Heating Reduces Proso Millet Protein Digestibility via Formation of

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Heating Reduces Proso Millet Protein Digestibility via Formation of Hydrophobic Aggregates Paridhi Gulati Gulati, Aixia Li, David Richard Holding, Dipak Santra, Yue Zhang, and Devin Jerrold Rose J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05574 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Journal of Agricultural and Food Chemistry

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Heating Reduces Proso Millet Protein Digestibility via Formation of Hydrophobic

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Aggregates

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Paridhi Gulati,a Aixia Li,b David Holding,b Dipak Santra,b Yue Zhang,a Devin J.

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Rosea,b,*

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a

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NE, USA

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b

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

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

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NE, USA

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*Corresponding author. 1901 N 21st St., room 268, Lincoln, NE 68588 USA; E-mail:

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[email protected]; phone: +1 402 472 2802; fax: +1 402 472 1693

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Short title: Heating Reduces Proso Millet Protein Digestibility

ACS Paragon Plus Environment


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ABSTRACT

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Proso millet protein has reported structural similarities with sorghum. In order to explore

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the potential of this crop as an alternative protein source for people with gluten

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sensitivity, in vitro protein digestibility was analyzed. De-hulled proso millet flour was

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subjected to various processing techniques (dry heating and wet heating). Regardless

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of the processing technique there was a significant decline in digestibility of protein in

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proso millet flour when compared with unprocessed flour (from 79.7±0.8% to

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42.0±1.2%). Reduced digestibility persisted even when cooking with reducing agents.

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Heating in the presence of urea (8 M) and guanidine HCl (4.5 M) prevented the

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reduction in observed digestibility (urea cooked: 77.4±0.8%; guanidine HCl cooked:

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84.3±0.9%), suggesting formation of hydrophobic aggregates during heating in water.

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This was supported by an increase in surface hydrophobicity upon cooking. Thus, the

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proso millet protein, termed Panicin, forms hydrophobic aggregates that are resistant to

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digestion when subjected to heat.

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Keywords: in vitro; prolamins; urea; sorghum; disulfide bonds


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INTRODUCTION Gluten-free foods are an important and expanding market. In a recent survey, it

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was found that the sales of gluten-free foods grew by 34% annually in the last five years1

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and is expected to continue to rise. The driving force for this rise is the increase in

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diagnosed gluten intolerant cases and changing mindsets towards gluten in the diet. In

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the US, this has created a demand for alternative gluten-free crops that can replace

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wheat in familiar foods with the added advantage of nutrition, cost effectiveness and

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availability.2 Among gluten-free grains (e.g., rice, millets, teff, sorghum), rice is the most

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widely produced and consumed crop, but has a low protein content compared with

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wheat. Lately millets (e.g., finger, pearl, proso, foxtail) have been utilized as gluten-free

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alternatives.

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Among different millet varieties, proso millet is the only type of millet grown on a

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commercial scale in the US. Proso millet (e.g., true millet, common millet, hog millet) is

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an important crop from an agricultural standpoint in the Great Plains of the US due to its

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short growing duration, low water requirement, and resistance to pests and diseases.

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Furthermore, millet can lead to improvement in yields of wheat, corn and sorghum when

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grown in rotation with these crops.3-4 The crop belongs to the genus Panicum and is

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more closely related to inedible grasses like switchgrass and panicgrass than to other

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millet species. Different millet types do not bear species or genus similarity (although

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pearl, proso, and foxtail millets belong to same subfamily, Panicoideae, which also

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includes maize and sorghum) and are grouped together based only on the basis of their

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small grain size and drought-resistance. Thus, different millets would be expected to

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have different physical and chemical properties.



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Nutritionally, proso millet is a good source of protein, vitamins and minerals and

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its nutritive parameters are comparable or better than common cereals.5 Reports have

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suggested that the quantities of nutrients in millet are very similar to the recommended

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ratio of protein, carbohydrate, and lipid.6 Also, research on proso millet protein has

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provided evidence for its beneficial role in cholesterol metabolism and liver injuries.7

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The protein content of proso millet (13%) is similar to wheat with the added

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advantage of being gluten-free. This makes proso millet a potential candidate as an

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alternative protein crop. However, there is dearth of information on the composition and

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quality of proso millet protein. While Lorenz8 found lower lysine content in proso millet

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protein compared to wheat, Kalinova6 reported the opposite results. Since sorghum and

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proso millet belong to the same subfamily a generalized statement is usually made for

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their protein structure,9 implying that these cereals contain similar types of proteins.

