Modification of glutenin and associated changes in digestibility due to

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Food and Beverage Chemistry/Biochemistry

Modification of glutenin and associated changes in digestibility due to methylglyoxal during heat processing Yaya Wang, Wang junping, Shujun Wang, Jun Guo, and Shuo Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04337 • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 3, 2019

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Journal of Agricultural and Food Chemistry

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Modification of glutenin and associated changes in

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digestibility due to methylglyoxal during heat processing

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Yaya Wanga, Junping Wang a*, Shujun Wanga, Jun Guoa, Shuo Wang ab*

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a

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Technology, State Key Laboratory of Food Nutrition and Safety

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29 The Thirteenth Road, Tianjin Economy and Technology Development Area,

College of Food Science and Engineering, Tianjin University of Science &

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Tianjin, 300457, P.R. China

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b

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University, Tianjin 300071, China

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*Corresponding Authors: [email protected]; Fax: (+86 22) 85358445; Tel:

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(+86 22) 85358445

Tianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai

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ACS Paragon Plus Environment


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Abstract

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Glutenin is the main protein of flour and is a very important source of protein

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nutrition for humans. Methylglyoxal (MGO) is an important product of the Maillard

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reaction that occurs during the hot-processing of flour products, and it reacts with

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glutenin to facilitate changes in glutenin properties. Here, the effects of MGO on

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glutenin digestion during the heating process were investigated using a simulated

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MGO-glutenin system. MGO significantly reduced the digestibility of glutenin. The

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structure MGO-glutenin and physicochemical properties were studied to understand

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the mechanism of the decrease of digestibility. These data suggest that changes in

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digestibility were caused by decreases in surface hydrophobicity and increases in

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disulfide bonds. MGO induces strong aggregation of glutenin after heating that led to

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the masking of cleavage sites for proteases. Moreover, carbonyl oxidation induced by

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MGO leads to intermolecular cross-linking of glutenin that increasingly masks or

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even destroys cleavage sites, further decreasing digestibility.

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Keywords: Glutenin; Methylglyoxal; Digestibility; α-dicarbonyl compounds;

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Hot-processing

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INTRODUCTION

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Wheat is the most widely planted and commonly consumed food crop globally.

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Glutenin is one of the primary components of wheat protein and also one of the main

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components of flour products, wherein it provides high nutrition for human

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consumption. Glutenin accounts for 47% of the total protein in wheat and is a protein

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aggregate of high molecular weight (HMW) and low molecular weight (LMW)

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subunits, with molar mass of approximately 200,000 to several million, which is

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stabilized by intermolecular disulfide bonds, hydrophobic interactions, and other

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forces1. Consequently, glutenin is a very important source of protein nutrition for

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humans. Some studies have investigated the physical and chemical changes of

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glutenin during processing, but the underlying mechanisms of such changes have not

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been fully clarified due to difficulties in the separation process, the complex structure

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of glutenin, and complex chemical reactions that occur during processing.1

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Heat processing is widely used to process foods. However, heat processing results

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in complex reactions within food matrices including the Maillard reaction and protein

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oxidation that lead to changes in protein structures within foods. The Maillard

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reaction is induced by reducing sugars and the alteration of protein structures due to

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sugar degradation products that alter the utilization of dietary proteins. α-dicarbonylic

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compounds (α-DCs) like methylglyoxal (MGO) and glyoxal (GO) are important

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intermediates of Maillard reactions that occur during heat processing.

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α-DCs can modify the side chain amino groups of lysine (Lys) and arginine (Arg)

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residues due to their high reactivity. Indeed, α-DC reactivity is much higher than that 3



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of glucose, resulting in the reaction of α-DC with side chain amino groups and

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subsequent potential risks to human health. Investigation of H3 histone modification

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by 3-Deoxyglucosone indicated that the reaction mainly happened on the ε-amino

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group of Lys and Arg residues, resulting in secondary structural transformations2.

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Among all of α-DCs, MGOs have the highest reactivity. MGO exists widely in

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hot processed foods. The concentration of MGO in honey is in the range of 0.8-33

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mg/kg3. The MGO content in espresso coffee is 230.9 μM, and the baking process

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affected the content of MGO in coffee beans due to the Maillard reaction4. MGO in

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fats and oils is formed by lipid degradation during processing. The amount of MGO

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formed in fish oil heated at 60 °C for 7 days was 2.03 to 2.89 mg/kg5. MGO also

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exists in fermented products, such as alcoholic drinks and vinegar, for example, the

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MGO content in wine and vinegar is 10 and 35 ppm, respectively6.

