BR obtained from extended DoM of different rice cultivars (Table 6). Perdon, Siebenmorgen, Mauromoustakos, Griffin, and Johnson, (2001) also showed that FV of Bengal rice harvested in 1995 and 1996 was increased with an increase in DoM. However, the average breakdown viscosity (BDV) of 54 cP, 37 cP and 45 cP, respectively, for HR obtained from 6%, 8% and 10% DoM of different rice cultivars was

Journal Pre-proof observed (Table 6). While average BDV of 42 cP, 48 cP, and 46 cP, respectively for BR obtained from 6%, 8% and 10% DoM of different rice cultivars was observed (Table 6). These results thus demonstrated that BDV of HR was largely influenced by DoM while BDV of BR by cultivars. These observations were further validated by F values, which revealed that effect of DoM on BDV was much higher for HR. F values indicated that cultivar effect on BDV was much larger for BR than HR (Table 7). Average setback viscosity (SBV) of 2093 cP, 2093 cP, and 2111 cP for HR and 2053 cP, 2079 cP, 2091cP reported for BR obtained from 6%, 8% and 10% DoM of different rice cultivars

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was observed (Table 6). Starch and particularly the amylose determined the PV. FV reflect the

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retrogradation property of starches that occurred due to the re-association followed by formation of a viscous mass after cooling of gelatinized starch (Noda, Nishiba, Sato, & Suda, 2003). BDV

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indicates the starch paste resistance to heat and shear. Higher BDV and lower SBV led to superior cooking characteristics, since rice with these attributes delay the retrogradation of

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cooked rice and inhibit stiffness of rice upon cooling. Therefore, higher BDV and lower SBV of

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rice milled to 6% DoM may have superior cooking attributes as compared to 8% and 10% DoM. These results also indicated that over-milling deteriorated both nutritional attributes as well as cooking properties of milled rice of different cultivars. F values revealed the significant effect of

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cultivars and DoM on different pasting properties of HR and BR (Table 7). F values further demonstrated that the effect of DoM on PV and FV of HR was much higher than for BR.

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Conversely, F values also revealed that the effect of varied DoM on SBV and BDV of BR were

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higher than that of HR (Table 7). These results were further strengthened by the fact that the interaction effect of DoM and cultivars on SBV and BDV were much larger for BR as compared to HR of different cultivars. The statistical analysis of different pasting attributes of HR and BR elaborated that BR had inferior cooking characteristics than HR. Albumin (water-soluble), globulin (salt-soluble) and glutelin (alkali-soluble) were major proteins in brown and milled rice, while contribution of prolamines was minor. Baxter, Blanchard, and Zhao (2014) reported that the exogenous incorporation of purified glutelin in rice starch resulted in an increase in PT but a decrease in the PV of rice starch. Whereas, exogenous application of purified rice globulin resulted in a decreased pasting and textural attributes other than gel hardness (Baxter, Blanchard, & Zhao, 2014). It was, therefore likely that the high level accumulation of 28 kDa glutelin typeD 1 (GLUD1_ORYSJ) PP and other PPs may have attributed to higher PV of HR obtained from

Journal Pre-proof 6% DoM than BR of different cultivars. Therefore, differential pasting properties of HR and BR with extended DoM may be influenced by major seed storage proteins since DoM also influenced the pasting properties of HR of different rice cultivars. Furthermore, an increase in average PV, FV and SBV of HR and BR obtained from 6%, 8% and 10% DoM of different rice cultivars may be attributed to over-milling of brown rice. Over-milling of brown rice led to breakage of medium-hard grains of rice as well as the marginal increase in apparent amylose content and λ max values, while a decrease in PC of HR and BR of different rice cultivars. Similar types of observations were also made by Singh, Sodhi, Kaur, and

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Saxena, (2003) and Chun, Song, Kim, and Lee, (2009). Studies of Singh et al. (2003)

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demonstrated that, as compared to translucent grains, chalky grains showed higher transition temperatures, enthalpy of gelatinization, peak height index, gelatinization range and lower values

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for cooking and textural parameters. Cheng, Zhong, Wang, and Zhang, (2005) also reported that chalky part of milled rice had a dramatically higher transition temperature (To, Tp, Tc), ΔH and

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hardness than translucent parts of rice endosperm, while, RVA properties and AC between the

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two parts was marginally or little varied. Lower cooking time of chalky kernels was correlated with lower kernel hardness, loosely packed structure and rapid water absorption, whereas, low amylose was correlated with poor eating quality of rice (Chun, Song, Kim, and Lee, 2009). Study

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of Sandhu, Singh, Kaler, Kaur, and Shevkani, (2018) also revealed that differences in protein, lipids and minerals in different layers of bran were associated with varied pasting, cooking and

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crystalline properties of different long-grain Indica rice cultivars. It was, therefore, likely that the

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increase in λmax and BV while decrease in the protein content was attributed to difference in peak viscosity of HR and BR of different rice cultivars.

3.7 Field emission scanning electron microscopy FESEM analysis revealed the remarkable differences in the morphology of HR and BR at low as well as at very high resolutions (Fig. 3A and 3B). HR showed highly condensed packaging of starch molecules with smooth edges, which were tightly imbibed in the proteins matrix, however, the inter-cultivar differences in the starch structure and packaging were also observed. PB1509 and PS5 revealed the deposition of proteins was differential from other cultivars (Fig 3A, H and M). Fissures and cracks in PB1 and PS44 were prominent. On the contrary, morphology of BR was dissimilar from HR (Fig 3B: plates: A to N). BR revealed lesser accumulation of starch granules with abnormal protein filling. Several fissures and cracks were observed in the starch

Journal Pre-proof granules of BR of PB2 and PS44 (Fig. 3B plates C and F). Low levels of starches in BR were further supported by the lower BV and λmax (Table 2). Chun, Song, Kim, and Lee, (2009) also reported similar kind of morphological characteristics for HR and chalky rice. 4.0 Conclusions HR of different cultivars revealed the higher accumulation of apparent amylose content, fat and protein content. PB1509 showed severe loss of HRY after extended DoM, which may be attributed to its short crop maturation time (120 days), as compared to other cultivars. The brown rice of PB1509 and PS5 was highly susceptible to breakage during extended DoM and

