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High pressure-high temperature processing reduces Maillard reaction and viscosity in whey protein-sugar solutions Geraldine Avila Ruiz, Bingyan Xi, Marcel Minor, Guido Sala, Martinus Van Boekel, Vincenzo Fogliano, and Markus Stieger J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01955 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 9, 2016

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Journal of Agricultural and Food Chemistry

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High pressure-high temperature processing reduces Maillard reaction and viscosity in

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whey protein-sugar solutions

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Geraldine Avila Ruiz 1,2, Bingyan Xi 2, Marcel Minor 1, Guido Sala 1, Martinus van Boekel 2,

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Vincenzo Fogliano 2 and Markus Stieger 3

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1

Food and Biobased Research, Wageningen University and Research Centre, P.O. Box 17,

6700 AA Wageningen, The Netherlands 2

Food Quality and Design Group, Wageningen University and Research Centre, P.O. Box

9101, 6700 HB Wageningen, The Netherlands 3

Division of Human Nutrition, Wageningen University and Research Centre, P.O. Box 17,

6700 AA Wageningen, The Netherlands

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Corresponding author: Markus Stieger

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E-mail address: [email protected]

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Tel. +31 317 481694

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ACS Paragon Plus Environment


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Abstract

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The aim of the study was to determine the influence of pressure in high pressure-high

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temperature (HPHT) processing on Maillard reactions and protein aggregation of whey

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protein-sugar solutions. Solutions of whey protein isolate containing either glucose or

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trehalose at pH 6, 7 and 9 were treated by HPHT processing or conventional high temperature

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(HT) treatments. Browning was reduced, and early and advanced Maillard reactions were

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retarded under HPHT processing at all pH values compared to HT treatment. HPHT induced a

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larger pH drop than HT treatments, especially at pH 9, which was not associated with

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Maillard reactions. After HPHT processing at pH 7, protein aggregation and viscosity of whey

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protein isolate-glucose/trehalose solutions remained unchanged. We conclude that HPHT

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processing can potentially improve the quality of protein-sugar containing foods, for which

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browning and high viscosities are undesired, such as high-protein beverages.

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Keywords

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High pressure-high temperature processing, whey protein, Maillard reactions, browning,

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protein aggregation

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1. Introduction

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The use of high pressure-high temperature (HPHT) processing to sterilize foods is a

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promising alternative to conventional retort heating 1. HPHT processing combines high

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temperatures (90-121°C) with pressures equal to or above 600 MPa to inactivate pathogens

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and spores. Compression heating allows reducing heating-up times leading to shorter

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processing times and lower heat loads compared to conventional retort sterilization. It was

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reported that lower heat loads are the main advantage of HPHT processing

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consequently improve sensorial and nutritional food properties

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unclear whether pressure itself or the lower heat load contributes to the improved sensory and

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nutritional properties of HPHT processed foods.

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Maillard reactions (MR) are an important factor contributing to sensory quality of foods and

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beverages 4. In sterilized foods, e.g. in dairy-based beverages, high-protein beverages,

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puddings, creams etc, MR are usually undesired. Studies on the effect of high pressure on MR

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are not extensive and were reviewed recently 5. The rates of some MR pathways can be

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increased or decreased by high pressures depending on the predominant mechanism and

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specific processing conditions. Some studies showed that pressure accelerated the

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condensation reaction and the formation of Amadori products, while other studies found that

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pressure decelerated amino acid-sugar conjugation, the Amadori rearrangement and the

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degradation of Amadori rearrangement products

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acid-sugar solutions, pressure retards or promotes the formation of advanced MR products

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and browning, depending on the pH

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pressure on MR products in protein-sugar solutions has been investigated only by two studies,

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whereas several studies have examined MR products in amino acid-sugar solutions. Proteins

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were found to denature and aggregate by a different mechanism under high pressure treatment

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compared to heat treatment 15. Changes in protein structure can be associated with the extent

6-10

1-3

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which can

. However, it remains

. Several studies reported that for amino

11-14

. To the best of our knowledge, the influence of

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of MR under HPHT

. In the current study three Maillard reaction products (furosine, Nε-

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(Carboxymethyl)-ι-lysine (CML) and Nε-(Carboxyethyl)-ι-lysine (CEL)) were monitored

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because they represent the most widely studied Maillard reaction products and are often used

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as biomarkers of food quality 19. Buckow et al. (2011) also studied physical properties of the

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solutions using SDS-PAGE. An increase in high molecular weight compounds after HPHT

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treatment (30 min, 200 and 600 MPa at 110°C) of BSA-glucose solutions compared to heat

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treatment (10 and 30 min, 0.1 MPa at 110°C) was found. Aggregation, and a potential change

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in rheological properties, in sterilized foods might be desirable or not, depending on the type

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of food. For liquid, sterilized foods containing protein, usually, viscosity increases are only

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desired to a certain extent, e.g. in high-protein beverages.