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MGO has also been found to modify protein. Modification of glutathione

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peroxidase (GSH-Px) by MGO demonstrated that the binding sites of MGO to

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glutathione peroxidase were Arg184 and Arg185 and that glutathione peroxidase

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activity declined after modification7.

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Protein modifications that are produced under physiological conditions are widely

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investigated as markers of oxidative stress that are associated with diseases such as

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diabetes, Alzheimer’s disease, and atherosclerosis8. However, few studies have

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evaluated the risk of consuming proteins with these modifications. Because proteins

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provide nutrients through digestion, digestibility is an important indicator when

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assessing protein nutrition that is affected by processing conditions. As indicated 4


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above, processing can result in very complex reactions among food matrices that can

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lead to significant changes in protein digestibility. Decreases in digestibility caused by

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structural changes of proteins from food processing have recently attracted increasing

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attention.9 In particular, changes in protein structure caused by Maillard reactions can

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reduce the nutritional value of proteins and the formation of undesirable byproducts.10

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For example, carbonylation of milk proteins results in the loss of essential amino

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acids while also reducing protein digestibility. Maillard reactions also affect the

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physicochemical properties of proteins by altering protein structures and triggering

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aggregation. The oxidative degradation of basic amino acids (e.g., lysine, arginine,

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and proline) due to protein carbonylation leads directly to changes in the

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physicochemical properties of dairy and meat proteins while decreasing the nutritional

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value of the corresponding products.11 A shotgun assay was previously used to

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characterize the number of sites and their locations in the lactosylation of cow’s milk

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proteins, which indicated that their numbers increased with increasing processing.

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These changes led to progressive modulation of physicochemical properties and

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decreases in digestibility.12 However, studies have not investigated the effects of

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reducing sugars and their degradation products during heat processing on the

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digestibility of glutenin.

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The goal of this study was to therefore investigate the influence of MGO on

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glutenin digestibility and evaluate the mechanisms of alteration at different heating

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temperatures within a simulated system. Digestibility was measured using the

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o-phthalaldehyde (OPA) method, while changes in the secondary structure, disulfide 5



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bonds (SS), endogenous fluorescence, and thermal stability of MGO-glutenin were

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evaluated to determine the mechanisms underlying decreases in its digestibility. The

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physicochemical properties of glutenin were also concomitantly measured to further

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deconvolute the mechanism underlying changes in glutenin digestibility. Moreover,

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the effects of thermal processing conditions on the nutritional value of wheat products

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were also evaluated based on the digestibility of glutenin.

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MATERIALS AND METHODS

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Materials

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Wheat was purchased from a local commercial market. All water used in the

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experiments was produced from a Milli-Q Ultrapure Water Systems, and all

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chemicals used were analytical grade, unless otherwise specified. Pepsin from porcine

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gastric mucosa (>2500 U/mg), trypsin from porcine pancreas (1655 U/mg), and

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chymotrypsin (>40 U/mg) were purchased from the Sigma-Aldrich Chemical

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Corporation.

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Separation of glutenin

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Glutenin was extracted from wheat flour, as previously described.13 Briefly,

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n-hexane was added to freeze-dried gluten at a 1:20 (n-hexane:gluten; w/v) ratio and

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then stirred for 1 h at room temperature to remove fat, followed by placing the gluten

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suspension in a fuming cupboard overnight to remove the n-hexane. A 0.4 mol/L

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NaCl solution was added to the gluten at a 1:20 (NaCl:gluten; w/v) ratio, followed by

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stirring again for 1 h at room temperature. The suspension was then centrifuged to 6


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collect the precipitate (A) and remove albumin and globulin. Ultrapure water was

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added to the precipitate (A) at a 1:20 (w/v) ratio and then stirred for 1 h at room

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temperature to remove NaCl, followed by centrifugation to collect precipitate (B).

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The precipitate (B) was again dissolved in 70% alcohol at a 1:40 (w/v) ratio and

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centrifuged at 10,000 g for 20 min to remove the prolamin. Each extraction step was

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repeated three times. The content of reducing sugar and MGO in glutenin powder was

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determined

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chromatography-tandem mass spectrometry (LC-MS), the content of which was

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220.05±34.24 ug/g and 67.12±1.06 ng/mg, respectively.

