Control of Maillard-Type Off-Flavor Development in Ultrahigh

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Control of Maillard-Type Off-Flavor Development in UltrahighTemperature-Processed Bovine Milk by Phenolic Chemistry Smaro Kokkinidou and Devin G. Peterson* Department of Food Science and Nutrition, Food Science and Nutrition Building, 1334 Eckles Avenue, University of Minnesota, St. Paul, Minnesota 55108, United States ABSTRACT: The application of phenolic compounds to suppress Maillard chemistry and off-flavor development in ultrahightermperature (UHT)-processed milk during processing and storage was investigated. Five phenolic compounds were examined for structure−reactivity relationships (catechin, genistein, daidzein, 1,2,3-trihydroxybenzene, and 1,3,5-trihydroxybenzene). The levels of key transient Maillard reaction (MR) intermediates (reactive carbonyl species) and select off-flavor markers (methional, 2-acetyl-2-thiazoline, 2-acetyl-1-pyrroline) were quantified by LC-MS/MS and GC-MS/ToF, respectively. The addition of phenolic compounds prior to UHT processing significantly reduced the concentration of MR intermediates and related off-flavor compounds compared to a control sample (p < 0.05). All phenolic compounds demonstrated unique structure reactivity and, notably, those with a more activated A-ring for aromatic electrophilic substitution (catechin, genistein, and 1,3,5trihydroxybenzene) showed the strongest suppression effect on the off-flavor markers and reactive carbonyl species. Sensory studies were in agreement with the analytical data. The cooked flavor intensity was rated lower for the recombination model samples of the catechin-treated UHT milk compared to the control UHT milk. Additionally, consumer acceptability studies showed catechin-treated UHT milk to have significantly higher liking scores when compared the control sample (Fisher’s LSD = 0.728). KEYWORDS: Maillard reaction, phenolic chemistry, reactive carbonyls species, thermal processing, UHT milk, off-flavor inhibition

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pounds that contributed to the “cooked” off-flavor of UHT milk, indicating that the MR is an important pathway of off-flavor development during thermal processing and a point of control to develop flavor improvement strategies for UHT milk and milkbased beverages. The MR is well-known to affect food flavor, color, toxicity, and nutritional value.10−16 Additionally, it is known to play an important role in regulatory biology, diabetes, and cardiovascular and age-related pathology.11,17 Therefore, a better understanding of the mechanisms that influence Maillard reaction product (MRP) generation in processed food systems would provide the food industry with the ability to more effectively control quality and also potentially improve the nutritional attributes of food. Phenolic compounds have been previously shown to inhibit Maillard-type flavor generation pathways and reduce off-flavors.2 Totlani and Peterson18 have shown that flavan-3-ols function as trapping agents (via electrophilic aromatic substitution reactions, primarily on the A-ring) of key transient Maillard precursors (reactive carbonyl species (RCSs) C2, C3, C4, and C6 sugar fragments) and consequently suppress the generation of MRPs. Flavanols such as catechin are found in several plant products, most notably in tea, cocoa, and wine, and their antioxidant capacity has been extensively studied. High consumption of those phenols has been associated with decreased risk of cardiovascular disease and cancer, as well as metabolic regulation and weight loss.19−24 Their use to control reactions that

INTRODUCTION Milk, like many foods and commodities, is mandated to undergo thermal processing during production as stated in the Code of Federal Regulations1 to improve microbial safety. Additional benefits of thermal processing are enhanced product stability and shelf life, which facilitates distribution and storage. For milk, a number of different pasteurization methods are available such as high temperature−short time (HTST), extended shelf life processing, and ultrahigh temperature (UHT) processing, each technology utilizing different time−temperature profiles. Aseptic UHT processing of milk yields a commercially sterile product, with the longest shelf life of 6 months to 1 year. It also allows for nonrefrigerated storage and transportation, widening distribution opportunities and reducing potential energy requirements. Despite the significant benefits of UHT products, hightemperature treatment also yields a product with reduced flavor and nutritional quality as well as undesirable color development, creating a barrier to consumer acceptance.2−4 Pasteurized milk (typically consumed in the U.S. market) has as a slightly sweet flavor, weak aroma attributes, and a pleasant mouthfeel and aftertaste.5 Any alteration to this “clean” flavor profile is considered a product defect. Research performed at Cornell University showed a direct correlation between the flavor quality of milk and the level of consumption.6 Blake et al.7 also reported UHT milk had a lower consumer liking compared to other less severe thermally processed samples (i.e., pasteurized milk). The negative flavor changes in UHT milk previously reported to develop during heat treatment and ambient storage have been associated with the Maillard reaction (MR) and lipid oxidation (LO).8,9 Recent work by Colahan-Sederstrom and Peterson2 identified three important Maillard-derived off-flavor com© 2014 American Chemical Society

Received: Revised: Accepted: Published: 8023

April 23, 2014 July 10, 2014 July 26, 2014 July 26, 2014 dx.doi.org/10.1021/jf501919y | J. Agric. Food Chem. 2014, 62, 8023−8033

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negatively affect flavor and nutrition would additionally benefit the food industry as consumers are increasingly aware of food labels and there is a greater demand for natural products. The objective of this study was to investigate phenolic structure−reactivity relationships on the mechanisms of offflavor development in 1% UHT milk.

