Effects of polyphenols on volatile profile and acrylamide formation in a model wheat bread system

Food Chemistry 297 (2019) 125008

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Effects of polyphenols on volatile profile and acrylamide formation in a model wheat bread system

T

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Sylwia Mildner-Szkudlarza, , Maria Różańskaa, Paulina Piechowskaa, Agnieszka Waśkiewiczb, Renata Zawirska-Wojtasiaka a b

Institute of Food Technology of Plant Origin, Faculty of Food Science and Nutrition, Poznań University of Life Sciences, Wojska Polskiego 28, 60-637 Poznań, Poland Department of Chemistry, Faculty of Wood Technology, Poznań University of Life Sciences, Wojska Polskiego 75, 60-625 Poznań, Poland

A R T I C LE I N FO

A B S T R A C T

Keywords: Phenolic compounds Bread Acrylamide GC–MS Maillard reaction Lipid oxidation

The formation of toxic and potentially carcinogenic acrylamide, alongside volatile aroma compounds, was studied after polyphenols ((+)-catechin, quercetin, gallic, ferulic, caffeic acids) were added to model bread. The addition of as little as 0.1% polyphenols to bread significantly reduced acrylamide (16.2–95.2%). In the case of quercetin, a promoting effect was observed (+9.8%) when its concentration increased. Of all the phenolic compounds, regardless of concentration, ferulic acid showed the highest level of acrylamide inhibition. This is probably due to the presence of 4-vinylguaiacol, a degradation derivative with strong antioxidant activity in heterogeneous systems. Although the phenolic compounds mitigate acrylamide, this adversely affected bread volatile profile. At the highest level (2.0%), caffeic acid most significantly suppressed Maillard-type volatiles (75.9%), followed by gallic acid (74.3%), ferulic acid (65.6%), (+)-catechin (62.4%), and quercetin (59.3%). Among polyphenols, ferulic acid decreased yeast fermentation products level the most (33.1%), simultaneously enhancing lipid oxidation product, probably due to inhibition of amylases and yeast activity.

1. Introduction

and potential carcinogen in humans. Moreover, volatile furan and furanic compounds have received considerable attention due to their hepatotoxic activity (Zheng et al., 2015). Existing strategies to reduce acrylamide formation include three basic aspects: (i) modification of raw materials, (ii) optimization of processing conditions, and (iii) addition of exogenous additives (Jin, Wu, & Zhang, 2013). Antioxidants have been proposed as effective inhibitors of acrylamide in thermally treated systems (Favreau-Farhadi, Pecukonis, & Barrett, 2015; Jin et al., 2013; Cheng et al., 2009). However, the literature presents confusing results, with some antioxidants either inhibiting or enhancing Maillard reaction-derived chemical hazards. For example, grape seed extract was not effective in inhibiting acrylamide in the study of Açar and Gökmen (2009), but was effective in the investigation of Xu et al. (2015). Since the mitigation mechanism still remains unclear, due to the chemical diversity of polyphenols and the complexity of composition in plant samples, the effect of pure phenolic compounds should be taken into consideration. Also, since reaction conditions, concentrations, and polarities might affect the final levels of acrylamide, research aimed at reducing its level during heat processing of real food matrices is of significant interest. Although phenolic compounds may reduce dietary contents of

Bread has been a central constituent in the diets of most populations for thousands of years. Among the different properties of bread that define its quality, aroma plays a key role in its acceptance by consumers (Moskowitz, Bin, Elias, & Peterson, 2012; Paraskevopoulou, Chrysanthou, & Koutidou, 2012). The aroma of bread crust, as one of the most important attributes, is critical to consumer acceptance (Pico, Bernal, & Gómez, 2015). A long list of compounds has been detected in the aroma fraction of bread, including aldehydes, alcohols, acids, esters, ketones, hydrocarbons, pyrazines, pyrroles, furans, and lactones originating in the Maillard reaction and lipid oxidation, with a third pathway resulting from yeast fermentation (Nor Qhairul Izzreen, Hansen, & Petersen, 2016; Pico et al., 2015; Poinot et al., 2008). Although thermal processing induces reactions that have significant effects on flavor and taste, several unfavorable Maillard reaction-derived chemical hazards—such as acrylamide, heterocyclic aromatic compounds, and advanced glycation end products—are simultaneously formed (Mildner-Szkudlarz et al., 2017; Nguyen, van der Fels-Klerx, & van Boekel, 2017; Zheng, Chung, & Kim, 2015; Oral, Dogan, & Sarioglu, 2014). One of the better known of these, acrylamide, is a neurotoxin

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Corresponding author. E-mail address: [email protected] (S. Mildner-Szkudlarz).

