Generation of Soluble Advanced Glycation End Products Receptor

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Generation of sRAGE-binding ligands during extensive heat treatment of whey protein/lactose mixtures is dependent on glycation and aggregation Fahui Liu, Malgorzata Teodorowicz, Harry J. Wichers, Martinus A.J.S. Van Boekel, and Kasper Arthur Hettinga J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02674 • Publication Date (Web): 27 Jul 2016 Downloaded from http://pubs.acs.org on August 6, 2016

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Journal of Agricultural and Food Chemistry

Graphical abstract 85x47mm (300 x 300 DPI)

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Generation of sRAGE-binding Ligands During Extensive Heat

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Treatment of Whey Protein/Lactose Mixtures is Dependent on Glycation

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and Aggregation

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(Generation of sRAGE-ligands is Dependent on Glycation and Aggregation)

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Fahui Liu a, Małgorzata Teodorowicz b, Harry J. Wichers b, c, Martinus A.J.S. van Boekel a, Kasper A. Hettinga a,*

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a Food Quality & Design Group, Wageningen University & Research Centre, 6700EV Wageningen, The Netherlands

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b

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c

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Cell Biology and Immunology Group, Wageningen University and Research Centre, 6708WD Wageningen, The Netherlands Food and Biobased Research, Wageningen University and Research Centre, 6700 AA Wageningen, The Netherlands

*

Corresponding Author: Dr Kasper Hettinga, Food Quality & Design Group, Wageningen University, Postbox 8129, 6700EV, Wageningen, The Netherlands E-mail: [email protected] Tel.: +31317482401 Fax: +31317483669 1


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ABSTRACT: Heating of protein- and sugar-containing materials is considered as the primary

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factor affecting the formation of advanced glycation end products (AGEs). This study aimed

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to investigate the influence of heating conditions, digestion, and aggregation on the binding

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capacity of AGEs to the soluble AGE receptor (sRAGE). Samples consisting of mixtures of

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whey protein and lactose were heated at 130 °C. An in vitro infant digestion model was used

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to study the influence of heat treatment on the digestibility of whey proteins. The amount of

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sRAGE-binding ligands before and after digestion was measured by an ELISA-based

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sRAGE-binding assay. Water activity did not significantly affect the extent of digestibility of

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whey proteins dry heated at pH 5 (range from 3.3% ± 0.2% to 3.6% ± 0.1% for gastric

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digestion and from 53.5% ± 1.5% to 64.7% ± 1.1% for duodenal digestion), but there were

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differences in cleavage patterns of peptides among the samples heated at different pH.

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Formation of sRAGE-binding ligands depended on the formation of aggregates and was

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limited in the samples heated at pH 5. Moreover, the sRAGE-binding activity of digested

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sample was changed by protease degradation and correlated with the digestibility of samples.

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In conclusion, generation of sRAGE-binding ligands during extensive heat treatment of whey

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protein/lactose mixtures is limited in acidic heating condition and dependent on glycation and

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aggregation.

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KEYWORDS: whey proteins, Maillard reaction, lactose, advanced glycation end products,

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digestion

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INTRODUCTION

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Advanced glycation end products (AGEs) can be generated from non-enzymatic glycation and

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oxidation of proteins. AGEs constitute a group of heterogeneous compounds (e.g. pentosidine,

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carboxymethyllysine (CML), imidazolone, pyrralin, GA-pyridine, etc.) 1. It occurs both in

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exogenous sources and slowly in biological systems in vivo where it contributes to tissue

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modifications 2. The exogenous source is mostly related to heat-processed food products that

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are rich in proteins and reducing sugars. These heat-processed foods, such as intensively

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heated milk and egg products, are intensely studied in clinical research on the resolution of

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allergy

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diseases, there is a need to optimize the heating conditions to limit the generation of AGEs

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during the preparation of such food products.

