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Green Tea Polyphenols Decrease Strecker Aldehydes and Bind to Proteins in Lactose-Hydrolyzed UHT Milk Therese Jansson, Valentin Maximilian Rauh, Bente Pia Danielsen, Mahesha Manjunatha Poojary, Sandra Stolzenbach Wæhrens, Wender Bredie, John Sørensen, Mikael Agerlin Petersen, Colin Andrew Ray, and Marianne N. Lund J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04137 • Publication Date (Web): 09 Nov 2017 Downloaded from http://pubs.acs.org on November 9, 2017
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Journal of Agricultural and Food Chemistry
Green Tea Polyphenols Decrease Strecker Aldehydes and Bind to Proteins in Lactose-Hydrolyzed UHT Milk
Therese Jansson1, Valentin Rauh2, Bente P. Danielsen1, Mahesha M. Poojary1, Sandra S. Waehrens1, Wender L. P. Bredie1, John Sørensen2, Mikael A. Petersen1, Colin A. Ray1, Marianne N. Lund1,3*
1
Department of Food Science, University of Copenhagen, Rolighedsvej 26, 1958
Frederiksberg C, Denmark 2
Arla Foods R&D, Agro Food Park 19, 8200 Aarhus N, Denmark
3
Department of Biomedical Sciences, University of Copenhagen, Blegdamsvej 3,
2200 Copenhagen N, Denmark
*Corresponding author: E-mail address:
[email protected], Phone: +45 3533 3547
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ABSTRACT
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The effect of epigallocatechin gallate enriched green tea extract (GTE) on flavor,
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Maillard reactions and protein modifications in lactose-hydrolyzed (LH) ultra-high
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temperature (UHT) processed milk was examined during storage at 40 °C for up to 42
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days. Addition of GTE inhibited the formation of Strecker aldehydes by up to 95%
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compared to control milk, and the effect was similar when GTE was added either
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before or after UHT treatment. Release of free amino acids, caused by proteolysis,
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during storage was also decreased in milk added GTE either before or after UHT
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treatment compared to control milk. Binding of polyphenols to milk proteins was
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observed in both fresh and stored milk samples. The inhibition of Strecker aldehyde
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formation by GTE may be explained by two different mechanisms; inhibition of
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proteolysis during storage by GTE or binding of amino acids and proteins to the GTE
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polyphenols.
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Keywords
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Maillard reaction, lactose-hydrolyzed milk, UHT, green tea extract, epigallocatechin
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gallate, storage stability, shelf life, Strecker aldehydes, flavor, α-dicarbonyls, protein-
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polyphenol binding
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INTRODUCTION
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Lactose-hydrolyzed (LH) milk has become an increasingly important dairy product
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during the last years due to an increased awareness of lactose intolerance.1 Ultra-high
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temperature (UHT) processing (e.g. 135-150 °C for 3-15 s) is often applied to
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increase the shelf life of milk by reducing the microbial load, however, the high
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temperature treatment may alter the color during storage as well as the flavor of the
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milk. The quality changes that are observed in UHT milk during storage are caused by
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oxidation of lipids and proteins as well as Maillard reactions.2–4 In LH milk, Maillard
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reactions are very pronounced due to the presence of glucose and galactose, which
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have been shown to react 10 times and 20 times faster, respectively, with amine
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groups compared to lactose.5 Additionally, proteolytic side activity from the lactase
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enzyme preparation used for lactose hydrolysis is responsible for release of free
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amino acids and thus an increased concentration of reactants for the Maillard reaction
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substrates.2,5–7
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The Maillard reaction is initiated by a condensation between a carbonyl group,
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usually from a reducing sugar, and an amine or guanidine group to form a Schiff base.