https://doi.org/10.1016/j.foodchem.2019.125008 Received 14 March 2019; Received in revised form 5 June 2019; Accepted 12 June 2019 Available online 13 June 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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analysis was performed using an Agilent 5975C gas chromatograph connected to a triple-axis detector, following a previously developed method (Mildner-Szkudlarz et al., 2017). For quantitative calculation, pyrazine-d4 was used as an internal standard. All samples were spiked with 100 µL of standard pyrazine-d4 solution, providing a sample concentration of 200 ng/g. Volatile compounds were identified by comparing their retention indices (RI) and mass spectra with standards from Sigma Aldrich, Steinheim, Germany or, in some cases, only tentatively through a search of the NIST MS Search 2.0 mass spectral library.

acrylamide, they may also adversely affect the volatile profiles of the final products. However, few studies have investigated the effects of antioxidants on flavor formation in real food systems (Sordini et al., 2019; Zhao, Yao, & Ou, 2017; Favreau-Farhadi et al., 2015; Zheng et al., 2015). This study was thus designed to determine the effects of different levels of phenolic compounds (0.1%, 0.5%, 1.0%, and 2.0%, w/w of gallic acid, ferulic acid, caffeic acid, (+)-catechin, and quercetin) on the formation of acrylamide and volatile compounds through the Maillard reaction, lipid oxidation, and yeast fermentation in model bread samples.

2.4. Statistical analysis 2. Materials and methods All data are reported as means ± standard deviations from at least triplicate measurements. One-way analysis of variance (p < 0.05) using Tukey’s HSD test was conducted to determine the differences between the mean values of the tested compounds. Cluster analysis was carried out to observe trends in the data. The data were statistically analyzed using Statistica 13.0 software (Dell Software, USA).

2.1. Preparation of bread model system Model bread was prepared according to a previously described procedure (Mildner-Szkudlarz et al., 2017). A basic bread formula for 500 g of wheat flour was: 300 g water, 15 g fresh yeast, and 7.5 g fine salt. Phenolic compounds—gallic acid, ferulic acid, caffeic acid, (+)-catechin, and quercetin (purity ≥ 95%; Sigma-Aldrich, Steinheim, Germany)—were added at levels of 0.1%, 0.5%, 1.0%, and 2.0% (w/w). After kneading, the dough was allowed to ferment for 60 min at 35 °C and at 75% relative humidity. The dough was then divided into pieces of equal weight (380 g), hand-molded, proofed for 20 min (35 °C and 75% RH), and baked for 30 min at 210 °C. After baking, bread was cooled to room temperature over 2 h. Subsequently, the bread was sliced about 1.5 cm thick, and the crust and crumb were carefully separated and kept frozen for later analysis.

3. Results and discussion 3.1. Effects of phenolic compounds on acrylamide formation in the model bread system The control wheat bread crust contained 65.4 µg/kg of acrylamide (Fig. 1, Supplementary data 1). Surdyk, Rosén, Andersson, and Åman (2004) reported a similar value for the crust of yeast-leavened wheat bread baked at 270 °C for 15 min (80 µg/kg), but significantly higher value was found by Wang, Yu, Xin, Wang, and Copeland (2017) for the crust of wheat bread baked at 220 °C for 25 min (570 µg/kg). The level of acrylamide in the crumb (24.2 µg/kg) was significantly smaller than that reported by Wang et al. (2017) (270 µg/kg). However, according to Surdyk et al. (2004) the inner temperature of the crumb does not exceed 100 °C, on account of low thermal conductivity of the dough; they suggested that the acrylamide detected in the crumb originated from parts of the crust remaining in the crumb due to the incomplete separation of the crust. As shown in Fig. 1, quercetin, (+)-catechin, and ferulic acid exhibited inhibitory effects at the lowest concentration (0.1%), while gallic acid and caffeic acid did so at the highest level (2.0%). Generally, at the lowest concentration (0.1%), the inhibition of acrylamide by phenolic compounds followed the sequence: gallic acid-formulated bread (54.8 µg/kg bread crust and 25.2 µg/kg bread crumb) was first, followed by quercetin-formulated bread (46.0 µg/kg bread crust and 19.8 µg/kg bread crumb); next came caffeic acid-formulated bread (18.9 µg/kg bread crust and 20.0 µg/kg bread crumb) and (+)-catechin-formulated bread (12.9 µg/kg bread crust and 15.5 µg/kg bread crumb), ending with ferulic acid-formulated bread (3.14 µg/kg bread crust and 3.12 µg/kg bread crumb). On the other hand, at the highest treatment level (2.0%) the ability of phenolic compounds to reduce acrylamide formation decreased in the following order: caffeic acidformulated bread (3.00 µg/kg bread crust and 5.83 µg/kg bread crumb) and ferulic acid-formulated bread (4.27 µg/kg bread crust and 5.18 µg/ kg bread crumb), then gallic acid-formulated bread (21.2 µg/kg bread crust and 16.1 µg/kg bread crumb), (+)-catechin-formulated bread (40.2 µg/kg bread crust and 22.9 µg/kg bread crumb), and quercetinformulated bread (76.3 µg/kg bread crust and 28.0 µg/kg bread crumb). Polyphenolics have been proven to be both positive and negative in acrylamide formation (Jin et al., 2013). Oral et al. (2014) found that the addition of caffeic acid reduced acrylamide formation by 30.8%, whereas oleuropein increased its formation at levels of 9.5% in a fructose–asparagine model system at 180 °C. Recently, Sordini et al. (2019) found that phenolic extract from olive mill waste water, rich in oleuropein, ligstroside, and demethylcarboxyoleuropein, reduced acrylamide production by about 40% in French fried potatoes after 6 h of the frying process.