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The aim of the study was to determine the influence of pressure in HPHT processing on

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Maillard reactions and protein aggregation of whey protein-sugar solutions. Browning, pH,

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Maillard reaction products (furosine, Nε-(Carboxymethyl)-ι-lysine (CML) and Nε-

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(Carboxyethyl)-ι-lysine (CEL)), viscosity and particle size of whey protein isolate solutions

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containing glucose (reducing sugar) or trehalose (non-reducing sugar) were quantified.

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Different HPHT treatment conditions (700 MPa, 0-15 min, 123°C) were compared with

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different high temperature (HT) treatments (0-15 min, 123°C). Processing times similar to

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those used in industry (3-5 min) were chosen 14.

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2. Materials and methods

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2.1. Materials

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Whey protein isolate (WPI) (BiPRO) was purchased from Davisco, Foods International, Inc.

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(Minnesota, USA). Glucose and trehalose were purchased from Sigma-Aldrich Chemie

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GmbH (Schnelldorf, Germany). MilliQ water was used.

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2.2. Preparation of WPI-glucose/trehalose solutions

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Aqueous solutions of 6% (w/w) WPI and 5% (w/w) glucose or 5% (w/w) trehalose were

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adjusted to pH 6, 7 and 9 by addition of 1 N HCl or 0.1 N NaOH, respectively, and stirred for

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3 h. WPI – glucose (WPI/G) and WPI – trehalose (WPI/T) solutions were stored overnight at

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4°C before processing to ensure dissolution of WPI.

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2.3. HPHT treatment of WPI/G and WPI/T solutions

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WPI/G and WPI/T solutions (10 ml) were sealed in small polyethylene bags after removal of

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air. Solutions were HPHT-treated using a Resato high-pressure apparatus (Resato FPU-100-

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50, Resato International B.V., Roden, The Netherlands). Pressure build-up rate was 4.5

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MPa/s. Water was used as pressure medium. Solutions were first preheated at 90°C for 3 min

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in a water bath and subsequently high-pressure treated at 700 MPa for 0, 1.5, 3, 9 and 15 min.

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The time point at which the solutions reached 123°C was taken as processing time zero.

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It was not possible to measure the temperature or pH of the solution during the HPHT

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treatment experimentally. To estimate temperature-time profiles for all processing times, two

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assumptions were made: 1) the adiabatic heat increase was uniformly transmitted to the

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solution without time delay; 2) the heat-transmitting properties of the WPI/G and WPI/T

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solutions were similar to those of water. To estimate the maximum temperature reached in the 5



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HPHT treatment, the temperature of the pressure medium during pressurization was measured

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using a lab-scale high-pressure unit (volume 180 ml, maximum pressure 1000 MPa, Resato

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International B.V., Roden, The Netherlands) (Figure S1). In previous studies the temperature

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of water after applying different pressures at various initial temperatures was measured

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When extrapolating the data of Esthiagi et al. (2001), a maximum temperature of 122.5°C

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during HPHT treatment at 700 MPa was obtained. The maximum temperature measured by

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Knoerzer et al. (2010) at 700 MPa was 125.0°C. Through combination of our experimental

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data and the data from literature, the maximum temperature in our study was estimated to be

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123°C ± 2°C.

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The temperature loss was determined by measuring the temperature of the pressure medium

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before pressure-build up and after pressure release. The difference in temperature was

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assumed to be equal to the temperature loss experienced during the processing times. The

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calculated temperature difference was linearly correlated to the initial temperature (Figure

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S2).

16, 17

.

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2.4. HT treatment of WPI/G and WPI/T solutions

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WPI/G and WPI/T solutions were heated in a heating block (Liebisch Labortechnik, type:

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53186301, Germany) to 123°C. Solutions were treated for 0, 1.5, 3, 9 and 15 min. To mimic

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the temperature-time profile of the HPHT treatment during the processing times, the heating

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block was set to lower temperatures during these times. Subsequently, solutions were cooled

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down to room temperature using a water bath at 15°C. The temperature of the solutions was

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monitored during the entire treatment. Temperature measurements were performed in

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triplicate.

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2.5. Determination of browning

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Browning intensity of undiluted HPHT and HT treated WPI/G and WPI/T solutions was

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determined by quantifying the absorbance at 420 nm with a spectrophotometer (Pharmacia

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Biotech, Uppsala, Sweden)

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regression of absorbance as a function of processing time was performed.

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. To compare browning rates between the treatments, linear

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2.6. Determination of Maillard reaction products

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Furosine, Nε-(Carboxymethyl)-ι-lysine (CML) and Nε-(Carboxyethyl)-ι-lysine (CEL) were

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used to monitor the Maillard reaction because they represent the most widely studied MR

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products and are often used as biomarkers of food quality 19. The compounds were quantified

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using a previously described method with small modifications 19.