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were considered to have poor milling quality. PV, FV, and SBV increased both for HR and BR

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from different cultivars with extended DoM while the PT was decreased. Therefore, extended DoM resulted in the loss of surface lipids, proteins, and other nutraceuticals, which thus affected

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the pasting properties of both HR and BR of different cultivars. Also, HR showed tighter packaging of protein-starch molecules than BR. The higher accumulation of 28 kDa, 24 kDa, and

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23 kDa PPs in HR than BR of different rice cultivars was also observed. These PPs seems to

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have some contribution to the mechanical strength of HR. The identity of 28, 24 and 23 kDa PPs was established as 60S ribosomal protein L10a/ glutelin type-D 1 (GLUD1_ORYSJ), pathogenesis-related protein 1 and 1-cys peroxiredoxin A by MALDI-ToF/MS. The levels of 28,

5.0 Acknowledgements

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24 and 23 kDa PPs was also low in PB1509 than other cultivars.

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NS greatly acknowledge DST, New Delhi for JC Bose National Fellowship. NS duly

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acknowledge the support of Dr. Sanjay Kumar, Director, IHBT Palampur for providing the proteomics facility at IHBT, Palampur. 6.0 References Balindong, J. L., Ward, R. M., Rose, T. J., Liu, L., Raymond, C. A., Snell, P. J., Ovenden, B. W., & Waters, D. L. E. (2018). Rice grain protein composition influences head rice yield. Cereal Chemistry, 95, 253-263. Baxter, G., Blanchard, C., & Zhao, J. (2014). Effects of glutelin and globulin on the physicochemical properties of rice starch and flour. Journal of Cereal Science, 60(2), 414-420. Buggenhout, J., Brijs, K., Celus, I., & Delcour, J. A. (2013). The breakage susceptibility of raw and parboiled rice: A review. Journal of Food Engineering. https://doi.org/10.1016/j.jfoodeng.2013.03.009 Cheng, F. M., Zhong, L. J., Wang, F., & Zhang, G. P. (2005). Differences in cooking and eating properties between chalky and translucent parts in rice grains. Food Chemistry, 90(12), 39-46.

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Chun, A., Song, J., Kim, K.-J., & Lee, H.-J. (2009). Quality of head and chalky rice and deterioration of eating quality by chalky rice. Journal of Crop Science and Biotechnology, 12(4), 239-244. Dinesh Babu, P., Subhasree, R. S., Bhakyaraj, R., & Vidhyalakshmi, R. (2009). Brown ricebeyond the color reviving a lost health food-A review. American-Eurasian Journal of Agronomy, 2(2), 67-72. Fitzgerald, M. A., McCouch, S. R., & Hall, R. D. (2009). Not just a grain of rice: The quest for quality. Trends in Plant Science, 14(3), 133-139. Igoumenidis, P. E., Zoumpoulakis, P., & Karathanos, V. T. (2018). Physicochemical interactions between rice starch and caffeic acid during boiling. Food Research International, 109, 589-595. Kaur, A., Kaur, P., Singh, N., Virdi, A. S., Singh, P., & Rana, J. C. (2013). Grains, starch and protein characteristics of rice bean (Vigna umbellata) grown in Indian Himalaya regions. Food Research International, 54(1), 102-110. Kawakatsu, T., Yamamoto, M. P., Hirose, S., Yano, M., & Takaiwa, F. (2008). Characterization of a new rice glutelin gene GluD-1 expressed in the starchy endosperm. Journal of Experimental Botany, 59(15), 4233-4245. Koistinen, K. M., Hassinen, V. H., Gynther, P. A. M., Lehesranta, S. J., Keinänen, S. I., Kokko, H. I., Kärenlampi, S. O. (2002). Birch PR-10c is induced by factors causing oxidative stress but appears not to confer tolerance to these agents. New Phytologist, 155(3), 381-391. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685. Larkin, P. D., & Park, W. D. (1999). Transcript accumulation and utilization of alternate and non-consensus splice sites in rice granule-bound starch synthase are temperaturesensitive and controlled by a single-nucleotide polymorphism. Plant Molecular Biology, 40(4), 719-727. Liu, K. l., Zheng, J. B., & Chen, F. S. (2017). Relationships between degree of milling and loss of vitamin B, minerals, and change in amino acid composition of brown rice, LWT Food Science and Technology, 82, 429-436. Lyon, B. G., Champagne, E. T., Vinyard, B. T., Windham, W. R., Barton, F. E., Webb, B. D., McClung, A. M., Moldenhauer, K. A., Linscombe, S., McKenzie, K. S., & Kohlwey, D. E. (1999). Effects of degree of milling, drying condition, and final moisture content on sensory texture of cooked rice. Cereal Chemistry, 76, 56-62. Noda, T., Nishiba, Y., Sato, T., & Suda, I. (2003). Properties of starches from several lowamylose rice cultivars. Cereal Chemistry, 80(2), 193-197. Pal, P., Singh, N., Kaur, P., & Kaur, A. (2018). Effect of parboiling on phenolic, protein, and pasting properties of rice from different paddy varieties. Journal of Food Science, 83, 2761-2771. Pal, P., Singh, N., Kaur, P., Kaur, A., Virdi, A. S., & Parmar, N. (2016). Comparison of composition, protein, pasting, and phenolic compounds of brown rice and germinated brown rice from different cultivars. Cereal Chemistry, 93(6), 584-592. Patindol, J., & Wang, Y. J. (2003). Fine structures and physicochemical properties of starches from chalky and translucent rice kernels. Journal of Agricultural and Food Chemistry, 51(9), 2777-2784.