2.2. Analysis of acrylamide by GC–MS Bread samples were prepared for acrylamide analysis using the method reported by Andrzejewski, Roach, Gay, and Musser (2004), with some modifications. About 1 g of sample was weighed into a 15mL centrifuge tube, and spiked with 1 mL (1 µg/mL) of 13C3-acrylamide (Sigma-Aldrich, Steinheim, Germany) as an internal standard; this was mixed with an appropriate amount of ultrapure water (9 mL). The mixture was homogenized for 5 min and centrifuged at 10,000g for 20 min at 5 °C (Universal 320 R, Hettich Zentrifugen) and filtered through a syringe filter (nylon membrane, 0.20 μm). The filtrate (2 mL) was gravitationally passed through an Oasis HLB cartridge (Waters, Milford, MA), and was followed with another 1.0 mL of deionized water. The eluent was discarded. The Oasis HLB cartridge, loaded with sample solution, was then connected in tandem to an Oasis MCX cartridge (Waters, Milford, MA). The eluent was collected in a 10-mL test tube and reduced by a gentle steam of nitrogen before being transferred to 2-mL autosampler vials for further LC–MS/MS analysis. LC–MS-MS analysis was performed using a Waters Acquity UPLC chromatograph connected to triple-quadrupole mass spectrometer equipped with an electrospray source (in positive ion mode) and Empower 2 software (Waters Corp., Milford, MA). The analytical column was a Waters Atlantis dC18 column (2.1 × 150 mm), the mobile phase was 10% methanol with 0.1% formic acid, and the flow rate was 0.2 mL/min. MS/ MS conditions were: capillary voltage of 2.5 kV, cone voltage of 20 V, source temperature of 120 °C, desolvation gas temperature of 250 °C; and flow rates of cone and desolvation gas (nitrogen) of 100 and 800 L/ h, respectively; the collision gas was argon. Multiple reaction monitored mode (MRM) was acquired with the characteristic fragmentation transitions m/z 72 → 55 for acrylamide and m/z 75 → 58 for 13C3-acrylamide. 2.3. Analysis of volatile compounds The volatile compounds of the bread crust were isolated using SPME fiber Carboxen/polydimethylsiloxane (CAR/PDMS; Supelco, Bellefonte, PA), as in Pacyński, Wojtasiak, and Mildner-Szkudlarz (2015). GC–MS 2