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2.6.1. Sample preparation

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A solution volume of 100 µl was mixed with HCl (6 N) in a screw capped flask with PTFE

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septa. The mixture was saturated with nitrogen (15 min at 2 bar) and hydrolyzed in a heating

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block (Liebisch Labortechnik, type: 53186301, Germany) for 20 h at 110°C. The mixture was

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centrifuged for 10 min at 4000 rpm at 4°C and the supernatant was subsequently filtrated

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using polytetrafluoroethylene filters (0.45μm, Phenomenex, USA). A volume of 200 µl

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filtered sample was dried under nitrogen flow in order to prevent the oxidation of the

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constituents. The sample was reconstituted in 190 µl of water and 10 µl of a mixed internal

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standard (d4-lys, d2-CML, d2-CEL and d2-furosine) was added. The sample was loaded onto

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equilibrated Oasis HLB 1 cc cartridges (Waters, Wexford, Ireland) and eluted according to the

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method previously described in detail

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overnight. Samples were dissolved in 150 µl of an acetonitrile – water (90:10) solution. Then,

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5 µl were injected into the LC-MS/MS system.

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. Eluted solutions were dried under nitrogen

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2.6.2. Liquid chromatography tandem mass spectrometry (LC–MS/MS)

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Separation of furosine, CML, CEL, lysine and their respective internal standards was

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achieved on a Hydrophilic Interaction Liquid Chromatography column using the following

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mobile phases: A) 0.1 % acetic acid in water, B) 50 mM ammonium acetate in water, and C)

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0.1% acetic acid in acetonitrile.

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The compounds were eluted and the chromatographic profile was recorded according to the

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method of Troise et al. (2015).

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2.6.3. Analytical performances

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CML, CEL and furosine were quantified using a linear calibration curve obtained with

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solutions of purified CML, CEL and furosine at different concentrations. The limit of

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detection (LOD) and the limit of quantitation (LOQ) were monitored according to Troise et al.

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(2015).

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2.7. Determination of pH

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pH of WPI/G and WPI/T solutions was determined at 20°C using a pH meter (Conductivity

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Proline Plus, QiS, The Netherlands). Measurements were performed in duplicate.

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2.8. Determination of viscosity

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Viscosity of the solutions was determined using an Ubbelohde viscometer (SI Analytics

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GmbH, Germany) at 25°C. The constant of the viscometer capillary was 0.004639 mm2s-2.

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Measurements were performed in triplicate. Viscosity was calculated using the following

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formula:

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ʋkin (m2s-1) = t (s) × capillary constant (mm2s-2) ×10-6

[1]

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where ʋkin is the kinematic viscosity and t is the flow-through time,

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η (Pa.s) = ʋkin (m2s-1) × ρ (kg.m-3)

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where η is the dynamic viscosity and ρ the density of the solutions.

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Density was determined using a density meter (DMA 5000, AntonPaar, Graz, Austria) at

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25°C. When setting equation 1 equal to equation 2, the viscosity was obtained and converted

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to mPa.s.

[2]

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2.9. Monitoring of particle size using SEC

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WPI/G and WPI/T solutions were diluted to a protein concentration of 0.5% (w/w) with

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MilliQ water and subsequently filtered using 0.2 µm RC filters. Particle size of the heated

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solutions was monitored by High Pressure – Size Exclusion Chromatography (HP-SEC) fitted

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with an Ultimate 3000 pump and a UV detector (Thermo Scientific, USA). The HP-SEC

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columns (TSKGel G3000SWXL, 5µm, 300 x 7.8 mm, and TSKGel G2000SWXL, 5 µm, 300

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x 7.8 mm) were equilibrated with 30% acetonitrile in MilliQ water and 0.1% trifluoroacetic

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acid as eluents. Samples were loaded and eluted at 1.5 ml/min at 30°C, and the eluates were

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monitored at 214 nm.

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3. Results and discussion

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3.1. Determination of processing conditions

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The temperature-time profiles of HT treatments were experimentally determined, while those

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of HPHT treatments were estimated (Figure 1). The main difference between the temperature-

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time profiles of HT and HPHT treatments was in the heating-up phase. The HT treatment took

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about 6 min to reach the target temperature (123°C), and it took about 3 min for the

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temperature to increase from 90 to 123°C. In contrast, the HPHT treatment took about 3 min

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to reach the target temperature (123°C), and it took only about 30 s for the temperature to

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increase from 90 to 123°C. This fast temperature rise in the HPHT treatments is due to

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adiabatic heating accompanied by pressure build-up

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the pressure-holding time was successfully matched in the HT treatments. During the cooling

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phase the temperature decreased from about 120 to 90°C faster for HPHT treatments

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compared to HT treatments.

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Due to the differences in the heating-up phase between the two treatment techniques, the time

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point at which the solutions reached 123°C (t = 6 min) was taken to compare HT and HPHT

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treatments in terms of heat load. The matching of the temperature-time profiles of the HT-

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treated solutions in the pressure-holding phase time also ensured a fair comparison between

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HT and HPHT treatments.

16, 17

. The temperature decrease during

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3.2. Browning

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The absorbance of undiluted WPI/G solutions treated with HT was higher compared to that of

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WPI/G solutions treated with HPHT for all processing times at pH 7 and 9 (Figure 2). At pH 7

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and 9, the browning rates were 15 times and 3.5 times higher for HT than for HPHT

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treatment, respectively. The difference in browning rate was also evident by eye (Figure S3).

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The absorbance of the WPI/G solutions at pH 6 could not be measured due to turbidity of the

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solutions. However, a reduced browning rate was observed by eye for the HPHT treatment

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compared to the HT solutions (Figure S3).