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Perdon, A. A., Siebenmorgen, T. J., Mauromoustakos, A., Griffin, V. K., & Johnson, E. R., (2001). Degree of milling effect on rice pasting properties. Cereal Chemistry, 78, 205209 Pinson, S. R. M., Jia, Y., & Gibbons, J. W. (2013). Three quantitative trait loci conferring resistance to kernel fissuring in rice identified by selective genotyping in two tropical Japonica populations. Crop Science, 53(6), 2434-2443. Reid, J. D., Siebenmorgen, T. J., Mauromoustakos, A. (1998). Factors affecting the slope of head rice yield vs. degree of milling. Cereal Chemistry, 75 (5), 738-741. Sandhu, R. S., Singh, N., Kaler, R. S. S., Kaur, A., & Shevkani, K. (2018). Effect of degree of milling on physicochemical, structural, pasting and cooking properties of short and long grain Indica rice cultivars. Food Chemistry, 260, 231-238. Sano, Y. (1984). Differential regulation of waxy gene expression in rice endosperm. Theoretical and Applied Genetics, 68(5), 467-473. Shobana, S., Malleshi, N. G., Sudha, V., Spiegelman, D., Hong, B., Hu, F. B., Mohan, V. (2011). Nutritional and sensory profile of two Indian rice varieties with different degrees of polishing. International Journal of Food Sciences and Nutrition, 62(8), 800-810. Siebenmorgen, T. J., Counce, P. A., Lu, R. & Kocher, M. F. (1992). Correlation of head rice yield with individual kernel moisture content distribution at harvest. Transactions of the ASAE, 35(6), 1879-1884. Siebenmorgen, T. J., Grigg, B. C., & Lanning, S. B. (2013). Impacts of preharvest factors during kernel development on rice quality and functionality. Annual Review of Food Science and Technology, 4(1), 101-115. Siebenmorgen, T. J., Nehus, Z. T., & Archer, T. R. (1998). Milled rice breakage due to environmental conditions. Cereal Chemistry, 75(1), 149-152. Singh, N., Singh, H., Kaur, K., & Bakshi, M. S. (2000). Relationship between the degree of milling, ash distribution pattern and conductivity in bran rice. Food Chemistry, 69, 147-151. Singh, N., Sodhi, N. S., Kaur, M., & Saxena, S. K. (2003). Physico-chemical, morphological, thermal, cooking and textural properties of chalky and translucent rice kernels. Food Chemistry, 82(3), 433-439. Singh, V., Singh, A. K., Mohapatra, T. S., Gopala, K. S., & Ellur, R. K. (2018). Pusa Basmati 1121 - a rice variety with exceptional kernel elongation and volume expansion after cooking. Rice (New York, N.Y.), 11, 19. doi:10.1186/s12284-018-0213-6. Somaratne, G. M., Prasantha, B. D. R., Dunuwila, G. R., Chandrasekara, A., Wijesinghe, D. G. N. G., & Gunasekara, D. C. S. (2017). Effect of polishing on glycemic index and antioxidant properties of red and white basmati rice. Food Chemistry, 237, 716-723. Song, X. J., Huang, W., Shi, M., Zhu, M. Z., & Lin, H. X. (2007). A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nature Genetics, 39(5), 623-630. Sreenivasulu, N., Butardo, V. M., Misra, G., Cuevas, R. P., Anacleto, R., & Kishor, P. B. K. (2015). Designing climate-resilient rice with ideal grain quality suited for hightemperature stress. Journal of Experimental Botany, 66(7), 1737-1748. Takeda, C., Takeda, Y. and Hizukuri, S. (1983) Physicochemical properties of lily starch. Cereal Chemistry, 60, 212-216.

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Legends to Figures: Fig. 1: SDS-PAGE analysis of seed storage proteins of head and broken rice of different rice cultivars. H: head rice; B: broken milled rice. Fig.2: Pasting profile of head (H) and broken (B) milled rice from PB1121 at different (6%, 8% and 10%) degree of milling (DoM). Fig. 3A and 3B: The field emission scanning electron microscopy analysis of head and broken rice of different rice cultivars.

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Fig. 1Sa and 1Sb: SDS-PAGE analysis showing effect of degree of milling (6% - 10%) on the protein profile of head and broken rice of different cultivars.

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Graphical Abstract

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A graphical abstract showing differences in head and broken rice of different long grain Indica r differences in the protein profile and internal starch granule structure, imbibed in protein matrix, are v PAGE and field emission scanning electron microscopic analysis.

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Fig 1

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Fig 2

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Fig 3A

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Table 1: MALDI-ToF/MS analysis of differentially accumulated proteins in head rice of long-grain Indica culti Accession no.

Theoretical pI/MW

Mascot Score

Glutelin type-D 1 (GLUD1_ORYSJ )

Q6K508

54827/9.34

86

Sequenc e coverage 20%

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Name of the protein

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Experimental MW (kDa) 28

26

E value

peptide matched

Matching pept

1.1e-05

15

R.LQAFEPLR.K R.LQAFEPLRK R.RVIEPQGLVV R.VIEPQGLVVP R.DEHQK.I K.IHEFR.Q K.SFANQLEPR K.SFANQLEPR R.QKEFLLAGN K.EFLLAGNNQ R.LQSQNDQRG R.VKHGLQLLK

39

36%

0.6

8

Pathogenesisrelated protein 1 [Oryza sativa Japonica Group] (REHYA_ORYS I) 1-Cys peroxiredoxin A

XP_01563 1105.1

17163/5.85

119

53%

2.2e-07

6

P0C5C8.1

24057

182

1.1e-13

12

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24467/9.83

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23

B8B9K6

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24

60S ribosomal protein L10a (R10A_ORYSI)

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27

66%

K.HGLQLLKPT R.QYQQVQYR K.FPILNLIGMG K.EAISQVVGE K.LPHIPRPK.M K.VCMLGDAQ K.MNKNK.K K.KYHAFLASE K.YHAFLASEA K.VLCMGVAV K.STMGKPIR.V R.AAVMDWHT K.IASHIVASAH K.AIETATSHIK K.DEITKAK.E K.ESLTGIFK.T