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study of Xu et al. (2015), who reported that ellagic acid most strongly inhibits acrylamide formation in an equimolar asparagine–glucose chemical model, followed by catechin, epicatechin, epicatechin gallate, caffeic acid, gallic acid, and quercetin, in that order. They suggested that higher antioxidant activity does not necessarily mean stronger acrylamide inhibiting ability, and the inhibition mechanism of antioxidants against acrylamide formation may need to be further elucidated. The same observation was made by Bassama et al. (2010), who suggested that the non-positive correlation between the antioxidant activity of phenolic compounds and the mitigation of acrylamide formation might be due to formation of phenolic compound degradation derivatives that possess antioxidant activity. Yuan, Shu, Zhou, Qi, and Xiang (2011) suggested that not only the type but also the concentration of phenolic compounds might play a significant role in formation pathway of acrylamide. They found that at lower concentrations of 0.2%, 0.5%, 0.8% and 1.2%, ascorbic acid inhibited acrylamide formation, with a highest inhibitory rate of 58%, while at a concentration of 1.5%, promoting effect was observed in a glucose–asparagine reaction model. In this study, quercetin significantly mitigated the acrylamide level, though only at the lowest level; at higher levels, a promoting effect was noticed (+9.8%). Zhang and Zhang (2008) reported a non-linear relationship between the reduction rate and the addition level of antioxidant, the so-called “antioxidant paradox” that might ascribe both antioxidant and pro-oxidant properties to phenolic extracts. This phenomenon could be linked to the fact that higher concentrations of polyphenols, especially catechol compounds, generate hydrogen peroxide involved in the formation pathway of acrylamide (Fujiwara et al., 2011). Moreover, it is known that, in heterogeneous systems, phenolic compounds are partitioned between the hydrophobic and hydrophilic phases, as well as interfacial environments, which might significantly influence their activity. The proportion of antioxidants in the aqueous phase of heterophasic systems reported by Schwarz, Frankel, and German (1996) was greatest for (+)-catechin (97.7%) and gallic acid (97.3%), followed by caffeic acid (90.4%) and ferulic acid (77.3%); approximately the reverse order held for acrylamide inhibition effectiveness. This suggests that affinities for the nonpolar phase might dominate the effectiveness of phenolic compounds in mitigating acrylamide in bread systems. As seen in the dendrogram in Fig. 2a, two different groups of results were obtained (70% cut-off point). The first cluster consists of the control bread and quercetin-formulated bread, indicating that quercetin is the weakest inhibitor of acrylamide level—in some cases, even increasing the net amount of acrylamide. The second cluster contains the remaining antioxidant-formulated samples, with two sub-groups formed by the ferulic acid and caffeic acid-formulated breads—the most potent inhibitors—as well as gallic acid- and (+)-catechin-formulated samples.

Fig. 1. Acrylamide concentration in model bread (a) crust and (b) crumb, with different concentrations of phenolic compounds. Sample codes: CB: control model bread; CB + Q: model bread with quercetin; CB + C: model bread with (+)-catechin; CB + CA: model bread with caffeic acid; CB + FA: model bread with ferulic acid; CB + GA: model bread with gallic acid. Results with different lowercase letters (comparing the same phenolic compounds at different concentrations), and different uppercase letters (comparing the same concentrations but different phenolic compounds; regular font for 0.1%, oblique font for 0.5%, bold font for 1.0%, and underlined font for 2.0%) are significantly different (p < 0.05).

Several mechanisms for the mode of action of phenolic compounds against the formation of acrylamide have been proposed, including trapping of reactive intermediates, antioxidant activity, and asparagine binding inhibition and competition (Oral et al., 2014; Bassama, Brat, Bohuon, Boulanger, & Günata, 2010; Cheng et al., 2009). Cheng et al. (2009) found that naringenin, a citrus flavonoid, significantly and dosedependently inhibited the formation of acrylamide by trapping asparagine-derived intermediates at the –C6 and –C8 positions of the A ring, forming two new derivatives, 8-C-(E-propenamide)-naringenin and 6-C(E-propenamide)-naringenin. It was hypothesized that polyphenols may also inhibit acrylamide formation, because of their strong antioxidant activity. For example, Kotsiou, Tasioula-Margari, Capuano, and Fogliano (2011) found that the mitigation effect could be correlated to the number of hydroxyl phenyl groups, in the decreasing order of tri, di, and mono hydroxyl group. In this study, regardless of concentration, ferulic acid showed the highest level of inhibition (∼89% reduction for crust and crumb), followed by caffeic acid (∼68% reduction for crust and crumb), (+)-catechin (∼42% reduction for crust and crumb), gallic acid (∼21% reduction for crust and crumb), and quercetin (∼1% increase for crust and crumb). Overall, there was no structure–antioxidant activity relationship for particular phenolic compounds with their reduction effects, which means that direct trapping of acrylamide precursors is a more probable mechanism of phenolic compound action at high temperatures in bread matrices. This is in line with the

3.2. Effects of phenolic compounds on the formation of volatiles generated through Maillard reaction A total of eleven Maillard-type volatile compounds at 3456 µg/kg were detected in the control bread crust (Fig. 3, Table 1). As demonstrated, phenolic compounds exhibited the strongest inhibitory activities on Maillard-type volatiles at the highest concentrations (2.0%). Gallic acid and (+)-catechin were the most effective antioxidants at reducing the levels of pleasant flavors, such as 2-methylpropanal, in the bread crust (∼68%), followed by caffeic acid (∼50%), ferulic acid (∼49%), and quercetin (∼47%) (Fig. 3a). This Strecker degradation product of valine with a malty-sweet note, can be derived from oxidative deamination and decarboxylation of α-amino acids in the presence of α-dicarbonyl compounds generated during the Maillard reaction (Zheng et al., 2015). Thus, phenolic compounds might react with dicarbonyls and form adducts, consequently inhibiting the formation of Strecker aldehydes. Additionally, the formation of furan derivatives—such as 23