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The browning kinetics of the WPI/G solutions treated with HT were comparable to those of

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casein (3% w/w) – glucose (150 mM) solutions (pH 6.8) heated to 120°C for up to 40 min 18.

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It is noteworthy that in our study the heating-up time was excluded from the reported

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processing times. After 4-15 min processing time, the absorbance of the HPHT-treated 10


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samples was always lower than the absorbance of the HT-treated samples. The temperature

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was the same for both treatments. Therefore, we conclude that pressure at high temperature

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had a retarding effect on browning. The higher browning rates of HT processed solutions

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indicate that the retarding effect of pressure was stronger than the promoting effect of heat on

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browning. The pressure effect was also stronger than the promoting effect of basic conditions,

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as Figure 2 shows a lower increase in absorbance after HPHT processing compared to HT

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processing.

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3.3. Maillard reaction products

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Concentrations of tested furosine, CML and CEL were higher in WPI/G solutions treated with

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HT than in solutions treated with HPHT at pH 6, 7 and 9, paralleling the browning

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development (Figure 3). The concentrations of furosine, CML and CEL increased about

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linearly with processing time for HPHT treatment, whereas for HT treatment, they first

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increased steeply and then approached a plateau value. At pH 9, the concentrations of MR

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products in HT solutions dropped at 15 min processing. At pH 3, no considerable differences

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in concentrations of furosine, CML and CEL were observed in WPI/G solutions treated with

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HT or HPHT (data not shown). At low pH (pH 3) the Maillard reactions were inhibited for

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HT and HPHT treatments.

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Furosine, CML and CEL concentrations at pH 6, 7 and 9 were comparable to those measured

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in UHT milk

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concentrations measured by Brands and van Boekel (2001) in casein (3%) – glucose (150

256

mM) solutions (pH 6.8) heated for 0-40 min at 120°C. CML and CEL concentrations were

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also comparable to results obtained in a previous study with heated casein (3%) – glucose

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(2.7%) solutions (pH 6.8) for 0-30 min at 120°C

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can be explained with the rate of MR product formation being equal to the rate of degradation,

19

. Furosine concentrations were in the same order of magnitude compared to

20

. The plateau observed for HT treatment

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after which the rate of degradation becomes dominant. Such a behavior has been previously

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reported for the Amadori products 21.

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The lower concentrations of MR products for HPHT treatment show that pressure had a

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retarding effect on the generation of furosine, CEL and CML. The difference in concentration

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profiles between the two treatment techniques was similar to that for furosine in heat-treated

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milk at different temperatures. At 130°C, furosine concentration increased linearly from 0 to

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18 min processing, whereas at 140°C, the concentration increased sharply from 0 to 8 min,

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after which it reached a plateau 22. Compared to previous studies, which investigated HPHT

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treatment using amino acids or purified proteins and long treatment times (0-24 h), in our

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study a retarding effect of HPHT treatment for a mixture of proteins with sugars was observed

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using treatment times closer to industrial applications. Previous studies ascribed this effect to

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pressure favoring the side of the reactants in Maillard reactions due to the smaller volume

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occupied compared to the volume occupied by the products (positive activation volume) 5. In

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our systems, the volume of native and denatured proteins might also play a role. It has been

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reported that pressure has a synergistic effect with heat on whey protein denaturation and

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unfolding 9, 23, 24. Buckow et al. (2011) reported that pressures of 600 MPa for up to 45 min at

276

70°C did not lead to significant unfolding of BSA. However, at higher temperatures, protein

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unfolding was accelerated, possibly exposing more lysine groups. In the same study, it was

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found that protein-sugar conjugation was decelerated under HPHT treatment compared to HT

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treatment. This could mean that although more reactive groups become available under high

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pressure at high temperature, they will not all react with the sugars, as a larger resulting

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volume through the formation of protein-sugar conjugates is not favorable.

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The differences in the concentration profiles between the HPHT and HT treatments,

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especially for furosine and CEL at pH 6 and 7, were in agreement with the observed

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differences in browning rates and indicate that pressure at high temperature had a stronger

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retarding effect on overall MR compared to the promoting effect of heat.

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3.4. pH change after HT and HPHT treatment

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The pH of WPI/G and WPI/T solutions decreased after HT and HPHT treatments (Figure 4).

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The pH decrease was larger at pH 9 compared to pH 7. For WPI/G solutions, the pH decrease

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was larger after HT treatment than after HPHT treatment, while the opposite was found for

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WPI/T solutions.

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A pH decrease after HT and HPHT treatment has been associated with enhanced MR at

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longer processing times and increasing temperatures, resulting in a production of higher

294

concentration of organic acids

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treatment compared to HT treatment can be directly ascribed to the effect of pressure on pH

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rather than to the effect of pressure on MR. This pressure-induced pH drop might be due to

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pressure promoting the dissociation of ionizable compounds such as salts, acids, bases and

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polyelectrolytes 5. According to a previous study, pressure shifts the dissociation equilibrium

299

to the dissociated species, resulting in a pH decrease. However, Hill et al. (1996) and Moreno

300

et al. (2003) could only explain the reduced MR at pH ≤ 8 on the basis of this mechanism. At

301

higher pH values, pressure was found to accelerate MR. In later studies, the mechanism of

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pressure influencing particularly acid-base reactions, leading to changes in pH and protein

303

reactions, has repeatedly been supported

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change in ionic strength, which would have an effect on ion activities.