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The conditions during heat treatment, including nutrient composition, temperature, heating

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time, amount of moisture, and pH of the food matrix, affect the generation of AGEs in foods

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during heating 5. Highest AGE levels were found in high-temperature processed nut and grain

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products 6, which are both rich in proteins and reducing sugars. In the thermal processing of

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proteins and reducing sugars, such as in the baking process of muffins or cookies, a

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substantial amount of AGEs may form. After the spray-drying of infant formulas, the amount

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of AGEs may exceed those present in human breast milk up to 670-fold 7. Another important

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factor that affects the generation of AGEs is the pH of the food matrix. Heat treatment of food

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products in acidic environment was recommended to limit the AGE formation 8. The products

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and pathways of the Maillard reaction are influenced by the initial pH of the solution with

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reactants and buffering capacity of the system. H+ ions are required to catalyse both the

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Amadori and Heyns rearrangements. The degradation of Amadori compounds is also affected

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by pH 9. To inhibit the formation of AGEs, apart from controlling the heating conditions

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during thermal processing, also various inhibitors have been proposed in the last few years 10.

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. However, due to the possible involvement of AGEs in the aetiology of several

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It is reported that such synthetic compounds are responsible for some adverse effects in in

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vivo tests

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polyphenols and flavonoid-rich extracts that have AGE inhibitory effects because of their

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antioxidant properties, have been extracted from green tea, mung bean, and grapes. However,

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the extracting efficiency needs to be improved before industrial applications. Alternatively,

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adjustment of pH of the food matrix represents a more simple, effective, and economical way

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to inhibit the formation of AGEs 12, 13.

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Before the AGEs or AGE-induced peptides reach the systemic circulation, GI digestion could

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affect their bioavailability and metabolic fate. Faist et al. reported that a large amount of

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advanced Maillard reaction products (MRPs) could be recovered in urine (16% of the

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consumed amount) and faeces (40-50% of the consumed amount) in rats 14. Similarly, most of

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the dietary pyrralline is absorbed and excreted through urine within 48 h

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weight (LMW) AGEs take up a large portion of excreted AGEs 16, indicating a high excretion

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rate of LMW AGEs and a comparatively short time in which they interact with functional

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proteins in the body of healthy individuals. Comparing to LMW AGEs, larger fragments of

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non-excreted AGEs bind non-specifically to proteins in the body, especially in liver and

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kidney. These larger fragments have comparatively higher affinity for proteins leading to

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extensive tissue retention, which would subsequently function in the development of disease

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11

. Therefore, instead of synthetic compounds, several natural products, such as

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. Low molecular

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. In terms of the digestion of proteins, prior glycation altered the peptide profiles after

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digestion. Some of these peptides formed after digestion may possess beneficial biological

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properties, such as antimicrobial, antihypertensive, and immuno-modulatory activities

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Dependently on the structural feature of the protein, Maillard reaction may either increase

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or decrease 19 the in vitro protein digestibility. These effects induced by the Maillard reaction

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may result from the reduction in the availability of lysine, structure changes in glycated

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proteins, or even from inhibited protease activity by MRPs 20.

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.

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In our previous work, we found that sRAGE-binding ligands were formed during intensive

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heating, especially in the samples with lower water activity (aw, (0.23), which is the ratio of

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the vapour pressure of water in a food divided by the vapour pressure of pure water at the

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same temperature 21. The present work aims at studying the feasibility of using controlled pH

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and water activity,aw which is the ratio of the vapour pressure of water in a food divided by

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the vapour pressure of pure water at the same temperature, to obtain intensively heated whey

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proteins containing limited amount of sRAGE-binding ligands. pH 5 and pH 7 were chosen to

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cover the diversity of commercial whey products

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known to change the protein unfolding process, certainly in combination with high

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temperature. Aw 0.23 was chosen to mimic the heating in dry state and 0.59 for semi-dry state,

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which are the water activities that reflect the baking process, which has been used in tolerance

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induction studies 4. An infant gastrointestinal digestion model was employed to study the

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digestive fate of AGEs. The amount of sRAGE-binding ligands before and after digestion was

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measured by an ELISA-based sRAGE assay. The peptide profile was investigated by HPLC

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to analyse the influence of heating conditions on proteolytic breakdown of the glycated whey

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proteins. To investigate the influence of aggregation on the sRAGE-binding capacity of

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and extended to pH 9 since a high pH is

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glycated proteins, the size of aggregates was measured by size exclusion chromatography.

