Effect of Processing on Mungbean ( Vigna radiata ) Flour Nutritional Properties and Protein Composition

Pulses are traditionally processed prior to consumption, providing opportunities for modifying nutritional composition, dependant on the type of pulse and method used. In this study, we investigated the effect of whole seed, dehulling (dahl), germination and roasting on changes in mungbean flour nutritional properties, protein composition and relative protein abundance. Processed flours were analysed and compared for protein content, moisture, fat, ash, dietary fibre, total starch and amylose. Significant differences were imparted on dietary fibre content, with roasting and germination increasing the ratio of insoluble/soluble fibre as well as resistant starch. Comparative proteomic analysis resulted in a combined total of 539 protein identifications, searching against the Mungbean reference genome (NCBI Vigna radiata Annotation Release 100). Normalised spectral abundance factors were used as a measure of relative abundance and statistical analysis was applied (Students’ T-Test), where proteins with a p-value of < 0.05 considered significantly different. Processing imparted considerable changes to nutritional composition and should be further exploited for food applications. The comparative proteomic analyses carried out in this study proved useful for investigating the effect of processing on subsequent changes in protein composition and relative abundance.


Introduction
Pulses can be used for enhancing the nutritional and functional properties of food, providing a source of protein, carbohydrate, dietary fibre, vitamins and minerals (Duranti & Gius, 1997;Prakash et al., 2001;Tharanathan & Mahadevamma, 2003;Boye et al., 2010;Nair et al., 2013;Vaz Patto et al., 2015).Major types of pulses grown in Australia include chickpea, faba bean, field pea, lentil, lupin and mungbean.The transformation of pulses from a commodity crop, to healthy value-added food ingredients, would benefit grain producers, processors and consumers.Promoting the health benefits of pulses, combined with improved nutritional qualities, functionality and diversity of food applications may lead to greater consumer acceptance, consumption and sustainable food production.
Processing of pulses prior to consumption provides an opportunity for modifying nutritional properties, reducing the level of anti-nutritional factors, increasing protein digestibility and bioavailability of nutrients, as well as improving functionality, flavour and aroma (Tharanathan & Mahadevamma, 2003;Vaz Patto et al., 2015;Patterson et al., 2017).Primary processing of pulses includes the more conventional methods such as soaking, dehulling, splitting and milling to flour for a range of applications.Secondary processing methods include a range of diverse treatments and include roasting, toasting, germination, fermentation and extrusion.Pulse flours can be further fractionated using wet or dry processes, for production of concentrated protein flours and isolates, which can also be modified to produce a range of functional ingredients (Fan & Sosulski, 1974;Thompson, 1977;Rahma et al., 2000;Li et al., 2010;Wang et al., 2011;Pelgrom et al., 2015).
Proteomic technologies provide a range of methods for characterising changes in protein composition, relative abundance and protein identification (Thelen & Peck, 2007;Matros et al., 2011).Aspects of this technology have previously been applied to mungbean for characterising changes in protein expression during seed development, germination and protein isolation (Ghosh & Pal, 2012;Kazlowski et al., 2013;Skylas et al., 2017).Advances in comparative proteomics include 'label-free' quantitation known as spectral counting (SC), based on counting the number of spectra identified for peptides of specific proteins, used as a proxy for protein abundance (Lundgren et al., 2010;Neilson et al., 2013).Resulting spectra is then used to interrogate protein sequence databases to infer identification.Abundantly expressed proteins, such as seed storage proteins, will produce more spectra, resulting in more peptides belonging to that particular protein being identified, which is then used as a measure relative abundance (Liu et al., 2004;Zhang et al., 2006;Zybailov et al., 2007;Neilson et al., 2013).Improvements in the quality of SC data were made with the application of normalised spectral abundance factors (NSAFs), which are applied to account for the length of individual proteins, enabling comparison and statistical analysis of relative protein abundance (Zybailov et al., 2006;Zybailov et al., 2007;Mosley et al., 2009;Podwojski et al., 2010;Neilson et al., 2013;Mirzaei et al., 2016).
The objective of this study was to determine the effect of selected primary and secondary processes such as dehulling, germination and roasting on the nutritional composition of mungbean flours, as well as subsequent changes in protein composition and relative abundance.This study provides further knowledge of mungbean protein composition and subsequent changes associated from processing.

Seed Material and Milling
Mungbean whole seed, raw dahl and roasted dahl were commercially processed and provided by the Blue Ribbon Group (Richlands, QLD 4077), produced from the large seeded Crystal variety.For germination of whole seed material, seed was cleaned in absolute ethanol for 1 min, then rinsed three times with water and drained.Fresh water was then added and seed material was allowed to soak and imbibe for 12 hours.Seeds were rinsed again, drained and germinated for 48 hours in an incubator (at 22 °C).Germinated seeds (including hulls) were oven dried (50 °C) and thrashed over a 2 mm sieve screen to dislodge seedling shoots, which were then separated and discarded.Seed material was milled to flour using an Alpine Pin Mill and designated herein as mungbean whole seed flour (MWF), raw dahl flour (MDF), roasted dahl flour (MRF) and germinated flour (MGF).