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by

3,5-dinitrosalicylic

acid

reagent

colorimetry

and

liquid

The final precipitate (C) was then lyophilized at -80°C and ground to a powder to

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obtain native glutenin.

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Glutenin suspension preparation

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A given mass of glutenin powder was placed in a mortar and fully ground. After

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grinding to ultrafine powders, 100 mg of glutenin powder was added to 100 ml of

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ultrapure water which was then homogenized at 10,000 rpm for 30 s using high-speed

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blender (Ika T18 Basic, Staufen, Germany) for several times until the glutenin powder

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was suspended stably in the ultrapure water.

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Carbonyl reactions between MGO and glutenin

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The glutenin suspension was homogenized using a high-speed blender to achieve

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uniform dispersion in water. MGO was added to the suspension to provide a mass

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ratio of glutenin to MGO of 1:8. The suspension was then heated to 100°C, 120°C,

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140°C, 160°C, and 180°C for 15 min each with a dry bath incubator (SBH200D/3, 7



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Stuart, England) to simulate hot processing. The glutenin suspension without MGO

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was heated to 100 °C, 120 °C, 140 °C, 160 °C, and 180 °C for 15 min each as the

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controls. Samples were then freeze-dried after ultrafiltration.

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Determination of digestibility

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Simulated gastric and intestinal fluids were produced based on US

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Pharmacopoeia formulae. Five milligrams of glutenin was dissolved in 5 mL of

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simulated gastric fluid which contains 2.5 mM CaCl2, 35 mM NaCl and pepsin (182

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U/mg proteins) and then digested at 37°C for 1 h in a constant temperature incubator.

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An in vitro intestinal digestion was then conducted with the gastric digestion

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products. A 1 mL solution of simulated intestinal fluid which contains 7.6 mM CaCl2

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and 20.3 mM Tris, 7.4 mM bile salts, trypsin (40 U/mg proteins) and chymotrypsin

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(0.5 U/mg proteins) was added to the gastric digestion products and then placed in a

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constant temperature incubator for 2 h of further digestion. The reaction was stopped

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by heating in boiling water for 5 min. The hydrolysate aliquots were then diluted at a

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1:20 (v/v) ratio with an OPA reagent. Subsamples of the dilution (200 μL) were added

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to a 96-well plate and incubated for 10 min, followed by measurement of fluorescence

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emission at an excitation wavelength of 340 nm and an emission wavelength of 450

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nm using a plate reader (Varioskan LUX, Thermo Scientific, Waltham, MA, USA).

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Different concentrations of tryptophan (0.01, 0.05, 0.1, 0.2, 0.5, and 0.6 mmol/mL)

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were used to establish a standard curve. Digestibility was then calculated as the ratio

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of free to total amino acid content after digestion of the protein. All measurements

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were conducted in triplicate. The extent of proteolytic hydrolysis (DH) was calculated 8


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using the following equation as previously described by Wenjun Wen et al. 14: DH (%) 

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hs  100% , htotal

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Where hs is the concentration (mmol) of free amine groups per gram of protein in the

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sample, and htotal is the concentration (mmol) of free amino groups per gram of

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protein, assuming complete hydrolysis of the protein (8.83 mmol/g protein). All

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measurements were made in triplicate.

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Surface hydrophobicity

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Hydrophobicity (H0) was measured as previously described

15.

Briefly, sample

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solutions at different concentrations (0.05, 0.1, 0.2, 0.5, 0.8, and 1.0 mg/mL) were

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prepared in sodium phosphate buffer solution (0.01 M, pH 7.0). Then, 20 μL of 8 mM

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8-Anilino-1-naphthalenesulfonic acid (ANS) solution was added to a 4 mL sample,

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and the ﬂuorescence intensity (FI) was immediately measured using a LUMINA

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fluorescence spectrometer (Thermo Scientific, Waltham, MA, USA) at an excitation

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wavelength of 390 nm and an emission wavelength of 470 nm. The initial slope from

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a plot of FI versus protein concentration was used as the H0 index.

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Foaming ability and stability

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Foaming ability and stability were evaluated as previously described

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Briefly,

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suspensions of glutenin samples in buffer were placed in glass measuring cylinders

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and vigorously mixed using a homogenizer operated at 20,000 rpm for 2 min at room

184

temperature. The volume of foam from each sample was then measured using a glass 9



185

measuring cylinder. The foam stability time was recorded as the time taken for the

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foam to completely disappear.

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Determination of solubility

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0.5 g of protein was accurately weighed and dispersed in 5 mL of ultrapure water.