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MATERIALS AND METHODS

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Chemicals. The spray-dried WPC that was used in this study (Wheyco GmbH, Hamburg,

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Germany) was reported to contain 79.8% proteins, 4.2% water, 9.5% lactose and 5.8% fat.

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NuPAGE® 12% Bis-Tris Gels, running buffer, washing buffer (20% methanol, 10% acetic

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acid, and 70% demineralized waterdemi water) and sample buffer contains lithium dodecyl

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sulphate at a pH of 8.4 for SDS-PAGE were provided by Life Technology (Carlsbad, USA).

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A molecular marker set of 5-245 kDa was obtained from Jena Bioscience (Jena, Germany).

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Pepsin (P6887), pancreatin (P1750), bile extract porcine (B8631), and Pefabloc (76307) were 5


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provided by Sigma (St Louis, MO). Beta-lactoglobulin (L3908) was purchased from Sigma

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(Zwijndrecht, The Netherlands). All the other chemicals were obtained from Sigma-Aldrich

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(Zwijndrecht, The Netherlands).

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Preparation of samples. Spray-dried WPC and lactose were dissolved in PBS at pH 5, 7,

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and 9 at a protein to lactose ratio of 1:1.5 (w/w), which is similar to the protein to lactose ratio

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in milk. The solutions were lyophilised. Three groups of such lyophilised samples (2.5 g) in

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plastic sample containers were stored in desiccators with saturated potassium acetate, sodium

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bromide, and ammonium chloride solution for two weeks at room temperature to obtain an aw

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of 0.23, 0.59, and 0.79, respectively. Powders in screw-cap test tubes (Schott GL18) were

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heated at 130 °C for 10 min in a heating block (Labtherm Graphit, Liebisch, Germany). As

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the Maillard reaction may take place during the two-week storage at room temperature for

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adjusting aw, although at a very limited rate, An an unheated whey protein/lactose mixture

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without adjusting aw was used as control. The samples were prepared in duplicate. Heated

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samples were put in an ice bath after the heat treatment to cool down immediately.

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Subsequently, all the powders were reconstituted in 30 ml PBS pH 7.4 .After that, the

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solutions were centrifuged (rotor JA 25.50, Avanti Centrifuge J-26 XP, Beckman Coulter,

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USA) at 10,000 g for 30 min to separate insoluble material from the soluble material. Lactose

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and salts in the samples were filtered out using an ultra-centrifugal device (Pall Life Science,

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Ann Arbor, USA) with a molecular weight cut-off of 3 kDa. The amount of remaining

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accessible amino groups in PBS-soluble protein was measured using the o-phthalaldehyde

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(OPA) method according to Nielsen et al.

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then the weight was measured. Protein content of the dried pellet was also measured by the

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Dumas (Thermo Quest NA 2100 Nitrogen and Protein Analyser, Interscience, Breda, the

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Netherlands), which measures the protein content by converting all nitrogen forms in the

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. The pellet was lyophilised again for 24 h and

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sample to nitrogen oxides through combustion at 800-1000 °C. In this study, a nitrogen to

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protein conversion factor 6.38 was chosen as recommended for milk and milk products 24.

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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of

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conjugates. SDS-PAGE was performed under non-reducing condition. A 2 µL sample with a

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protein concentration of 2 mg/mL was diluted in 5 µL 4 × concentrated sample buffer and 15

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µL MilliQ water. Then the mixture was centrifuged at 2000 rpm for 1 min and heated at 70 °C

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in a heating block (Labtherm Graphit, Liebisch, Germany) for 10 min. Samples were then

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loaded onto 12% Bis-Tris Gels. Gels were run at 200 V and stained with Coomassie Blue R-

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250 0.1% (w/w), followed by destaining with washing buffer. A prestained protein marker (5-

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245 kDa) was used for molecular weight calibration. The scanned gels were analysed by

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Image Lab version 4.1 (Bio-Rad).