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Another mechanism associated with the pressure-induced pH drop might be irreversible

306

changes in the protein structure caused by pressure. Pressures beyond 150 MPa, 400 MPa and

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800 MPa cause irreversible loss of native structure for β-LG, α-LA and BSA, respectively 27.

14

. For WPI/T solutions, the larger pH drop after HPHT

25, 26

. However, the pH drop may also be due to a

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Such irreversible changes in protein structure and conformation might affect ion charges and

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ion-solvent interactions leading to permanent pH changes 25, 26.

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3.5. Protein aggregation

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WPI/G solutions at pH 7 treated with HT contained larger particles and displayed higher

313

viscosities compared to WPI/G solutions treated with HPHT (Figure 5 and 6). At pH 9,

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particle size and viscosity of samples treated with HT and HPHT did not differ considerably.

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With respect to particle size and viscosity, WPI/T solutions displayed similar behavior as

316

WPI/G solutions (data not shown). The viscosity at pH 6 could not be measured due to the

317

presence of large, coagulated particles.

318

At pH 9, pressure did not have an effect on particle size and viscosity of WPI/G and WPI/T

319

solutions. At pH 7, the smaller particle size and lower viscosity of WPI/T and WPI/G

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solutions treated with HPHT compared to WPI/T and WPI/G solutions treated with HT show

321

that pressure at high temperature inhibited protein aggregation, hence viscosity development.

322

A linear dependence of viscosity on particle size has been described previously for protein-

323

enriched liquids 28. The inhibitory effect of HPHT could have been, at first glance, associated

324

with the retardation of MR. Reduced crosslinking of proteins and sugars might have been

325

responsible for reduced aggregate formation. However, the similar trends of WPI/G (glucose

326

= reducing sugar) and WPI/T (trehalose = non-reducing sugar) solutions with regards to the

327

effect of processing time on particle size and viscosity suggest that MR did not play a major

328

role in aggregate formation and viscosity development. However, a positive correlation

329

between protein glycation and aggregate formation was found by Buckow et al. (2011) at pH

330

9. HT and HPHT treatments resulted in increased protein-sugar conjugation and formation of

331

high molecular weight compounds in BSA-glucose solutions. In contrast to our results at pH

332

9, increased protein aggregation was reported after HPHT treatment (30 min, 200 and 600 14


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MPa, 110°C) compared to HT treatment (10 and 30 min, 0.1 MPa, 110°C). The increased

334

protein aggregation was associated with changes in the protein conformation under HPHT.

335

Another study showing a positive correlation between protein-sugar conjugation and

336

molecular weight stands in contrast to our results at pH 7

337

higher molecular weights in casein-glucose solutions after HT treatment (4 h, 95°C), no

338

differences in particle size were found in the WPI/G solutions treated with HT in our study.

339

This difference can be due to the different types of treatments used and the different proteins

340

used. Casein does not denature and unfold in contrast to whey protein.

341

The larger particle size of WPI/T and WPI/G solutions treated with HT at pH 7 compared to

342

pH 9 is in line with the finding from a previous study 30. When heating β-LG solutions at pH

343

6.5, high molecular weight aggregates were formed compared to pH 7.5. This seemed to be

344

associated with different degrees of hydrophobic interactions and disulfide bond formation.

345

The smaller particle size and lower viscosity of the solutions treated with HPHT might be thus

346

associated with a reduced degree of such phenomena. As mentioned in section 3.3, pressure

347

has been found to act synergistically with heat on whey protein denaturation and unfolding.

348

To the best of our knowledge, no study has investigated protein aggregation during HPHT

349

treatment at and above 100°C and whether the synergistic effect of pressure and heat on

350

protein denaturation and unfolding also leads to protein aggregation. However, it can be

351

anticipated that the particle size and viscosity of solutions treated with HPHT and HT are

352

associated with pH-dependent differences in protein conformation, protein-protein

353

interactions as well as with differences in the pressure and heat sensitivity of whey proteins 15,

354

29

. While Hofmann (1998) found

31

. Other phenomena connected to protein aggregation, and which might play a role in the

355

present findings, involve protein glycation and solubility. There are two main phenomena

356

connected to the protein glycation during thermal treatment: 1) introduction of the polar

357

groups at protein surface which increases the solubility, and 2) carbohydrate-mediated cross

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linking of proteins which reduces the solubility. Under mild thermal treatment especially

359

those run at low temperature the first phenomenon is dominant and it has been actually used

360

to improve the solubility of different type of proteins 32. Under severe thermal treatment the

361

crosslinking become more relevant: browning of the solution is a good indication of the

362

prevalence of this phenomenon, which would decrease protein solubility. Thus, there is not a

363

simple relationship between the extent of MR, protein glycation, solubility and protein

364

aggregation. However, our data showed that protein glycation played a minor role with

365

respect to protein aggregation.