M.PGLTIGDTV R.GVKLLGISCD R.VTYPIMADP R.VTYPIMADP K.QLNMVDPDE R.ALHIVGPDK K.LSFLYPSCV R.NMDEVVRA R.AVDALQTAA K.HAVATPVNW

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Table 2: Effect of extended degree of milling on head and broken rice yield of different cultivars. Head rice Broken rice Source Cultivar DoM DoM 6% 8% 10% 6% 8% cd d d b 60±0.34 55±0.45 51±0.42 40±0.12 45±0.22a PB1 58±0.31c 51±0.22b 46±0.36bc 42±0.43bc 49±0.12b PS44 Rice 50±0.51a 47±0.15a 43±0.11a 50±0.45de 53±0.34cd PB1509 yield de bc c a 61±0.32 52±0.33 47±0.12 39±0.32 48±0.51b PB1121 (%) 55±0.21b 49±0.41b 42±0.34a 45±0.21c 51±0.24c PS5 Average 57 51 46 43 49

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DoM: degree of milling. Means with similar letters in a column are not differ significantly (at P ≤ 0.05).

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10% 49±0.23a 54±0.34b 57±0.42c 53±0.21b 58±0.46cd 54

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Table 3: Proximate composition of flour of head rice and broken rice of different cultivars. Head rice Source

Cultivar

Brown rice

Broken rice

DoM

DoM

6% 8% 10% 6% 8.46±0.13a 8.02 ±0.23a 6.22 ±0.22a 4.23±0.12a 7.55±0.21b PB1 8.70b±0.14b 8.22 ±0.21a 6.32 a±0.11a 4.11±0.44a 7.23±0.42a PS44 bc bc b bc a 8.90 ±0.10 8.63 ±0.42 6.69 ±0.13 4.36±0.24 7.59±0.45b Protein PB1509 (%) 9.60cd±0.12cd 9.12 ±0.41cd 6.80±0.24c 4.82±0.35bc 7.96±0.12cd PB1121 b b b a ab 8.80 ±0.15 8.55±0.12 6.32±0.12 4.44±0.23 7.02±0.15a PS5 Average 8.90 8.51 6.47 4.39 7.47 1.99d±0.05 1.23±0.01c 1.02±0.02b 0.89±0.03ef 1.11±0.02c PB1 1.87d±0.08 1.10±0.03b 1.01±0.01b 0.77±0.05d 0.98±0.05b PS44 cd b b b 1.77 ±0.06 1.14±0.05 1.04±0.03 0.53±0.02 0.96±0.02b PB1509 Fat e de cd a (%) 2.10 ±0.03 1.58±0.04 1.14±0.06 0.49±0.05 1.36±0.04de PB1121 1.90d±0.02 1.02±0.05a 0.97±0.03a 0.65±0.02c 0.89±0.03a PS5 Average 1.90 1.21 1.04 0.67 1.06 0.1586d±0.0001 0.1984±0.0001d 0.2872±0.0001c 0.3855±0.0002de 0.1247±0.0002b PB1 0.1374c±0.0003 0.1518±0.0002b 0.2574±0.0004b 0.3596±0.0003c 0.1118±0.0003b PS44 0.1139b±0.0005 0.1778±0.0002c 0.2228±0.0003a 0.2976±0.0001b 0.1048±0.0005a Blue PB1509 e d e c value PB1121 0.1738 ±0.0002 0.1956±0.0006 0.3321±0.0002d 0.3541±0.0005 0.1548±0.0002c a a ab a 0.1048 ±0.0003 0.1303±0.0003 0.2358±0.0003 0.2874±0.0004 0.9870±0.0005d PS5 Average 0.1377 0.1708 0.2671 0.3368 0.2966 599.4±0.13b 599.9±56c 600.2±23b 600.9±76b 587.5±32b PB1 599.3±0.15b 598.4±34b 599.8±34a 600.5±65b 585.6±64a PS44 b b a a 599.1±0.17 598.2±24 598.6±54 599.4±43 587.4±24b λmax PB1509 (nm) PB1121 600.1±0.19c 596.9±53ab 600.4±57b 600.7±56b 590.1±63cd a a a a 598.6±0.23 595.7±64 598.7±62 599.6±33 585.4±45a PS5 Average 599.3 597.8 599.5 600.2 587.2 DoM: degree of milling. Means with similar letters in a column are not differ significantly (at P ≤ 0.05).

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8% 5.63±0.32c 5.43±0.42b 5.39±0.34b 5.99±0.35d 5.22±0.24a 5.53 0.86±0.02c 0.79±0.03b 0.74±0.01a 0.99±0.04d 0.74±0.07a 0.82 0.2517±0.0003c 0.2254±0.0001b 0.1874±0.0005a 0.2836±0.0005d 0.2014±0.0004b 0.2299 596.7±45b 596.1±34b 595.7±53a 598.8±67c 598.4±32c 597.1

10% 3.31±0.21a 3.22±0.34a 3.01±0.31a 3.86±0.32bc 3.12±0.23a 3.30 0.75±0.04e 0.56±0.05d 0.51±0.03c 0.33±0.05a 0.49±0.05b 0.53 0.3417±0.0004d 0.3343±0.0003c 0.2534±0.0005b 0.2471±0.0002b 0.1968±0.0004a 0.2747 598.9±43b 598.4±64a 598.8±45b 599.7±66d 598.2±54c 598.8

Journal Pre-proof Table 4a: Effect of extended degree of milling on the amino acids composition of milled rice of different rice cultivars. Amino acid (%)

PB1

6%

PS44

PB1509

Head rice

Broken rice

Head rice

Broken rice

Head rice

DoM

DoM

DoM

DoM

DoM

8%

10%

6%

8%

10%

6%

8%

10%

6%

8%

10%

6%

8%

Broken rice DoM 10%

6%

8%

10%

Asp

5.06±0.23d 4.55±0.19d 3.96±0.33c 4.99±0.18de 4.11±0.41d 3.47±0.19d 7.14±0.43g 6.13±0.45f 5.86±0.24e 6.53±0.45g 6.01±0.34f 5.51±0.21e 7.49±0.45f 6.96±0.23f 6.36±0.23g 7.11±0.11h 6.66±0.32g 6.11±0.31g

