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Unique Pattern of Protein-Bound Maillard Reaction Products in Manuka (Leptospermum scoparium) Honey Michael Hellwig, Jana Rückriemen, Daniel Sandner, and Thomas Henle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00797 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 18, 2017

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Journal of Agricultural and Food Chemistry

Unique Pattern of Protein-Bound Maillard Reaction Products in Manuka (Leptospermum scoparium) Honey

Michael Hellwig, Jana Rückriemen, Daniel Sandner, Thomas Henle

Chair of Food Chemistry, Technische Universität Dresden, D-01062 Dresden, Germany

Corresponding author: T. Henle Tel.: +49-351-463-34647 Fax: +49-351-463-34138 Email: [email protected]

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Abstract

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As a unique feature, honey from the New Zealand Manuka tree (Leptospermum scoparium)

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contains substantial amounts of dihydroxyacetone (DHA) and methylglyoxal (MGO). Though

5

MGO is a reactive intermediate in the Maillard reaction, very little is known about reactions

6

of MGO on honey proteins. We hypothesized that the abundance of MGO should result in a

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particular pattern of protein-bound Maillard reaction products (MRPs) in Manuka honey. A

8

protein-rich high-molecular weight fraction was isolated from 12 Manuka and 8 non-Manuka

9

honeys and hydrolyzed enzymatically. By HPLC-MS/MS, 8 MRPs, namely N-ε-

10

fructosyllysine, N-ε-maltulosyllysine, carboxymethyllysine, carboxyethyllysine (CEL),

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pyrraline, formyline, maltosine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1),

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were quantitated. Compared to non-Manuka honeys, the Manuka honeys were characterized

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by high concentrations of CEL and MG-H1, while the formation of N-ε-fructosyllysine was

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suppressed, indicating concurrence reactions of glucose and MGO at the ε-amino group of

15

protein-bound lysine. Up to 31% of the lysine and 8% of the arginine residues, respectively,

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in the Manuka honey protein can be modified to CEL and MG-H1, respectively. CEL and

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MG-H1 concentrations correlated strongly with the MGO concentration of the honeys.

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Manuka honey possesses a special pattern of protein-bound MRPs, which might be used to

19

prove the reliability of labelled MGO levels in honeys and possibly enable the detection of

20

fraudulent MGO or DHA addition to honey.

21 22

Keywords

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Maillard reaction; glycation; Manuka honey; methylglyoxal; methylglyoxal-derived

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hydroimidazolone 1 (MG-H1); N-ε-carboxyethyllysine (CEL); honey protein

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Introduction

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Honey shows natural antibacterial activity that mainly originates from hydrogen peroxide. In

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addition, honey can contain further substances that may account for additional “non-peroxide”

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antibacterial activity.1,2 The outstanding non-peroxide antibacterial activity of honey

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originating from the nectar of the New Zealand Manuka tree (Leptospermum scoparium)3 is

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due to the occurrence of the 1,2-dicarbonyl compound methylglyoxal 1 (MGO, Figure 1),4

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which is generated by water elimination from dihydroxyacetone 2 (DHA).5,6 The amount of

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DHA in Manuka flower nectar depends on the cultivar, geographical origin and harvest year

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and can reach up to 3 µg/mg of total sugars.6,7

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While the majority of foods contain MGO in concentrations not exceeding 70 mg/kg (for non-

36

Manuka honey maximum values were 5.7 mg/kg),4,8,9 between 25 and 1178 mg/kg were

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quantitated in Manuka honey.4,5,6,10

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MGO is an important intermediate in the Maillard reaction. This reaction starts with the

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addition of a reducing sugar to an amino or imino group. The first step of the reaction leads to

40

the formation of Amadori rearrangement products (ARPs) such as N-ε-fructosyllysine 3 or N-

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ε-maltulosyllysine 4, from the reaction of the ε-amino group of lysine with glucose and

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maltose, respectively (Figure 1).11,12,13 In the second step, ARPs are degraded to reactive 1,2-

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dicarbonyl compounds such as 3-deoxyglucosone 5 (3-DG), MGO 1, and glyoxal 6 (GO).

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Degradation of reducing sugars also occurs in the absence of amino compounds during

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caramelization, where the disaccharide-specific compounds 3-deoxypentosone 7 (3-DPs) and

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maltol 8 are generated.14,15 The β-glycosidic maltol derivative 8a has already been described

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in Manuka honey.16 In the third step of the Maillard reaction, dicarbonyl compounds react

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with amino or guanidino groups of proteins to form “advanced glycation end products”

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(AGEs). N-ε-carboxymethyllysine 9 (CML) and N-ε-carboxyethyllysine 10 (CEL),

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respectively, are formed by reaction of the ε-amino group of lysine with GO 6 and MGO 1, 3 ACS Paragon Plus Environment


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respectively.17,18 With the 1,2-dicarbonyl compounds 5 and 7, the pyrrole compounds

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pyrraline 11 and formyline 12 are formed.19,20 The iron-chelating 3-hydroxy-4(1H)pyridinone

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derivative maltosine 13 is an AGE specific for reactions of disaccharides, and it is generated

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mainly by condensation of maltol 8 and maltol precursors with the ε-amino group of

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lysine.14,21 Characteristic hydroimidazolone structures are formed through reaction with 1,2-

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dicarbonyl compounds at the guanidino group of arginine, e.g., “methylglyoxal-derived

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hydroimidazolone 1” 14 (MG-H1) with MGO.22,23 Argpyrimidine 15 has been described as a

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characteristic fluorophore resulting from the reaction of arginine with two molecules of

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MGO.24

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Due to the large amounts of reducing mono- and disaccharides, sugar degradation reactions

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are important during production and storage of honey. 5-Hydroxymethylfurfural 16 (HMF),

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formed from fructose and glucose via the intermediate 5 (Figure 1), is an important marker for

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honey quality.8,25 Beyond MGO 1, also the dicarbonyl compounds 3-DG 5 (119-1641 mg/kg),

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3-deoxygalactosone (14-46 mg/kg), diacetyl (0-4.3 mg/kg), and GO 6 (0.2-1.3 mg/kg) were

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quantitated in honey.4,8,9,26 However, very limited information is available in the literature on

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protein glycation resulting from 1,2-dicarbonyl compounds in honey. The concentrations of

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protein and free amino acids in honeys both range between 0.3 and 3 g/kg, and the ratio

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between individual free and protein-bound amino acids is between 0.5 and 3.4.27,28 Up to now,

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the impact of Maillard and sugar degradation reactions on chemical modifications of the

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honey protein has only been assessed with regard to ARPs, collectively measured after

71

conversion to furosine through acid hydrolysis of honey samples. Between 0.3 and 1.8 g

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furosine per 100 g of protein was quantitated in samples of different floral origins.29,30,31

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Besides furosine, furoyl methyl amino acids of other free amino acids such as proline and γ-

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amino butyric acid were quantitated in honey (0.1–1.4 g/100 g protein), indicating that

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reactions at the N-termini of free amino acids, peptides and proteins do also play a significant 4 ACS Paragon Plus Environment

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role in honey.30 Correspondingly, the concentrations of free amino acids in honey generally

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decrease during storage.32 Due to its high reactivity, MGO can also react with free amino

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acids of Manuka honey. The concentration of the aroma compound 2-acetyl-1-pyrroline 17 as

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a reaction product of proline and MGO increases when the concentration of MGO in honey

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exceeds 250 mg/kg.33 Thus, we hypothesized that also proteins should be modified by MGO

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in a concentration-dependent manner. Knowledge about specific reactions of methylglyoxal

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could prove useful for the characterization of Manuka honey, and possibly might allow

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indications on fraudulent MGO or DHA addition. Therefore, protein-rich high-molecular

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weight fractions from 12 Manuka honeys and 8 non-Manuka honeys were isolated by dialysis

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and precipitation, and the concentrations of two ARPs (N-ε-fructosyllysine 3 and N-ε-

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maltulosyllysine 4, Figure 1) and 6 advanced glycation end products (CML 9, CEL 10,

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pyrraline 11, formyline 12, maltosine 13, MG-H1 14, Figure 1) were analyzed in honeys for

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the first time.