366

367

To summarize, the influence of pressure at high temperature on Maillard reaction products,

368

browning and physicochemical properties of whey protein isolate glucose/trehalose solutions

369

was evaluated comparing HPHT and HT treatments. A pressure of 700 MPa at about 123°C

370

had a significant influence on browning, MR, pH, particle size and viscosity by acting on its

371

own or in combination with heat. The novelty with regards to previous studies is that pressure

372

at high temperature retarded browning and MR under conditions closer to application, namely

373

the use of protein-sugar mixtures and shorter processing times. The retarding effect of

374

pressure on MR development was stronger than the promoting effect of heat on MR

375

development. Interestingly, pressure initially induced a pH decrease in WPI/G solutions via a

376

mechanism not related to MR. Pressure at high temperature inhibited protein aggregation and,

377

thereby, viscosity development. These findings suggest that HPHT treatment can improve

378

food quality when browning and high viscosities are undesired. We showed that the

379

uniqueness and added value of HPHT treatment lies in the impact of pressure on the MR itself

380

rather than the smaller heat load resulting from the mere presence of pressure. HPHT

381

processing of liquid products containing protein and sugar, where browning and viscosity

382

increases are undesired, could be introduced in the future. 16


Page 17 of 30


383 384

4. Abbreviations

385

HPHT, high pressure-high temperature; HT, high temperature; WPI, whey protein isolate;

386

WPI/G, whey protein isolate – glucose; WPI/T, whey protein isolate – trehalose; MR,

387

Maillard reactions

388 389

5. Acknowledgments

390

The authors gratefully acknowledge the technical assistance of Geert Meijer in the LC-

391

MS/MS experiments and the financial support of the “IPOP Customized Nutrition” program

392

of Wageningen University and Research Centre.

393 394

6. Supporting Information description

395

396

Fig. S1. Temperature of pressure medium at 700 MPa for different initial temperatures.

397

398

Fig. S2. Temperature difference before pressure build-up and after pressure release for

399

different processing times using a pre-treatment at 90°C.

400

401

Figure S3. WPI/G solutions prepared at different pH values and treated for various times

402

using HT and HPHT treatment.

403

17



7. References

404 405

Page 18 of 30

1.

Sevenich, R.; Bark, F.; Crews, C.; Anderson, W.; Pye, C.; Riddellova, K.; Hradecky, J.;

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Moravcova, E.; Reineke, K.; Knorr, D., Effect of high pressure thermal sterilization on

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the formation of food processing contaminants. Innovative Food Science & Emerging

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Technologies 2013, 20, 42-50.

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2.

Kebede, B. T.; Grauwet, T.; Mutsokoti, L.; Palmers, S.; Vervoort, L.; Hendrickx, M.;

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Van Loey, A., Comparing the impact of high pressure high temperature and thermal

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sterilization on the volatile fingerprint of onion, potato, pumpkin and red beet. Food Res.

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Int. 2014, 56, 218-225.

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3.

Vervoort, L.; Van der Plancken, I.; Grauwet, T.; Verlinde, P.; Matser, A.; Hendrickx, M.;

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Van Loey, A., Thermal versus high pressure processing of carrots: A comparative pilot-

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scale study on equivalent basis. Innovative Food Science & Emerging Technologies 2012,

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Reaction. Angewandte Chemie - International Edition 2014, 53 (39), 10316-10329.

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Martinez-Monteagudo, S. I.; Saldaña, M. D. A., Chemical Reactions in Food Systems at High Hydrostatic Pressure. Food Engineering Reviews 2014, 6 (4), 105-127.

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Hellwig, M.; Henle, T., Baking, Ageing, Diabetes: A Short History of the Maillard

6.

Bristow, M.; S. Isaacs, N., The effect of high pressure on the formation of volatile

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products in a model Maillard reaction. Journal of the Chemical Society, Perkin

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Transactions 2 1999, (10), 2213-2218.

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reaction. Journal of Physical Organic Chemistry 1996, 9 (9), 639-644.

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Isaacs, N. S.; Coulson, M., Effect of pressure on processes modelling the Maillard

8.

Schwarzenbolz, U.; Klostermeyer, H.; Henle, T., Maillard-type reactions under high

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hydrostatic pressure: Formation of pentosidine. Eur. Food Res. Technol. 2000, 211 (3),

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208-210.

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9.

Buckow, R.; Wendorff, J.; Hemar, Y., Conjugation of Bovine Serum Albumin and

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Glucose under Combined High Pressure and Heat. J. Agric. Food Chem. 2011, 59 (8),

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3915-3923.

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10. Devi, A. F.; Buckow, R.; Singh, T.; Hemar, Y.; Kasapis, S., Colour change and

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proteolysis of skim milk during high pressure thermal–processing. J. Food Eng. 2015,

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147, 102-110.

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11. Tamaoka, T.; Itoh, N.; Hayashi, R., High Pressure Effect on Maillard Reaction. Agric. Biol. Chem. 1991, 55 (8), 2071-2074.