Glut

5.11±0.21d 4.12±0.41d 3.93±0.26c 4.39±0.13d 4.03±0.31d 3.78±0.39d 6.87±0.23f 5.55±0.48e 5.25±0.13e 5.96±0.48f 5.43±0.12e 5.03±0.23e 6.99±0.34f 6.23±0.54f 5.98±0.41h 6.85±0.24g 6.15±0.23g 5.54±0.32f

Gln

4.63±0.13c 10.26±0.31i 9.36±0.37g 4.55±0.43d 9.69±0.36i 12.5±0.51k 2.99±0.34b 10.33±0.23h 9.63±0.41g 1.86±0.16b 9.89±0.49i

Ser

11.01±0.32i

8.06±0.26h

6.95±0.39e

18.01±0.24j

7.93±0.39g

Gly

1.22±0.45a

1.03±0.45a

11.63±0.17h

1.12±0.35a

Thr

7.14±0.11f

6.89±0.38f

6.23±0.31e

7.01±0.51g

13±0.34k 1.14±0.03a 10.89±0.28h 9.86±0.21f 1.01±0.32a 10.66±0.21i 14±0.31h

f o

6.74±0.29g

14.26±0.21i 10.36±0.32h

9.65±0.25g

20.87±0.31j

9.96±0.21i

9.55±0.39h 14.17±0.26h 11.47±0.27i 10.88±0.28i 20.61±0.14k 11.02±0.34j 10.2±0.34k

6.01±0.42f

13.5±0.36l

2.39±0.35b

2.11±0.26b

11.68±0.21g

2.14±0.23c

7.12±0.42g

13.4±0.13k 1.79±0.24a 1.32±0.36ab 12.31±0.36k 1.46±0.38b 6.11±0.21g

6.43±0.38f

5.98±0.41f

7.18±0.45g

6.69±0.37f

6.42±0.25h

6.89±0.19g

6.56±0.41f

6.14±0.29f

o r p

7.42±0.39g

5.89±0.26e

5.44±0.32f

6.89±0.41g

5.69±0.11f

13±0.12j 5.23±0.21f

Cit

8.14±0.12g 7.89±0.47g 7.18±0.26f 7.96±0.38g 7.49±0.29g 6.98±0.39g 5.66±0.32e 4.86±0.34d 4.29±0.27f 5.01±0.44f 4.81±0.45d 4.04±0.31d 2.89±0.26b 2.1±0.21h 1.84±0.21b 2.14±0.12c 1.98±0.24b 1.61±0.24b

Arg

6.15±0.34e 5.26±0.45e 4.98±0.52d 5.36±0.47e 5.14±0.41e 4.78±0.41e 6.27±0.25f 6.12±0.42f 5.78±0.28d 6.14±0.21g 5.93±0.12e 5.65±0.21e 6.27±0.28f 6.03±0.45f 5.49±0.31f 6.13±0.42g 5.98±0.15f 5.41±0.14f

His

5.1±0.21d 4.61±0.42c 4.15±0.46d 4.96±0.41d 4.36±0.51d 4.01±0.51e 6.33±0.52f 5.59±0.43e 4.84±0.39e 5.96±0.42f 5.23±0.41e 4.41±0.32d 6.14±0.32f 5.52±0.32e 4.83±0.34e 5.98±0.38f 5.23±0.12f 4.61±0.23e

GABA

1.89±0.24a 1.4±0.31a 1.03±0.45a 1.49±0.52a 1.12±0.36a 0.97±0.41a 0.98±0.23a 0.74±0.37a 0.49±0.32d 0.86±0.01a 0.69±0.04a 0.24±0.05a 1.02±0.01a 0.83±0.37a 0.71±0.31a 0.99±0.31a 0.8±0.41a 0.69±0.31a

Tyr

3.25±0.31b 2.11±0.35b 1.96±0.12a 2.14±0.49b 2.01±0.29b 1.47±0.27b 2.56±0.29b 2.23±0.45b 2.01±0.36a 2.41±0.27c 2.11±0.13b 1.93±0.32a 3.26±0.12c 3.11±0.21c 2.87±0.36c 3.12±0.17d 3.01±0.13d 2.27±0.24c

Ala

5.23±0.25d 4.02±0.38d 3.88±0.38c 4.12±0.34c 3.94±0.34c 3.67±0.39d 4.12±0.36d 4.03±0.42d 3.55±0.26b 4.11±0.38e 3.83±0.26c 3.41±0.18c 6.2±0.23f 5.69±0.25e 5.23±0.41f 5.88±0.23f 5.38±0.34f 4.47±0.35e

Cys

4.36±0.27c 10.56±0.32i 9.98±0.33g 3.87±0.37j 10.03±0.45j 9.47±0.41i 3.89±0.39c 9.69±0.51g 8.51±0.29c 3.78±0.23d 8.76±0.16h 8.26±0.17g 4.01±0.38d 7.25±0.12g 6.63±0.45g 3.56±0.28d 7.03±0.12h 6.54±0.15g

Val

5.36±0.31d 4.99±0.35d 4.53±0.36d 5.12±0.42d 4.96±0.26d 4.01±0.23e 5.14±0.38e 4.69±0.32d 4.16±0.17f 5.03±0.34f 4.55±0.28d 4.04±0.35d 5.66±0.41e 4.91±0.42d 3.74±0.32d 5.23±0.13f 3.98±0.32d 3.69±0.22d

Met

5.01±0.37d 4.75±0.23d 4.11±0.43d 4.78±0.31d 4.29±0.32d 3.87±0.21d 5.02±0.32e 4.33±0.36d 3.87±0.41d 4.75±0.17e 4.11±0.31d 3.59±0.24c 5.96±0.45e 4.89±0.23d 4.67±0.33e 5.12±0.39f 4.69±0.42e 4.59±0.32e