90 91 92

Materials and Methods

93 94

Chemicals. The following substances were from commercial suppliers: nonafluoropentanoic

95

acid (NFPA), leucine aminopeptidase (18 U/mg protein), prolidase (208 U/mg protein), γ-

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globulin (Sigma-Aldrich, Steinheim, Germany); trichloroacetic acid (Roth, Karlsruhe,

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Germany); heptafluorobutyric acid (HFBA) (Alfa Aesar, Karlsruhe, Germany); phosphoric

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acid, ammonium formate (Grüssing, Filsum, Germany); pepsin (10 FIP-U/mg protein),

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pronase E (4000 PU/mg protein) (Merck, Darmstadt, Germany); hydrochloric acid, methanol

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gradient grade, acetonitrile (VWR, Darmstadt, Germany); Coomassie brilliant blue G-250

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(Fluka, Buchs, Switzerland). All further chemicals were of the highest purity available. The 5 ACS Paragon Plus Environment


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water used for the preparation of buffers and solutions was obtained by a Purelab Plus

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purification system (USF Elga, Ransbach-Baumbach, Germany). Double-distilled water was

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used for HPLC-MS/MS solvents. Standards of N-ε-fructosyllysine 3,34 N-ε-maltulosyllysine

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4,13 CML 9,35 CEL 10,35 pyrraline 11,36 formyline 12,20 maltosine 13,21,37 MG-H1 14,35 and

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argpyrimidine 1535 were prepared in our laboratory and purified via semi-preparative ion

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exchange chromatography according to the literature stated. Evaluation of identity and purity

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of these compounds by nuclear magnetic resonance spectroscopy, mass spectrometry,

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elemental analysis, and amino acid analysis revealed that they met the characteristics reported

110

in the literature.

111 112

Honey samples. Twelve commercially available Manuka honeys and 8 non-Manuka honeys

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were included in the study. Four non-Manuka honeys were of monofloral origin (European

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chestnut, castanea sativa; lime, tilia europea; ulmo, eucryphia cordifolia; heather, calluna

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vulgaris) and four were of multifloral origin. After purchase, all honeys were stored at 4 °C

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until analysis. A subset of these honeys was utilized in a preparatory study in order to

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evaluate the efficiency of protein isolation.

118 119

Protein determination. In a variation of the Bradford method,27 the respective reagent was

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prepared by dissolving 20.1 mg Coomassie brilliant blue G-250 in a mixture of ethanol (10

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mL) and phosphoric acid (85%, w/v, 20 mL). The solution was transferred to a volumetric

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flask (200 mL) and filled up with water. For protein determination, 800 µL of an aqueous

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honey solution (1%, w/v) was mixed with 200 µL of the Bradford reagent. After 10 min, the

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absorbance of the solution was read at 595 nm using a photometer (Ultrospec 1000;

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Pharmacia Biotech, Uppsala, Sweden). Calibration was performed using γ-globulin as a

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standard at concentrations between 4 and 28 mg/L.


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Preparation of honey samples for MRP analysis. High molecular-weight (HMW) fractions

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were isolated from honey either by dialysis or by precipitation with trichloroacetic acid

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(TCA). For the former approach, ca. 3 g of honey was dissolved in 10 mL of water and

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transferred to a dialysis tube (MWCO, 14 kDa, Sigma). The samples were dialyzed against

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distilled water for 2 days with the water changed twice per day. The retentates were then

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lyophilized and stored at -18 °C. For TCA precipitation, 10 mL of TCA (20%, w/v) was

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added to 10 g of honey, and the suspension was stored at -20 °C for 30 min after mixing.

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Following centrifugation (4 °C, 5000 rpm, 10 min), the supernatant was decanted and

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discarded, and the precipitate was suspended in water (10 mL). After centrifugation (4 °C,

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5000 rpm, 10 min), the supernatant was discarded, and the precipitate was washed once more

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in the same way. Finally, the precipitate was suspended in water (5 mL), lyophilized, and

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stored at -18 °C.

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Acid hydrolysis of protein isolates was performed by adding 2 mL of 6 M HCl to 2-3 mg of

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the fractions and heating of the mixture at 110 °C for 23 h in a pre-heated oven. Then, 500 µL

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of the hydrolyzate was evaporated to dryness in a vacuum concentrator (SPD Speed Vac;

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Thermo Fisher Scientific, Karlsruhe, Germany). The residue was reconstituted in 1 mL of

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sample buffer for amino acid analysis (0.12 N lithium citrate, pH 2.2). For enzymatic

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hydrolysis, 1.05 mL of 0.02 M hydrochloric acid containing 1 FIP-U of pepsin was added to

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3-4 mg of HMW fractions.13,21 After incubation (37 °C, 24 h), 300 µL of 2 M TRIS buffer,

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pH 8.2, containing 400 PU of pronase E was added. After further incubation (37 °C, 24 h), 0.4

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U of leucine aminopeptidase and 1 U of prolidase were added. After incubation (37 °C, 24 h),

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the hydrolyzate was subjected to HPLC-MS/MS. For amino acid analysis, 100 µL of the

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hydrolyzate was diluted with 300 µL of 0.12 M lithium citrate buffer, pH 2.2.

151 152

High-Pressure Liquid Chromatography with UV-detection (HPLC-UV). All analyses

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were performed using a low-pressure gradient system consisting of a solvent organizer (K7 ACS Paragon Plus Environment


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1500; Knauer, Berlin, Germany), an autosampler (Basic Marathon; Spark Holland, Emmen,

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The Netherlands) a pump (Smartline 1000, Knauer), an online degasser (Knauer), a column

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oven, and a diode array detector (DAD 2.1L, Knauer). Chromatograms were evaluated using

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the software ClarityChrom version 6.1.0.130. HMF 16,38 argpyrimidine 15,35 and MGO

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14,8,9,33 were analyzed by RP-HPLC-UV according to the respective literature methods.

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Argpyrimidine was quantitated using excitation and emission wavelengths of 320 nm and 380

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nm, respectively, on the fluorescence detector RF-10AXL (Shimadzu, Duisburg, Germany),

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which was connected in series to the DAD.