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The protein dispersion was stirred with a magnetic stirrer for 1 h at room temperature

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and centrifuged at 5000 g for 20 min. The protein level in the supernatant was

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measured by Coomassie brilliant blue method. Solubility (%) is determined17

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according to the following formula:

193 194

Solubility (%) =

𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝑙𝑒𝑣𝑒𝑙 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑢𝑝𝑒𝑟𝑛𝑎𝑡𝑎𝑛𝑡 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 𝑙𝑒𝑣𝑒𝑙 𝑖𝑛 𝑡ℎ𝑒 𝑠𝑢𝑠𝑝𝑒𝑛𝑠𝑖𝑜𝑛

× 100

Evaluation of emulsifying properties

195

Samples were dispersed well in a phosphate buffer solution to achieve a final

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concentration of 1 mg/mL. Five milliliters of peanut oil was then added to the samples

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and homogenized at 24,000 rpm for 1 min using an Ultra-Turrax homogenizer (Ika

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T18 Basic, Staufen, Germany) to generate an emulsion. Then, 50 μL of the emulsion

199

was diluted (1:100, v/v) into 0.1% (w/v) SDS solution using micropipettes. The

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absorbance of the emulsion was measured at 500 nm using a plate reader (Varioskan

201

LUX, Thermo Scientific, Waltham, MA, USA), with duplicate measurement after

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letting the emulsion stand for 10 min. EAI and ESI values were calculated18 using the

203

following formula:

204

205

EAI (m2 / mg ) 

2  2.303  A0  DF c φ (1 θ) 10000

ESI (min ) 

A0  10 , A0  A10 10


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207

Where DF is the dilution factor (100), C is the protein concentration (g/mL), ψ is the

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optical path (1 cm), θ is the oil volume fraction (0.25), and A0 and A10 are the

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absorbance values of the emulsion at 0 and 10 min, respectively.

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Fourier-transform infrared (FT-IR) spectroscopy

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Samples were fully dried with P2O5 for preservation. A total of 2.00 mg of dried

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samples and 150.00 mg of KBr powder were pre-dried to a constant weight using

213

rapid grinding with an agate pestle, followed by pressing into a pellet. A scanning

214

band of 4000400 cm-1 was then used for FT-IR spectroscopy using 32 scanning

215

frames. The corresponding resolution of the spectra was 4 cm-1.19

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Sulfhydryl (SH) and disulfide bond (SS) contents

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The total and free sulfhydryl (SH) contents of glutenin were determined, as 20,

218

previously described

with slight modifications. To determine free SH content, 15

219

mg of protein was suspended into 10 mL of Tris-glycine buffer containing 8 M urea

220

for a total of 1 h. The protein concentration was then diluted to 0.1 mg/mL using

221

Tris-glycine buffer. A 1 mL aliquot of the dilution was then reacted for 25 min with

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10 μL of Ellman’s reagent within 10 mM Tris-glycine buffer, followed by absorbance

223

measurement at 412 nm. The SS content was then measured by dissolving 5 mg of

224

glutenin in 10 mL of Tris-glycine buffer (pH 8.0) containing 8 M urea. A 1 mL

225

aliquot of the sample solution was then diluted with 4 mL of Tris-glycine buffer (pH

226

8.0) containing 50 μL of 2-mercaptoethanol and then maintained at room temperature

227

for 1 h. Then, 10 mL of 12% (w/v) trichloroacetic acid (TCA) was added to the 11



228

mixture and allowed to stand for 1 h. The mixture was centrifuged at 5000 g for 15

229

min to obtain a precipitate that was suspended in 2 mL of Tris-glycine buffer (pH 8.0)

230

containing 8 M urea and maintained at room temperature for 1 h. Glutenin solutions

231

were then diluted with the Tris-glycine buffer to achieve a final concentration of 0.1

232

mg/mL. A 1 mL aliquot of the solution was reacted with and without 10 μL of

233

Ellman’s reagent for 10 min at room temperature, followed by absorbance

234

measurement at 412 nm.

235

236

The SH contents (µmol SH/g) were then calculated using the following equation: SH ( mol / g ) 

73.53  A421  D , C

237

238

Where A412 is the absorbance at 412 nm, C is the glutenin concentration (mg/mL), and

239

D is the dilution factor (i.e., 1 in our experiment). The SS contents (µmol SS/g) were

240

calculated using the following equation:

241

SS ( mol / g ) 

SHtotal  SHfree , 2

242

243

Where A412 is the absorbance at 412 nm, C is the glutenin concentration (mg/mL), and

244

D is the dilution factor (i.e., 1 in our experiment).