Try

4.12±0.32c 3.98±0.43c 3.79±0.29c 4.01±0.45c 3.86±0.29c 3.48±0.38d 4.25±0.41d 3.69±0.32c 3.14±0.49c 4.01±0.36e 3.55±0.37c 2.79±0.15b 4.59±0.41d 4.11±0.41d 3.77±0.21d 4.16±0.27e 3.98±0.46d 3.21±0.21d

Phe

4.01±0.45c 3.56±0.54c 3.05±0.36c 3.66±0.35c 3.49±0.21c 2.79±0.27c 4.23±0.45d 3.99±0.27c 3.57±0.21c 4.12±0.28e 3.76±0.38c 3.44±0.21c 4.57±0.23d 3.98±0.51c 3.47±0.28d 4.14±0.12e 3.55±0.12d 3.11±0.24d

ILeu

3.04±0.23b 2.78±0.51b 2.59±0.42b 2.89±0.21b 2.73±0.38b 2.44±0.29c 3.47±0.28c 2.87±0.29b 2.44±0.22b 3.11±0.21d 2.59±0.32b 2.19±0.11b 3.41±0.43c 3.03±0.31c 2.67±0.21c 3.23±0.14d 2.98±0.44c 2.49±0.12c

Lys

3.1±0.41b 2.87±0.41b 2.13±0.45b 2.99±0.23b 2.49±0.18b 2.02±0.13c 3.56±0.39c 3.15±0.35c 2.97±0.21b 3.43±0.45d 3.02±0.28c 1.67±0.15a 4.01±0.25d 3.43±0.24c 1.14±0.45b 3.9±0.39d 2.91±0.34c 1.09±0.23b

Pro

7.07±0.17f 6.33±0.36f 4.58±0.41d 6.55±0.39f 5.89±0.28e 4.15±0.43e 3.69±0.31c 2.85±0.12b 1.89±0.12a 3.03±0.42d 2.09±0.24b 1.69±0.12a 3.01±0.23c 2.36±0.42b 2.11±0.23c 2.49±0.28c 2.21±0.42ck 2.07±0.23c

Acidic

10.17±0.34h 8.67±0.17h 7.89±0.38f 9.38±0.42h 8.14±0.45h 7.25±0.42h 14.01±0.48i 11.68±0.25i 11.11±0.31h 12.49±0.23h 11.44±0.41j 10.5±0.23i 14.48±0.21h 13.19±0.23j 12.34±0.32k 13.96±0.21i 12.81±0.24l 11.7±0.12i

Basic

13.13±0.43j 12.74±0.12j 11.26±0.26h 13.34±0.34i 11.99±0.42k 10.8±0.41j 10.2±0.32h 14.86±0.23j 13.59±0.49i 15.53±0.34i 14.18±0.29k 11.7±0.36j 7.92±0.29g 14.98±0.41k 11.46±0.39j 16.01±0.24j 14.12±0.41g 11.1±0.32i

l a n

Aromatic 7.26±0.32f 5.67±0.41e 5.01±0.38de 5.8±0.41e

e

r P

r u o

J

5.5±0.28e 4.26±0.34e 6.79±0.36f 6.22±0.37f 5.58±0.28e 6.53±0.12g 5.87±0.46e 5.37±0.34e 7.83±0.31g 7.09±0.45g 6.34±0.12g 7.26±0.34h 6.56±0.43 5.38±0.23f

DoM: Degree of milling; Asp: asparagine; Glut: glutamic acid; Gln: glutamine; Ser: serine; Gly: glycine; Thr: threonine; Cit: citrulline; Arg: arginine; His: histidine; GABA: gammaaminobutyric acid; Tyr: tyrosine; Ala: alanine; Cys: cysteine; Val: valine; Met: methionine; Try: tryptophan; Phe: phenylalanine; ILeu: isoleucine; Lys: lysine; Pro: proline. Means with similar letters in a column are not differ significantly (at P ≤ 0.05).

30

Journal Pre-proof

Table 4b: Effect of extended degree of milling on the amino acids composition of head rice and broken rice of different cultivars. Amino acid (%)