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previously described by Totlani and Peterson.27 A 50 mL saline phosphate buffer (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) reaction system containing 10 mM catechin and methylglyoxal, glyoxal, or 3-deoxyglucosone was incubated in a shaking water bath at 37 °C for 1 h. After incubation, 5 mL of the reaction mixture was spiked with an internal standard (5 μL of 2% solution of butyl paraben in methanol) and an SPE clean up and concentration step followed (DSC-18 cartridge; Supelco, Bellefonte, PA, USA). Isolates were then purified using a 2D-RP-HPLC/MS-ESI fraction collection system coupled with a Varian Dynamax Pursuit 5 Diphenyl (250 × 10.0 mm) and a Dynamax Pursuit 5 C18 (250 × 10.0 mm). Chromatography was performed with a flow rate of 3.7 mL/min with the following linear gradient conditions (solvent A, 1 mM ammonium acetate, pH 5; solvent B, methanol): solvent B at 20% (0−15 min), increased to 55% (15−61 min), then to 80% (61−66 min), then to 100% (66−72 min), and finally reduced to 20% (72−81 min). Target analytes were collected by MS guided fractionation (m/z reported in Table 1).

MATERIALS AND METHODS

Chemicals. The following chemicals were obtained from the sources given: (+)-catechin ≥97% (TCI America); genistein ≥98% (Shaanxi Schipar Biotechnology Co. Ltd., China); daidzein ≥98% (Shaanxi Schipar Biotechnology Co. Ltd., China); phloroglucinol 97% (1,3,5trihydroxybenzene) (Aldrich, St. Louis, MO, USA); pyrogallol (Fluka, St. Louis, MO); acetoin ≥96% FCC, food grade (FG) (Aldrich); acetol 90% (Aldrich); 2,3-butanedione 97% (Aldrich); 3-deoxyglucosone (TRC, Toronto Research Chemicals, Ontario, Canada); glyoxal 40% solution in water (Sigma, St. Louis, MO, USA); methylglyoxal 40% solution in water (Sigma); methional 97% FG (Aldrich); 2-acetyl-2thiazoline ≥96% FG (Aldrich); o-phenylenediamine 99.5% (Aldrich); oethylhydroxylamine hydrochloride (Aldrich); and 99.9+% anhydrous diethyl ether (Fisher Chemicals). 2-Acetyl-1-pyrroline was not commercially available but was extracted from pandan leaves (Pandanus amaryllifolius Roxb.), obtained from a local market. The purification procedure is described below. Pilot Plant Scale UHT Milk Processing. Raw skim and whole milk were obtained from the University of Minnesota Joseph Warthesen Food Processing Center (St. Paul, MN, USA) and mixed to yield raw milk with 1% fat content. Samples underwent aseptic UHT processing using a MicroThermics HTST/UHT Indirect Tubular Processing System with water final heat (DIP-W), a homogenizer, and aseptic filling station. Processing conditions were as follows: the unit was first sanitized while passing through a chlorine solution (100 ppm, sodium hypochlorite at pH 7.00) for 1 h at 40 °C followed by a clean water wash at 121.1 °C for 1 h. The milk was homogenized, then processed at final temperature of 140 °C, holding for 6 s to ensure commercial sterilization, immediately cooled to 15 °C, and filled in a sterilized laminar flow hood into previously sterilized 1 L amber bottles. Small-Scale Benchtop Simulated UHT Processing. A simulated UHT method reported by Kokkinidou and Peterson25 was used to process milk, utilizing small sample volumes for cost and time efficiency to monitor the development of off-flavor compounds in milk spiked with RCSs (Figure 9). Briefly, 5 mL of 1% raw milk in headspace vials was placed in a cooling vial holder held at 8 °C. Vials were transferred to an agitator (150 °C) and held for 265 s to allow the sample to reach a final temperature of 140−141 °C for 6 s and subsequently returned to the cooler. Characterization of Off-Flavor Generation in UHT Processed Milk. Identification and quantitation of MR precursors methylglyoxal, glyoxal, 3-deoxyglucosone, and diacetyl (dicarbonyls) as well as glycolaldehyde, acetoin, and acetol (hydroxycarbonyls) were performed using the synthesized stable isotopes [13C4]-acetoin and [13C4]-diacetyl26 as internal standards. Each internal standard (0.5 mM) was added to 5 mL of milk, followed by 500 μL of 10% trichloroacetic acid. The samples were then vortexed for 1 min and centrifuged at 3904g for 20 min at 4 °C (Beckman Coulter, Allegra X-22R), and the supernatant was collected. A solid phase extraction (SPE) method was used to isolate the compounds of interest and avoid interference from phenolic compounds and adducts according to the method of Kokkinidou and Peterson.25 A derivatization method followed, and samples were analyzed using an Acquity UPLC system coupled with a Quattro Premier XE micromass mass spectrometer (Waters Co. Milford, MA, USA). An Acquity UPLC 2.1 × 100 mm BEH Phenyl 1.7 μm column with a VanGuard 2.1 × 5 mm BEH Phenyl 1.7 μm precolumn was used for separation, and all experiments were performed in triplicate. Analytes were detected using electrospray positive ionization−multiple reaction monitoring (MRM) using methods previously developed and reported by Kokkinidou and Peterson.25 Generation of Phenolic−RCS Adduct Standards. Model reactions were used to generate standards of Maillard−phenolic products as

Table 1. Mass Fragmentation Ions of Main Detected Adducts between Catechin (CAT) and Glyoxal (GO), Methylglyoxal (MGO), and 3-Deoxyglucosone (3DG) in Model Systemsa adduct/phenolic

parent ion (m/z)

cone voltage (V)

collision energy (V)

daughter ion (m/z)

CAT−GO CAT2−GO CAT−MGO CAT−MGO2 CAT−3DG

347 619 361 433 451

35 35 35 35 32

21 20 20 27 22

167 329 181 235 289

a

Optimum multiple-reaction monitoring (MRM) conditions used for their identification based on Mass Lynx (Waters, Milford, MA, USA).