162 163

High-Pressure Liquid Chromatography with tandem mass-spectrometric detection

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(HPLC-MS/MS). Protein-bound MRPs were quantitated on an HPLC-MS/MS system

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consisting of a binary pump (G1312A), an online degasser (G1379B), an autosampler

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(G1329A), a column thermostat (G1316A), a diode array detector (G1315D), and a triple-

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quadrupol mass spectrometer (G6410A; all from Agilent Technologies, Böblingen,

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Germany). At the ESI source, nitrogen was utilized as the nebulizing gas (gas flow, 11 L/min;

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gas temperature, 350 °C; nebulizer pressure, 35 psi), and the capillary voltage was at 4000 V.

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For chromatographic separation, the column Zorbax 100 SB-C18 (2.1 × 50 mm, 3.5 µm;

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Agilent) was used at a column temperature of 35 °C. HPLC solvent A was a solution of 10

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mM nonafluoropentanoic acid (NFPA) in water, and solvent B was a solution of 10 mM

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NFPA in acetonitrile. The solvents were pumped at a flow rate of 0.25 mL/min in the gradient

174

mode (0 min, 5% B; 15 min, 32% B; 16 min, 85% B; 20 min, 85% B; 21 min, 5% B; 27 min,

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5% B). The injection volume was 5 µL. Data were acquired and evaluated with the software

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Mass Hunter B.02.00 (Agilent).

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The standard addition method was used for quantitation of MRPs. All samples were analyzed

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at least in duplicate. In the sample without addition, 100 µL of enzymatic hydrolyzate was

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mixed with 20 µL of water. In the second run, 100 µL of hydrolyzate was mixed with 10 µL 8 ACS Paragon Plus Environment

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of water and 10 µL of a standard solution. In the last run, 100 µL of hydrolyzate was mixed

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with 20 µL of standard solution. All samples were centrifuged before injection (10,000 rpm,

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10 min). In the standard solution added to Manuka honeys, N-ε-fructosyllysine (87.5 µg/mL),

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N-ε-maltulosyllysine (35.2 µg/mL), CML (13.4 µg/mL), CEL (19.9 µg/mL), formyline (0.4

184

µg/mL), MG-H1 (5.0 µg/mL), maltosine (0.11 µg/mL), and pyrraline (2.0 µg/mL) were

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dissolved in water. In the standard solution added to non-Manuka honeys, the same

186

concentrations were used, except for CEL (1.0 µg/mL), and MG-H1 (0.25 µg/mL).

187 188

Amino acid analysis. Proteinogenic amino acids were quantitated with an amino acid

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analyser (S 433; Sykam, Fürstenfeldbruck, Germany) using a PEEK column filled with the

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cation exchange resin LCA K07/Li (150 × 4.6 mm, 7 µm). The respective lithium buffers

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were also obtained from Sykam and employed for custom gradient programs utilized

192

previously.35 The effluent was derivatized with ninhydrin, and detection of products was

193

performed with an integrated two-channel photometer (λ = 440 nm, 570 nm). External

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calibration was performed with an amino acid mixture (Sigma-Aldrich, Steinheim, Germany).

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The injection volume was between 50 and 100 µL. Leucine was taken as an internal reference

196

for the evaluation of amino acid concentrations due to its abundance in the honey protein.28

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Amino acid analysis was also used to calculate the protein content of HMW isolates and the

198

efficiency of enzymatic hydrolysis. The release of amino acids during enzymatic hydrolysis

199

was calculated by regarding the release during acid hydrolysis as 100%.

200 201

Statistical treatment. The significance of differences between the medians of the groups of

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Manuka and non-Manuka honeys was determined with the two-tailed Mann-Whitney U test

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using the software PASW statistics 18. In this test, MRP and amino acid concentrations

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between LOD and LOQ were considered with the concentration of the LOQ, while MRP and

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amino acid concentrations below the LOD were considered with the concentration of the 9 ACS Paragon Plus Environment


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LOD. Correlations between MGO concentrations and the percentage release of amino acids

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during enzymatic hydrolysis were determined by Spearman’s rank correlation analysis.

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Differences and correlations were considered significant at P < 0.05 (*), and strongly

209

significant at P < 0.01 (**), otherwise not significant (n.s.).

210 211 212

Results

213 214

Isolation of a protein-rich high molecular-weight fraction from honeys. Despite the

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extreme ratio between reducing sugars and reactive 1,2-dicarbonyl compounds on the one

216

hand to protein on the other, the extent of formation of AGEs in honey protein has not yet

217

been assessed. Based on the hypothesis that the high amount of methylglyoxal in Manuka

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honey leads to a characteristic pattern of protein-bound Maillard reaction products, a survey

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of protein-bound MRPs in honey was intended. Twelve Manuka honeys and 8 non-Manuka

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honeys were included in the study. All honeys had similar concentrations of 5-

221

hydroxymethylfurfural (HMF), but the MGO concentrations of all Manuka honeys were

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significantly higher than those of all non-Manuka honeys (Figure 2).

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Dialysis with tubings of an MWCO of 14 kDa was chosen as the method for the isolation of

224

the protein fraction of honey. In a preparatory study with a subset of 8 honeys (5 Manuka and

225

3 non-Manuka), the efficiency of dialysis for protein isolation was assessed by comparing the

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protein concentrations in honeys with those in the retentates. As measured by the Bradford

227

method, the honeys had a mean protein concentration of 0.12%, which matches literature

228

values.27,28 The retentate yield was not dependent on the MGO concentration of the honey.

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From the amount of honey subjected to dialysis, 0.24% was obtained as the retentate. The

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protein content of the retentates was calculated as the sum of amino acids measured by amino 10 ACS Paragon Plus Environment

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acid analysis after acid hydrolysis. A mean protein content of the retentates of 26.8%

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indicates that the retentates represent at least 50% of the protein-bound amino acids in honey.

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The “missing” protein fraction must have been removed during dialysis. The pattern of amino

234

acids after acid hydrolysis of the 12 Manuka and the 8 non-Manuka honeys was very similar

235

and matched the general pattern described in the literature28 with aspartic and glutamic acids,

236

leucine, and valine as the dominating acids. However, one strong difference was discerned in

237

the concentration of lysine: The mean content of lysine in acid hydrolyzates of Manuka

238

honeys was 25% smaller than that in non-Manuka honeys and negatively correlated with the

239

MGO concentration. The arginine concentration was not influenced in this way (data not

240

shown).

241

The analysis of peptide-bound MRPs such as pyrraline, formyline and MG-H1 requires

242

enzymatic hydrolysis.39 The efficiency of the enzymatic hydrolysis procedure was assessed by

243

comparing the concentrations of amino acids in enzymatic hydrolyzates with those in acid

244

hydrolyzates of the same sample. The mean release of serine, alanine, valine, isoleucine,

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leucine, and phenylalanine was slightly lower for Manuka (77-93%) than for non-Manuka

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honeys (88-97%), with the release of individual amino acids being reduced by 3-12%. These

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values are consistent with release patterns determined earlier for different food items.13,21

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Roughly, the increase of MGO by 100 mg/kg led to a decrease in the release of the

249

aforementioned amino acids of 0.5-1.2%, but the respective correlations between MGO and

250

the release of these amino acids during enzymatic hydrolysis were not significant (data not

251

shown).