245

Fluorescence spectroscopy

246

The intrinsic fluorescence spectra of the samples were determined, as previously

247

described21. Prior to measurement, sample concentrations were adjusted to 1 mg/mL 12


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with 10 mM phosphate buffer (pH 7.0). Measurements were made with an excitation

249

wavelength of 295 nm and emission wavelengths from 320 to 550 nm using a

250

fluorescence spectrophotometer (LUMINA Fluorescence Spectrometer, Thermo

251

Scientific, Waltham, MA, USA). The scanning speed was set to 60 nm/min, and the

252

slit-width was set as 5 nm. Native glutenin was used as the control, and a buffer

253

solution alone was used to measure background fluorescence. Samples were measured

254

in triplicate.

255

Heat stability of glutenin

256

Thermal gravimetric (TG) analysis was performed using a Thermogravimetric

257

Analyzer (TGA, Netzsch STA409PC, Germany). For TG analyses, the heating rate

258

was set at 10°C/min, and the temperature was varied over 50600°C, with nitrogen as

259

the heating gas. The first derivative of the TGA curve was plotted to determine the

260

degradation temperatures (Td) using the STAR software program (version 9.01).22

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Statistical analyses

262

Statistical analysis of differences in measurements among the SH and S-S group

263

abundances, surface hydrophobicity, secondary structure variation, and in vitro

264

digestibility was conducted by one-way analysis of variance (ANOVA) using the

265

SPSS 19.0 software (IBM Corporation, New York, USA). Statistically significant

266

differences were set at P < 0.05.

267 268

RESULTS AND DISCUSSION

269

Results 13



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Variation in digestibility of glutenin products

271

272 273

Fig. 1. Changes in gastric (a) and intestinal (b) digestibility of the control and heated

274

MGO-glutenin samples with increasing temperature. Data show the means ± SD of triplicate

275

independent experiments. Bars represent the standard error of the mean.

276 277

The gastric (Fig. 1a) and intestinal (Fig. 1b) digestibility were measured for the

278

resulting glutenin products. The gastric digestibility of the control and heated

279

MGO-glutenin products decreased with increased heating, but the gastric digestibility

280

of heated MGO-glutenin was lower than that of the control. A similar trend was

281

observed for intestinal digestibility via treatment with trypsin and α-chymotrypsin.

282

Differences in physicochemical properties of glutenin

283

The surface hydrophobicity index, H0, was measured for all samples (Table 1). H0

284

of native glutenin was 32.49 ± 0.40, while H0 of the control first increased with

285

increasing temperature and then subsequently decreased. In contrast, H0 of heated

286

MGO-glutenin decreased with increasing temperature and the H0 was not detected 14


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after heating at 180°C.

288

The foaming ability (Fig. 2a) and stability (Fig. 2b) at different temperatures

289

were also measured. The foaming ability of MGO-glutenin first increased with

290

temperature but then subsequently decreased, while that of the control group did not

291

vary. The foaming stability of the control and heated MGO-glutenin samples

292

decreased with increasing temperature exposure, while the foaming stability of the

293

control was higher than that of heated MGO-glutenin under the same conditions.

294 295

Table 1. The surface hydrophobic indices and degradation temperatures of native glutenin, control

296

proteins, and heated MGO-glutenin at increasing temperatures.

Reaction condition

H0; X ± SD

Glutenin

Td (°C)

MGO-glutenin

Glutenin

32.50 ± 0.41d

Glutenin

MGO-glutenin 271.70 ± 1.18a

100°C

33.99 ± 0.05d

28.85 ± 1.27b

271.11 ± 0.63a

104.66 ± 1.14b

120°C

36.87 ± 0.98c

26.12 ± 1.07c

272.05 ± 0.76a

98.11 ± 0.20c

140°C

41.90 ± 1.10b

14.24 ± 0.14d

271.04 ± 0.66a

89.35 ± 1.22d

160°C

42.46 ± 1.20b

6.78 ± 0.22e

271.65 ± 1.00a

82.40 ± 2.59e

180°C

34.65 ± 0.01a

n.d.

272.48 ± 0.73a

76.99 ± 3.80e

297

Values are given as mean ± standard deviation (SD). Different superscript letters within the same column indicate

298

statistically significant differences (P

Modification of glutenin and associated changes in digestibility due to

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