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the fermentation process (Poinot et al., 2008). Furfural, a compound with a characteristic almond, and bread-like note (Nor Qhairul Izzreen et al., 2016), was present in the blank bread crust at a level of 565 µg/ kg. At the highest treatment, caffeic acid decreased the level of furfural the most (79%), followed by (+)-catechin (74%), gallic acid (70%), and quercetin and ferulic acid (∼59%). Similarly, in the study of Oral et al. (2014), the addition of phenolic compounds like caffeic acid, ellagic acid, chlorogenic acid, epicatechin, punicalagin, and tyrosol significantly reduced furfural formation in a glucose–glycine model system at 180 °C. Apart from their contribution to bread aroma, furan derivatives are also capable of further reaction, subsequently leading to the formation of many important classes of flavor compounds characterized by a low detection threshold. It is known that the cyclization of α-aminocarbonyls from Strecker degradation forms pyrazines. A total of seven pyrazines: 2-methylpyrazine, 2,6-dimethylpyrazine, 2-ethylpyrazine, 2-vinylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3-methylpyrazine, and 5-methyl-2-vinylpyrazine were noted in the control bread (Table 1). At the highest treatment level (2.0% w/w), caffeic acid most significantly decreased the levels of total pyrazines (81%), followed by gallic acid (75%), ferulic acid (70%), (+)-catechin (62%), and quercetin (60%). According to Pozo-Bayón, Ruíz-Rodríguez, Pernin, and Cayot (2007) 2,6-dimethylpyrazine and 2-vinylpyrazine are among the greatest contributors to the characteristic roasted cake aroma. In this study, the levels of 2,6-dimethylpyrazine with its green spicy odor, and of 2-vinylpyrazine with its boiled-potato-like note, were 125 µg/kg and 48.4 µg/kg in the blank crust, respectively (Table 1). However, 2-vinylpyrazine was absent, while 2,6-dimethylpyrazine was decreased by almost 70% after the addition of phenolic acid in the highest concentration. Apart from these, 2-ethyl-6-methylpyrazine and 2-ethyl-3methylpyrazine were also perceived as significant contributors to the wheat bread crust aroma (Paraskevopoulou et al., 2012). Our results indicate that the presence of antioxidants at the highest treatment level inhibited the formation of these two key odorants by about 52%. Favreau-Farhadi et al. (2015) postulated that pyrazines can only be formed when carbon fragments are intact, meaning that antioxidants might react with carbonyl precursors to inhibit further reactions. The results of Shao et al. (2014) point to the crucial influence of the number and position of the hydroxyl group on this trapping activity, showing that the more hydroxyl groups on the phenyl ring, the stronger the trapping efficiency, due to the nucleophilic reaction between the phenol and methylglyoxal. Thus, the structure–activity relationship of phenolic acid in targeting α-dicarbonyl species should be gallic acid > caffeic acid > ferulic acid. On the other hand, the structural ability of flavonoids to trap carbonyl is as follows: (i) the 5 –OH group in the A ring and (ii) the double bond between C-2 and C-3 on the C ring (Shao et al., 2014). In this regards, (+)-catechin belongs to flavan-3-ol with its 3 –OH group, but as it lacks a double bond on the C ring it does not fulfill the structural requirements of the latter; it should thus have a lower trapping ability than quercetin. The results demonstrate that the suppression of Maillard-type volatiles is not only the result of their trapping efficacy. Also, Wang (2000) did not establish a structure–activity relationship and found that, of the hydroxycinnamic acids, ferulic acid shows the highest reactivity for suppressing the flavor formation, followed by chlorogenic acid, and caffeic acid. It has been suggested that phenolic acid might scavenge Maillard-derived radicals (such as enaminols and pyrazinium radical cations) and thereby suppress Maillard product generation (Wang, 2000). It is well known that the pH at which the Maillard reaction occurs greatly influences the nature of the volatiles formed, and hence the flavor of the final product. The pH of control bread (5.85) slightly decreased to between 5.14 and 5.69 as a result of the addition of phenolic compounds (data not shown). According to the study of Martins, Marcelis, and Van Boekel (2003), an increase in pH from 5.5 to 6.8 had almost no influence on the formation of methylglyoxal, which is a reactive carbonyl species that plays an important role as a precursor of