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Furthermore, at times the protein behavior of different millet types (finger, foxtail, pearl

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etc) are assumed to be same despite belonging to different genera.10 There is also

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contradiction pertaining to proso millet protein digestibility. Ravindaran11 reported an

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improvement in digestibility of proso millet proteins on cooking, while Kovalev et al.12

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found a reduction in protein digestibility upon cooking. These discrepancies demand a

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thorough understanding of proso millet protein quality.

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In vitro protein digestibility techniques are often a first step in measuring cereal

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protein quality due to their rapidity and sensitivity. A number of in vitro techniques with

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varying protease types and concentration, incubation conditions and end product

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analysis techniques have been used to measure protein digestibility of various foods.13-14

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Among these techniques, the residue method, which employs pepsin as the main


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proteolytic enzyme,15 has been successfully used for measuring protein quality of mainly

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sorghum, but has also been applied to other cereals.10 Thus, the established technique

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for sorghum digestibility was also employed as a first step studying digestibility of proso

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millet flour.

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Since all cereals are subjected to some kind of processing technique before

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being consumed, this study was mainly focused on understanding the effect of different

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processing techniques on the in vitro protein digestibility of proso millet flour. The

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secondary objective was to determine the cause of any changes in digestibility of proso

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millet protein upon cooking.

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MATERIALS AND METHODS

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Materials. Commercially available de-hulled proso millet was obtained from

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Clean Dirt Farms (Sterling, CO, USA). Eight pure cultivars of proso millet (Early Bird,

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Sunrise, Cope, Snobird, Plateau, Horizon, Sunup and Huntsman) were also used in this

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study. These samples were grown at Scottsbluff, Nebraska, USA. For comparison,

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whole finger millet, [University of Nebraska-Lincoln (UNL) NeFm #1], pearl millet (UNL

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NM-4B), and foxtail millet (UNL N-Si-7), grown in 2013 or 2014 at Mead, NE, USA, were

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used in this study. Whole white sorghum (UNL3016) and wheat (variety McGill) were

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grown in Lincoln, NE USA. Sorghum grains were decorticated using a laboratory scale

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decorticator (Venables Tangential Abrasive Dehulling Device, Venables Machine

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Works, Ltd, Saskatoon, Canada) for 60 seconds before being milled. All grains were

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milled using a pilot scale hammer mill (20SSHMBD, C.S. Bell, Tiffin, OH, USA) with

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screen size of 0.7 mm.



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

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amylase (3,000 U/mL) and amyloglucosidase (3,260 U/mL), each from Megazyme

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(Bray, Ireland); pullulanase (E2412), pepsin (P7000), pancreatin (8X USP) sodium

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azide, sodium hydroxide, phosphoric acid (85%), sodium bisulfite, 2-mercaptoethanol,

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

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2,4,6-trinitrobenzenesulfonic acid (TNBS, P-2297), Folin-Ciocalteu phenol reagent (2

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N), sodium dihydrogen phosphate, 8-anilinonaphthalene-1-sulfonic acid (ANS), urea,

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phenol, sodium bicarbonate, and sodium carbonate, each from Sigma-Aldrich (St.

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Louis, MO USA); and guanidine HCl, tetramethylethylenediamine (TEMED), ammonium

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persulphate, and coomassie brilliant blue (R-250), each from Thermo Fisher (Waltham,

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MA USA).

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Extraction of millet proteins. Millet proteins were separated from starch and

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fiber components by a wet milling method.16 Modifications and additions to the

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published method to improve purity of millet protein were made based on preliminary

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experimentation. Briefly, 250 g de-hulled millet grains were steeped in distilled water for

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3 h at 40 °C followed by overnight steeping at 4 °C. The steeped grains were washed

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and then blended with 500 mL fresh water in a Waring blender (Dynamic Corp. of

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America, New Hartford City, CT, USA) with the blades reversed for 4 min. The blender

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was attached to an autotransformer (Staco Energy Products Co., Dayton, OH, USA)

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and power (120 V) was adjusted to 80%. The slurry obtained after blending was filtered

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through a #16 mesh sieve (1.18 mm openings; Air Jet Sieve, Hosokawa micron powder

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systems, NJ, USA). The slurry passing through the filter was passed through a plate mill

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(Quaker City model no. 4-E, The Straub Co., Warminster, PA). The finely ground slurry


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was then filtered through #100 mesh sieve (150 µm openings). The filtrate obtained was

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centrifuged at 5,000 x g for 15 min at 4 °C. The supernatant obtained was discarded

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and the top gray layer (protein) was scarped from white (starch) bottom layer of pellet.

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The scraped off protein fractions were mixed from different centrifugations and washed

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with water followed by repeated centrifugation and protein layer separation. When there

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was no more visible distinction left between protein and starch layers, the mixture was

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subjected to hydrolysis by α-amylase (0.05 mL / g solids), amyloglucosidase (0.7 mL/g

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solids) and pullulanase (0.12 mL/g solids) for 48 h at 37 °C and pH 5 (sodium acetate

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buffer) with 0.01% sodium azide. The tube was then washed twice and freeze dried and

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the protein content was measured by combustion.