Fragmentation spectra of the purified analytes were determined by LCMS/MS techniques and are shown in Table 1. Fragmentation spectra were obtained to monitor these analytes in milk by MRM methods as well as to support the hypothesized structures of these adducts. Isolation and Quantitation of Off-Flavor Compounds in Milk. Three off-flavor compounds previously identified in UHT milk2 were monitored and included methional, 2-acetyl-2-thiazoline (2A2T), and 2acetyl-1-pyrroline (2AP). Quantitation was conducted by stable isotope surrogate standards of each compound. The labeled compounds were synthesized as previously described [2H3]-methional,28 [2H3]-2-acetyl1-pyrroline,29 and [2H4]-2-acetyl-2-thiazoline.30 The identity and purity of each labeled standard were confirmed by MS analysis in both electron impact (EI, 70 eV) and chemical ionization (CI, at 115 eV with isobutane as reagent gas), analyzed with a Hewlett-Packard GC (HP 5890 series II) coupled with an MS detector (HP 5972). The concentration of each labeled standard was determined based on GC analysis in comparison to authentic compounds. Solvent-Assisted Flavor Evaporation. Volatile isolates of the samples were prepared by solvent extraction that was adapted from the method of Colahan-Sederstrom and Peterson.2 Prior to extraction [2H4]-2A2T, [2H3]-2AP, and [2H3]-methional were added as internal standards at levels of 5 μg standard/kg milk. The concentrated extract was then subjected to high-vacuum distillation using a solvent-assisted flavor evaporation (SAFE) apparatus.31 The isolate was further fractionated into an acidic (A) and a neutral/basic (N/B) fraction as described by Colahan-Sederstrom.2 The N/B fraction was prepared and stored at −80 °C prior to analysis by an Agilent 7890A GC (Agilent Technologies, Inc., Wilmington, DE, USA) coupled with a TruTOF HT time-of-flight mass spectrometer (Leco Co., St. Joseph, MI, USA) and a DB-5MS column (30 m × 0.250 mm × 0.25 μm, Agilent Technologies). The column heating profile was at 40 °C for 2.00 min, then ramped to 160 °C at 3 °C/min and ramped to 250 °C at 30 °C/min, and held for 5 min. Injection mode was splitless, and temperature was 250 °C. Dynamic Headspace GC-MS. Off-flavor analysis of the milk samples spiked with RCSs (Figure 9) was performed using a 6890 GC coupled with a DB-5MS (30 m × 0.250 mm × 0.25 μm, Agilent Technologies) 8024

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Figure 1. Example chromatogram and MS spectra of purified 2AP isolated from pandan leaf extract after multidimensional chromatography, heartcutting, and fraction collection. column and equipped with a 5973 mass selective detector (Agilent Technologies), thermal desorption unit (TDU, Gerstel), PTV inlet (CIS 3, Gerstel), and MPS 2 with headspace and dynamic headspace (DHS) option (Gerstel). Briefly, 5 mL of UHT processed milk was placed in headspace vials (20 mL) along with 1 mL of nanopure water and sodium chloride (1 M). Prior to dynamic headspace analysis, [2H4]2A2T, [2H3]-2AP, and [2H3]-methional were added as internal standards at levels of 5 μg standard/kg milk. The headspace vials were then incubated at 40 °C for 10 min and subsequently purged with nitrogen at a rate of 40 mL/min for a total time of 12.5 min while agitated at 500 rpm to increase volatility. The trap (Tenax TA) was then dry purged for 4 min at a rate of 30 mL/min and transferred to a thermal desorption unit. The trapped volatiles were then desorbed, cryofocused at −125 °C, and injected in solvent vent mode. The column heating profile was at 40 °C for 2.00 min, ramped to 160 °C at 3 °C/min, then ramped to 250 °C at 30 °C/min, and held for 5 min. Identification of Odorants. Positive identifications were made by linear retention indices (LRI) and mass spectra compared to authentic compounds analyzed under identical conditions. Generation of 2-Acetyl-1-pyrroline Standard. A 2AP standard was prepared from an extract of pandan leaves (P. amaryllifolius Roxb.) as previously described by Colahan-Sederstrom and Peterson.2 The reference compound was further purified using a two-dimensional GC system coupled with a fraction collector. The analytical system consisted of an Agilent 6890N GC equipped with two Dean switch devices, two Gerstel modular accelerated column heaters (MACH), and an Agilent 7683 autosampler and injector and was additionally coupled with an Agilent 5973N MSD and a Gerstel preparative fraction collector (PFC). An RTX-5S ILMS (30 m × 0.25 mm × 0.25 μm) was used as a primary column, and a DB-Wax (30 m × 0.25 mm × 0.25 μm) was used as a secondary column. GC conditions for all runs were as follows: inlet temperature, 200 °C; 10 μL of sample injected in solvent vent mode (0 kPa, 0.15 min); helium carrier gas ramp pressure mode (initial pressure or 357 kPa held for 7.1 min and then ramped to 10 Pa at 999 kPa/min); and a total flow of 3.9 mL/min with a split 1:4 to MSD and 3:4 to PFC. MSD conditions were as follows: capillary direct interface temperature, 250 °C; mass range, 35−300 amu; The MACH heating profile for the RTX-5S ILMS capillary column was 40 °C for 1 min, then ramped to 280 °C at 10 °C/min, and held for 2.35 min, whereas for the DB-Wax the oven profile was 40 °C for 7.60 min, ramped to 230 °C at 10 °C/min, and held for 2 min. The valve-switching program for heart-cutting and isolation of 2AP was as follows: valve 1 switched on at 6.29 min, directing the flow to the secondary column, and