Fig. 2. Hierarchical cluster analysis of the model bread samples, showing levels of (a) acrylamide and (b) volatile compounds. Normalization of the scale tree was performed to (Dlink/Dmax)*100 (D: distance; link: linkage; max: maximum linkage Euclidean distance). Amalgamation rule: Ward’s method; distance metric: Euclidean distances. Sample codes: CB: control model bread; CB + Q: model bread with quercetin; CB + C: model bread with (+)-catechin; CB + CA: model bread with caffeic acid; CB + FA: model bread with ferulic acid; CB + GA: model bread with gallic acid.

furanmethanol, 2-acetylfuran and 5-methylfurfural—was suppressed significantly by phenolic compounds (Fig. 3b,c,d). Maillard-type furanic compounds are formed through the breakdown of glycosylamine in the early stages of the reaction. Phenolic acids showed stronger inhibitory effect on furanic compounds formation than did flavonoids. At the highest concentration (2.0%), the order of phenolic compound reactivity in suppressing the formation of Maillard-type furan derivatives in model bread crust was gallic acid (85%) and caffeic acid (85%), followed by ferulic acid (71%), (+)-catechin (61%), and quercetin (58%). In the study of Zheng et al. (2015), chlorogenic acid was the most effective antioxidant at reducing the furan level in the coffee model systems (by 67%), followed by ferulic acid (58%), Trolox (50%), caffeic acid (48%), and (+)-catechin (45%). Additionally, 5-methylfurfural, a compound with a characteristic almond odor, was present in the blank crust at a level of 38.4 µg/kg but absent from all phenolic acid-formulated breads when the concentration was increased to 2.0% (Fig. 3d). The high level of this particular furanic compound might be sensorially important, due to its low odor threshold value (OT value 6 µg/L; Nor Qhairul Izzreen et al., 2016). All the phenolic compounds also exerted considerable inhibitory effects on the furfural level (Table 1). This odor-impact compound is mainly generated by the 1,2enolisation pathway via 3-deoxyosone, though it may also come from 4

Food Chemistry 297 (2019) 125008

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Fig. 3. Effect of phenolic compounds on the concentration (µg/kg) of volatile compounds resulting from Maillard reaction in the bread crust. Sample codes: CB: control model bread; CB + Q: model bread with quercetin; CB + C: model bread with (+)-catechin; CB + CA: model bread with caffeic acid; CB + FA: model bread with ferulic acid; CB + GA: model bread with gallic acid. Results with different lowercase letters (comparing the same phenolic compounds at different concentrations), and different uppercase letters (comparing the same concentrations but different phenolic compounds; regular font for 0.1%, oblique font for 0.5%, bold font for 1.0%, and underlined font for 2.0%) are significantly different (p < 0.05).

(Birch, Petersen, & Hansen, 2013), significantly increased with the addition of ferulic acid and gallic acid (Fig. 4 c,d). When ferulic acid was added at the highest concentration to the model bread, heptanal and nonanal increased by 17% and 91%, while the addition of gallic acid increased their levels by 32% and 14%, respectively. The results of Zhao et al. (2017) showed that the addition of feruloylated oligosaccharides at 50 mg/mL increased nonanal formation by a factor of 2.2 in a glucose–asparagine reaction model. Overall, at the highest treatment level (2.0%), quercetin and (+)-catechin were the most effective antioxidants, reducing heptanal and nonanal levels by approximately 32% and 63%, respectively; they were followed by caffeic acid (12% and 33%). Those components with low OT values (8 µg/L for heptanal and 1.1 µg/L for nonanal; Nor Qhairul Izzreen et al., 2016) could come from non-enzymatic oxidation of lipids during fermentation or from the decomposition during baking of hydroperoxides generated by lipoxygenases. According to Jensen, Oestdal, Skibsted, Larsen, and Thybo (2011), wheat bread stored for two and three weeks has greater intensities of chemical, acid and dusty aromas, as well as astringency and bitter taste, which is linked with the presence of nonanal and heptanal, as well as other aldehydes originating from lipid oxidation. It thus seems that these aldehydes confer unpleasant aromas when their concentration increases. 2-Heptanone reached a level of 62.1 µg/kg in the blank crust—close to the value reported by Paraskevopoulou et al. (2012) for wheat flour bread crust (67.6 µg/kg) (Fig. 4 b). Methyl ketones are formed by βoxidation of fatty acids followed by decarboxylation of β-ketoacids during thermal processes (Kellard, Busfield, & Kinderlerer, 1985). This lipid oxidation product was reduced by all phenolic compounds by 15–31%. However, due to relatively high OT value (140 mg/L; Nor

heterocyclic aroma compounds. On the other hand, deoxyosones, which are furan precursors, increased more rapidly at pH 5.5 than at pH 6.8 during heating at 120 °C (Martins et al., 2003). Moreover, according to Yu and Zhang (2010), low pH (pH 5.0) favored the formation of furans, while high pH values (from pH 8.00 to 9.55) promoted the production of pyrazines in a model reaction of L-threonine or L-serine with L-ascorbic acid. However, in this study, even though the pH of the bread with antioxidants was close to that of the control (or slightly lower), significant reduction of Maillard-type volatiles was nonetheless observed. It thus seems that the observed reduction is not pH-dependent.