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The Schiff base is rearranged to an aminoketose, an Amadori product, which is
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further degraded to highly reactive α-dicarbonyls, such as glyoxal, methylglyoxal and
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deoxyosones. These can react further to produce flavor compounds, browning and
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advanced glycation endproducts (AGEs), such as N-ε-carboxymethyllysine, pyrraline,
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and pentosidine.8 Alongside with the Amadori degradation, the reducing sugars can
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also undergo different fragmentations reactions to form the reactive carbonyl
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compounds.9 The concentration of 3-deoxyglucosone (3-DG) and 3-deoxygalactosone
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(3-DGal) have been found to increase up to 0.3 mM in LH UHT milk stored for 4 3 ACS Paragon Plus Environment
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months at 37 °C.10 α-Dicarbonyls will also react with free amino acids, ultimately
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resulting in Strecker degradation and formation of flavor-active Strecker aldehydes,
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which are known to be responsible for off-flavor in UHT milk.11 Jansson et al. (2014)
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observed an increased level of free amino acids and Strecker aldehydes in LH UHT
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milk compared to conventional UHT milk during storage at room temperature after
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nine months.7 The increase in free amino acids was caused by proteolytic side-activity
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of the lactase preparation used for lactose hydrolysis of LH UHT milk.2,6
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Development of feasible strategies for inhibition of Maillard reactions in LH UHT
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milk would be favorable for prolonging shelf life.8 Previous studies have shown that
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polyphenols from plants can inhibit the formation of Maillard reaction products and
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certain undesired aroma compounds (e.g. methional, pyrazine, and furfural) in
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conventional UHT milk during storage.12–14 However, few studies have reported on
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the effect of plant polyphenols on LH UHT milk stability. Totlani & Peterson15,16
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showed that epicatechin, which is a flavonoid found in plant-based foods such as
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grapes and green tea, is capable of trapping α-dicarbonyls through aromatic
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electrophilic substitution and thereby preventing them from further reaction. Today, it
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is generally believed that this is the major mechanism by which polyphenols inhibit
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Maillard reactions in food.8,17,18 Nevertheless, polyphenols may also react with
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components other than α-dicarbonyls in foods such as furfural19 and Strecker
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aldehydes.20,21 Furthermore, under oxidative conditions present in UHT milk,7
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polyphenols are oxidized to quinones, which may undergo Michael additions with
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nucleophilic groups, such as amines and thiols,22 or form Schiff bases with amines
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and thereby cause changes in milk proteins.23–25
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In most of the previous studies polyphenols have been added to milk before heat
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treatment.12,13,26,27 In the present study, the aim was to investigate the inhibitory effect
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of a green tea extract (GTE) containing 95% epigallocatechin gallate (EGCG) on the
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delay of Maillard and Strecker reactions in LH UHT milk during storage at 40 °C for
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42 days. GTE was added either before or after UHT treatment to examine the role of
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the UHT treatment on the reactions investigated. It was hypothesized that addition of
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polyphenols to milk would create protein-polyphenol conjugates. Mechanisms for
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how EGCG may react with milk components are proposed and discussed.
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MATERIALS AND METHODS
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Chemicals
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Teavigo® green tea extract (GTE) was obtained from Taiyo International
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(Minneapolis, USA), and was reported to contain 95.3% EGCG based on
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chromatographic analysis. EZ:Faast Kit® for amino acids analysis, including a
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standard amino acid solution containing 26 amino acids and norvaline was obtained
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from Phenomenex (Torrance, CA, USA). LDS sampling buffer, MOPS running buffer
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(20x), NuPAGE transfer buffer (20x), NuPAGE antioxidant and SYPRO® ruby
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protein gel stain were obtained from Invitrogen (Carlsbad, CA, USA). Pierce™
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unstained protein MW marker was purchased from Thermo Fisher Scientific Inc.
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(Waltham, MA, USA). Acetic acid, ammonium bicarbonate, ammonium sulfate, 6-
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aminocaproic acid, benzaldehyde, Brilliant Blue G acid blue 90, diethylentriamine
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pentaacetic acid (DETAPAC), dithiothreitol (DTT), 5-dithio-bis(2-nitrobenzoic acid)
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(DTNB), fluorescamine, glycine, methanol, 2-methylbutanal, 3-methylbutanal, 4-
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methyl-1-pentanol, nitroblue tetrazolium (NBT), 2-furfural, o-phenylenediamine
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(OPD), ortho-phosphoric acid, potassium hexacyanoferrate (II), trichloroacetic acid 5 ACS Paragon Plus Environment
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(TCA), tris-base, trifluoroacetic acid, and zinc acetate were obtained from Sigma
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Aldrich Inc. (Steinheim, Germany). Glacial acetic acid was purchased from VWR
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(Radnor, PA, USA). Ethanol was obtained from Kemetyl AB (Jordbro, Sweden).
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Urea, ethylenedinitrilotetraacetic acid (EDTA), and sodium citrate dihydrate were
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obtained from Merck KGaA (Darmstadt, Germany). All chemicals were of analytical
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grade. Deionized water (Milipore, Bedford, MA) was used throughout.
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Determination of sensory relevant GTE concentration
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The maximal acceptable dosage of GTE was determined based on the ‘difference
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from control’ method.28 Skim milk samples (0.1% fat) with GTE (0.1-0.6%) were
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tasted and compared against a reference skim milk without GTE to assess the level
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where GTE had a minor overall taste contribution to UHT milk. Differences from
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control were rated on a 15-cm unstructured line scale ranging from “No difference” to
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“Extremely different” by an internal sensory panel with 7 members (4 females and 3
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males). At a concentration of 0.275% GTE the panel could not detect taste differences
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from the control milk (p