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Amino acid profile. For amino acid analysis, samples were hydrolyzed for 24 h

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using 6N HCl containing 0.5% phenol and derivitized.17 Briefly, the hydrolyzed samples

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were dried and amino acids were reconstituted with 1 mL of 20 mM HCl. The

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constituted amino acids and hydrolysate standard amino acid mixture were reacted with

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the AccQ-Tag derivatization chemistry (Waters, Milford, MA, USA), following this they

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were separated detected and quantified using reversed phase HPLC (Agilent Infinity

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1290 HPLC-DAD). The concentrations were calculated using a series of standard

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dilutions run alongside the samples and reported as g/kg protein. Under the acid

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hydrolyzed conditions cysteine is converted into cysteic acid and methionine into

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methionine sulfone. During the process tryptophan is destroyed, hence tryptophan was

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not determined.



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Phenolics. Phenolics in acidified methanol extracts from different samples were

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quantified using Folin-Ciocalteu reagent.18 Results were expressed as mg of gallic acid

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equivalents per gram of sample.

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Heating in excess water. Samples (400 mg) of de-hulled proso millet, whole

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proso millet, wheat, de-hulled sorghum, finger millet, pearl millet, and foxtail millet were

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suspended in 5 mL of water and then heated at 25-100 °C (in 15 °C increments) for 20

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min. Following the holding time all flours were first cooled to room temperature

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(approximately for 10 min) and then prepared for pepsin digestibility by warming at 37

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°C (see ‘In vitro protein digestibility’).

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Heating in limited water. The moisture contents of de-hulled millet flour,

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sorghum and wheat flour were adjusted at 10, 20 and 30% by the addition of a

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calculated volume of water. The flours were then hermetically sealed in a glass vial (2

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mL) and subjected to oven heating (VWR Scientific, 1350F Forced Air Oven, IL) or

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pressure-cooking (Deni Electric Pressure Cooker, Model 9780, Keystone Mfg company,

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Buffalo, NY) at 120 °C for 20 min. After cooling, 400 mg of sample was subjected to

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digestibility measurements (see ‘In vitro protein digestibility’).

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De-hulled millet flour, sorghum flour, and wheat flour were extruded using a

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laboratory scale co-rotating conical twin screw extruder at a screw speed of 210 rpm

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(CTSE-V, C.W. Brabender, Hackensack, NJ, USA).19 The extruder barrel had 4

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temperature zones with temperatures of the ﬁrst two zones maintained at 50 and 80 °C,

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respectively and last two zones at 120 °C. The moisture of the flours was adjusted to

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17% (wet basis) and equilibrated overnight before extruding. The extrudates were

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ground using cyclone sample mill (UDY, Fort Collins, CO, USA) with a screen size of 1


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mm and subjected to pepsin digestibility measurement (see ‘In vitro protein

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digestibility’).

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Heating in the presence of reducing agents. Millet, sorghum and wheat flour

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(400 mg) were heated (100 °C/ 20 min) in 5 mL of either 0.1 M sodium bisulfite or 0.1 M

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2-mercaptoethanol,20 and then subjected to in vitro digestion (see ‘In vitro protein

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digestibility’).

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Heating in the presence of chaotrops. In order to explore any hydrophobic

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interactions, millet flour (400 mg) was heated (100 °C/ 20 min) in 5 mL of 1 M or 8 M

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urea or 4.5 M guanidine hydrochloride. Following cooking, urea and guanidine

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hydrochloride were removed by dialysis (MW 12-14 kDa) overnight against water. After

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dialysis, the samples were subjected to in vitro digestion (see ‘In vitro protein

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digestibility’).

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In vitro protein digestibility. Pepsin digestibility was measured using residue

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method developed by Mertz et al., 1984.15 with slight modifications.21 After digestion,

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the nitrogen remaining in the pellet was measured and 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 concentration of N in the sample before digestion and Nf was the concentration

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of N in the recovered pellet after digestion.