252

For the expression of MRP concentrations, leucine was taken as an internal reference, which

253

enables better comparison of Manuka and non-Manuka honeys by circumventing the

254

drawback of reduced amino acid release. Moreover, as not the whole protein is retained

255

during dialysis, only a fraction of the whole Manuka honey protein is considered. All amino

256

acid concentrations are therefore given as leucine equivalents. Leucine is not modified in the 11 ACS Paragon Plus Environment


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Maillard reaction, and it is very abundant in the honey protein.28 There are only small

258

variations in the leucine concentrations of all honey proteins in the HMW fractions (molar

259

concentrations generally 9.6 ± 0.3 mol-% of whole amino acids). Other abundant amino acids

260

such as asparagine and glutamine are converted to aspartic and glutamic acids during acid

261

hydrolysis. Thus, leucine has the advantage to be adoptable for comparisons with amino acid

262

concentrations after acid hydrolysis. From our data, it can be estimated that 1 mmol of leucine

263

in the HMW fraction is equivalent to ca. 10 g of HMW fraction (corresponding to ca. 2.7 g of

264

HMW protein) or 4 kg of honey.

265 266

Determination of protein-bound Maillard reaction products in a protein-rich HMW

267

fraction isolated from honey. Individual MRPs of beer proteins have been analyzed recently

268

by an HPLC-MS/MS method and quantitated using the standard addition method.13 This

269

method, especially the use of quantifier and qualifier ions, was optimized for honey analysis

270

by measurement of enzymatic hydrolyzates of Manuka and non-Manuka honeys. CML and

271

CEL were included in the study, and LODs and LOQs were determined by evaluation of

272

signal-to-noise ratios at decreasing concentrations of MRPs added to the enzyme blank (Table

273

2). The respective concentrations are in good agreement with earlier published data for MRPs

274

in different foods.13,21 Standard addition had to be applied, because the MS signal was

275

influenced by the honey matrix. In the concentration range covered during standard addition,

276

a linear response of the signal was always given (Table 2). This is a prerequisite for the

277

feasibility of quantitation by this method. After the first measurements of HMW hydrolyzates

278

from different honeys, it was possible to discern strong differences in the abundance of CEL

279

and MG-H1 in Manuka and non-Manuka honeys. Both compounds only play a minor role in

280

non-Manuka honeys—the CEL concentration was always below the LOQ, and only for one

281

non-Manuka honey could a concentration above the LOQ be determined (0.7 µmol/mmol

282

Leu). The concentrations increased with increasing MGO content in Manuka honeys up to 12 ACS Paragon Plus Environment

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values of 102.8 µmol/mmol Leu for CEL and 24.2 µmol/mmol Leu for MGH1 (Figure 3). At

284

a slightly higher retention time than MG-H1, a second peak with similar fragmentation

285

behavior emerged in compliance with the MGO concentrations. It is possible that this peak

286

corresponds to MG-H1 isomers such as MG-H2 or MG-H3.40 Similar correlations were not

287

obvious from the chromatograms of other MRPs such as CML (Figure 3). The concentrations

288

of the standards in the solution added for quantitation were adjusted to the pattern of MRPs in

289

the enzymatic hydrolyzates of HMW fractions from honey, and different solutions were

290

employed for Manuka and non-Manuka honeys due to the differences in the concentrations of

291

CEL and MG-H1. Further chromatograms of MRPs showing standard addition are available

292

in the supporting information (Figures S1-S5).

293

The ARP N-ε-fructosyllysine was the most abundant MRP in the honey protein ranging

294

between 10 and 134 µmol/mmol leucine (Figure 4). A lysine blockage by N-ε-fructosyllysine

295

of 3-21% in Manuka honeys and 10-32% in non-Manuka honeys can be estimated under

296

consideration of lysine and the modified lysine species after enzymatic hydrolysis. This is

297

similar to the range of lysine blockage in heated milk products and crumbs of bakery

298

products.41,42 The ARP N-ε-maltulosyllysine was included in the study, because maltose was

299

described as the most important sugar in the fraction of di- and oligosaccharides in Manuka

300

honey.43 However, its concentrations were much lower than those of N-ε-fructosyllysine and

301

generally below the LOQ. ARPs have already been analyzed in honey after conversion to

302

furosine by acid hydrolysis of whole honeys. Furosine was determined between 5.9 and 11.3

303

g/kg protein after hydrolysis with 7.95 M HCl with protein being calculated through nitrogen

304

determination of whole honeys (N × 6.25).29,30 Taking into account that only 46.1% of N-ε-

305

fructosyllysine is converted to furosine under these conditions,34 this corresponds to a N-ε-

306

fructosyllysine concentration of 15.8-30.2 g/kg protein which is in good agreement with our

307

data (1.1-15.3 g/kg protein) regarding the considerable differences in the analytical workflow.



Page 14 of 39

308

CML and pyrraline were further important AGEs in the honey protein whose concentrations

309

tended to be higher in Manuka honeys, but a clear distinction between Manuka and non-

310

Manuka honeys was not possible (Figure 4). No difference was determined in the formyline

311

concentrations of the honey classes. As hypothesized, MG-H1 and CEL, the two AGEs

312

resulting from the reaction of MGO with amino-acid side chains, were nearly exclusively

313

formed in Manuka honeys (Figure 4). The range of the differences is more pronounced than

314

that found in our previous study for 2-acetyl-1-pyrroline.33 In the majority of Manuka honeys,

315

CEL turned out to be the most important MRP, with concentration even higher as compared

316

to N-ε-fructosyllysine. In Manuka honeys, up to 31% of protein-bound lysine residues can be

317

modified to CEL, and up to 8% of protein-bound arginine residues may have reacted to MG-

318

H1. The concentrations of MG-H1 and CEL in the HMW fractions are equivalent to ca. 1% of

319

total MGO having reacted with the protein. CEL and MG-H1 contents correlated very well

320

with the MGO concentration in Manuka honeys (Figure 5). Moreover, in the 11 Manuka

321

honeys, where both compounds could be quantitated, the ratio of CEL to MG-H1 was

322

constantly at 5.1 ± 0.9. The amount of CEL was always higher than that of CML. In other

323

food products, MG-H1 normally predominates over CEL by 10- to 20-fold, and CML and

324

CEL concentrations are in the same order of magnitude.39,44 Thus, not only the high

325

abundance of CEL in the protein, but also the extreme excess of this MRP over MG-H1 is a

326

special feature of Manuka honeys.

