Glucose Glycation of Î±-Lactalbumin and Î²-Lactoglobulin in Glycerol

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Food and Beverage Chemistry/Biochemistry

Glucose Glycation of #-Lactalbumin and #-Lactoglobulin in Glycerol Solutions Xiaoxia Chen, Lina Zhang, Bhesh Bhandari, and Peng Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03544 • Publication Date (Web): 19 Sep 2018 Downloaded from http://pubs.acs.org on September 22, 2018

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Journal of Agricultural and Food Chemistry

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TITLE:

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Glucose Glycation of α-Lactalbumin and β-Lactoglobulin in Glycerol Solutions

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AUTHORSHIP:

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Xiaoxia Chen1,2, Lina Zhang1,2, Bhesh Bhandari3, Peng Zhou1,2

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State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,

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Jiangsu Province 214122, People’s Republic of China

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Jiangnan University, Wuxi, Jiangsu Province, 214122, China

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Queensland, 4072, Australia

International Joint Research Laboratory for Functional Dairy Protein Ingredients,

School of Agriculture and Food Science, University of Queensland, Brisbane,

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Running Title: Glycation in glycerol solutions

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Corresponding author:

ACS Paragon Plus Environment


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Peng Zhou

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Phone: 86-510-8532-6012

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Fax:

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E-mail: [email protected]

86-510-8532-9625

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Co-corresponding author:

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Bhesh Bhandari

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Phone: 61-7-33469192

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Fax:

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E-mail: [email protected]

61-7-33469192


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Abstract

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The glucose glycation of α-lactalbumin and β-lactoglobulin at 50 oC in glycerol-based

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liquid system was investigated to evaluate the effect of water activity on glycation and

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site-specificity in glycerol matrix. Glycation extent during the reaction was

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determined using o-phthalaldehyde (OPA) method as well as ultra-performance liquid

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chromatography

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(UPLC-ESI-MS). Glycation sites were identified by data-independent acquisition

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LC−MS (LC-MSE). The surface potential achieved by PyMOL and tertiary structure

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determined by circular dichroism (CD) were used to assist the analysis of the

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glycation site-specificity in glycerol matrix. The water activity of glycerol solutions

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was negatively correlated to the glycerol concentration. Results showed that the initial

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glycation rate in glycerol matrix was fitted to a linear equation in the first 48 h.

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Glycation accelerated with the increase of glycerol concentration, namely the

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decrease of water activity, regardless native structure of protein. The glycation sites

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were identical at a similar DSP although achieved at different water activity, with 4

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and 7 sites detected in α-lactalbumin and β-lactoglobulin, respectively. However,

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compared with the glycation sites in water based matrix, the site-specificity of

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glycation was affected by the glycerol matrix, depending on the native structure of

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proteins. Glycation was prone to occur at the reactive sites distributed on the surface

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of the proteins, particularly the region with positive potential.

combined

with

electro-spray

ionization


mass

spectrum


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Keywords

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Maillard glycation, glycerol, α-lactalbumin, β-lactoglobulin, water activity


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Introduction

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Maillard reaction is one of the most common reactions during food processing and

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storage, which occurs between the free amino groups of proteins and carbonyl groups

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of reducing sugars.1 Factors including temperature, pH and water activity have been

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known as the key environmental parameters influencing the Maillard reaction.2,3 The

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influence of temperature and pH have been well-studied without controversy.

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However, the effect of water activity on Maillard reaction remained debatable.

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Initially, a conclusion was reached that maximum Maillard reaction rate occurs

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around water activity of 0.4~0.5.4-7 Subsequently, the Maillard reaction was studied in

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a model system in which water activity was dominated by glycerol. It was observed

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that browning at water activity 0.11 was 1.5 times faster than that at water activity

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0.65.8 Lately, it was observed that the Maillard reaction between glycine and sugars

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slowed down with the increase of water content in a matrix dominated by glycerol.9

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However, most of the previous studies focused on the browning, which involved a

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series of complicated reaction and resultant products.

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The initial stage of Maillard reaction, also called glycation, involves an attack on

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carbonyl groups by nucleophilic amino groups, forming Schiff base and water.10 The

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Schiff base is unstable and rapidly rearranges to the Amadori product in the case of

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aldose. Water plays a significant role at the beginning of Maillard reaction.