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Gastrointestinal proteolysis was also simulated using the method described by

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Mandalari et al.22 with some modifications. Individual tubes were maintained in duplicate

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for various time periods between 0-120 min for gastric digestion and 0-240 min for

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intestinal digestion (following 2 h of gastric digestion; 120-360 min total digestion time)

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along with a blank (no enzyme tube). In each tube, 100 mg of flour was suspended in



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simulated gastric fluid (SGF; 4 mL, 0.5 M NaCl, pH:2.5) to achieve a pH of 2.5

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(approximately 4 mL) and incubated at 37 °C for 10 min. To analyze the effect of

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heating, flour samples (100 mg) were first suspended in water (1 mL) and heated at 100

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°C for 20 min prior to adding SGF and proceeding with the gastric digestion phase. The

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contents were then mixed with pepsin dissolved in SGF to give an activity of 200 U of

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pepsin/mg of protein in sample and incubated for the specified time. Gastric digestion

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was stopped by raising the pH to 7 using 0.5 M sodium bicarbonate. For intestinal

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digestion, pepsin hydrolyzed samples were mixed with 1 mL simulated intestinal fluid

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(SIF) (0.05 M KH2PO4, pH: 7.0) and warmed at 37 °C. Meanwhile, pancreatin (1.5

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mg/mL) was suspended in SIF and centrifuged. The supernatant (3 mL) was added to

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flour mixture and incubated for the specified time. Intestinal digestion was stopped by

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plunging the tubes into a boiling water bath for 5 min and then placing on ice. Gastric

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and intestinal digested tubes were centrifuged and the supernatants were used to

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quantify degree of hydrolysis (DH) while the pellets were analyzed for insoluble nitrogen

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(protein).

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The DH, or extent of proteolytic hydrolysis, was determined by the reaction of

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free amine groups with TNBS using leucine as the standard.23 DH was then calculated

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using the following equation: DH (%) = (hs/htotal) ×100%, where hs was defined as the

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mmol of free amine groups (leucine equivalents) per gram of protein (initial) in the

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sample and htotal was the mmol of free amino groups per gram of protein assuming

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complete hydrolysis of the protein (7.96 mmol/g protein). This was calculated by

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summing the molar concentrations of individual amino acids per gram of protein (see

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‘Amino acid profile’ section and Supporting Information). The pellets were analyzed for


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insoluble nitrogen and results were expressed as a fraction of the total initial nitrogen.

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All tubes (representing duplicate samples from above) were measured thrice.

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Electrophoresis. Proteins before and after digestion or with and without heating

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were extracted from 50 mg of sample. If samples were wet, they were dried overnight at

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50 °C. First, samples were heated at 100 °C for 20 min in 1.5 mL of 0.0125 M sodium

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tetraborate buffer (pH 10) containing 1% SDS and 2% 2-mercaptoethanol and then

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extracted for 2 h at room temperature followed by centrifugation.24 Alternatively, 8 M

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urea was added to the extraction buffer when testing the effects of urea on protein

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extraction. The supernatant was subjected to SDS-PAGE analysis using a vertical mini

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

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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 12% polyacrylamide in 1.9 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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done at 100 V for 60 min in tank buffer consisting of 1.9 M Tris, and 1% SDS (w/v). After

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

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

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washing gels several times in a solution of 10% acetic acid and 30% methanol in water.

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Gel images were captured and analyzed using Image Analyser (BioRad Molecular

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Imager, Gel Doc-XR system, CA).



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Surface hydrophobicity. Fluorescence intensity of millet proteins were

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measured both intrinsically and using an external fluorescence probe (8-

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anilinonaphthalene-1-sulfonic acid, ANS) as described.25 Intrinsic fluorescence of

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proteins is mainly attributed to tryptophan, a hydrophobic amino acid when the

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excitation wavelength is higher than 290nm. Two sets of samples of millet proteins were

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dispersed in 0.01 M phosphate buffered saline (PBS; pH 7, 10% NaCl, 2.5% sodium

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dihydrogen phosphate) with concentrations ranging from 0.01% to 0.1%. One set of

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millet proteins were heated at 100 °C for 20 min while other was maintained at room

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temperature. For measuring intrinsic fluorescence, both heated and unheated samples

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were excited at 295 nm and the fluorescence intensity recorded at 415 nm using a

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spectrofluorometer (LS55, PerkinElmer Inc., Walthman, MA). This was the maximum

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intrinsic fluorescence intensity based on a scan from 300-700 nm. For extrinsic

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fluorescence, the heated and unheated samples were mixed with 20 µL of ANS (8 mM

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in 0.01 M PBS) and incubated for 2 h at room temperature in dark. The fluorescence

248

intensity was recorded at an excitation wavelength of 365 nm and emission wavelength

249

of 520 nm.

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Statistical analysis. The overall treatment effects were first analyzed using

251

ANOVA. Following ANOVA, the specific difference among treatments were assessed

252

using either t-test, Tukey’s test or Dunnett test at a significance level of 0.05. The

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multiple comparison test used was specified in footnotes. All descriptive statistics were

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computed using JMP statistical software (JMP version 12.0.1, SAS Institute Inc.)