327

Glycation by MGO is also reported to lead to the formation of the fluorescent AGE

328

argpyrimidine,24 which could possibly explain at least a part of the specific fluorescence of

329

Manuka honey.45 Due to the known difficulties in the quantitation of argpyrimidine in the

330

matrix from enzymatic hydrolysis, HPLC with fluorescence detection of the hydrolyzates was

331

performed. Argpyrimidine was not detectable even in honeys with high MGO and MG-H1

332

concentrations (LOD, 7.8 µmol/mmol leucine). In a Manuka honey with 100 mg/kg MGO,

333

there is a ca. 10-fold excess of MGO over HMW-protein bound arginine or lysine residues. 14 ACS Paragon Plus Environment

Page 15 of 39


334

On the contrary, maltosine was detectable in all Manuka honeys, whereas it was not

335

detectable in 5 out of 8 non-Manuka honeys. Higher maltosine concentrations were

336

accompanied by higher MGO concentrations. This might be caused by reaction of two

337

molecules of MGO and/or DHA at lysine residues. Similar reactions leading to the formation

338

of heterocyclic derivatives such as pyrroles have been described.46 The formation of traces of

339

maltol 8 (Figure 1) in model reactions of DHA has also been demonstrated.47 Moreover, as

340

maltosine is also formed from isomaltol and substituted isomaltol derivatives,14,21 formation

341

from the Manuka honey constituent β-glucosyl maltol 8a (Figure 1) can be expected. The

342

latter compound has not yet been quantitated.16

343 344

Assessment of an alternative protein preparation method. The advantage of dialysis is the

345

defined molecular weight cut-off, which allows clear separation of a high- and a low-

346

molecular weight fraction. Any interference from possible glycation-induced changes in the

347

solubility of honey proteins in protein precipitation reagents such as ethanol28 or TCA could

348

thereby be avoided. The disadvantage is the long time needed for work-up of the samples.

349

Therefore, preliminary experiments were performed with regard to the use of TCA for protein

350

isolation. The yield of TCA isolates was lower; however, the protein content of the

351

preparations was higher (41-48%). HPLC-MS/MS and amino acid analysis revealed AGE

352

concentrations in the same range as those obtained in the dialysis retentates (60-120%).

353

However, in the TCA isolates, a mean fourfold increase in the apparent concentration of N-ε-

354

fructosyllysine and a mean twofold increase in the apparent concentration of N-ε-

355

maltulosyllysine was observed. The apparent lysine concentration was reduced concomitantly.

356

We conclude that the acidic conditions during sample-workup might invoke a rearrangement

357

of intermediate Schiff bases of protein-bound lysine and glucose or maltose. The Amadori

358

rearrangement is known to be acid-catalyzed,48 and Schiff bases should be present in honey

359

due to the strong excess of reducing sugars. This could also explain the slightly lower 15 ACS Paragon Plus Environment


Page 16 of 39

360

amounts of protein-bound ARPs found in our study by direct measurement of enzymatic

361

retentate hydrolyzates as compared to acid treatment and subsequent analysis.29,30 Thus, for

362

future experiments, dialysis and the examination of the retentate protein probably gives the

363

best information about the extent of the Maillard reaction on proteins in honey. When only

364

AGEs, especially MG-H1 and CEL, are required, it should be possible to benefit from the

365

easier sample work-up by TCA precipitation.

366 367

Discussion

368 369

The pattern of AGEs in Manuka honey protein is clearly different from that of non-Manuka

370

honey protein but also from that of other food items because CEL predominates over CML

371

and MG-H1. Further work is needed to elucidate the mechanisms by which lysine and

372

arginine are modified during reaction with MGO and DHA and the kinetics of formation of

373

both compounds. While the role of MGO in the Maillard reaction in foods and in

374

physiological systems has been studied and reviewed thoroughly,49,50 a particular reactivity of

375

DHA besides its dehydration to MGO has only scarcely been addressed.46,47 Knowledge about

376

the formation of CEL and MG-H1 during maturation and their stability during storage of

377

Manuka honey would also allow conclusions on whether these protein-bound structures

378

represent suitable indicators for the originality of Manuka honey, potentially allowing to

379

prove fraudulent MGO or DHA addition. We propose that the reactivity of DHA and MGO

380

changes during maturation of honey, possibly when the reaction conditions turn from the

381

aqueous nectar system to the conditions in honey with a water activity between 0.5-0.65.51

382

MGO and/or DHA impose a special glycation impact on proteins in Manuka honey: With

383

ascending MGO concentrations, not only the CEL and MG-H1 concentrations rise, but the

384

ARP concentrations decrease. The content of N-ε-fructosyllysine is significantly lower in 16 ACS Paragon Plus Environment

Page 17 of 39


385

Manuka than in non-Manuka honeys. While the concentrations of N-ε-maltulosyllysine were

386

between the LOD and LOQ in all non-Manuka honeys, the compound was not detected in

387

50% of the Manuka honeys. Roughly, an increase in the MGO concentration of 100 mg/kg

388

leads to an increase in the CEL concentration by 8 µmol/mmol leucine, whereas the

389

concentration of N-ε-fructosyllysine decreases by 6 µmol/mmol leucine (Figure 6). At an

390

MGO concentration of ca. 415 mg/kg, the lysine modification caused by N-ε-fructosyllysine

391

equals that caused by CEL as can be seen from the intercept of the respective regression lines

392

in Figure 6. Regarding the 1000-fold excess of glucose (ca. 400 g/kg) over MGO at that point,

393

the reactivity of MGO at lysine residues can be estimated to ca. 1000-fold that of glucose

394

under the reaction conditions in Manuka honey. An increase in the concentration of MGO

395

leads to a decrease in the concentration of lysine in the honey protein (Figure 6). However,

396

regarding that the summarized concentrations of N-ε-fructosyllysine and CEL are quite

397

constant at increasing MGO concentrations while the lysine contents keep decreasing, we

398

conclude that a significant part of lysine derivatization in Manuka honeys remains

399

unexplained. Interestingly, the retentates of Manuka honeys tended to be darker than those of

400

non-Manuka honeys. A correlation between the concentration of 1,2-dicarbonyl compounds

401

in and the color of honeys has been described.26 Our results indicate that MGO might promote

402

the formation of honey melanoidins and that part of the special color of Manuka honey is due

403

to protein-bound structures. At this point, it would be interesting to gain further insight into

404

the molecular structures of the HMW fractions and possible particularities of Manuka honey.

405

The highest concentrations of the carboxyalkylated amino acids CML and CEL have been

406

determined in cereal products such as bread and biscuits up to now.44 The lysine blockage by

407

both compounds in these food items can be estimated to about 3-5% with CML

408

predominating. Largely due to the high CEL concentrations of up to 102.8 µmol/mol leucine,

409

which is equivalent to a lysine derivatization of ca. 31%, the protein of Manuka honey is the

410

most strongly carboxyalkylated protein as yet found in food. N-ε-carboxyalkylation of lysine 17 ACS Paragon Plus Environment


Page 18 of 39

411

involves an increase in negatively charged residues at the expense of positively charged

412

residues with an overall drop in the isoelectric point of the protein. As protein acidification

413

affects the properties of the proteins, e.g., in terms of solubility and digestibility, it would be

414

interesting to study the effect of such proteins on gastrointestinal physiology but also on

415

microbial homeostasis and metabolism. Carboxyalkylated proteins or acidic peptides

416

hydrolyzed from them could be novel carriers of antimicrobial activity.