switched off at 7.10 min; then valve two switched on at 15.4 min directing the flow to cryotrap of PFC for 2AP collection and switched off at 15.75 min. The PFC switching device was operated at 250 °C, and the trap was kept at −70 °C. 2AP was extracted from the trap using food grade ethanol and further quantified using vinylpyrrolidine as a standard using an HP 5890 series II GC coupled with an FID. An example chromatogram of 2AP is presented in Figure 1. Sensory Analysis of UHT Milk Samples. Both consumer acceptability and descriptive analysis including off-flavor recombination models were conducted. For the consumer acceptability test, 49 panelists were recruited on the basis of regular milk consumption. Aseptically processed UHT milk samples with and without added 1.7 mM catechin and a commercial UHT milk (Parmalat, Agropur Inc., Division Natrel, USA) were evaluated for acceptability after storage at 30 °C for 1 month. The samples were presented at room temperature in coded 3 oz cups in randomized order. The panelists were asked to rate them on a ninepoint hedonic scale (ranging from dislike extremely to like extremely). Differences among treatments were evaluated by analysis of variance (ANOVA) followed by Tukey’s HSD pairwise comparison. All analyses were performed with Statistix software (version 9). Significance was established at p < 0.05. For the descriptive analysis trained panelists evaluated the intensity of cooked off-flavor in milk samples. Seven graduate students from the Department of Food Science and Nutrition at the University of Minnesota were recruited for the analysis and were selected on the basis of availability and willingness to participate. They received eight 1 h training sessions focused on the cooked attribute of the milk samples. A cooked reference was prepared by heating 1% milk (Schroder Milk Co.) to 90 °C and holding for 45 min. This cooked reference was mixed with various amounts of pasteurized milk to create a range of cooked intensities. The panel agreed to use the following intensities as references: 100% cooked = 15; 75% cooked/25% pasteurized = 8; 50% cooked/50% pasteurized = 4; 30% cooked/70% pasteurized = 2. Samples were evaluated on a 15-point intensity scale, anchored on the left with “none” and on the right with “extreme”. Panelists participated in three evaluation sessions, occurring over the period of 1 day. The four samples were evaluated in triplicate in a randomized block design. The four samples evaluated were (1) pasteurized 1% milk (Schroder Milk Co.); (2) commercial 1% UHT milk (Parmalat); (3) off-flavor recombination model for simulation of control UHT milk prepared using pasteurized 1% milk (Schroder Milk Co.) spiked with 2AP (1.35 μg/kg), 2A2T (0.95 μg/kg), and methional (4.05 μg/kg); and (4) off-flavor recombination model for simulation of 8025

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flavor suppression would be beneficial for the optimization of the dose response. For example, select phenolic structures might be more reactive (effective in suppressing off-flavor) during thermal processing (ca. 140 °C), whereas others could be more reactive during ambient storage. Five phenolic compounds (see Figure 2) were selected to examine phenolic structure function reactivity on the MR

UHT milk with added catechin prepared using pasteurized 1% milk (Schroder Milk Co.) spiked with 2A2T (0.41 μg/kg) and methional (0.88 μg/kg) to simulate UHT milk (Figure 3) . The spiked levels of offflavor markers in recombination models were determined after quantitation in pasteurized 1% milk (Schroder Milk Co.) and addition of appropriate amounts for simulation of UHT processed milk samples. Differences among treatments were evaluated by analysis of variance (ANOVA) followed by Tukey’s HSD pairwise comparison. ANOVA was also used to monitor panelist performance. All analyses were performed with Statistix Software (version 9). Significance was established at p < 0.05. Microbial Testing. Microbial analysis was conducted on both control and treatment samples using 3 M Petrifilm aerobic count (AC) plates and coliform count (CC) plates (3M Co., St. Paul, MN, USA) according to protocol. For counts in 3M AC and CC plates, 1 mL of each dilution 1:10, 1:100, and 1:1000 of milk samples was plated using phosphate buffer, KH2PO4 (0.0425 g/L, adjusted to pH 7.2, Butterfield’s buffer; Weber Scientific, Hamilton, NJ, USA). Colonies were enumerated after incubation at 37 °C for 24 h for coliforms and at 35 °C after 48 h for aerobic bacteria.

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RESULTS AND DISCUSSION Milk with 1% fat content was selected for evaluation, as it is the most consumed fluid milk product in the United States. Although overall fluid milk consumption has steadily decreased over the past three decades, consumption of low-fat milk has increased (in comparison to whole milk) as consumers become more aware of caloric intake and negative effects of saturated fat intake.32 As previously reported 6,7 UHT milk receives significantly lower liking scores than pasteurized milk or other less severely thermally processed milk samples in the domestic marketplace. Consequently, the negative flavor attributes of aseptic UHT milk are a hurdle to utilization and product acceptability. Colahan-Sederstrom and Peterson2 had previously reported the addition of a phenolic compound prior to UHT processing reduced the cooked flavor intensity of UHT milk suggesting an improved product quality. Therefore as an initial step, a consumer acceptability study was performed to assess the effectiveness of phenolic compound addition on consumer liking. UHT milk samples with and without catechin were stored at 30 °C for 30 days, for accelerated aging (estimated 2−3 months at ambient storage conditions), prior to evaluation. A commercial UHT sample was also included as an internal anchor. The results of the consumer panel are presented in Table 2. The UHT milk