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RESULTS AND DISCUSSION


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In vitro protein digestibility of grains heated in excess water. Proso millet,

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sorghum, and wheat flours were heated in excess water at temperatures ranging from 25

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°C to 100 °C for 20 min. Following the holding time, flours were warmed or cooled to 37

259

°C and pepsin digestibility was assayed. Holding the flour-water slurries at temperatures

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above 40 °C before digestion resulted in a significant decrease in digestibility of sorghum

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and proso millet proteins while no change in wheat protein digestibility was observed

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(Figure 1). At the higher temperatures (85 °C and 100 °C), the digestibility of proso millet

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protein was significantly less than for sorghum. At 100 °C, a 50% decline in digestibility of

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millet flour was observed when compared with unheated flour.

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Poor protein digestibility may be positive or negative depending on perspective.

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Poor protein digestibility of foods consumed as staples in a limited diet can have negative

267

consequences on health. In contrast, in the developed world, where protein is often

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consumed far in excess of dietary requirements, low protein digestibility could constitute a

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lower calorie food that could help reduce energy intake. Under these circumstances,

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however, the same undigested protein may serve as substrate for large bowel

271

fermentation resulting in toxic metabolites that can trigger disease.26

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In general, cooking cereal flours or proteins in excess water has little impact on

273

pepsin digestibility, 27 as seen for wheat flour. However, reduction in pepsin digestibility

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upon cooking has been reported for sorghum.20 This unusual property of sorghum grain

275

has been attributed mainly to extensive disulfide cross-linking of proteins upon heating. 28

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A decrease in pepsin digestibility upon heating proso millet flour has not been reported

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previously. On the contrary, one report suggested an increase in digestibility upon heating



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proso millet9 These contradictory results could be due to varietal differences or differences

279

in assay technique. Therefore, both possibilities were explored.

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Eight cultivars of whole proso millet flour were assayed for pepsin digestibility

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along with three other types of millet (Table 1). All proso millet cultivars showed similar

282

dramatic decreases in digestibility upon cooking. Heating also reduced the digestibility of

283

pearl millet and foxtail millet flour but the reduction was not as drastic as observed for

284

proso millet. In contrast, cooking actually improved the digestibility of finger millet protein.

285

These results were in accordance with previous studies.29-31

286

Protein digestibility was also assayed using a sequential digestion procedure

287

mimicking both gastric and small intestinal digestion (Figure 2). This showed that heating

288

reduced digestibility of proso millet flour during both the gastric (pepsin) and small

289

intestinal phases (pancreatin). Thus, both the pepsin digestibility assay and the sequential

290

digestion assay confirmed that heating of proso millet flour had a significantly detrimental

291

effect on its protein digestibility.

292

In vitro protein digestibility of grain heated in limited water. Previous studies

293

have suggested that heating sorghum flour in limited water has less of a negative impact

294

on protein digestibility than heating in excess water.21,32 The above experiments

295

demonstrated that, like sorghum, proso millet protein digestibility also decreases when

296

heating in excess water. Therefore, proso millet, sorghum, and wheat flours were

297

adjusted to 10%, 20%, or 30% moisture (wet basis) and then heated in sealed containers

298

in an oven or autoclave and protein digestibility was subsequently assayed.

299 300

Oven heating and autoclaving of proso millet flour, even at low moisture contents, gave similar results to heating in excess water with a 28% to 46% reduction in digestibility


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(Table 2). It is remarkable that the digestibility of millet flour reduced so dramatically even

302

when heated with as little as 10% moisture. Heating sorghum flour with limited water also

303

decreased digestibility, but the effect was not nearly as dramatic as for proso millet.

304

Extrusion, being a common low-moisture food processing technique, was also

305

tested. Extrusion resulted in a dramatic decrease in digestibility of proso millet proteins

306

(Table 2). In accordance with previous studies, only minimal reduction in digestibility was

307

observed when sorghum was extruded. It has been suggested that the lower impact of

308

dry heating on sorghum protein digestibility is due to limited formation or hydrolysis of

309

disulfide linkages during heating or high shear.13 As expected, there was no change in

310

digestibility of wheat flour on processing.

311

Effects of exogenous factors on protein digestibility of proso millet protein.

312

Poor digestibility of sorghum protein in general has been attributed to protein interactions

313

with dietary fiber, starch, polyphenols, phytic acid, and other cell wall constituents.33 The

314

de-hulled proso millet flour used in this study had the lowest concentration of extractable

315

phenolics among the grains studied herein (see Supporting Information). This was

316

expected because the grain was de-hulled. To determine if interactions among other flour

317

components were affecting proso millet protein digestibility, proso millet protein was

318

partially purified using a wet-milling procedure. The focus of wet-milling is usually to

319

isolate the starch from a grain; however, it is also an efficient way to isolate protein

320

without denaturing it (as happens when extracting with aqueous alcohols). Wet-milling of

321

proso millet resulted in a fraction that contained 80% protein (wet basis) (%N X 6.25).