PB1121

PS5

Head rice Broken rice Head rice Broken rice DoM DoM DoM DoM 6% 8% 10% 6% 8% 10% 6% 8% 10% 6% 8% 10% 6.02±0.35f 5.13±0.27e 4.88±0.21d 5.56±0.24e 5.11±0.15d 4.59±0.23e 7.56±0.23h 7.03±0.35h 6.11±0.26g 7.13±0.37h 6.33±0.32g 5.96±0.21f Asp 6.14±0.27e 6.63±0.23f 6.23±0.25e 5.79±0.34f 5.71±0.12f 4.96±0.21e 4.19±0.21e 5.13±0.22f 4.51±0.21g 4.12±0.28e Glut 7.89±0.14g 6.56±0.39f a i g a h k e i h k i 1.15±0.04 10.66±0.34 15±0.12 4.14±0.32 10.36±0.32 9.98±0.35 22.9±0.41 10.23±0.37 14.1±0.21j Gln 1.79±0.24 11.03±0.36 10.44±0.42 g j h i i i k j j d j 7.36±0.32 12.55±0.12 11.59±0.21 21.59±0.23 11.98±0.23 11.5±0.34 14.76±0.43 11.69±0.21 11.21±0.45 3.15±0.28 11.41±0.28 10.90±0.32h Ser b b i b g j b b k f 12.86±0.32 2.46±0.43 8.09±0.32 13.9±0.43 1.25±0.23 1.06±0.32 12.77±0.39 5.59±0.21 8.98±0.19h 13.4±0.31i Gly 2.19±0.23 2.21±0.31 f e d e c e h g f b g 6.89±0.34 5.13±0.45 4.36±0.15 5.54±0.23 4.95±0.21 4.26±0.12 7.14±0.32 6.36±0.34 5.88±0.42 1.11±0.24 6.01±0.23 5.51±0.25f Thr e e d e d e f f e g e 5.87±0.24 5.29±0.31 4.84±0.32 5.36±0.45 5.12±0.23 4.58±0.34 5.23±0.24 5.03±0.31 4.87±0.25 6.53±0.25 4.96±0.21 4.78±0.32e Cit g d d d c d g f f f f 4.10±0.16 4.89±0.32 4.24±0.32 3.79±0.42 6.86±0.12 5.86±0.21 5.56±0.32 5.21±0.32 5.63±0.34 5.08±0.23f Arg 7.01±0.31 4.59±0.25 g e c e c d g f e g f 7.16±0.41 5.23±0.32 3.87±0.24 5.69±0.21 4.02±0.12 3.78±0.21 6.19±0.43 5.41±0.23 4.87±0.26 6.23±0.45 5.01±0.37 4.59±0.31e His a a a a a a a a a e a 0.74±0.26 1.01±0.01 0.87±0.32 0.59±0.43 0.93±0.23 0.69±0.39 0.47±0.21 4.78±0.35 0.55±0.36 0.28±0.21a GABA 1.07±0.21 0.98±0.31 c c c c b c d c c d c 3.78±0.13 3.46±0.21 3.11±0.27 3.56±0.32 3.14±0.21 2.68±0.32 3.2±0.32 2.86±0.38 2.01±0.25 3.02±0.24 2.19±0.39 1.98±0.34b Tyr d c b c b c f e e a e 4.76±0.45 3.12±0.12 2.91±0.27 3.33±0.38 3.02±0.32 2.17±0.32 5.19±0.12 4.73±0.31 4.41±0.35 0.78±0.38 4.55±0.28 4.15±0.23e Ala d g e c f g d j i d i 6.78±0.12 3.86±0.25 7.16±0.21 6.74±0.32 3.69±0.32 11.96±0.32 10.03±0.31 3.23±0.28 10.22±0.28 9.87±0.12g Cys 4.68±0.31 7.56±0.23 e c b c b c f e e f e 5.89±0.32 3.11±0.12 2.85±0.45 3.89±0.23 3.06±0.43 2.75±0.43 5.14±0.43 4.37±0.32 4.10±0.21 5.02±0.21 4.21±0.31 3.78±0.23d Val f e c e c d f d d e d 3.99±0.34 5.06±0.45 4.87±0.31 3.49±0.25 5.47±0.12 3.86±0.26 3.12±0.31 4.12±0.32 3.47±0.24 3.04±0.21d Met 6.01±0.21 5.03±0.34 d c c d b d e d c e c 3.41±0.26 4.19±0.34 3.63±0.31 3.09±0.23 4.12±0.32 3.32±0.32 2.74±0.33 4.01±0.27 2.96±0.33 2.54±0.22c Try 4.99±0.12 3.98±0.45 d d d d c e e d c d c 4.89±0.32 4.56±0.34 4.27±0.31 4.84±0.41 4.36±0.42 4.17±0.22 4.02±0.23 3.12±0.21 2.48±0.36 3.66±0.21 2.87±0.12 2.17±0.02c Phe c c b c b c c b b c b 2.78±0.23 3.72±0.32 3.11±0.36 2.54±0.21 2.15±0.54 1.89±0.32 1.22±0.37 2.11±0.24 1.58±0.11 1.14±0.02b ILeu 3.78±0.45 3.46±0.32 d c c d b c d c c d c 4.18±0.15 3.86±0.21 3.11±0.19 4.01±0.12 3.27±0.43 2.97±0.43 3.84±0.43 2.85±0.37 2.11±0.32 3.17±0.32 2.21±0.12 1.07±0.32b Lys c c b c b b d c b d c 3.79±0.35 3.16±0.32 2.97±0.27 3.66±0.23 3.11±0.32 1.69±0.32 3.41±0.12 2.59±0.23 1.87±0.24 3.12±0.22 2.12±0.09 1.57±0.22b Pro j i h h i h j j i i i 10.3±0.25 12.26±0.32 10.84±0.43 10.10±0.15h Acidic 13.91±0.25 11.69±0.42 11.02±0.32 12.19±0.34 11.34±0.32 10.4±0.32 13.27±0.34 11.99±0.12 i k h i i h i m k 10±0.23 14.12±0.43 12.54±0.25 14.99±0.24j 12.85±0.23k 10.7±0.22h Basic 11.12±0.24 13.68±0.32 11.08±0.34 14.59±0.42 11.53±0.21 10.5±0.23 h h f g f g h f 7.38±0.15 8.4±0.23 7.5±0.41 6.85±0.34 7.22±0.34 5.98±0.23 4.49±0.35e 6.68±0.23g 5.06±0.14f 4.15±0.34e Aromatic 8.67±0.21 8.02±0.23 DoM: Degree of milling; HR: head rice; BR: broken rice; Asp: asparagine; Glut: glutamic acid; Gln: glutamine; Ser: serine; Gly: glycine; Thr: threonine; Cit: citrulline; Arg: arginine; His: histidine; GABA: gamma-aminobutyric acid; Tyr: tyrosine; Ala: alanine; Cys: cysteine; Val: valine; Met: methionine; Try: tryptophan; Phe: phenylalanine; ILeu: isoleucine; Lys: lysine; Pro: proline. Means with similar letters in a column are not differ significantly (at P ≤ 0.05).