Figure 2. Select phenolic compounds evaluated in UHT milk.

pathways and off-flavor development in UHT processed milk, and the effectiveness of each structure on off-flavor suppression was evaluated immediately after processing as well as after storage. Three common food phenolic compounds, catechin (flavanol), genistein, and daidzein (isoflavones), along with two simple phenols, 1,2,3-trihydroxybenzene and 1,3,5-trihydroxybenzene, were selected. The food phenolic compounds differ in the degree and position of hydroxyl substitution on rings A and B as well as the presence of a keto group on the C-ring (isoflavones). The simple phenols were used to simulate different ring chemistry, more redox reactive (123THB) or more “trapping” reactive of carbonyl compounds by electrophilic aromatic substitution reactions (135THB) to investigate the mechanisms by which phenolic compounds alter MR pathways. Among the foodderived phenolic compounds, catechin would have the highest trapping reactivity, followed by genistein and then daidzein, based on the position of the hydroxyl groups on the A-ring and electron-withdrawing groups on the neighboring C-ring (keto group). Phenolic compounds were added at dose levels of 1.7 mM, which was selected as this amount of epicatechin was reported2 to be effective in suppressing off-flavor development and not result in a detectable bitterness (a sensory attribute associated with polyphenols). Sensory Recombination Analysis: Markers of UHT Cooked Flavor. To quantitatively monitor off-flavor progression in UHT milk, the off-flavor compounds as well as reaction intermediates of these compounds were targeted. For target off-flavor compounds, Colahan-Sederstrom and Peterson2 identified three Maillard-type reaction products related to the cooked off-flavor profile of 1% UHT milk. These included methional, 2-acetyl-1-pyrroline (2AP) and 2-acetyl-2-thiazoline (2A2T). In the current study the contribution of these off-flavor compounds to the “cooked” off-flavor attributes of UHT milk was further defined by sensory recombination analysis in milk systems.

Table 2. Consumer Acceptability Rating of 1% UHT Milk Manufactured with and without Catechin Plus a Commercial UHT Sample sample

mean liking (9-point hedonic scale)

1% UHT-catechin containing (1.72 mM)a 1% UHT milka commercial UHT (Parmalot)

6.04a 5.12b 4.73b

a

Samples prepared and produced at University of Minnesota Joseph Warthensen Processing Center facilities (processed to 140 °C for 6 s) and stored at 30 °C for 1 month. Different letters indicate statistically significant differences. Fisher’s LSD = 0.728 (5% significance level).

with catechin added prior to processing received a significantly higher liking score then both the control and commercial UHT milk samples, demonstrating the application of phenolic compounds to improve UHT milk quality. Phenolic Structure Reactivity. A mechanistic understanding of the reactivity of phenolic chemistry related to off8026

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as predictive chemical markers of cooked off-flavor associated with UHT processed milk. Effect of Phenolic Compounds on Levels of Off-Flavor Markers. The influence of different phenolic structures on the generation of cooked off-flavor both during thermal treatment (day 0) and after storage (30 days at 30 °C) is further illustrated in Figure 3; the concentration in the raw milk prior to UHT processing was also included for comparison. Overall, the concentration of all three marker compounds (methional, 2AP, and 2A2T) increased during UHT processing and storage at 30 °C in direct comparison to raw and pasteurized milk (Figure 3). Further comparison of the influence of phenolic addition on the suppression of off-flavor compounds indicated the effects reported were variable and dependent on the type of off-flavor compounds, the phenolic structure added, and the temperature of the system (UHT processing temperature, 140 °C, versus storage, 30 °C). For example, catechin and daidzein slightly inhibited the amount of methional generated during UHT processing, whereas the other three phenolics had no substantial effect. However, during storage, all phenolic compounds were reported to suppress its generation, with genistein and catechin being more effective, reducing methional by 80 and 75%, respectively. This observation indicated that catechin was more effective for suppressing the generation of methional during both processing and storage. However, for methional, daidzein is more reactive during processing, whereas genistein was more reactive during storage conditions. For 2A2T, the addition of phenolic compounds had little impact on off-flavor development during processing temperatures as compared to control UHT, but all effectively suppressed generation during storage. Addition of catechin, genistein, or daidzein resulted in concentrations of 2A2T after storage 42− 53% lower than the control, notably at concentrations below the odor threshold of this compound (1 μg/kg). Genistein was the most reactive phenolic compound for off-flavor suppression under storage conditions. Finally for 2AP, most phenolic compounds had negligible influence on off-flavor development during processing. However, during storage, genistein and catechin were the most reactive and effective in suppressing off-flavor generation, whereas daidzein, 123THB, and 135THB had negligible effects. In general 135THB, which is more activated toward aromatic electrophilic substitution, was more effective in reducing the concentration of off-flavor markers as compared to 123THB, supporting the idea that one of the mechanisms by which phenolic compounds inhibit MR pathways and off-flavor generation is reactive carbonyl trapping. On the basis of the observed unique structure reactivity of these phenolic compounds, utilizing mixtures of phenolic compounds as an off-flavor inhibition strategy was subsequently investigated in a further study.25 Effect of Phenolic Compounds on RCS Levels. To further elucidate the influence of the phenolic structure reactivity on the generation of the Maillard-type off-flavor compounds, the RCSs (key transient sugar fragments) were also quantified. The concentrations of dicarbonyl and hydroxycarbonyl sugar fragments as influenced by UHT processing, phenolic addition, and storage are illustrated in Figures 4 and 5, respectively. Overall, UHT processing resulted in increased concentrations of RCSs when compared to both raw and pasteurized milk. This was expected, as the UHT conditions would catalyze the MR and subsequently the generation of MR intermediates such as RCSs. Examination of the sugar fragments in the control UHT sample