322

Upon heating this material, a decline in digestibility similar to heated proso millet flour was

323

observed (Figure 3). These results suggested that the reduction in digestibility of proso



Page 16 of 37

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millet protein upon heating was not due to exogenous interactions. Therefore, further

325

experiments focused on intermolecular interactions among proso millet proteins as the

326

cause for the decreased digestibility.

327

In vitro protein digestibility of grain heated in the presence of reducing

328

agents. Proso millet, sorghum, and wheat flours were heated in the presence of 2-

329

mercaptoethanol or sodium bisulfite and their pepsin digestibility was measured. There

330

was an improvement in digestibility of sorghum flour when heated in presence of either 2-

331

mercaptoethanol or sodium bisulfite (Figure 4). In contrast, heating in the presence of

332

these reducing agents did not improve the digestibility of proso millet protein. There was

333

no significant change observed in digestibility of wheat flour when heated in reducing

334

agents compared to water.

335

The improvement in digestibility upon heating sorghum with reducing agents has

336

been reported previously.20 Sorghum kafirins are more resistant to proteases than other

337

prolamins due to extensive disulfide linkages that form during heating.28 Thus, treating

338

sorghum flour with reducing agent like 2-mercaptoethanol improved the digestibility during

339

heating by reducing internal di-sulfide bond formation. However, because these reducing

340

agents had no effect on the digestibility of proso millet, the reduction in digestibility of

341

proso millet proteins upon heating was probably not due to disulfide bond formation.

342

In vitro protein digestibility of millet flour heated in the presence of

343

chaotropes. We hypothesized that heating proso millet flour denatures protein thereby

344

exposing hydrophobic amino acids that form compact hydrophobic aggregates at partial

345

state of denaturation that limit proteolytic hydrolysis. At high concentrations chaotropes

346

are known to prevent aggregation of proteins by binding to exposed hydrophobic amino


Page 17 of 37


17 347

acids.34 Therefore, millet flour was heated in the presence of 1 M and 8 M urea followed

348

by extensive dialysis to remove the urea and then digestibility was assayed.

349

While heating in the presence of 1 M urea had no effect on digestibility, heating in

350

the presence of 8 M urea resulted in a significant improvement in digestibility when

351

compared to heating in water. This was likely due to the interaction of urea with less polar

352

amino acid residues by H-bonding, which stabilizes the partially unfolded protein

353

conformation during denaturation by heating and impedes hydrophobic interactions

354

between protein chains.35 With continuous heating the denaturation of millet proteins is

355

completed and on removal of urea they go back to original conformation which is

356

digestible by in-vitro assays.

357

Similar to urea, guanidine HCl is also a known denaturant involving hydrophobic

358

interactions but with a different mechanism. It is thought that guanidium ions coat protein

359

hydrophobic surfaces preventing the aggregation of hydrophobic amino acid residues.34

360

When millet flour was heated in presence of 4.5 M guanidine HCl it was found that the

361

digestibility was same as uncooked flour (Table 3). Thus, two reagents that prevent

362

hydrophobic aggregation of proteins supported our hypothesis of formation of

363

hydrophobic aggregates in millet proteins during heating in presence of water.

364

Surface hydrophobicity. To evaluate the change in surface hydrophobicity upon

365

heating, proso millet protein was mixed with ANS, a fluorescent probe that binds to the

366

exposed hydrophobic regions on a protein which leads to an increase in its fluorescence

367

quantum. For unheated proso millet protein there was no change in fluorescence intensity

368

of ANS as protein concentration increased (Figure 5). In contrast, when millet protein was



Page 18 of 37

18 369

heated there was an increase in fluorescence intensity of ANS, suggesting more

370

hydrophobic groups were exposed on heating and could readily bind with ANS.

371

Intrinsic fluorescence (IF) of millet protein was also measured, which is mostly due

372

to exposed tryptophan residues on the protein surface. Though the IF value for the

373

system is very low but the IF intensity was greater for heated millet protein compared with

374

unheated millet protein. This suggests that there is a change in millet protein on heating

375

which is potentially due to the exposure of tryptophans to aqueous phase due to opening

376

of protein structure. Cumulatively both these results supported the hypothesis that the

377

reduced digestibility could be due to formation of hydrophobic aggregates in millet

378

proteins during heating in presence of water.

