Accepted Manuscript Intact cellular structure in cereal endosperm limits starch digestion in vitro Rewati R. Bhattarai, Sushil Dhital, Andrew Mense, Michael J. Gidley, Yong-Cheng Shi PII: S0268-005X(17)32085-4 DOI: 10.1016/j.foodhyd.2018.02.027 Reference: FOOHYD 4286 To appear in: Food Hydrocolloids Received Date: 17 December 2017 Revised Date: 12 February 2018 Accepted Date: 14 February 2018 Please cite this article as: Bhattarai, R.R., Dhital, S., Mense, A., Gidley, M.J., Shi, Y.-C., Intact cellular structure in cereal endosperm limits starch digestion in vitro, Food Hydrocolloids (2018), doi: 10.1016/ j.foodhyd.2018.02.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT MANUSCRIPT ACCEPTED ACCEPTED MANUSCRIPT 1 Intact cellular structure in cereal endosperm limits 2 starch digestion in vitro ab a b a 3 Rewati R. Bhattarai , Sushil Dhital , Andrew Mense , Michael J. Gidley and b* 4 Yong-Cheng Shi 5 aARC Centre of Excellence in Plant Cell Walls, Centre for Nutrition and Food Sciences, 6 Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St 7 Lucia, Qld 4072, Australia. 8 bDepartment of Grain Science and Industry, Kansas State University, Manhattan, Kansas 9 66506, United States. 10 11 *Corresponding Author: Prof. Yong-Cheng Shi, Ph.D. 12 E-mail: [email protected] MANUSCRIPT 13 Phone: 785-532-6771 ACCEPTED 1 ACCEPTED MANUSCRIPT 14 Abstract 15 Limiting the rate and extent of starch digestion is a major target for increasing the nutritional 16 value of cereal-based foods. One mechanism that could be exploited is the ability of intact 17 cell walls to protect intracellular starches from enzyme hydrolysis, but the extent to which 18 this mechanism is valid for cereal endosperm cells is not well understood. This study aimed 19 to isolate individual intact cellular structures from cereals, viz. wheat and sorghum, in order 20 to elucidate the effect of intactness of cell walls on enzymic hydrolysis of entrapped starch. 21 Intact cells were isolated from dry milled flour obtained using three grinding rolls coupled 22 with a wet sieving technique using selected sieves having varying apertures. The intact 23 cellular structure in wheat and sorghum hindered the hydrolysis of entrapped starch as 24 observed from the lower extent of digestion (9 and 7%) compared to deliberately broken cells 25 (19 and 17% under the same conditions). The extent of digestion was markedly increased 26 once the intact cells were cooked (33 and 26% forMANUSCRIPT wheat and sorghum cooked cells), but this 27 was less than half the digestion extent of non-enca psulated cooked starches (77 and 62% 28 respectively). Microscopic observations coupled with fluorescence labelling of enzyme, cell 29 walls and starch suggest a) wheat and sorghum cell walls are effective barriers for access of 30 amylase; and b) both an extensive protein matrix (particularly in sorghum) and non-catalytic 31 binding of amylase on cell wall surfaces can limit the amylolysis of starch within intact cells. 32 Furthermore, the presence of incompletely gelatinised starch inside cooked intact cells, 33 suggests limited swelling of granules trapped inside the cells. This study shows how 34 preservation of ACCEPTEDcellular matrices in cereal-based foods could be beneficial for increasing the 35 amount of enzyme resistant starch in cereals with added nutritional benefits. 36 Keywords: Wheat, sorghum, intact cells, starch digestion, non-specific binding 37 2 ACCEPTED MANUSCRIPT 38 1. Introduction 39 Evidence from both in vitro and in vivo experiments shows that the structure of plant-based 40 foods can have a major effect on the digestion of dietary macronutrients. More specifically, 41 enzyme susceptibility of starch, proteins and lipids in intact cells and tissues of cereals, 42 legumes, nuts and oilseeds are reduced as the macronutrients are encapsulated inside cells 43 walls that are not hydrolysed by human digestive enzymes (Berry, et al., 2008; Bhattarai, 44 Dhital, Wu, Chen, & Gidley, 2017; Dhital, Bhattarai, Gorham, & Gidley, 2016; Edwards, 45 Grundy, et al., 2015; Ellis, et al., 2004; Livesey, et al., 1995; Roman, Gomez, Li, Hamaker, & 46 Martinez, 2017). Edwards et al (2015) reported the role of intact cellular structure in reducing 47 the in vivo human glyceamic response based on a feeding trial of porridges made from coarse 48 wheat endosperm particles (2 mm) and fine flour particles (<0.2 mm). More recently, Roman 49 et al., (2017) showed that the lower starch digestibility in maize flour from cooked hard 50 endosperm compared to a soft counterpart was relateMANUSCRIPTd to the food matrix. These and other 51 studies used fragments of plant tissues consisting of many cells, whereas the contribution of a 52 single cellular structure to the digestion of starch in cereals has not been defined to date. Our 53 previous studies provided evidence that individual intact cells from legumes are essentially 54 impervious to amylase and protease (Bhattarai, et al., 2017; Dhital, et al., 2016), however, 55 analogous information for the case of cereals is lacking. 