The concentrations of methional, 2AP, and 2A2T were determined after 30 days of storage at 30 °C in UHT milk as well as in the UHT milk with catechin (see Figure 3). On the basis of

Figure 3. Concentration of off-flavor markers 2-acetyl-1-pyrroline (2AP), methional, and 2-acetyl-2-thiazoline (2A2T) in raw (R), pasteurized (P), control UHT milk (CNTRL), and UHT milk with added catechin (CAT), genistein (GEN), daidzein (DAID), 1,3,5trihydroxybenzene (135THB), and 1,2,3-trihydroxybenzene (123THB) as determined right after UHT processing (day 0) and after 30 days of storage in 30 °C (day 30). The same letters indicate no statistical significant differences (p < 0.05). Lower case letters correspond to day 0 treatments, and upper case letters correspond to day 30 treatments.

these quantitative off-flavor fingerprints, the corresponding aroma recombination models of UHT and UHT with catechin samples were made in pasteurized (HTST) milk and evaluated for the cooked flavor intensity (see Table 3). The UHT aroma recombination model was not significantly different in cooked flavor intensity in comparison to commercial UHT milk and was significantly higher in cooked flavor intensity compared to both commercially pasteurized (HTST) milk and the model UHT milk that contained catechin. Overall these findings supported the utilization of these three Maillard-type off-flavor compounds Table 3. Average Cooked Flavor Intensity of Commercial Milk and Off-Flavor Recombination Model Systems sample commercial 1% UHT milk (Parmalat) off-flavor recombination model of 1% UHT milk commercial 1% pasteurized milk off-flavor recombination model of UHT-catechin containing (1.72 mM) a

mean rating

homogenous groupsa

5.5 5.3 1.1 0.8

A A B B

Fisher’s LSD = 0.319 (5% significance level), n = 8 in duplicate. 8027

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Figure 4. Concentrations of α-diacarbonyls (glyoxal, methylglyoxal, 3-deoxyglucosone, and diacetyl) in raw (R), pasteurized (P), control UHT milk (CNTRL), and UHT milk with added catechin (CAT), genistein (GEN), daidzein (DAID), 1,3,5-trihydroxybenzene (135THB), and 1,2,3trihydroxybenzene (123THB), as determined immediately after UHT processing (day 0) and after 30 days of storage in 30 °C (day 30). Each compound was monitored as quinoxaline derivatives and run in triplicate. The same letters indicate no statistical significant differences (p < 0.05)). Lower case letters correspond to day 0 treatments, and upper case letters correspond to day 30 treatments. All treatments were significantly different at day 30 when compared to day 0 (p < 0.05).

Figure 5. Concentration of α-hydroxycarbonyls (glycolaldehyde, acetol, and acetoin) in raw (R), pasteurized (P), control UHT milk (CNTRL), and UHT milk with added catechin (CAT), genistein (GEN), daidzein (DAID), 1,3,5-trihydroxybenzene (135THB), and 1,2,3-trihydroxybenzene (123THB), as determined immediately after UHT processing (day 0) and after 30 days of storage in 30 °C (day 30). Each compound was monitored as ethoxylamine derivatives and run in triplicates. The same letters indicate no statistical significant differences (p < 0.05). Lower case letters correspond to day 0 treatments, and upper case letters correspond to day 30 treatments. Unless otherwise indicated, all treatments were significantly different at day 30 when compared to day 0 (p < 0.05).

after storage indicated that two of the dicarbonyl compounds, namely, methylglyoxal and diacetyl, increased, whereas glyoxal and 3-deoxylglucosone decreased; all hydroxylcarbonyls in-

creased (glycolaldehyde and acetol) or maintained similar levels (acetoin). The observed reduction in the concentration of glyoxal and 3-deoxyglucosone could be indicative of the high 8028