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12. Hill, V. M.; Ledward, D. A.; Ames, J. M., Influence of High Hydrostatic Pressure and pH

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on the Rate of Maillard Browning in a Glucose−Lysine System. J. Agric. Food Chem.

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1996, 44 (2), 594-598.

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13. Moreno, F. J.; Molina, E.; Olano, A.; López-Fandiño, R., High-Pressure Effects on

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Maillard Reaction between Glucose and Lysine. J. Agric. Food Chem. 2003, 51 (2), 394-

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400.

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14. De Vleeschouwer, K.; Van der Plancken, I.; Van Loey, A.; Hendrickx, M. E., The Effect

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of High Pressure−High Temperature Processing Conditions on Acrylamide Formation

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and Other Maillard Reaction Compounds. J. Agric. Food Chem. 2010, 58 (22), 11740-

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11748.

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15. Considine, T.; Patel, H. A.; Anema, S. G.; Singh, H.; Creamer, L. K., Interactions of milk

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proteins during heat and high hydrostatic pressure treatments — A Review. Innovative

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Food Science & Emerging Technologies 2007, 8 (1), 1-23.

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16. Eshtiagi, M. N., High Pressure 2001.

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17. Knoerzer, K.; Buckow, R.; Versteeg, C., Adiabatic compression heating coefficients for

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high-pressure processing – A study of some insulating polymer materials. J. Food Eng.

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2010, 98 (1), 110-119.

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18. Brands, C. M. J.; van Boekel, M. A. J. S., Reactions of Monosaccharides during Heating

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of Sugar−Casein Systems: Building of a Reaction Network Model. J. Agric. Food Chem.

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2001, 49 (10), 4667-4675.

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19. Troise, A. D.; Fiore, A.; Wiltafsky, M.; Fogliano, V., Quantification of Nε-(2-

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Furoylmethyl)-l-lysine

(furosine),

Nε-(Carboxymethyl)-l-lysine

(CML),

Nε-

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(Carboxyethyl)-l-lysine (CEL) and total lysine through stable isotope dilution assay and

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tandem mass spectrometry. Food Chem. 2015, 188, 357-364.

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20. Nguyen, H. T.; van der Fels-Klerx, H. J.; van Boekel, M. A. J. S., Kinetics of Nε-

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(carboxymethyl)lysine formation in aqueous model systems of sugars and casein. Food

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Chem. 2016, 192, 125-133.

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21. van Boekel, M. A. J. S., Kinetic aspects of the Maillard reaction: a critical review. Food / Nahrung 2001, 45 (3), 150-159. 22. Van Boekel, M. A. J. S., Effect of heating on Maillard reactions in milk. Food Chem. 1998, 62 (4), 403-414. 23. Huppertz, T.; Fox, P. F.; Kelly, A. L., High pressure treatment of bovine milk: effects on casein micelles and whey proteins. J. Dairy Res. 2004, 71 (1), 97-106. 24. Hinrichs, J.; Rademacher, B., Kinetics of combined thermal and pressure-induced whey protein denaturation in bovine skim milk. Int. Dairy J. 2005, 15 (4), 315-323. 25. Stippl, V. M.; Delgado, A.; Becker, T. M., Ionization equilibria at high pressure. Eur. Food Res. Technol. 2005, 221 (1), 151-156.

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26. Oey, I., Effects of High Pressure on Enzymes. In High Pressure Processing of Food:

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Principles, Technology and Applications, Balasubramaniam, V. M.; Barbosa-Cánovas, V.

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G.; Lelieveld, L. M. H., Eds. Springer New York: New York, NY, 2016; pp 391-431.

477

27. Patel, H. A.; Creamer, L. K., High-Pressure-Induced Interactions Involving whey

478

Proteins. In Milk Proteins, 2008; pp 205-238.

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28. Amin, S.; Barnett, G. V.; Pathak, J. A.; Roberts, C. J.; Sarangapani, P. S., Protein

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aggregation, particle formation, characterization & rheology. Current Opinion in

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Colloid & Interface Science 2014, 19 (5), 438-449.

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29. Hofmann, T., Studies on the Relationship between Molecular Weight and the Color

483

Potency of Fractions Obtained by Thermal Treatment of Glucose/Amino Acid and

484

Glucose/Protein Solutions by Using Ultracentrifugation and Color Dilution Techniques.

485

J. Agric. Food Chem. 1998, 46 (10), 3891-3895.

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30. Laligant, A.; Dumay, E.; Casas Valencia, C.; Cuq, J. L.; Cheftel, J. C., Surface

487

hydrophobicity and aggregation of .beta.-lactoglobulin heated near neutral pH. J. Agric.

488

Food Chem. 1991, 39 (12), 2147-2155.

489

31. Yang, J.; Powers, J. R., Effects of High Pressure on Food Proteins. In High Pressure

490

Processing of Food: Principles, Technology and Applications, Balasubramaniam, V. M.;

491

Barbosa-Cánovas, V. G.; Lelieveld, L. M. H., Eds. Springer New York: New York, NY,

492

2016; pp 353-389.