379

Changes in proso millet protein profiles upon heating. Change in proso millet

380

protein profiles upon cooking and digestion were visualized on SDS-PAGE. After

381

digestion of uncooked proso millet flour, a decrease in the prolamin band (20 kDa)

382

intensity was evident (Figure 6; lanes 1 and 3). This indicated good digestibility of

383

unheated proso millet flour. In contrast, only faint prolamin bands were seen in the

384

cooked proso millet protein extracts both before and after digestion, and did not show

385

noticeable changes upon digestion (Figure 6; lanes 2 and 4). The low intensity of the

386

prolamin band in the cooked samples was not due to loss of the protein; it was due to the

387

inability of the reducing buffer to extract the protein after cooking. This was evident

388

because 91% of the protein remained in the pellet after extraction of the cooked flour

389

(data not shown). The inability of the extraction buffer, which contained reducing agents,

390

to extract proso millet prolamins after cooking supported our previous results that

391

suggested the changes in millet protein upon cooking are not driven by disulfide linkage


Page 19 of 37


19 392

formation. Furthermore, when urea was added to the extraction buffer, extraction of the

393

prolamin fraction was greatly improved in heated proso millet flour (Figure 6; lanes 2 and

394

6); thus, conforming the ability of urea to prevent hydrophobic aggregation. Finally, when

395

the sample was cooked in urea and then digested (following removal of urea), digestibility

396

was good for both unheated and heated samples as evidenced by lack of prolamin bands

397

(Figure 6; lanes 7 and 8).

398

Amino acid profile. On comparing the amino acid profile of proso millet flour,

399

sorghum flour and wheat flour it was found that hydrophobic amino acids contributed to

400

51% of total amino acids in proso proteins while for sorghum and wheat, hydrophobic

401

amino acids contributed 48 and 37%, respectively, of total amino acids (see Supporting

402

Information). The results obtained for sorghum and wheat are consistent with previous

403

findings.36 Although the proportion of hydrophobic amino acids in sorghum and millet

404

proteins were similar, the different causes for lower digestibility after cooking could be due

405

to the sequence of amino acids in the primary structure of the protein

406

Proso millet storage proteins (prolamins) have been reported to be structurally

407

similar to sorghum prolamins, or kafirins, and thus have been commonly grouped

408

together. However, while both sorghum and proso millet proteins possess the unique

409

property of low digestibility upon cooking, the present study has shown that although the

410

observed effect is similar the mechanism involved is different. Thus, proso millet

411

prolamins are unique among the cereal proteins and deserve their own nomenclature. In

412

following the pattern used for some other cereal proteins, including corn (zein), rye

413

(secalin), and barley (hordein), we propose that proso millet prolamins should be named



Page 20 of 37

20 414

‘panicin’. This name is particularly appropriate because some old manuscripts from India

415

indicate that proso millet as a staple crop in the Neolithic era was called ‘Pani’.37-38

416

The objective of the present study was to assess the potential of proso millet as an

417

alternate crop for gluten intolerant people with prime focus on protein digestibility as

418

affected by processing. The results indicated that regardless of processing technique,

419

there was a 50% decline in digestibility of proso millet protein when compared with

420

unprocessed flour. Heating the flour in the presence of reducing agents did not improve

421

the digestibility but denaturants like urea and guanidine HCl did. The improvement in

422

protein digestibility of proso millet in the presence of these agents suggested formation of

423

hydrophobic aggregates in proso protein upon cooking. This indicates a unique property

424

of proso millet protein among other cereal proteins.

425

ACKNOWLEDGEMENT

426

This project was partially supported by US Department of Agriculture-National

427

Institute of Food and Agriculture, NC-213: Marketing and Delivery of Quality Grains and

428

Bioprocess Coproducts (#NEB-31-140). The authors are grateful to the Proteomics and

429

Metabolomics Facility at the Center for Biotechnology at the University of Nebraska-

430

Lincoln for running the amino acid profiles.

431

SUPPORTING INFORMATION

432

Amino acid composition of different samples

433

Total extractable phenolics in different grains


Page 21 of 37


21 434 435

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quality, selection. Food Navigator-USA, Newsletter. 2015, http://www.foodnavigator-

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Table 1. Effect of heating in excess water on protein digestibility (%) of millets.a Sample Unheated Heated* % change Proso millet Early Bird -56 84.6 ± 0.3 37.5 ± 1.1 Sunrise -57 75.8 ± 1.5 32.8 ± 0.9 Cope -53 83.4 ± 0.8 39.1 ± 2.7 Snobird -54 81.6 ± 0.9 37.9 ± 0.4 Plateau -46 77.9 ± 1.3 42.8 ± 0.6 Horizon -54 81.5 ± 1.1 37.7 ± 0.9 Sunup -54 80.9 ± 0.2 37.7 ± 1.2 Huntsman -46 77.4 ± 1.8 42.1 ± 2.3 Other millets Finger millet (UNL NeFm #1) +24 62.3 ± 2.0 77.1 ±1.6 Pearl millet (UNL NM-4B) -11 89.4 ± 0.8 79.7 ±0.8 Foxtail millet (UNL N-Si-7) -23 79.6 ± 1.3 60.7 ± 1.2 a Heating at 100 °C for 20 min; mean ± SD (n=3); *all heated samples were significantly different from their unheated counterpart (t-test, α=0.05).