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Hydroxycarbonyls. The effect of phenolic treatment on the levels of hydroxycarbonyl compounds also reported unique structure reactivity, but the observed suppression effects were not as pronounced. All phenolic compounds significantly (p < 0.05) reduced the levels of glycolaldehyde with the more reactive phenols being catechin, genistein, and 1,3,5-trihydroxybenzene. Only catechin, genistein, and 1,3,5-trihydroxybenzene significantly reduced acetoin, and for acetol no significant (p > 0.05) changes were observed for the phenolic treatments when compared to control UHT milk. The overall noted reduction in phenolic reactivity for the hydroxycarbonyl compounds was anticipated. Hydroxylcarbonyl compounds are relatively weaker electrophiles and, thus, are less likely to be trapped by phenolic compounds. From the three hydroxycarbonyls investigated, glycolaldehyde is the most electrophilic and, as predicted, was the most influenced by phenolic addition. Phenolic Trapping Reactivity. Overall, on the basis of the predicted reactivity of the selected phenolic compounds toward aromatic electrophilic substitution reactions and RCS trapping, catechin, as a flavanol, was expected to be significantly more reactive as compared to isoflavones such as genistein and daidzein. However, due to the complexity of food systems, phenolic-carbonyl reactions are anticipated to be influenced by multiple factors such as phenolic stability and competing reactions and hence why this general trend (based on aromatic electrophilic substitution reactions) was not always observed. In this study, genistein demonstrated overall similar reactivity toward trapping RCSs as compared to catechin. This observation could potentially be explained by the increased autoxidation reactivity of catechin resulting in the formation of quinones, thus leading to greater reactivity with milk proteins and other milk constituents and reduced RCS trapping reactivity. Among the simple phenolic compounds, 135THB was more effective in reducing the levels of RCSs, which is in agreement with previously findings.33 1,3,5-Trihydroxybenzene would be expected to be a more reactive trapping agent of sugar fragments than 123THB on the basis of the polyhydroxyl ring configuration. 135THB has three electron-donating hydroxyl groups in the meta configuration on a benzene ring, which activate the ortho and para positions for electrophilic aromatic substitution reactions at the three substituted carbon sites (e.g., RCSs). Consequently, the noted higher reactivity of 135THB suggests that one of the mechanisms by which phenolic compounds alter the flavor generation pathways in UHT milk is the formation of adducts with these precursors as previously hypothesized by Totlani and Peterson.34 In general, the observed reduction in the concentration of RCSs correlated with both analytical and sensorial data regarding the inhibition of off-flavor generation in UHT milk as influenced by the addition of phenolic compounds when compared with traditional UHT milk. The noted improvement in the consumer acceptability of UHT milk treated with phenolic compounds (catechin) could also be due to, in part, the reduction in the concentrations of the RCS. Many of the RCS have unique (off)-flavor attributes. Diacetyl has been identified as a contributor to the heated, cooked, and fermented notes of UHT milk with a flavor threshold of 12 ppb (0.14 μM),35−37 and notably diacetyl concentration in control UHT milk after 30 days of storage was above this flavor threshold value. Glyoxal and methyglyoxal have mild sour, pungent, and slightly nutty aromas. Acetol is characterized as sweet and roasted or as having a yogurt-like flavor in emulsions, and acetoin has a sweet, buttery, creamy aroma.38−41

reactivity of glyoxal entering several reaction pathways leading to off-flavor formation, and for 3-deoxyglucosone its further fragmentation to shorter chain sugar fragments further contributed to MRP generation. In general, the addition of phenolic compounds prior to UHT processing resulted in reduced levels of these MR precursors (RCSs) compared to the control UHT milk samples. The influence of phenolic compounds on the reduction of RCSs indicated that the different structures have unique reactivity and was dependent on the structure of the dicarbonyl or hydroxycarbonyl compound, as well as on the temperature of the system (UHT processing temperature, 140 °C, or storage, 30 °C). Dicarbonyls. Overall, all of the food phenolic compounds (catechin, genistein, and daidzein) reported a significant effect (p < 0.05) compared to control UHT milk on the levels of dicarbonyl compounds during processing and storage. Catechin, genistein, and 135THB were the most effective phenolic treatments in reducing the concentration of glyoxal after 30 days of storage followed by diadzein. These observations were expected on the basis of the reactivity of the phenolic structures toward aromatic electrophilic substitution (Figure 2). Genistein and 135THB were the most reactive phenolic compounds during UHT processing, demonstrating the highest reduction of glyoxal as compared to control UHT milk. Similarly for methylglyoxal, addition of catechin, genistein, or 135THB significantly reduced the concentration of this RCS after 30 days of storage. Additionally, both catechin and genistein were effective in reducing the concentration of methylglyoxal during storage to concentrations lower than observed right after processing (day 0), and it is noteworthy these levels are not significantly different from concentrations found in pasteurized milk demonstrating unique reactivity as compared to other phenolic treatments. The most reactive phenolic compound for suppressing the levels of methylgloxyal during UHT processing was 135THB. Similarly, 135THB was also the most reactive phenolic treatment in reducing the concentration of 3-deoxyglucosone during both UHT processing and storage, followed by catechin. Notably, these two phenolic treatments resulted in 3deoxyglucosone levels that were not significantly different from or lower than those found in pasteurized and raw milk. The increased reactivity of 135THB as compared to catechin could also be explained by reduced steric hindrances. Overall, genistein had reduced reactivity toward C6 sugar fragments. The reduced reactivity of both genistein and diadzein as compared to catechin and 135THB was expected due to the presence of a keto group at the C-5 position on ring C that can decrease nucleophilicity of the activated ortho positions of ring A (C-8 and C-6) (Figure 2) and thus its sugar fragment trapping reactivity. The increased reactivity of daidzein as compared to genistein could potentially be attributed to reduced steric hindrance due to the absence of the hydroxyl group at the C-5 position of the A-ring (Figure 2). Diacetyl was effectively reduced during UHT processing by all phenolic treatments except diadzein, which was not significantly different from the control UHT milk demonstrating negligible reactivity for this C4 sugar fragment. Catechin was the most reactive phenolic treatment in reducing the concentration of diacetyl during storage to levels below those observed at day 0. Findings again demonstrate unique phenolic structure reactivity. For all other phenolic treatments, the concentration of diacetyl continued to increase during storage, albeit at reduced rates when compared to the control. 8029

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Figure 6. MS/MS fragmentation spectra for catechin−sugar fragment adduct reaction products. Spectra correspond to catechin−glyoxal, catechin− glyoxal−catechin, catechin−methylglyoxal, methylglyoxal−catechin−methylglyoxal, and catechin−3-deoxyglucosone adducts from top to bottom. ∗ denotes alternative position activated for aromatic electrophilic substitution. RCSs substitution can occur at either the C-6 or C-8 position of the A-ring. Only one structure is presented for simplicity.