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Specifically, water serves as reaction medium but an excessive amount of water could

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dilute the concentration of reactants leading to a depressive effect on glycation. It was

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noteworthy that recently the glycation during freeze-drying had been reported, in

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which the temperature was -80 oC and the water exist in form of crystal with very low

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water activity.44,45

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Glycation has been widely studied and proved to be a significant path to modify

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proteins.11 The Amadori product formed between aldose and protein is called glycated

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protein. The functionality of glycated proteins exhibited an improvement in some

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way.12 Chevalier et al. reported the increased of solubility of β-lactoglobulin at an

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acidic condition close to pI because of glycation.13 The thermal stability of glycated

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β-lactoglobulin was significantly enhanced under acidic and neutral conditions in

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respect of the increased solubility during heat treatment.14 The β-lactoglobulin

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glycated with various sugars showed superior emulsifying and foaming properties to

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the native β-lactoglobulin, depending on the structure of sugars.15-17 In addition,

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glycation was reported as a potential way to achieve proteins with high radical

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scavenging and ferric reducing activities.18,19 With respect to immunology, glycation

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contributed to the reduction of β-lactoglobulin allergenicity because of the decreased

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IgE binging ability.20 As a result, glycation was considered as a promising way to

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prepare emulsifiers and wall materials applied in the field of bioactive product

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encapsulation. Among the existing researches, glycation was commonly studied in a

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solid-matrix-based system because it was much slower in an aqueous-matrix-based ACS Paragon Plus Environment

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system.21 In latter conditions, not only the diluting effect on reactant but also

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suppression of water were responsible for the slow-down of glycation. Currently, in

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lab scale, Maillard glycated protein was prepared in a solid-matrix-based system, also

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called as “dry” condition.14,22

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In a solid-matrix-based system, the effect of water activity on glycation has attracted

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lots of attentions.22,43 To prepare the glycated protein in the solid-matrix-based system,

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proteins and reducing sugars were initially dissolved in water and then freeze-dried.

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The freeze-dried mixture was heated under different water activity of environment

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was controlled by supersaturated salt solution. Low glycation has been found at low

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water activity while the maximal glycation rate was reported occurring at water

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activity ranging from 0.4 to 0.8.42,43 As our preliminary experiment indicated, the

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glycation rate at water activity 0.53 was roughly 2 times faster than at 0.23. The

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limitation of reactants’ mobility was attributed to the failure of glycation at low water

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activity. Accordingly, it was more proper to say that the effect of water activity in the

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solid matrix was attributed to the enhancement of mobility of reactants.22 Thus, it is

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likely gave a false negative feedback to study the effect of water on glycation,

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particularly at low water activity. In order to investigate the effect of water activity

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itself, it would be more persuasive to use a system without or with less restriction of

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reactants’ mobility. Glycerol, as a naturally liquid humectant, was widely used to

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control the water activity in a liquid state. Hence glycerol is one of the appropriate

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matrix to study the effect of water activity on glycation in liquid matrix system. ACS Paragon Plus Environment


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In this research, the glycation in a glycerol-matrix-based system was studied with

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respects of reaction extent and site-specificity influenced by water activity and solvent

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matrix. Whey proteins including α-lactalbumin and β-lactoglobulin were used,

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avoiding the specificity due to the protein native structure. Glycerol is the solvent

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matrix of glycation meanwhile controls the water activity of system. The degree of

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glycation

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liquid-chromatography-mass-spectrometry (LC-MS). Protein conformation may be

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influenced by solvent matrix, possibly resulting in the preferable accessibility of

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carbonyl groups to the free amino groups. Hence, the glycation sites of two proteins

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were also identified by mass spectrometry.

was

estimate

by

o-phthalaldehyde


(OPA)

method

and

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Materials and Methods

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Materials

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The α-lactalbumin (JE-022-6-414) and β-lactoglobulin (JE-001-0-45) were provided

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by Davisco (Davisco Foods International, Inc, MN, USA). Anhydrous glucose and

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glycerol were purchased from Alfa Aesar (Thermo Fisher Scientific Co., Shanghai,

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China). The OPA reagent and TPCK-trypsin was purchased from Sigma-Aldrich (St.

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Louis, USA). Other chemicals used in this study were of analytical grade.