417

One of the concerns linked to the Maillard reaction is the reduction in the nutritional value of

418

proteins due to lysine blockage.41 Therefore, the amounts of MRPs ingested with honeys were

419

calculated (Table 3). With a 10-g serving of honey, which contains ca. 12 mg of protein,

420

comparable amounts of CML, formyline, and pyrraline are ingested. However, these amounts

421

are not higher than 3 µg, which is far lower than the mean daily intakes estimated for

422

pyrraline (20-40 mg), and formyline (2-3 mg).52,53 Differences in the MRP concentrations

423

between Manuka and non-Manuka honeys (Figure 4) are also reflected in the ingested

424

amounts per serving with more CEL and MG-H1 and less N-ε-fructosyllysine being taken up

425

with Manuka honeys. The abundance of N-ε-fructosyllysine, the most important MRP

426

measured in this study, is also far from reaching the daily intake of this Amadori product,

427

which was estimated to an amount up to 1200 mg.52 CEL and MG-H1 are predominantly

428

found in cereal foods such as bread, biscuits, and cooked pasta.39,44 An inclusion in the diet of

429

300 g of bread, 100 g of biscuits, and 300 g of cooked pasta would imply the intake of 1.5-5

430

mg of CEL and 15-50 mg of MG-H1. The amounts ingested with honey would then be

431

equivalent to max. 5% of the daily CEL intake and 0.1% of the daily MG-H1 intake. As

432

estimated above, ca. 50% of the honey protein was retained by dialysis in this study, implying

433

that the total MRP intake would rise to approximately twice the amounts given in Table 3.

434

Taken together, honey-derived protein-bound MRPs only contribute to a very low extent to

435

the daily intake of MRPs.


Page 19 of 39


437 438

Abbreviations used

439

3-DG, 3-deoxyglucosone; AGE, advanced glycation end product; ARP, Amadori

440

rearrangement product; CEL, N-ε-carboxyethyllysine; CML, N-ε-carboxymethyllysine; DHA,

441

dihydroxyacetone;

442

hydroxymethylfurfural; HMW, high molecular-weight; LOD, limit of detection; LOQ, limit

443

of quantitation; MG-H1, methylglyoxal-derived hydroimidazolone 1; MGO, methylglyoxal;

444

MRM, multiple reaction monitoring; MRP, Maillard reaction product; MWCO, molecular-

445

weight cut-off; NFPA, nonafluoropentanoic acid; TCA, trichloroacetic acid

GO,

glyoxal;

HFBA,

heptafluorobutyric

acid;

HMF,

5-

446 447

Acknowledgements

448

The authors wish to thank Karla Schlosser, Institute of Food Chemistry, TU Dresden, for

449

performing the amino acid analyses.

450 451

Supporting Information Description

452

Supporting information available: HPLC-MS/MS chromatograms (MRM mode) of

453

enzymatically hydrolyzed HMW fractions of Manuka honeys without and with addition of

454

fructosyllysine (Figure S1), maltulosyllysine (Figure S2), pyrraline (Figure S3), formyline

455

(Figure S4), and maltosine (Figure S5). This material is available free of charge via the

456

Internet at http://pubs.acs.org.

457 458

Funding

459

Financial support to J.R. from Manuka Health Ltd., New Zealand, is gratefully acknowledged.

460

This support did not prevent the authors from publishing both positive and negative results.

461

Publishing this research was possible without the prior approval of the sponsor. 19 ACS Paragon Plus Environment


Page 20 of 39

462 463

Notes

464

The authors declare no competing financial interest.


Page 21 of 39


465 References [1] White, J.W.; Subers, M.H.; Schepartz, A.I. The identification of inhibine, the antibacterial factor in honey, as hydrogen peroxide and its origin in a honey glucose-oxidase system. Biochim. Biophys. Acta 1963, 73, 57–70. [2] Weston, R.J. The contribution of catalase and other natural products to the antibacterial activity of honey: a review. Food Chem. 2000, 71, 235–239. [3] Allen, K.L.; Molan, P.C.; Reid, G.M. A survey of the antibacterial activity of some New Zealand honeys. J. Pharm. Pharmacol. 1991, 43, 817–822. [4] Mavric, E.; Wittmann, S.; Barth, G.; Henle, T. Identification and quantification of methylglyoxal as the dominant antibacterial constituent of Manuka (Leptospermum scoparium) honeys from New Zealand. Mol. Nutr. Food Res. 2008, 52, 483–489. [5] Atrott, J.; Haberlau, S.; Henle, T. Studies on the formation of methylglyoxal from dihydroxyacetone in Manuka (Leptospermum scoparium) honey. Carbohydr. Res. 2012, 361, 7–11. [6] Adams, C.J.; Manley-Harris, M.; Molan, P.C. The origin of methylglyoxal in New Zealand manuka (Leptospermum scoparium) honey. Carbohydr. Res. 2009, 344, 1050–1053. [7] Williams, S.; King, J.; Revell, M.; Manley-Harris, M.; Balks, M, Janusch, F.; Kiefer, M.; Clearwater, M.; Brooks, P.; Dawson, M. Regional, annual, and individual variations in the dihydroxacetone content of the nectar of Manuka (Leptospermum scoparium) in New Zealand. J. Agric. Food Chem. 2014, 62, 10332–10340. [8] Weigel, K.U.; Opitz, T.; Henle, T. Studies on the occurrence and formation of 1,2dicarbonyls in honey. Eur. Food Res. Technol. 2004, 218, 147–151. [9] Degen, J.; Hellwig, M.; Henle, T. 1,2-Dicarbonyl compounds in commonly consumed foods. J. Agric. Food Chem. 2012, 60, 7071–7079. 21 ACS Paragon Plus Environment


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[10] Oelschlaegel, S.; Gruner, M.; Wang, P.-N.; Boettcher, A.; Koelling-Speer, I.; Speer, K. Classification and characterization of Manuka honeys based on phenolic compounds and methylglyoxal. J. Agric. Food Chem. 2012, 60, 7229–7237. [11] Ledl, F.; Schleicher, E. New aspects of the Maillard reaction in foods and in the human body. Angew. Chem. Int. Ed. Engl. 1990, 29, 597–626. [12] Hellwig, M.; Henle, T. Baking, ageing, diabetes: a short history of the Maillard reaction. Angew. Chem. Int. Ed. Engl. 2014, 53, 10316–10329. [13] Hellwig, M.; Witte, S.; Henle, T. Free and protein-bound Maillard reaction products in beer: method development and a survey of different beer types. J. Agric. Food Chem. 2016, 64, 7234–7243. [14] Ledl, F.; Osiander, H.; Pachmayr, O.; Severin, T. Formation of maltosine, a product of the Maillard reaction with a pyridone structure. Z. Lebensm.-Unters. Forsch. 1989, 188, 207– 211. [15] Hollnagel, A.; Kroh, L.W. 3-Deoxypentosulose: An α-dicarbonyl compound predominating in nonenzymatic browning of oligosaccharides in aqueous solution. J. Agric. Food Chem. 2002, 50, 1659–1664. [16] Adams, C.J.; Grainger, M.N.C.; Manley-Harris, M. Isolation of maltol glucoside from the floral nectar of New Zealand Manuka (Leptospermum scoparium). Food Chem. 2015, 174, 306-309. [17] Ahmed, M.U.; Thorpe, S.R.; Baynes, J.W. Identification of Nε-carboxymethyllysine as a degradation product of fructoselysine in glycated protein. J. Biol. Chem. 1986, 261, 4889– 4894. [18] Ahmed, M.U.; Brinkmann Frye, E.; Degenhardt, T.P.; Thorpe, S.R.; Baynes, J.W. Nε(carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem. J. 1997, 324, 565–570. 22 ACS Paragon Plus Environment

