In Vitro Pepsin Digestibility of Cooked Proso Millet (Panicum

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In Vitro Pepsin Digestibility of Cooked Proso Millet (Pancium miliaceum L.) and Related Species from Around the World Paridhi Gulati, Shangang Jia, Aixia Li, David Richard Holding, Dipak Santra, and Devin Jerrold Rose J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02315 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

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Journal of Agricultural and Food Chemistry

In Vitro Pepsin Digestibility of Cooked Proso Millet (Pancium miliaceum L.) and Related Species from Around the World

Paridhi Gulati,a Shangang Jia,b Aixia Li,b David R. Holding,b Dipak Santra,b Devin J. Rosea,b,*

a

Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln,

NE, USA b

Department of Agronomy and Horticulture, University of Nebraska-Lincoln, Lincoln,

NE, USA *Corresponding author. E-mail: [email protected]; phone: +1 402 472 208

ACS Paragon Plus Environment


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ABSTRACT

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Thirty-three accessions of proso millet (Panicum miliaceum) with different

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countries of origin were screened for their pepsin digestibility after cooking to identify

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samples with high digestibility. The pepsin digestibility of all samples ranged from 26 to

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57% (average 32%). There were no apparent differences in protein profiles (SDS-

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PAGE) of samples with the lowest, intermediate, and highest digestibilities. However,

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LC-MS/MS analysis revealed a negative correlation between pepsin digestibility and

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peptides that matched to high molecular weight proteins (24 kDa) from Panicum hallii

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with regions of contiguous hydrophobic amino acids. Low digestibility upon cooking was

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also observed for other species from the Panicum genus, such as little millet,

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switchgrass and panicgrass, which suggests a unique inherent property of the genus.

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The obtained results from this study may form a basis for in-depth analysis of proso

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proteins that may help in developing new cultivars with higher digestibility upon cooking.

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Keywords: accessions, protein identification, switchgrass, panicins, gluten-free


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INTRODUCTION

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Panicum is a large plant genus that includes more than 400 species of grasses,1

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with common species being P. miliaceum (proso millet); P. sumatrense (little millet); P.

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virgatum (switchgrass), P. capillare (witchgrass); P. halli; and P. hirticaule (panicgrass).

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Among these, proso millet, and to some extent little millet, have economic importance

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given their application mostly as bird feed and some share in the human food industry.2

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Proso millet is one of the oldest domesticated crops with recorded origin in China

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and wide culinary presence in Asian countries.3 From Asia, the cultivation of proso millet

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spread to Eurasia and eastern Europe and was introduced to the US by German-

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Russian immigrants at the end of 19th century.4

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Presently, the US Department of Agriculture-National Plant Germplasm System

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(USDA-ARS GRIN Global)5 has record of almost 700 accessions of proso millet grown

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in almost every country in the world. This suggests the excellent adaptability of proso

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millet to grow in diverse conditions. Apart from that, proso millet is an excellent

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agricultural aid especially for wheat, corn and sorghum. It is often employed as a

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rotational crop as it can replenish the soil nutrients, and preserve deeper soil water. It is

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also used to manage grass weeds and diseases and has been a common choice as an

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emergency cash crop.6 Although the grain is gluten-free and contains an appreciable

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amount of protein, fiber, and antioxidants, its limited human consumption currently

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restricts its demand.

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In order to promote proso millet for human consumption, our previous work

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focused on evaluation of proso millet protein quality. We found a substantial decline in



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protein digestibility during thermal processing due to hydrophobic aggregation.7,8 This

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could prove to be a significant hurdle in promoting the crop as human food.

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One way to combat this issue is to develop proso millet-based products using

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novel processing techniques that could prevent the loss in digestibility. Alternatively,

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there may be proso millet cultivars with protein constituents that are less prone to the

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formation of hydrophobic aggregates upon cooking, which could be used to modify the

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commonly grown cultivars. The current research was focused on the latter, i.e.,

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identification of proso millet lines that are naturally highly digestible after cooking. We

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expected that variations in protein structure of proso millet samples from around the

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world could result in variation in pepsin digestibility upon cooking.