3.3. Effects of phenolic compounds on the formation of volatiles generated through lipid oxidation Overall, in the blank bread crust, the lipid oxidation components hexanal, 2-heptanone, heptanal, and nonanal were observed at a level of 283 µg/kg (Fig. 4). According to Guinet and Godon (1994), hexanal, followed by heptanal and pentanal, is the major aliphatic aldehyde in wheat flour. Hexanal is a characteristic product of the cleavage of linoleate 13-OOH and 9-OOH hydroperoxide, generated by lipoxygenase (Pico et al., 2015). At the lowest concentration (0.1%), all phenolic compounds slightly reduced the generation of hexanal (∼12%) (Fig. 4 a). However, further increasing the antioxidant level resulted in an increase in its level by ferulic acid (∼32%) and gallic acid (∼66%). Due to its low OT value (4.5 µg/L; Nor Qhairul Izzreen et al., 2016) and its negative impact on wheat bread sensory quality, a high level of hexanal might not correlate positively with bread aroma. Similarly, the level of heptanal with its fatty/rancid and citrus/ malty odor (Pico et al., 2015), and of nonanal with its citrus odor 5

Food Chemistry 297 (2019) 125008

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Table 1 Effect of phenolic compounds on the concentration (µg/kg) of bread crust volatiles, grouped by origin. CB

CB + Q

CB + C

CB + CA

CB + FA

CB + GA

0.1% 0.5% 1.0% 2.0% 0.1% 0.5% 1.0% 2.0% 0.1% 0.5% 1.0% 2.0% 0.1% 0.5% 1.0% 2.0% 0.1% 0.5% 1.0% 2.0% 0.1% 0.5% 1.0% 2.0% 0.1% 0.5% 1.0% 2.0%

571 ± 12a,A 235 ± 9b,D 233 ± 2b,B 221 ± 6b,A 121 ± 3a,A 81.7 ± 2.6b,C 53.7 ± 3.0c,C 48.9 ± 2.6d,A 145 ± 6b,A 71.0 ± 3.8c,C 65.2 ± 2.3c,B 57.2 ± 2.7d,A 47.5 ± 2.4a,A 44.1 ± 1.5b,A 40.5 ± 1.4c,A 15.3 ± 0.1e,B 46.3 ± 2.2a,A 24.8 ± 1.1b,C 25.2 ± 1.1b,C 21.1 ± 0.2c,B 78.3 ± 3.1a,A 40.6 ± 1.4b,C 42.0 ± 1.6b,B 38.5 ± 1.4b,C 57.3 ± 2.8a,A 31.0 ± 1.5b,D 31.0 ± 1.7b,B 29.0 ± 0.9b,B

442 ± 7b,C 319 ± 8c,C 306 ± 3c,A 209 ± 3d,B 97.5 ± 3.8b,B 56.3 ± 1.8c,D 48.8 ± 2.6d,C 35.9 ± 2.0e,B 107 ± 6b,C 79.7 ± 4.7c,C 69.7 ± 4.7d,B 58.3 ± 3.4e,A 32.6 ± 1.3b,C 25.8 ± 1.2c,C 23.6 ± 0.5c,B 17.0 ± 0.6d,A 50.0 ± 2.8a,A 35.2 ± 0.4b,B 36.1 ± 1.3b,A 16.9 ± 0.7c,C 78.7 ± 2.8a,A 62.9 ± 0.2b,B 48.3 ± 0.3c,A 40.5 ± 0.4d,BC 51.7 ± 2.9b,B 36.3 ± 0.9c,C 34.2 ± 1.5c,B 34.2 ± 1.8c,A

579 ± 7a,A 572 ± 8a,A 106 ± 1b,C 65.3 ± 1.5c,E 122 ± 1a,A 124 ± 9a,A 26.7 ± 1.8b,D 23.3 ± 1.5b,C 149 ± 6a,A 146 ± 8a,A 66.7 ± 1.9b,B 33.6 ± 1.6c,B 47.8 ± 2.1a,A 43.4 ± 0.8b,A 7.72 ± 0.42c,D NDd,C 43.7 ± 2.2b,B 43.1 ± 1.9b,A 20.1 ± 0.9c,D 17.4 ± 0.9c,C 78.9 ± 2.9a,A 79.3 ± 2.6a,A 48.0 ± 1.6b,A 42.3 ± 0.6c,AB 57.1 ± 2.9a,A 55.6 ± 2.6a,A 31.0 ± 1.5b, B 27.5 ± 1.3b,B