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Preparation of glycerol solutions

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Glycerol was mixed with double distilled water into solutions at concentration of 30%,

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50%, 70%, 90% and 95% by volume. The glycerol reagent with purity over 99.5%

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according to the manufacture was taken as ~100% glycerol. The density of glycerol

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and water were 1.26

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calibrated based on the weight and density.

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and 1.00 g·m-3, respectively. The volume of solutions was

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Water activity of glycerol solutions

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Water activity of glycerol solutions was measured using water activity meter

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(LabSwift-aw, Novasina, Horsham, UK). The glycerol solutions of different

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concentrations were kept in the accessory containers and equilibrated to 25 oC before

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the measurements. Each sample was conducted in duplicates.

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Preparation of glycated proteins

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Protein powder was suspended in 30~100% glycerol solutions with concentration of

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α-lactalbumin and β-lactoglobulin at 0.77 mM and 0.63 mM, respectively, and stirred

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at speed of 400 rpm for 4 h (RO10 Magnetic Stirrers, IKA Co., Staufen, Germany).

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Afterwards, the bubbles in above solution were removed using a vacuum chamber.

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The protein-glycerol solutions were then stored at 4 oC to equilibrate for 48 h. The

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concentration of Maillard reactive amino groups in the protein-glycerol parent

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solutions were, theoretically, 10 mM, including the N-terminal and lysine residues.

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Anhydrous glucose was suspended in 30~100% glycerol solutions at concentration of

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20 mM and then heated at 90 oC in sealed containers until dissolved. Afterwards, the

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glucose-glycerol solutions cooled down to room temperature and was stored at 4 oC

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before use (no crystallization of glucose observed). Due to the high viscosity of

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glycerol, the volume of each glycerol solution was taken by mass based on the density

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of glycerol solution.

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showed little changes compared with corresponding glycerol solutions.

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The water activity of above solution was monitored and


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The protein-glycerol solutions and the glucose-glycerol solutions were mixed at a

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mass ratio of 1:1, with the mole ratio of amino and carbonyl groups at 1:2, and then

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vortexed for 30 min at room temperature using vortex-genie 2 (Scientific Industry,

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Inc., Bohemia, NY, USA). The containers were tightly sealed and wrapped by

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Parafilm (Bemis Company, Neenah, WI, USA). The protein-glycerol solutions and the

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glycerol solutions without glucose were mixed in the same way and considered as

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heated controls. The samples and heated controls were incubated at 50 oC for 12, 24,

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48, 72 and 96 h using water bath (Precision GP 28, Thermo Scientific, Waltham, MA

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USA). To stop the reaction, water was added with final weight reaching 2.5 times of

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each sample. The diluted samples were stored at -18 oC before further treatment. Each

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sample was conducted in duplicates.

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Degree of glycation determined by OPA method

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The amount of free amino groups was determined to evaluate the glycation extent

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using the OPA method according to the description by Goodno et al.24 The OPA

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reagent was prepared by diluting a mixture of 80 mg OPA (dissolved in 2 mL ethanol),

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5 mmol sodium tetraborate, 1 g sodium dodecyl sulfate (SDS) and 0.2 mL

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2-mercaptoethanol into 100 mL. The OPA reagent was used freshly within 3 h after

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preparation.



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Samples were unfrozen and diluted 2 folds. Afterwards, 200 µL of diluted samples

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were added into 4 mL OPA reagent and mixed by a vortex-generator (vortex-genie 2,

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Scientific Industry, Inc., Bohemia, NY, USA). The mixture was incubated for 5 min at

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room temperature. The absorbance at 340 nm was read by UV-VIS spectrophotometer

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(UV-2700, Shimadzu Co., Tokyo, Japan). The unheated protein-glycerol solution was

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diluted in the same way and considered as control with 100 % free amino group. The

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remained free amino acid after glycation is calculated as following: Free amino groups %=

Asample × 100% Acontrol

(1)

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For the purpose to estimate the glycation rate, the percentages of free amino groups

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over the first 48 h were fitted to a linear equation:

= −

(2)

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where t refers to the reaction time. The f(t) is the percentage of free amino groups

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after heating for t h. The f0 refers to the free amino groups at the beginning which is

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supposed to be 100%. The v is the glycation rate.