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Further, there are many other species of grasses within the Panicum genus that

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are mostly used for bio-fuel production. Comparing pepsin digestibility of these species

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to proso millet may reveal if the observed poor digestibility property of proso millet

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proteins is restricted to P. miliaceum or is a characteristic of the genus. Also, since

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proso millet is usually grouped with other millet varieties (e.g., finger, pearl, foxtail), we

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investigated the pepsin digestibility of other millets to understand their protein behavior

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after cooking. Thus, the principal objective of this study was to determine the variation

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in digestibility of proso millet accessions after cooking, specifically to identify samples

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with higher digestibility. A secondary objective was to compare digestibility of cooked

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proso millet to other related species and other types of millet.

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MATERIALS AND METHODS

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Millet seed procurement, preparation and growth in the greenhouse. In total,

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35 samples of proso millet and 10 samples of related species were used in this study


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(Table 1). Specifically, 33 of the proso millet samples with different countries of origin

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were obtained from the USDA-ARS North Central Regional Plant Introduction Station

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(Ames, IA, USA). Two commercial de-hulled samples of proso millet were also used in

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the study. One of them was obtained from Clean Dirt Farms (Sterling, CO, USA) and

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the other was obtained from a local market in the Ukraine. Three samples of related

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species (little millet, panicgrass, and witchgrass) were obtained from the USDA-ARS

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North Central Regional Plant Introduction Station. Additionally, three samples of

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switchgrass were obtained from the USA (two from Nebraska and one from Illinois), and

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whole finger millet, pearl millet, and foxtail millet samples were obtained from the USA

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(Nebraska). Finally, a commercial sample of de-hulled foxtail millet was obtained from

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China.

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The 33 proso millet accessions that were obtained from USDA-ARS were grown

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in a greenhouse at the University of Nebraska-Lincoln (first 33 samples in Table 1). Ten

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seeds of each cultivar were planted in a potting mixture containing peat moss (85%)

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along with vermiculite and perlite (Sunshine MVP, Sun Gro Horticulture, Agawam, MA).

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The seeds started germinating one week after planting. Four germinated seeds were

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moved to four different pots and grown under 16 h day length cycle with day time

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temperature of 28-30 °C and night time temperature of 20-23 °C. The plants were

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watered every alternate day and fertilized once a week (12-2-14, Optimum, Master

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Plant-Prod, Ontario, Canada). The flowering stalks of each plant were covered with

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paper bags at the first sign of anthesis to facilitate self-pollination.

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seeds were allowed to dry on the plant and the seeds were harvested 110-120 d after

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planting. Only the seeds from the earliest flowering main branch were used for analysis.


The developed


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Seeds collected from the four plants growing for each accession were pooled and then

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stored at 4 °C until analysis. Plant height (cm), flowering days (days after planting), and

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any special morphological trait (e.g., panicle characteristic, seed color) observed were

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recorded for plants growing in the greenhouse (Table 1).

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The original seeds and greenhouse-grown seeds were milled using a ball mill

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(Genogrinder; Thermo Scientific, Waltham, MA) for 120 s at 1600 rpm. The milled

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samples were saved in polyethylene bags at 4 °C until further analysis.

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Chemicals. The following chemicals and enzymes were used in the study:

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pepsin (P7000), trypsin (T1426), sodium hydroxide, phosphoric acid (85%), 2-

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mercaptoethanol, potassium phosphate dihydrate, sodium dodecyl sulfate, acrylamide,

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sodium

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iodoacetamide, formic acid, acetonitrile each from Sigma-Aldrich (St. Louis, MO USA);

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tetramethylethylenediamine (TEMED), ammonium persulphate, and coomassie brilliant

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blue (R-250), each from Thermo Fisher (Waltham, MA USA), and chymotrypsin from

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Promage (Madison, WI, USA).

dihydrogen

phosphate,

sodium

tetraborate,

1,4-dithriothreitol

(DTT),

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Protein concentration. Protein concentration of all samples (original and

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greenhouse grown) were analyzed by combustion using a nitrogen analyzer (FP 528,

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Leco, St. Joseph, MI, USA) with a protein factor of 6.25.

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In vitro pepsin digestibility. Two hundred milligrams of milled sample were

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cooked in 10 mL water at 100 °C for 20 min (time recorded after temperature was

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reached) in a water bath. The cooked samples were cooled to room temperature

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(approximately 10 min) and immediately used for digestibility measurements.