Page 26 of 37

Page 27 of 37


Table 2. Effect of dry heating and extrusion on protein digestibility (%) of selected grain samples.a Sample

Autoclave (121 °C, 20 min) Unheated 10% moisture 20% moisture

Proso millet 79.7 ± 0.8 52.2 ± 1.8* Sorghum 76.1 ± 2.8 66.6 ± 0.3* Wheat 94.2 ± 0.6 92.4 ± 0.0 a

34.4 ± 1.3* 40.6 ± 2.7* 89.3 ± 0.8*

30% moisture

Oven (121 °C, 20 min) 10% moisture 20% moisture

30% moisture

Extrusion

44.3 ± 0.8* 39.2 ± 1.9* 89.3 ± 1.3*

49.1 ± 1.4* 49.7 ± 0.4* 91.6 ± 1.2

45.5 ± 1.2* 38.6 ± 0.8* 88.6 ± 2.2*

38.3 ± 1.1* 69.9 ± 0.9* 93.5 ± 0.9

34.4 ± 1.6* 37.3 ± 2.4* 89.3 ± 0.8*

Mean ± SD (n=3); *Means marked with asterisks are significantly different from their unheated counterpart (Dunnett Test at α=0.05).



Table 3. Effect of heating in urea and guanidine-HCl on protein digestibility (%) of proso millet.a Additive in heating water Unheated proso millet Heated proso millet Urea (1M) 82.3 ± 0.8 37.9 ± 2.0* Urea (8M) 82.0 ± 1.9 78.6 ± 0.2 Guanidine-HCl (4.5 M) 83.0 ± 1.4 84.3 ± 1.1 a Heating at 100 °C for 20 min; mean ± SD (n=3); *means marked with asterisks are significantly different from their unheated counterpart (t-test at α=0.05).


Page 28 of 37

Page 29 of 37


FIGURE CAPTIONS Figure 1. Effect of heating in excess water on protein digestibility of proso millet, sorghum, and wheat flours; error bars show standard deviation (n=3); some error bars were too small to plot; a,b,cMeans marked with different letters indicate significant differences among grains within temperature (Tukey’s adjustment; α=0.05); *Means marked with asterisks indicate significant difference within grain type with its previous temperature. Figure 2. Sequential in vitro digestion of uncooked and cooked proso millet flour; a) degree of hydrolysis; b) insoluble nitrogen; dotted line indicates the cut-off between gastric and small intestinal phase of digestion; error bars show standard deviation (n=2); *means marked with asterisks indicate significant difference within heating condition with its previous temperature (t-test; α=0.05). Figure 3. Effect of heating in excess water on protein digestibility of proso millet flour and semi-purified proso millet protein; error bars show standard deviation (n=3); *means marked with asterisks indicate significant difference with its unheated counterpart. Figure 4. Effect of reducing agents on protein digestibility of proso millet, sorghum and wheat flour; A) heating with 2-mercaptoethanol; B) heating with sodium bisulfite; open bars: flour in water; solid bars: flour in reducing agents; error bars show standard deviation (n=3); *bars marked with asterisks indicate a significant difference from the corresponding sample heated in water only (t-test; α=0.05). Figure 5. Effect of cooking on A) intrinsic and B) ANS based fluorescence of proso millet protein.



Figure 6. SDS-PAGE gels of proso millet flour; lane 0, MW marker; 1, uncooked before digestion; 2, cooked before digestion; 3, uncooked after digestion; 4, cooked after digestion; 5, uncooked before digestion, extracted with buffer containing 8 M urea; 6, cooked before digestion, extracted with buffer containing 8 M urea; 7, uncooked placed in 8M urea and digested; 8, cooked in 8M urea and then digested (urea removed before digestion).


Page 30 of 37

Page 31 of 37


Figure 1.



Figure 2.


Page 32 of 37

Page 33 of 37


Figure 3.



Figure 4.


Page 34 of 37

Page 35 of 37


Figure 5.



kDa

0

1

2

3

4

5

6

7

8

250 150 100 75 50 37 25 20 15 10

Figure 6.


Page 36 of 37

Page 37 of 37


Table of Contents Abstract.


Heating Reduces Proso Millet Protein Digestibility via Formation of

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