56 The fragile and thin nature of cereal endosperm cells makes them comparatively difficult to 57 isolate, particularly under the extended aqueous conditions that are used to separate 58 individual cells ACCEPTEDfrom legume cotyledons (Dhital et al., 2016). To avoid lengthy exposure to 59 aqueous conditions, we hypothesise that the separation of individual intact cereal cells from 60 wheat and sorghum should be possible using dry milling (gradual reduction process) with 61 tight control of the particle size. The selection of wheat and sorghum for study is based on 62 their high nutritional and functional potential as well as their distinct anatomical structures. 3 ACCEPTED MANUSCRIPT 63 Whilst sorghum endosperm is harder than wheat (Zhao & Ambrose, 2016), endosperm in 64 both grains consists of cells surrounded by cell walls and containing starch granules 65 embedded in a protein matrix (Corke, 2015; Koehler & Wieser, 2013; Rooney, Miller, & 66 Mertin, 1981; Stefoska-Needham, Beck, Johnson, & Tapsell, 2015; Vu, Bean, Hsieh, & Shi, 67 2017). We hypothesized that with the addition of an appropriate amount of moisture, wheat 68 and sorghum endosperm could be rendered softer and more friable, such that samples can be 69 gradually reduced using a series of selected roller mills to obtain fractions containing 70 individual intact cells. 71 In this study, we isolated intact cells from wheat and sorghum and investigated the enzymic 72 susceptibility of intact cells compared with flour, broken cells and isolated starches in both 73 raw and cooked forms. Identification of how to process cereals into single cell forms provides 74 a new paradigm that could lead to enhanced nutritional quality of derived foods. 75 2. Materials and methods MANUSCRIPT 76 Hard red winter (HRW, Everest, 2015) wheat was obtained from Foundation Seed (Kansas 77 State University, Manhattan, KS, USA). White pearled sorghum grain was obtained from Nu 78 Life Market LLC (Scott City, Kansas, USA). Purified α-amylase (Sigma A6255 from porcine 79 pancreas), fluorescein isothiocyanate isomer I (FITC, F7250, Sigma), calcofluor-white stain 80 (Sigma, 18909) and phosphate buffered saline buffer (PBS; P4417, Sigma, pH 7.2) were 81 purchased from Sigma-Aldrich. Enzyme glucose reagent (glucose oxidase/peroxidase; 82 GOPOD) (TR15221) was purchased from Fisher Scientific, USA, and amyloglucosidase (E- 83 AMGDF100), totalACCEPTED starch assay kit (K-TSTA), α-amylase assay kit (K-CERA) and integrated 84 total dietary fibre assay kit (K-INTDF 01/17) were obtained from Megazyme, Bray, Ireland. 85 2.1 Optimisation of milling-sieving process 4 ACCEPTED MANUSCRIPT 86 A combination of dry milling-sifting and wet sieving methods were used to isolate individual 87 intact cells from cereals (Fig. 1). HRW wheat (50 g at a time) was debranned using a 88 laboratory barley pearler (Model 17810, Strong Scott, Webster, WI) for 3 min to remove 89 most of the germ and bran fractions. The debranned wheat samples were then sifted by hand 90 for about one minute on a 300 µm sieve to remove all the dust. Debranning value was 91 calculated as the weight percentage of remaining wheat. The pearled sorghum as obtained 92 from the supplier was free from bran as indicated by its clear white endosperm. A detailed 93 description of milling-sieving techniques used to obtain intact cells is presented in 94 Supplementary section S1. MANUSCRIPT ACCEPTED 5 ACCEPTED MANUSCRIPT MANUSCRIPT 95 96 Fig. 1 . Schematic diagram showing the dry milling and wet sieving technique used for 97 separation of intact wheat cells. 98 2.2 PreparationACCEPTED of broken cells and isolated starch fractions 99 In order to investigate the role of cell walls, intact cells were deliberately broken to obtain 100 broken cell fractions which were devoid of intact cell walls by applying a shear mixing force 101 using magnetic stirrer bars as described previously (Bhattarai, et al., 2017). Due to the 6 ACCEPTED MANUSCRIPT 102 relatively fragile nature of cereal cells, all the intact cells were broken within 6 h, whereas 103 legume cells require 16 h. 104 In order to obtain starch fractions, the broken cells were allowed to settle in excess water and 105 sieved through a 32 µm sieve. The sedimented material that passed through the 32 µm sieve 106 contained starch and trace amount of proteins released from the breakage of intact cells. Light 107 microscopy was used to confirm the purity of starch fractions.