Elsevier

Food Chemistry

Volume 183, 15 September 2015, Pages 36-42
Food Chemistry

Glycation of β-lactoglobulin and antiglycation by genistein in different reactive carbonyl model systems

https://doi.org/10.1016/j.foodchem.2015.02.122Get rights and content

Highlights

Abstract

Advanced glycation end products (AGEs), which are formed in β-lactoglobulin (β-lg) glycation systems via the Maillard reaction, have been implicated in diabetes-related long-term complications. In the present study, we found that reaction conditions, including temperature, time, pH, reactant type and molar ratio of beta-lg to a sugar/MGO/GO, can significantly affect the formation of AGEs. Using SDS–PAGE, we further demonstrated that genistein, a natural isoflavone found in a number of plants including soybeans and kudzu, can efficiently inhibit cross-links of the glycated β-lg, and suppress the formation AGEs in a dose-dependent manner by trapping reactive dicarbonyl compounds. The products formed from genistein and methylglyoxal (MGO) in the β-lg–MGO assay were analyzed using LC/MS. Both mono-MGO and di-MGO adducts of genistein were detected with this method.

Introduction

β-Lactoglobulin (β-lg) is the major whey protein of ruminant species, and its properties are regularly studied (Broersen, Voragen, Hamer, & de Jongh, 2004). β-lg is often used in various foodstuffs, including infant formulas, baked products and beverages. As it is a lipocalin, it can bind small hydrophobic ligands and act as specific transporters, in the same way serum retinol binding protein does (Kontopidis, Holt, & Sawyer, 2004). β-lg is also one of the main allergens in milk (Docena et al., 1996, Gaudin et al., 2008, Natale et al., 2004), and has consequently caught worldwide attention. In order to reduce the sensitization to milk at the present time (Hattori et al., 2000), the high temperature method (Kim et al., 2007) and glycation method (Meltretter, Seeber, Humeny, Becker, & Pischetsrieder, 2007) have been investigated. Heat treatments, such as pasteurization and ultrahigh-temperature (UHT) processes, required to reduce the bacterial load and increase the milk’s shelf life, can change some characteristics of the beta-lg (Fox, 1981). Furthermore, milk proteins are modified by the Maillard reaction or glycation during heating (Maillard, 1912), as milk contains high amounts of lactose and low quantities of other reducing sugars. Glycation is one of the most frequent chemical modifications during industrial processing (Ames, 1990, Chevalier et al., 2002, Chuyen, 1998). It is a complex reaction, leading to the formation of advanced glycation end products (AGEs). The reacting sugar is degraded and may undergo a retro-aldol cleavage of the carbon chain, leading to several α-dicarbonyl compounds, such as methylglyoxal (MGO) and glyoxal (GO) (Hofmann & Schieberle, 2002). Once generated by the thermal treatment of carbohydrates, some intermediates of the Maillard reaction accelerate further reactions. Consequently, free amino-group residues, especially lysine, arginine and cysteine (both Lys, Arg or Cys side chains and the N-terminal amino group can form adducts with α-dicarbonyl compounds) (Lo et al., 1994, Zeng and Davies, 2005) are blocked and no longer available for digestion, thus their nutritional values are considered to be severely decreased. The formation of AGEs have also been implicated in a wide and seemingly disparate range of pathologies, such as connective tissue disease, particularly in rheumatoid arthritis, and neurological conditions, such as diabetic microvascular disease, Alzheimer’s disease and end-stage renal disease (Baynes, 2001, Goldin et al., 2006, Singh et al., 2001).

The rate of the Maillard reaction in milk products depends on many factors, including temperature, duration, pH, as well as milk composition (e.g., lactose) (Guyomarc’h et al., 2000, Taheri-Kafrani et al., 2009, Van Boekel, 1998, Van Boekel, 2001). Conditions that favor the Maillard reaction may also favor cross-linking reactions involving the formation of AGEs in the milk proteins (Gerrard, 2002). Endogenous inhibitors of AGEs formation may be one of the significant factors to decrease the level of AGEs. The inhibitory effect of naturally occurring flavonoids, such as luteolin, quercetin, rutin, EGCG, phloretin and phloridzin, on the formation of AGEs are well-studied (Sang et al., 2007, Shao et al., 2008, Wu and Yen, 2005). Our previous study also suggested that genistein can inhibit AGEs formation in human serum albumin by trapping MGO (Lv, Shao, Chen, Ho, & Sang, 2011).

The aim of this study was to investigate the effect of different glycation parameters (type of sugar, temperature, pH and glycation time) on the extent of glycation reaction in different β-lg models. We also studied the inhibitory effect of genistein on β-lg glycation by determining the level of fluorescence AGEs and cross-linking protein using SDS–PAGE. In addition, LC–MS was used to analyze the mechanism by which genistein inhibits glycation of β-lg.

Section snippets

Materials

β-lg (⩾92%) was purified from whey protein by our lab (methods: water extraction, salting-out, isoelectric point precipitation and Sephadex G-50 column chromatography were used). All reducing sugars (⩾99%) were purchased from Shanghai Sangon Biological Co. Genistein (⩾99%) was purchased from Nanjing Guangrun biological Products Co., Ltd. MGO and GO were purchased from Sigma, St Louis, MO, USA. All other reagents were of analytical grade.

Preparation of β-lg glycation systems for influencing factors analysis

β-lg with different reactants (lactose, MGO and GO) was

Effect of reactant type and molar ratio on AGEs formation in β-lg glycation systems

The levels of AGEs produced by β-lg with various reactants are presented in Fig. 1A. The β-lg–MGO system produced the highest levels of AGEs i.e. more than threefold higher compared to the other glycation systems. Among the seven kinds of sugars used, ribose induced the highest level of AGEs. Lactose, fructose, and galactose were less reactive, while glucose generated the lowest level of AGEs. The activity of reactants to produce AGEs was as follows: MGO > GO > ribose > lactose > fructose > galactose > 

Discussion

In this study, we investigated the effect of reactants’ molar ratio, reactant type, temperature, time and pH on the formation of AGEs in β-lg glycation. Among these factors, temperature influenced the formation of AGEs most. This increase of AGEs levels can be attributed not only to an increase in reactivity between the carbonyl group and amino group, but also to a greater unfolding of the protein structure, thereby exhibiting a larger number of reactive functional groups (lysine residues)

Conflict of interest

All authors report no conflict of interest.

Acknowledgements

This work was supported by the NSF of Jiangsu province of China (Project BK2012850), the Program of Natural Science Research of Jiangsu Higher Education Institution of China (Project 12KJB550005), and the Natural Science Foundation of Zhejiang province of China (Project LY12C15001). Our thanks to Aaron Yerke from North Carolina Agriculture and Technical University for his suggestions on revisions and editing.

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    Xiaoming Li contributed equally with Yanghui Kong, and is the co-first authors for this paper.

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