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In vitro gastrointestinal digestion. The digestion was performed following the methods 25

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previously reported

. The following modifications were done to specifically mimic infant

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gastrointestinal digestion

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pancreatin concentration was decreased by a factor of 10; the amount of pancreatin inhibitor

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was reduced by a factor of 10 accordingly; the bile salts concentration was decreased by a

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factor of 4. Briefly, 30 ml of the reconstituted sample with protein concentration of 32.7

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mg/mL was mixed well with 22.5 mL simulated gastric fluid (SGF

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added to reach an enzyme activity of 61.25 U/mg protein in SGF. After that, 15 µL 0.3 M

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CaCl2 was added, followed by an adjustment of pH to 3 with 0.2 M HCl. The mixture was

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then incubated at 37 °C for 2 hours in a shaking water bath. The enzymatic hydrolysis was

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stopped by adjusting the pH to 7 and subsequent snap-freezing in liquid nitrogen.

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: the pepsin concentration was decreased by a factor of 8; the

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). Porcine pepsin was

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In duodenal digestion, 20 mL gastric digesta was mixed with 11 mL simulated intestinal

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fluid (SIF 25) electrolyte stock solution. Porcine pancreatin (4 USP specifications) was added

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to reach an enzyme activity of 200 U/mL as measured by the method of Vandermeers et al. 27,

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followed by addition of 2.5 mL freshly prepared bile salts at a concentration of 160 mM as

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measured following the reported method by Collins et al. 28. After that, 40 µL of 0.3M CaCl2

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was added. The pH of the chyme was adjusted to 7 with 1 M HCl. After incubation at 37 °C

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for 2 hours in a shaking water bath, the enzyme was inactivated by addition of the protease

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inhibitor Pefabloc® SC to reach a concentration of 1 mM in the duodenal chyme.

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The digestibility of the samples was measured by the formation of amino groups detected

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by the OPA method according to Nielsen et al. with modifications 23. The dissolved protein

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samples were diluted to a protein concentration of 0.82 mg/mL in PBS pH 7.4. Then 26 mg of

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L-leucine was dissolved in 10 mL MilliQ water, which corresponds to 20 mM L-leucine. A

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sequence of samples with L-leucine concentration of 0, 0.156, 0.625, 1.25, 2.5 mM were used

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to obtain a standard curve. Freshly prepared OPA reagent (0.1 M sodium tetraborate, 3.5 mM

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SDS, 6.0 mM OPA, and 5.7 mM DTT) was mixed with diluted samples in a ratio of 20:3

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(v/v), followed by incubation at room temperature for 20 min. The absorbance was measured

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at 340 nm against a control containing PBS and the OPA reagent. All the samples were

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measured in triplicate. The amount of the free amino groups in unknown samples was

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calculated according to the standard curve. The calculation of degree of enzymatic hydrolysis

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(DH) was according to the following formula: DH= h/ htot * 100%

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Where htot is the number of peptide bonds in equivalents/g protein and was estimated

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following the method of Adler-Nissen et al., and the htot used for whey proteins in this study

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was 8.8

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protein.

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. The value of h was obtained from the OPA method as meqv L-leucine NH2/g

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RP-HPLC chromatography of duodenal digesta. The duodenal digesta were

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centrifuged at 10,000 g for 10 min. The supernatant was collected and filtered through a 0.45

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µm syringe filter. Ten microliter of the filtered samples was loaded onto the Aeris PEPTIDE

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3.6u XB-C18 250 × 4.6 mm column attached to a Dionex Ultimate 3000 HPLC system with

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RS Diode Array UV detector operating at 204 nm. The column was eluted using 0.1% formic

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acid in MilliQ water as solvent A and 0.1% formic acid in acetonitrile as solvent B. The

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elution gradient was: 0 - 3 min 3% solvent B, 3 - 33 min gradient from 3% to 65% solvent B,

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33 - 38 min gradient from 65% to 3% solvent B, 38 - 41 min 3% solvent B. All the samples

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were eluted with a flow rate of 1.2 mL/min.

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Size exclusion chromatography (SEC). Separation by size of glycated whey protein

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aggregates was carried out by SEC on an AKTA micro system (GE Healthcare, Uppsala,

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Sweden) equipped with a Superdex 75 HR 10/300 column (GE Healthcare) as described by

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Butré, et al

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buffer with 150 mM NaCl at pH 8.0 (filtered through a 0.2 µm membrane), and a flow rate of

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800 µL/min. The elution of the injected samples (50 µL) was monitored at 280 nm.