Nutritional Composition
Nutritional testing was carried out at the NATA accredited AEGIC Analytical Laboratory (Sydney) using approved standard methods of analysis.Testing was carried out in duplicate and the averaged result was reported.Nitrogen content was determined by the Dumas method using a LECO TruMac protein analyser (AOAC 992.23) and converted to protein (N × 6.25).Standard methods used included ash; AOAC 923.03 and AACC moisture;fat;and

Comparative Proteomic Analysis
The comparative proteomic methodology used in this study was the same as previously reported by the authors (Skylas et al., 2017).Flour samples were solubilised in 50 mM TEAB containing 0.5% SDS and probe sonicated, reduced (using dithiothreitol) and alkylated (iodoacetamide).Samples were digested with trypsin for 16 hours at 37°C and SDS was removed from the digested samples using a detergent removal kit followed by a C18 clean up.Samples were dried down, resuspended in 0.1% formic acid and used for analysis.Analysis was carried out by reversed phase nano-LC directly coupled in line with a MS/MS system (LC-MS/MS).Samples from each fraction were separated over 90 minute gradients using an Easy Nano LC 1000 (Thermo Scientific).Samples (10 L) were injected onto an 'in house' packed solid core Halo C18 100 m × 3 cm peptide trap column and desalted with 20 L of 0.1% formic acid.The peptide trap was switched on line with the C18 75 m × 10 cm analytical reversed phase column.Peptides were eluted from the column using a linear solvent gradient, step-wise from 5-25% of buffer [99.9% (v/v) acetonitrile, 0.1% (v/v) formic acid] for 80 min, 25-85% of buffer for 2 min and then held at 85% for 8 min at a flow rate of 300 L/min across the gradient.
The column eluate was directed into a nanospray ionization source of the QExactive mass spectrometer (ThermoScientific) and a 1.5 kV electrospray voltage was applied via a liquid junction upstream of the column.Resulting spectra were scanned over the range 350-2000 amu.Automated peak recognition, dynamic exclusion, and MS/MS of the top ten most intense precursor ions at 30% normalised collision energy were performed.The LC-MS/MS spectra were searched using the MS software Mascot (Matrix Science, London, UK), against the Mungbean reference genome (NCBI Vigna radiata Annotation Release 100) containing 35143 entries (Kang et al., 2014).Peptides were identified with a 1% false-discovery rate from a concatenated forward-reversed database search.Significant peptide matches were exported and samples compared using NSAF with the program referred to as "SCRappy" (Neilson et al., 2013).Proteins with p-values < 0.05 following Student's T-Test of NSAF were considered significantly different between groups.

Nutritional Composition of Processed Mungbean Flours
Nutritional composition of respective mungbean flours (MWF, MDF, MRF and MGF) were analysed in duplicate and the average result for each nutritional component is reported in Table 1.The process of dehulling removes the outer seed coat from the cotyledon, which reportedly comprises ~12% of dry seed weight (Singh et al., 1968), producing dahl with improved palatability and cooking time, used in a range of food applications.The effect of dehulling on nutritional composition was determined by comparison of MWF and MDF, primarily resulting in decreased dietary fibre content, from 10.6 to 4.6 g 100 g -1 , respectively.Dehulling also reduced ash content, by removal of mineral content present in the outer seed coat.The effect of germination on nutritional composition was determined by comparison of MWF and MGF, with germination increasing protein and dietary fibre content, altering the ratio of insoluble (IDF) to soluble dietary fibre (SDF).Changes in the dietary fibre content most likely resulting from enzymatic modification of cell wall polysaccharides during germination, consistent with previous reports of increased crude fibre in lupins and dietary fibre in peas (Martín-Cabrejas et al., 2003;James et al., 2012).However, changes in nutritional composition can be partly attributed to decreased starch content, resulting from enzyme hydrolysis during germination, required to provide a source of energy for the emerging seedling.The proportion of amylose in starch also decreased in MGF, with similar findings observed for germinated lentil and horsegram flours (Ghumman et al., 2016).Roasting is often used for enhancing nutritional qualities, flavour and aroma, with the effect on nutritional composition determined by comparison of MDF and MRF.Roasting imparted significant changes in the ratio of IDF/SDF, increasing from 2.5 to 16, for MDF and MRF, respectively.The increased resistant starch content of MRF is most likely due to high-temperature induced modification of starch structure, increasing resistance to starch-degrading enzymes (Li et al., 2011).Table 1.Nutritional composition of processed mungbean flours (g 100 g -1 ).Protein and starch were corrected for moisture content and reported on a dry basis