f o

l a n

e

o r p

r P

r u o

J

31

Journal Pre-proof

Jo

ur

na

lP

re

-p

ro

of

Table 5: F values obtained from the analysis of variance of head and broken rice data shown in table 4a and table 4b. Source Head rice Broken rice F value F value Amino Acid DoM Variety Variety x DoM Variety Variety DoM x DoM 56168** 99893** 772** 48529** 94748** 518** Aspartic acid 93640** 1156** 33270** 82678** 512** Glutamic acid 80374** 197266** 216253** 138002** 5720870** 260508** 6960** Serine 2666449** 10963** 32117** 5084769** 11293** 45718** Glutamine 105123** 14202** 6597** 71388** 13910** 2051** Histidine 5612431** 15867** 6324** 5209700** 29899** 16689** Glycine 85407** 25806** 5146** 49666** 37792** 1181** Threonine 34240** 337535** 1339** 20975** 351698** 626** Citrulline 70115** 13349** 8621** 24555** 40321** 815** Arginine 48351** 61193** 3363** 29295** 71445** 1292** Alanine 25203** 20834** 1957** 23107** 22588** 935** Tyrosine 9005** 8050** 393** 8367** 5997** 201** GABA 1176030** 119244** 43630** 1135249** 87852** 26606** Cysteine 94447** 13188** 9993** 54901** 29770** 1415** Valine 90306** 15994** 3598** 41445** 19476** 2045** Methionine 41161** 9341** 1901** 41770** 6069** 1443** Tryptophan 35731** 23943** 1118** 33702** 30054** 1102** Phenylalanine 25964** 32743** 609** 27229** 30418** 623** Isoleucine 80764** 14420** 7606** 114109** 19324** 5272** Lysine 85522** 191186** 4420** 88810** 181065** 5600** Proline 268810** 248918** 3087** 162157** 250296** 526** Acidic 742824** 58197** 31566** 572133** 61565** 9921** Basic 120877** 74200** 3858** 112538** 85468** 3035** Aromatic *= p≤ 0.05; **= p ≤ 0.005; DoM: degree of milling; NS: non-significant; HR: head rice; BR: broken rice.

32

Journal Pre-proof Table 6: Pasting properties of flour of milled rice of different cultivars obtained after 6% to 10% degree of milling (DoM). Cultivars

Head rice DoM 6% 8% 10% PB1 87±3a 84±2a 84±1a 88±2b 84±2a 86±3b PS44 b b 88±4 86±4 86±2b PB1509 b c 88±3 88±3 84±2a PB1121 a b 87±2 86±4 86±3b PS5 Average 88 86 85 1917±45de 1932±73d 2014±45e PB1 1509±32c 1535±32c 1622±63c PS44 b b 1321±89 1345±43 1389±44b PB1509 d e 1907±78 1939±54 1983±57d PB1121 a a 940±12 966±11 1003±22a PS5 Average 1519 1543 1602 4407±324f 4444±345f 4540±134ef PB1 3252±132b 3285±211b 3408±231b PS44 c c 3652±312 3699±131 3789±123c PB1509 de de 3750±121 3801±142 3884±121d PB1121 a a 2731±432 2770±231 2815±114a PS5 Average 3558 3600 3687 69±6d 20±4a 37±3b PB1 25±3a 36±2b 48±4c PS44 b d 33±5 56±6 61±5e PB1509 ef c 87±7 37±2 55±7d PB1121 c b 54±3 36±4 25±8a PS5 Average 54 37 45 2559±321 2532±121de 2563±54ef PB1 1768±23a 1786±78a 1738±67a PS44 de c 2364±54 2410±115 2461±87d PB1509 c b 1930±32 1899±83 1956±34c PB1121 b b 1845±27 1840±73 1837±26b PS5 Average 2093 2093 2111 Means with similar letters in a column are not differ significantly (at P ≤ 0.05).

Setback viscosity (cP)

Breakdown viscosity (cP)

Final Viscosity (cP)

Peak Viscosity (cP)

Pasting temperature (°C)

Source

l a n

r u o

J

33

f o

o r p

e

r P

6% 82±2b 81±1a 82±4b 82±3b 82±3b 82 1833±42d 1503±45c 1288±64b 1873±25de 909±19a 1481 4328±245ef 3238±231b 3632±134c 3740±214d 2639±234a 3515 41±3c 18±5a 63±2e 56±4d 34±4b 42 2454±112de 1717±67a 2407±74c 1923±31b 1764±28a 2053

Broken rice DoM 8% 81±2a 81±3a 82±4b 81±1a 81±3a 81 1906±34e 1515±47c 1298±64b 1821±25d 934±13a 1495 4402±234e 3266±134b 3653±543c 3722±133d 2727±152a 3554 55±9c 80±14ef 56±8d 23±4a 28±5b 48 2551±132ef 1671±98a 2411±117d 1924±45c 1839±38b 2079

10% 81±3a 82±2b 81±1a 81±3a 81±4a 81 1970±43de 1559±35c 1328±54b 1932±53d 969±18a 1552 4467±345ef 3344±211b 3693±324c 3792±126d 2755±267a 3610 48±11d 31±8a 39±7b 43±11c 70±14e 46 2545±126ef 1746±76a 2404±98d 1903±53c 1856±47b 2091

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Table 7: F values obtained from the analysis of variance of head and broken rice data shown in table 2, table 3 and table 6. Source Head rice Broken rice Cultivar DoM Cultivar x DoM Cultivar DoM Cultivar x DoM 110** 455** 7.50** 110** 455** 7.50** RY 8331.20** 635329** 620** 8680** 651885** 553** PC 641.30** 11722** 869** 749** 10658** 650** FC 4.30* 24.8** 4.30** NS NS NS PT 1596327** 41391** 2383** 1454685** 17209** 3085** PV 3524547** 72919** 1341** 3443928** 33702** 3032** FV 1210234** 2442** 1870** 1187687** 5639* 3805* SBV 1033497** 10428753** 168007** 11087539** 1734314** 17652605** BDV *= p≤ 0.05; **= p ≤ 0.005; DoM: degree of milling; NS: non-significant; PC: protein content; FC: fat content; PT: pasting temperature; PV: peak viscosity; FV: final viscosity; SBV: setback viscosity; BDV: breakdown viscosity; RY: rice yield.

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Graphical Abstract

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A graphical abstract showing differences in head and broken rice of different long grain Indica r differences in the protein profile and internal starch granule structure, imbibed in protein matrix, are v PAGE and field emission scanning electron microscopic analysis.

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Journal Pre-proof Evaluation of head and broken rice of long grain Indica rice cultivars: Evidence for the role of starch and protein composition to head rice recovery Amardeep Singh Virdi1, Narpinder Singh1*, Priyanka Pal1, Parmeet Kaur1, Amritpal Kaur1 1

Department of Food Science & Technology, Guru Nanak Dev University, Amritsar, Punjab-

143005, India *

Author for correspondence Email: [email protected] (N. Singh).

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Authors declared no conflict of interest.

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Figure 1

Figure 2

Figure 3A

Figure 3B