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Ovalbumin (45 KDakDa), β-lactoglobulin (BLG, 18.4 KDakDa), bovine serum albumin (69

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KDakDa), and α- lactalbumin (14.1 KDakDa) were used as standards.

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. The experiments were performed at 20 °C with 10 mM sodium phosphate

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Circular dichroism (CD). BLG and lactose were dissolved in PBS pH 7.4 at a protein to

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lactose ratio of 1:1.5 (w/w) to study the influence of dry heating on the secondary structures

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of BLG. The solution was lyophilised. Two groups of such lyophilised samples in plastic

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sample containers were stored in desiccators for two weeks at room temperature to obtain an

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aw of 0.23. Powders in screw-cap test tube (Schott GL18) were heated at 130 °C for time

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periods of 0, 2, 4, 6 and 8 min in a heating block (Labtherm Graphit, Liebisch, Germany).

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Far-UV CD spectra were recorded at 20 ˚C using a J-715 spectropolarimeter (JASCO)

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equipped with a temperature controlled cuvette holder. The CD-spectra, ranging from 185 nm

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to 260 nm, of each sample was acquired using a 1 mm quartz cuvette (Starna, type 21) at a

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scan speed of 100 nm/min. Each sample was diluted to a protein concentration of 0.2 mg/ml

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using 10 mM potassium phosphate buffer pH 6.9 before the measurement. The far-UV CD-

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spectra of 10 mM potassium phosphate buffer pH 6.9 was acquired to correct the baseline.

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sRAGE binding assay. The competition ELISA-based RAGE binding assay was used to

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measure the binding between the soluble form of RAGE receptor and the heated WPC, both

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before and after digestion. The specificity was tested by creating competition between a

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known high affinity ligand coated on the plate and the possible ligand - dry heated WPC

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samples. The transparent ELISA plates (Geiner Bio-One, cat nr 655061) were coated with

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glycated 90 (soy protein extract glycated with glucose for 90 minutes), which showed highest

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binding capacity to human sRAGE receptor as measured by direct ELISA assay, in 1.5 mM of

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sodium carbonate buffer pH 9.6 overnight at 4 °C. Next day the plate was washed with PBS

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containing 0.05% (v/v) Tween-20 and the plate was blocked with 3% BSA for 1 hour at room

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temperature. WPC samples in the number of dilutions (0.25, 2.5, 25, 250, 2500 µg/mL) were

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pre-incubated (45 minutes, 37 °C) with sRAGE (recombinantly produced in E. coli.

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Biovendor RD172116100, 1 µg/mL) in the dilution buffer (1.5% BSA and 0.025% Tween-20

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in PBS). After blocking, the coated ELISA plate was washed and then the pre-incubation

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mixture was added followed by 1 hour of incubation at 37 °C. The plate was washed and anti-

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RAGE antibody (monoclonal mouse IgG2B human RAGE antibody MAB11451; 1 µg/mL)

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was added and incubated for 30 minutes on the shaker at room temperature. After washing the

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detection antibody (polyclonal goat anti-mouse HRP-conjugated P0447; 1:1000) was added

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and incubated for 30 minutes on the shaker at room temperature. The plate was washed and

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TMB substrate (3,3’,5,5’-tetramethylbenzidine) was added and incubated for 15 minutes. The

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colouring was stopped using 2% HCl. The plate was read using the Filtermax F5 at 450 nm

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with 620 nm as reference. The percentage of inhibition was calculated using the following

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formula: (

ை஽௦ோ஺ீாିை஽௦௔௠௣௟௘ ை஽௦ோ஺ீா

)*100, where OD sRAGE was the signal obtained for sRAGE

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incubated without the competition agent (WPC sample) in the dilution buffer (maximal

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signal). All tests were performed two times in triplicate.

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Statistics. Two independent experiments for two batches of treated WPI/lactose samples

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were performed in duplicate or triplicate. Data were expressed as mean ± SD. Two-way

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ANOVA was used to determine the statistical significance of the data. Significance was

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defined at p

Generation of Soluble Advanced Glycation End Products Receptor

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