493 494

32. Liu, J.; Ru, Q.; Ding, Y., Glycation a promising method for food protein modification: Physicochemical properties and structure, a review. Food Res. Int. 2012, 49 (1), 170-183.

495

496

8. Figure captions

497

Figure 1. Temperature-time profiles of (a) HPHT treatments estimated using experimental and

498

literature data, and (b) HT treatments experimentally determined. Profiles of HPHT

499

treatments start at 3 min to show the starting point for processing when 123°C is reached.

500

21



Page 22 of 30

501

Figure 2. Absorbance of WPI/G solutions at (a) pH 7 and (b) pH 9 as a function of processing

502

time after HT and HPHT treatments. Means of two measurements are shown with standard

503

deviations.

504

505

Figure 3. Concentrations of furosine, CML and CEL as a function of processing time in

506

WPI/G solutions prepared at pH 6, 7 and 9 and treated with either HT () or HPHT (□).

507

Means of two measurements are shown with standard deviations. a = single measurements.

508

509

Figure 4. pH difference as a function of processing time for WPI/G and WPI/T solutions

510

treated with either HT or HPHT prepared at (a) pH 7 and (b) pH 9 . As the standard deviations

511

were smaller than the data point markers, they are not shown.

512

513

Figure 5. Viscosity as a function of processing time for WPI/G and WPI/T solutions treated

514

with HT or HPHT prepared at (a) pH 7 and (b) pH 9. As the standard deviations were smaller

515

than the data point markers, they are not shown.

516

517

Figure 6. Size-exclusion chromatograms of (a) WPI/G solutions and (b) WPI/T solutions

518

prepared at pH 7 treated for various times by HT and HPHT.

519

520

521

22


Page 23 of 30


522

523

524

525

526

9. Graphic for table of contents

527

HT

Furosine concentration (μm/g protein)

16 12 8

HPHT

4

High pressure – high temperature (HPHT) processing retards Maillard reactions in whey protein – glucose solutions compared to high temperature (HT) processing.

0 0

5

10

15

20

Processing time (min)

528

23



Temperature (°C)

a

Page 24 of 30

120 0 min

90

1.5 min 60

3 min 9 min

30

15 min

0 0

Temperature (°C)

b

5

10 15 Processing time (min)

20

25

120 0 min 90

1.5 min 3 min

60

9 min 15 min

30 0 0

5


20


25

Page 25 of 30


Absorbance at 420 nm

a

2.5 2.0 y = 0.0788x + 0.4141

1.5 1.0 0.5

Series1

y = 0.0053x + 0.1813

HPHT 0.0

Untreated 0

5

10

15

20


Absorbance at 420 nm

b

Linear (Series1) Linear (HPHT) HT

2.5

HPHT

y = 0.1142x + 0.564

2.0

Untreated Linear (HT)

1.5

Linear (HPHT)

1.0

y = 0.0326x + 0.1376

0.5 0.0 0

5

10

15

20




Furosine (μg/mg protein)

pH 6

pH 7 16

16

12

12

12

8

8

8

4

4

4

0 0

CML (ng/mg protein)

pH 9

16

0 5

10

15

0 0

20

5

10

15

20

0

400

400

400

300

300

300

200

200

200

5

10

15

20

a

a 100

100

0

100

0 0

CEL (ng/mg protein)

Page 26 of 30

5

10

15

20

0 0

5

10

15

20

0

160

160

160

120

120

120

80

80

40

40

a

80 40

5

10

15

20

5

10

15

20

a

a 0

0 0

5

10

15

20

0 0

5

10

15

20



0

Page 27 of 30


a

0.5

HT WPI/G HPHT WPI/G

0.0 pH difference

HT WPI/T -0.5

HPHT WPI/T

-1.0 -1.5 -2.0 -2.5 0

b

5


20

0.5

pH difference

0.0 -0.5 HT, glu

-1.0

HPHT, glu HT, tre

-1.5

HPHT tre -2.0 -2.5 0

5



20


a

HT WPI/G

3.5

HPHT WPI/G

Viscosity (mPa.s)

3.0

HT WPI/T

2.5

HPHT WPI/T

2.0

Untreated WPI/G Untreated WPI/T

1.5 1.0 0

b

5 10 15 Processing time (min)

20

Series3

3.5

HPHT, Glu

Viscosity (mPa.s)

3.0

HT, tre HPHT, tre

2.5

Untreated, glu 2.0

Untreated, tre

1.5 1.0 0

5 10 15 Processing time (min)

20


Page 28 of 30

Page 29 of 30


3 min HT

a

15 min HT

900

3 min HPHT

Signal (mAU)

15 min HPHT 600

300

0 6

b

8

10 Elution time (min)

12

3 min HT 900

15 min HT

Signal (mAU)

3 min HPHT 15 min HPHT 600

300

0 6

8

10 Elution time (min)

12



HT

Furosine concentration (μm/g protein)

16 12 8

HPHT

4

High pressure – high temperature (HPHT) processing retards Maillard reactions in whey protein – glucose solutions compared to high temperature (HT) processing.

0 0

5

10

15

20



Page 30 of 30

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