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[19] Nakayama, T.; Hayase, F.; Kato, H. Formation of ε-(2-formyl-5-hydroxymethyl-pyrrol1-yl)-L-norleucine in the Maillard reaction between D-glucose and L-lysine. Agric. Biol. Chem. 1980, 44, 1201–1202. [20] Hellwig, M.; Henle, T. Formyline, a new glycation compound from the reaction of lysine and 3-deoxypentosone. Eur. Food Res. Technol. 2010, 230, 903–914. [21] Hellwig, M.; Kiessling, M.; Rother, S.; Henle, T. Quantification of the glycation compound 6-(3-hydroxy-4-oxo-2-methyl-4(1H)-pyridin-1-yl)-L-norleucine (maltosine) in model systems and food samples. Eur. Food Res. Technol. 2016, 242, 547–557. [22] Henle, T.; Walter, A.W.; Haeßner, R.; Klostermeyer, H. Detection and identification of a protein-bound imidazolone resulting from the reaction of arginine residues and methylglyoxal. Z. Lebensm.-Unters. Forsch. 1994, 199, 55–58. [23] Lo, T.W.C.; Westwood, M.E.; McLellan, A.C.; Selwood, T.; Thornalley, P.J. Binding and modification of proteins by methylglyoxal under physiological conditions. J. Biol. Chem. 1994, 269, 32299–32305. [24] Shipanova, I.N.; Glomb, M.A.; Nagaraj, R.H. Protein modification by methylglyoxal: Chemical nature and synthetic mechanism of a major fluorescent adduct. Arch. Biochem. Biophys. 1997, 344, 29–36. [25] Codex Alimentarius Commission. Revised Codex Standard for Honey; Codex Alimentarius Commission: Geneva, Switzerland, 1981. [26] Marceau, E.; Yaylayan, V.A. Profiling of alpha-dicarbonyl content of commercial honeys from different botanical origins: Identification of 3,4-dideoxyglucosone-3-ene (3,4DGE) and related compounds. J. Agric. Food Chem. 2009, 57, 10837–10844. [27] Bogdanov, S. Bestimmung von Honigprotein mit Coomassie Brilliantblau G 250. Mitt. Gebiete Lebensm. Hyg. 1981, 72, 411–417. [in German]



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[28] Bosi, G.; Battaglini, M. Gas chromatographic analysis of free and protein amino acids in some unifloral honeys. J. Apicult. Res. 1978, 17, 152–166. [29] Villamiel, M.; del Castillo, M.D.; Corzo, N.; Olano, A. Presence of furosine in honeys. J. Sci. Food Agric. 2001, 81, 790–793. [30] Sanz, M.L.; del Castillo, M.D.; Corzo, N.; Olano, A. 2-Furoylmethyl amino acids and hydroxymethylfurfural as indicators of honey quality. J. Agric. Food Chem. 2003, 51, 4278– 4283. [31] Morales, V.; Sanz, M.L.; Martín-Álvarez, P.J.; Corzo, N. Combined use of HMF and furosine to assess fresh honey quality. J. Sci. Food Agric. 2009, 89, 1332–1338. [32] Iglesias, M.T.; Martín-Álvarez, P.J.; Polo, M.C.; De Lorenzo, C.; González, M.; Pueyo, E. Changes in the free amino acid contents of honeys during storage at ambient temperature. J. Agric. Food Chem. 2006, 54, 9099–9104. [33] Rückriemen, J.; Schwarzenbolz, U.; Adam, S.; Henle, T. Identification and quantitation of 2-acetyl-1-pyrroline in Manuka honey (Leptospermum scoparium). J. Agric. Food Chem. 2015, 63, 8488–8492. [34] Krause, R.; Knoll, K.; Henle, T. Studies on the formation of furosine and pyridosine during acid hydrolysis of different Amadori products of lysine. Eur. Food Res. Technol. 2003, 216, 277–283. [35] Hellwig, M.; Geissler, S.; Matthes, R.; Peto, A.; Silow, C.; Brandsch, M.; Henle, T. Transport of free and peptide-bound glycated amino acids: Synthesis, transepithelial flux at Caco-2 cell monolayers, and interaction with apical membrane transport proteins. ChemBioChem 2011, 12, 1270–1279.


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[36] Hellwig, M.; Geissler, S.; Peto, A.; Knütter, I.; Brandsch, M.; Henle, T. Transport of free and peptide-bound pyrraline at intestinal and renal epithelial cells. J. Agric. Food Chem. 2009, 57, 6474–6480. [37] Geissler, S.; Hellwig, M.; Markwardt, F.; Henle, T.; Brandsch, M. Synthesis and intestinal transport of the iron chelator maltosine in free and dipeptide form. Eur. J. Pharm. Biopharm. 2011, 78, 75–82. [38] Jeuring, H.J.; Kuppers, F.J.E.M. High-performance liquid-chromatography of furfural and hydroxymethylfurfural in spirits and honey. J. Assoc. Off. Anal. Chem. 1980, 63, 1215– 1218. [39] Scheijen, J.L.J.M.; Clevers, E.; Engelen, L.; Dagnelie, P.C.; Brouns, F.; Stehouwer, C.D.A.; Schalkwijk, C.G. Analysis of advanced glycation endproducts in selected food items by ultra-performance liquid chromatography tandem mass spectrometry: Presentation of a dietary AGE database. Food Chem. 2016, 190, 1145–1150. [40] Ahmed, N.; Argirov, O.K.; Minhas, H.S.; Cordeiro, C.A.A.; Thornalley, P.J. Assay of advanced glycation endproducts (AGEs): surveying AGEs by chromatographic assay with derivatization by 6-aminoquinolyl-N-hydroxysuccinimidyl-carbamate and application to Nεcarboxymethyllysine and Nε-(1-carboxyethyl)lysine-modified albumin. Biochem J. 2002, 364, 1–14. [41] Finot, P.-A.; Deutsch, R.; Bujard, E. The extent of the Maillard reaction during the processing of milk. Prog. Food Nutr. Sci. 1981, 5, 345–355. [42] Johnson, J.M.; Harris, C.H. Effects of acidulants in controlling browning in cakes prepared with 100% high-fructose corn syrup or sucrose. Cereal Chem. 1989, 66, 158–161. [43] Weston, R.J.; Brocklebank, L.K. The oligosaccharide composition of some New Zealand honeys. Food Chem. 1999, 64, 33–37.