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Degree of glycation determined by UPLC-MS

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In order to remove the glycerol and unreacted glucose, the samples were dialyzed at 4

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o

C against 20 folds deionized water by volume, using cellulose membrane (Union


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Carbide Co., Danbury, CT, USA) with 7000 Da cut-off molecular weight for 48 h.

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The deionized water was changed every 12 h for a total of 48 h period. Then the

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samples were freeze-dried for 48 h (Bench top Pro, SP Scientific, Warminster, PA,

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USA).

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UPLC-ESI -MS (LCZ/2690 XE/996, Waters Co., Milford, MA, USA) was used to

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measure the glycation extent. The freeze-dried samples were dissolved in MilliQ

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water at a concentration of 1 mg/mL. A 2.1×100 mm BEC C4 column (Ethylene

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Bridged Hybrid, Waters, Milford, MA, USA) packed with 1.7 µm particles was used.

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A gradient elution at 0.3 mL/min by formic acid (0.1%) and acetonitrile was carried

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out with the proportion of 0.1% formic acid from 98 to 60% in the initial 8 min and

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then to 20% during the next 2 min. The mass data were analyzed using MassLynx V

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4.1 software (Waters Co., Milford, MA, USA). The glycation extent is chartered by

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the weighted average degree of substitution per protein (DSP), calculated as

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following:

Average DSP=

∑ × ∑

(3)

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where i refers to the number of glucose reacted with amino groups on each protein

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and Ii refers to the intensity of the peaks of glycated protein molecule with

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corresponding amount (i) of glycose attached.

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Identification of glycation sites

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The glycated protein powder was dissolved into 1 mg/mL in the presence of 0.32 mg

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TPCK-trypsin (BAEE ≥ 10000 unit/mg according to the manufacturer). The pH of

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solution was adjusted to 8 by ammonia and incubated at 37 oC for 20 h. Hydrochloric

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acid was used to inactivate the trypsin by adjusting pH to 2. The hydrolyzed samples

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were filtered through 0.45µm hydrophilic PTFE syringe filters (Millex, Merck

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MilliporeCo., Darmstadt, Germany).

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Glycation sites of protein were determined using data-independent acquisition

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LC−MS (LC-MSE) with a Waters SYNAPT MS system (Waters Co., Milford, MA,

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USA).13 A 2.1×150 mm BEH 130 column packed with 1.7 µm particles with a pore

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width of 130 Å was used. A gradient elution at 0.3 mL/min by formic acid and

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acetonitrile was carried out with the proportion of formic acid from 100 to 80% in the

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initial 40 min, from 80 to 60% in the next 10 min, from 60 to 40% in the following 5

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min and reaching 0 in the last 5 min. A positive ionization mode was used. The mass

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determination was conducted under collision energy at 6 eV followed by 25 eV. The

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results were analyzed using MassLynx V 4.1 software equipped with MassEnt (Waters

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Co., Milford, MA, USA).

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Site-specificity analysis


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The visualized distributions of glycation site on α-lactalbumin and β-lactoglobulin

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were analyzed based on crystal structure from protein data base (PDB) using the

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protein ID of 1f6s25 and 3blg26, respectively. PyMOL (open-source community)

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created by Warren Lyford DeLano was used to graph the 3-dimensional model and

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label the glycated sites on proteins. Besides, the protein contact potential was

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analyzed by PyMOL based on Poisson-Boltzmann equation.

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Circular dichroism measurements

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The tertiary structure of glycated protein was measure by circular dichroism (CD)

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(Jasco-710, Jasco Co., Tokyo, Japan). Samples were dissolved in water at

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concentration of 1 mg/mL for near-UV measurements. Near-UV CD spectra were

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recorded in the range from 320 nm to 250 nm using a cuvette with 10 mm path length.

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The scanning was performed in a continuous mode with speed at 100 nm/min. The

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contribution of water was subtracted. The results were shown as the average of three

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independent scans. Each sample was conducted in duplicates.

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Statistical analysis

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The significance analysis was done using SPSS (PASW Statistics 18, IBM Co., NY,

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USA) using one way ANOVA to determine significant differences between means

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(p

Glucose Glycation of Î±-Lactalbumin and Î²-Lactoglobulin in Glycerol

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