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Pepsin digestibility of milled seeds from originally procured samples and

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greenhouse-grown samples were analyzed after cooking using the residue method

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described in Gulati et al.7 This method measures changes in solubility of cereal proteins

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after hydrolysis with pepsin, which are normally largely insoluble, and matched closely

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with multi-enzyme digestion in our previous publicaiton.7 Pepsin digestibility was

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calculated according to the following equation: PD (%) = [(Ni − Nf)/Ni] × 100%, where Ni

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was the total N in the sample before digestion and Nf was the amount of N recovered

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after digestion.

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Electrophoresis. Total proteins from milled un-cooked samples (50 mg) were

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extracted with 1.5 mL of 0.0125 M sodium tetraborate buffer (pH 10) containing 1%

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SDS and 2% 2-mercaptoethanol for 2 h at room temperature followed by

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centrifugation.9 The supernatant was subjected to SDS-PAGE analysis using a vertical

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mini gel system (Mini protein II cell tetra system, Biorad, CA). Protein extract was mixed

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with sample buffer in the ratio of 4:1 and loaded along with molecular weight markers

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(BioRad Precison Plus Dual color protein standard, 10-250 KDa) on to gel system with

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following specifications. The resolving gel consisted of 15% polyacrylamide in 1 M Tris

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HCl buffer (pH 8.8), and 1% SDS (w/v). The stacking gel contained 5% polyacrylamide

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in 0.63 M Tris HCl buffer (pH 6.8), and 1% SDS (w/v). TEMED (0.05% v/v) and

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ammonium persulfate (0.1 v/v) were used to polymerize the gels. Electrophoresis was

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performed at 70 V for 120 min in tank buffer consisting of 1.9 M Tris, and 1% SDS (w/v).

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After electrophoresis, the gels were stained with 20 mL of coomassie brilliant blue

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reagent (0.25%) containing isopropanol and acetic acid for 60 min. De-staining was

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achieved by washing gels several times in a solution of 10% acetic acid and 30%



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methanol in water. Gel images were captured and analyzed using Image Analyser

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(BioRad Molecular Imager, Gel Doc-XR system, CA).

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In-gel protein hydrolysis and LC-MS/MS analysis. The storage protein band

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(23 kDa) from SDS-PAGE gels were used for in-gel chymotrypsin-trypsin hydrolysis and

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then subjected to LC-MS/MS analysis in duplicate. Gel bands were excised from the

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gels with a razor blade, reduced with 10 mM DTT, alkylated with 20 mM iodoacetamide

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and then fully de-stained before hydrolysis. The solutions were removed by

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centrifugation. For hydrolysis, 200 ng of chymotrypsin was added to each sample and

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incubated overnight at 37 °C. The peptides were then liberated from the gel pieces

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using 2% acetonitrile/1% formic acid solution and then 60% acetonitrile. To increase

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coverage, the extracted peptides and the gel pieces left from the chymotrypsin

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hydrolysis were re-combined and 200 ng of trypsin was added and the tubes incubated

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at 37 °C overnight. The proso millet peptides obtained from chymotrypsin-trypsin

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hydrolysis were pooled and separated on a rapid separation liquid chromatography

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system (Dionex U3000 nano) equipped with a C18 column (0.075 mm x 250mm Waters

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CSH). The peptides were eluted using a gradient of mobile phase A as 0.1% formic acid

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in MilliQ water and mobile phase B as 0.1% formic acid in 80% acetonitrile at a flow rate

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of 300 nL min-1 for 1 h and detected using a mass spectrometer (Q-Exactive HF;

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Thermo Fisher Scientific).

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Data were analyzed with Mascot software (Matrix Science, London, UK; version

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2.6.1), which was set up to search the cRAP_20150130 and NCBI databases (selected

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for Viridiplantae, January 2018, 5845301 entries). Mascot was searched with a fragment

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ion mass tolerance of 0.060 Da and a parent ion tolerance of 10.0 PPM. De-amidation


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of asparagine and glutamine, oxidation of methionine and carbamidomethyl of cysteine

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were specified in Mascot as variable modifications. Scaffold (version 4.7.5, Proteome

153

Software Inc., Portland, OR) was used to validate the MS/MS based peptide and protein

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identifications from both replicates. Protein identifications were accepted if they could be

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established at greater than 99.0% probability and contained at least 2 identified

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peptides using a false discovery rate of

In Vitro Pepsin Digestibility of Cooked Proso Millet (Panicum

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