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[44] He, J.; Zeng, M.; Zheng, Z.; He, Z.; Chen, J. Simultaneous determination of Nε(carboxymetyhl)lysine and Nε-(carboxyethyl)lysine in cereal foods by LC-MS/MS. Eur. Food Res. Technol. 2014, 238, 367–374. [45] Bong, J.; Loomes, K.M.; Schlothauer, R.C.; Stephens, J.M. Fluorescence markers in some New Zealand honeys. Food Chem. 2016, 192, 1006–1014. [46] Adams, A.; Polizzi, V.; van Boekel, M.; De Kimpe, N. Formation of pyrazines and a novel pyrrole in Maillard model systems of 1,3-dihydroxyacetone and 2-oxopropanal. J. Agric. Food Chem. 2008, 56, 2147–2153. [47] Popoff, T.; Theander, O.; Westerlund E. Formation of aromatic compounds from carbohydrates. VI. Reaction of dihydroxyacetone in slightly acidic, aqueous solution. Acta Chem. Scand. B 1978, 32, 1–7. [48] Isbell, H.S.; Frush, H.L. Mutarotation, hydrolysis, and rearrangement reactions of glycosylamines. J. Org. Chem. 1958, 23, 1309–1319. [49] Wang, Y.; Ho, C.-T. Flavour chemistry of methylglyoxal and glyoxal. Chem. Soc. Rev. 2012, 41, 4140–4149. [50] Nemet, I.; Varga-Defterdarović, L.; Turk, Z. Methylglyoxal in food and living organisms. Mol. Nutr. Food Res. 2006, 50, 1105–1117. [51] Gleiter, R.A.; Horn, H.; Isengard, H.-D. Influence of type and state of crystallisation on the water activity of honey. Food Chem. 2006, 96, 441–445. [52] Henle, T. AGEs in foods: Do they play a role in uremia? Kidney Int. 2003, 63, S145– S147. [53] Hellwig, M.; Henle, T. Quantification of the Maillard reaction product 6-(2-formyl-1pyrrolyl)-L-norleucine (formyline) in food. Eur. Food Res. Technol. 2012, 235, 99–106.


Page 27 of 39


Figure captions

Fig. 1. Pathways of caramelization and Maillard reaction in Manuka honey (Literature see text). Highlighted substances are formed from methylglyoxal.

Fig. 2. Concentrations of HMF and MGO in 8 non-Manuka honeys (NMH) and 12 Manuka honeys (MH). Dotted lines indicate the medians.

Fig. 3. RP-HPLC-MS/MS (MRM mode) of enzymatically hydrolyzed HMW fractions (dialysis retentates) of Manuka honeys with ascending MGO concentration (a-e). A, quantifier transition for CML; B, quantifier transition for CEL; C, quantifier transition for MG-H1.

Fig. 4. Concentrations of protein-bound MRPs in enzymatically hydrolyzed HMW fractions (dialysis retentates) of 8 non-Manuka honeys (NMH) and 12 Manuka honeys (MH). Dotted lines indicate the medians. Significance of differences between MH and NMH medians was assessed by the Mann-Whitney U test.

Fig. 5. Correlation of the protein-bound MRPs MG-H1 (A) and CEL (B) with the concentration of MGO and correlation of protein-bound CEL and MG-H1 (C) in 11 Manuka honeys.

Fig. 6. Correlation of the individual and summed concentrations of the protein-bound amino acids lysine, N-ε-fructosyllysine, and CEL with the concentration of MGO in 11 Manuka honeys.



Page 28 of 39

Tables Table 1. Transitions Recorded during MRM Measurement of MRPs in Enzymatically Hydrolyzed HMW Fractions of Honey.[a]

Compound

N-ε-

Precursor ion Product

ion Fragmentor

Collision

Q/q[b]

[m/z]

[m/z]

voltage [V]

energy [eV]

471

128

140

20

q

471

225

140

20

Q

309

84

120

30

Q

309

225

120

10

q

255

148

80

20

q

255

175

80

10

Q

255

84

120

20

Q

255

126

120

10

q

229

70

120

20

q

229

114

120

10

Q

225

134

80

20

Q

225

161

80

10

q

219

130

100

10

q

219

84

100

20

Q

205

130

100

10

q

205

84

100

20

Q

Maltulosyllysine

N-εFructosyllysine

Pyrraline

Maltosine

MG-H1

Formyline

CEL

CML


Page 29 of 39


[a] General conditions: Positive mode; dwell time, 70 ms; transitions recorded between 5 and 20 min. [b] Q, transition used for quantitation; q, transition used for the confirmation of the presence of the analyte.



Page 30 of 39

Table 2. Performance parameters of the HPLC-MS/MS method for the Measurement of MRPs in Enzymatically Hydrolyzed HMW Fractions of Honey.

Standard addition MRP

LOD[a]

LOQ[a]

cV[b]

Mean R2 Intercept

range[c]

[d]

[µM]

accuracy[e]

[µmol/m

[µmol/m

mol Leu]

mol Leu]

1.8

5.1 (3.9%) 17.0

2–100

0.9981

103 ± 10

1.3

3.8 (9.3%) —

0.3–14

0.9997

108 ± 10

CML

0.5

1.9 (7.2%) 18.5

1.7–20

0.9986

101 ± 4

CEL

0.9

4.0

10.6

0.5–76

0.9971

101 ± 5

10.2

0.2–2

0.9986

102 ± 4

8.4

0.1–0.5

0.9982

101 ± 3

14.1

0.02–0.34

0.9751

95 ± 7

4.1

0.1–17

0.9975

102 ± 8

N-ε-

[%]

Linear

[%]

Fructosyllysine N-εMaltulosyllysine

(10.7%)

Pyrraline

0.07

0.23 (3.3%)

Formyline

0.04

0.12 (3.7%)

Maltosine

0.02

0.06 (11.3%)

MG-H1

0.16

0.55 (3.5%)


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[a] Limits of detection (LOD) and limits of quantitation (LOQ) are based on a protein amount of 4 mg per enzymatic hydrolysis. The relative standard deviation at the LOQ is given in parentheses. [b] Coefficients of variation (cV) were determined on Manuka honeys (n = 5-6). No cV is given for N-ε-maltulosyllysine due to its concentration < LOD. [c] Linear range is the range between the lowest concentration in samples after enzymatic hydrolysis and the highest concentration after standard addition. [d] R2 is given for the regression function of standard addition. [e] Intercept accuracy is calculated as the intercept of the regression line (peak area vs. concentration) obtained by standard addition divided by the peak area of the analyte measured in the sample without standard addition and expressed in percent.



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Table 3. Amounts (µg) of Protein-Bound MRPs in Manuka and Non-Manuka Honey, Calculated for a Serving Size of 10 g.

Manuka honey

Non-Manuka honey

CML

3.2 (2.3-5.5)

2.3 (1.6-3.8)

CEL

18.0 (n.d.-56.1)

tr

MG-H1

3.5 (n.d.-13.8)

tr

Formyline

0.2 (0.1-0.2)

0.2 (tr-0.2)

Pyrraline

0.5 (0.2-1.0)

0.4 (0.2-2.4)

N-ε-Fructosyllysine

31 (8-69)

60 (43-103)

Data are given in µg, based on the median levels (Figure 4) and the ranges given in parentheses. n.d., not detectable, below LOQ; tr, traces, between LOD and LOQ.


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Figures Figure 1



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Figure 2


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Figure 3



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Figure 4


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Figure 5



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Figure 6


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TOC graphic


Unique Pattern of Protein-Bound Maillard Reaction ... - ACS Publications

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