Phenolics−RCSs Adducts. To further support the hypothesis of adduct formation as a mechanism for off-flavor inhibition, model reaction systems were prepared and adducts between catechin and glyoxal, methylglyoxal, and 3-deoxyglucosone were purified to develop analytical methods for their identification in UHT milk with added catechin. The most prominent products of these reactions were identified as catechin−glyoxal, catechin− glyoxal−catechin, catechin−methylglyoxal, methylglyoxal−catechin−methylglyoxal, and catechin−3-deoxyglucose adducts. The proposed reaction product structures shown in Figure 7 were based on prior literature27,42,43 and MS/MS fragmentation patterns (Figure 6). The parent ions of all purified products/ adducts generated daughter ions of m/z 289, which corresponds to a catechin moiety along with fragments of m/z 245, 205, and 179, which are characteristic daughter ions of catechin fragmentation44 confirming its presence in these products. The m/z 245 fragment may be generated after a neutral loss of CO2, characteristic of negative ionization mode.45 The m/z 205 fragment is most likely generated by loss of rings B and C,46 and m/z 179 is produced after an A-ring loss. This particular fragmentation pattern (loss of A-ring) supports the adduct formation between catechin and RCSs via electrophilic aromatic substitution at C-8 or C-6 of ring A as well as the proposed adduct structures because fragments that correspond to a loss of

ring A with RCS moieties were detected for all adducts. Namely, m/z 167 (A-ring−glyoxal), m/z 181 (A-ring−methylglyoxal), m/ z 253 (A-ring−methylglyoxal2), and m/z 271 (A-ring−3deoxyglucosone) were detected as daughter ions of catechin− glyoxal and catechin−glyoxal−catechin, catechin−methylglyoxal, methylglyoxal−catechin−methylglyoxal, and catechin−3deoxyglucosone adducts, respectively. MRM methods were developed, and the detection of all adducts in aseptically processed UHT milk with added catechin was achieved. An example chromatogram of the detection of catechin−glyoxal and catechin−methylglyoxal adducts is shown in Figure 8. Multiple peaks in chromatograms were anticipated to be due to four diastereoisomers of catechin forming during thermal processing reacting with RCSs. Catechin−methylglyoxal adducts resulted in more than four peaks as the addition of methylglyoxal added another chiral center to the molecule, thus increasing the number of possible products. RCSs and Off-Flavor Causality. To demonstrate causality between increased levels of RCSs and off-flavor generation, RCSs were additionally added to milk prior to UHT processing and the levels of off-flavor volatiles were monitored. More specifically, pasteurized milk (control) and pasteurized milk spiked with levels of RCSs reported in UHT milk glyoxal (GO) 60 μM, methylglyoxal (MGO) 20 μM, diacetyl 0.4 μM, and 38030

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Figure 7. Hypothesized MS/MS fragmentation pattern of adducts between catechin and glyoxal, methylglyoxal, and 3-deoxyglucosone. ∗ denotes predicted main alternative position for aromatic electrophilic substitution based on prior literature.27,42,43 RCSs substitution can occur at either the C-6 or C-8 position of the A-ring. Only one structure is presented for simplicity.

Figure 9. Concentration of off-flavor markers 2-acetyl-1-pyrroline (2AP), methional, and 2-acetyl-2-thiazoline (2A2T) in control UHT milk and UHT milk with added glyoxal (65 μM), methylglyoxal (20 μM), diacetyl (0.4 μM), and 3-deoxyglucosone (12 μM), as determined right after UHT processing (day 0). Figure 8. LC-MS/MS (ESI negative mode) chromatograms of detection catechin adducts with glyoxal (top,m/z 347 → 167) and methylglyoxal (bottom, m/z 361 → 181) in UHT milk with catechin. A suggested reaction product is illustrated.

In summary, phenolic compounds were reported to significantly suppress MR off-flavor pathways in UHT milk during thermal processing and storage, resulting in a product with increased consumer acceptability. Furthermore, the results of this study support phenolic trapping reactions of Maillardgenerated sugar fragments as an important mechanism of offflavor suppression. This work further defined the interplay between phenolic chemistry and the mechanisms of the MR in foodstuffs. Unique phenolic−structure reactivity was also reported for the suppression of Maillard chemistry in UHT milk; different phenolic structures were more reactive during thermal processing versus storage in reducing both RCSs and offflavor markers. Overall, these results support the potential of phenolic compound mixtures or food extracts rich in phenolic compounds (e.g., cocoa, tea, coffee, grape, soy) as treatments to

deoxyglucosone (3DG) 10 μM) were subsequently UHT processed, and the concentrations of the selected off flavor markers were determined immediately after processing. The results are shown in Figure 9 and demonstrate that increased levels of RCSs resulted in significantly higher levels of off-flavor markers, as compared to the control. More specifically, 2AP, methional, and 2A2T were increased by 77.5, 74.2, and 42.1%, respectively, when milk was spiked with RCSs prior to UHT processing, thus demonstrating causality and further supporting the formation of phenolic−RCS adducts as one of the main mechanisms of phenolic off-flavor inhibition. 8031

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improve the flavor stability and nutritional value of thermally processed foods and in this case milk and milk-based beverages. Self-affirmed generally regarded as safe (GRAS) phenolic ingredients such as Teavigo (flavanol) and geniVida (isoflavone) are currently commercially available (DSM, NL).

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AUTHOR INFORMATION

Corresponding Author

*(D.G.P.) E-mail: [email protected]. Phone: (612) 624-3201. Fax: (612) 625-5272. Funding

This work was supported in part by Dairy Management, Inc., as managed by Dairy Research Institute (Rosemount, IL, USA). Notes

The authors declare no competing financial interest.

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