Effect of Germination on Protein Composition and Relative Abundance
During the germination process, increased enzyme activity leads to modification of nutritional composition, resulting from hydrolysis of macronutrients such as starch and protein (Nout & Ngoddy, 1997).This process can be used to enhance nutritional composition, resulting in increased protein solubility and digestibility (James et al., 2012).The effect of germination on protein composition and relative abundance was determined by comparison of proteins identified for MWF (95 proteins) and MGF (169 proteins).Of these proteins, 85 were classified as common, in which, 27 were found to be significantly different in relative abundance, reported in Table 3, sorted in descending order of NSAFs.Identified proteins classified as specific for either MWF or MGF are listed in Appendix B.
Of those proteins that were significantly different between MGF and MDF, there were 6 enzymes, involved in metabolism, which were more abundant in MGF.These included enzymes involved in starch degradation and carbon metabolism, including alpha-1,4 glucan phophorylase, UTP-glucose-1-phosphate uridylyltransferase, fructose-bisphosphate aldolase and ATPase (subunit 1).The other two enzymes included seed linoleate 9S-lipoxygenase, involved in fatty acid metabolism (Aanangi et al., 2016) and nudix hydrolase 3-like, which is involved in hydrolysis of a wide range of organic pyrophosphates and has been implicated to play a role in germination of Arabidopsis (Zeng et al., 2014).Other enzymes involved in metabolism, detected only in MGF (Appendix B), were glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, fructokinase-2-like and enolase.
Heat shock proteins (HSP) were also found to be significantly different between MGF and MDF.These proteins act as molecular chaperones, assisting in cellular processes including folding, assembly and degradation of proteins, as well as stabilisation and refolding of proteins in response to stress related conditions (Wang et al., 2004).HSPs found to be more abundant in MGF included HSP 70 kDa, HSP cognate 70 kDa protein 2 and HSP 83.The HSP cognate 70 kDa protein had the second highest ratio of NSAFs between MGF/MDF (at 7.28714), with the luminal-binding protein, also thought to function as a chaperone, having the highest ratio of NSAFs (at 7.64959).This is indicative of these proteins playing a crucial role in protein synthesis and degradation during the early stages of germination.Small HSPs (17.6 kDa) have previously been identified in germinated mungbean cotyledons, also thought to play a protective role during this process (Ghosh & Pal, 2012).

Effect of Roasting on Protein Composition and Relative Abundance
High temperatures required for roasting can partially denature, aggregate and modify protein structures, including glycation of lysine and free amines, resulting from Maillard reactions (Walker & Kochhar, 1982;Sun-Waterhouse et al., 2014;Wang et al., 2016).Such protein modifications may reduce the potential number of tryptic peptides generated during the protein digestion step, carried out prior to LC-MS/MS, potentially leading to an under-estimation of the relative abundance of these proteins, compared to raw or non-roasted samples.The effect of roasting on protein composition and relative abundance was determined by comparison of proteins identified for MDF (164 proteins) and MRF (111 proteins).Of these proteins, 105 were classified as common, in which, 14 were found to be significantly different in relative abundance, reported in

Conclusion
The main objectives of this study were to expand our knowledge and understanding of the effect of specific processing conditions on nutritional composition of mungbean flours, as well as providing significant and comprehensive analyses of mungbean protein composition and relative abundance using a comparative proteomic approach.Processing methods used in this study imparted significant changes to mungbean nutritional composition, leading to altered functionality and potential end-use applications.Investigating the effect of processing conditions on protein composition and relative abundance is important for the production of functional, value-added high protein fractions for food applications.Innovative processing methods applied to mungbean and other pulse flours, combined with advanced proteomic tools for characterising protein composition and relative abundance, will provide an effective platform for developing concentrated protein flours and isolates.This study paves the way for further work focussing on the production of functional flours with enhanced digestibility and bioavailability of nutrients.Enhanced nutritional qualities and promotion of the health benefits of pulse products could potentially lead to wider consumer acceptance and increased sustainability for future food production.

Table 2 .
Table 4, sorted in descending order of NSAFs.Identified proteins classified as specific for either MWF or MRF are listed in Appendix C. Inferred identity and relative abundance of those proteins classified as common for MDF and MWF.NSAF ratios of MDF/MWF that are statistically significant (p < 0.05) are highlighted in bold

Table 3 .
Inferred identity and relative abundance of those proteins classified as common for MGF and MWF.NSAF ratios of MGF/MWF that are statistically significant (p < 0.05) are highlighted in bold

Table 4 .
Inferred identity and relative abundance of those proteins classified as common for MDF and MRF.NSAF ratios of MDF/MRF that are statistically significant (p < 0.05) are highlighted in bold