433 ± 13b,C 423 ± 30b,B 322 ± 15c,A 146 ± 2d,C 104 ± 6b,B 104 ± 5b,B 72.9 ± 4.1c,B 49.6 ± 2.6d,A 114 ± 6b,BC 111 ± 4b,B 94.9 ± 3.0c,A 33.0 ± 0.5d, B 43.2 ± 1.7b,B 30.9 ± 1.1c,B NDd,E NDd,C 44.0 ± 2.4b,B 42.8 ± 2.3b,A 24.6 ± 1.2c,C 23.2 ± 1.2c,A 78.2 ± 3.1a,A 76.6 ± 2.9a,A 45.2 ± 1.5b,AB 44.8 ± 0.9b,A 52.0 ± 2.8b,AB 51.2 ± 2.6b, AB 31.4 ± 1.6c,B 28.6 ± 1.4c,B

486 ± 10b,B 454 ± 16b,B 122 ± 4c,C 108 ± 4c,D 126 ± 4a,A 119 ± 6ab,A 110 ± 6b,A 38.7 ± 2.0c,B 119 ± 2b,B 47.1 ± 1.8c,D 40.6 ± 1.6d,C 35.6 ± 1.4d,B 30.4 ± 0.4b,C 13.4 ± 0.5c,D 9.4 ± 0.3d,C NDe,C 43.9 ± 2.2b,B 43.9 ± 2.2b,A 30.3 ± 1.6c,B 22.0 ± 0.9d,AB 79.1 ± 2.1a,A 78.6 ± 2.9a,A 48.0 ± 1.7b,A 42.9 ± 1.6b,AB 50.1 ± 2.5b,B 48.3 ± 2.4b,B 45.3 ± 2.3b,A 22.5 ± 1.1c,C

Compounds resulting from Maillard reaction and lipid oxidation 2-Pentylfuran 194 ± 11a 0.1% 0.5% 1.0% 2.0% Benzyl alcohol 35.8 ± 1.8a 0.1% 0.5% 1.0% 2.0%

188 ± 6a,AB 158 ± 4b,C 155 ± 5b,C 105 ± 1c,E 36.4 ± 2.0a,B 30.7 ± 1.1b,C 30.4 ± 1.3b,C 24.0 ± 1.3c,C

173 ± 1ab,B 158 ± 9bc,C 149 ± 7 cd,C 133 ± 8d,D 37.2 ± 1.7a,B 31.5 ± 1.5b,C 29.2 ± 1.6 bc,C 25.2 ± 1.3c,C

192 ± 8a,A 189 ± 8a,B 183 ± 10ab,C 166 ± 1b,C 35.8 ± 2.2 a,B 19.5 ± 1.0b,D 12.7 ± 5.0c,D 8.39 ± 0.50d,D

172 ± .6a,B 386 ± 19b,A 386 ± 23b,A 382 ± 14b,A 46.9 ± 2.6 d,A 61.3 ± 2.5c,B 104 ± 3b,B 106 ± 6b,B

134 ± 8c,C 125 ± 1c,D 212 ± 10b,B 240 ± 13b,B 10.6 ± 0.6d,C 69.3 ± 3.8c,A 123 ± 7b,A 132 ± 8b,A

Compounds resulting from Maillard reaction and fermentation 3-Methylbutanal 1443 ± 85a 0.1% 0.5% 1.0% 2.0% a 2-Methylbutanal 1831 ± 114 0.1% 0.5% 1.0% 2.0% Furfural 565 ± 30a 0.1% 0.5% 1.0% 2.0%

1439 ± 83ab,A 1295 ± 50abc,AB 1269 ± 46bc,A 1147 ± 41c,A 1572 ± 59b,A 1542 ± 71b,A 1209 ± 72c,A 1109 ± 45c,AB 507 ± 24ab,B 453 ± 28b,A 248 ± 14c,B 218 ± 12c,A

1439 ± 21a,A 1158 ± 44b,B 998 ± 3c,B 847 ± 18d,BC 1538 ± 37b,A 1473 ± 73b,A 1140 ± 65c,A 1116 ± 63c,AB 350 ± 1b,C 321 ± 3b,B 269 ± 15c,B 146 ± 3d,BC

1487 ± 64a,A 1159 ± 26b,B 931 ± 39c, B 774 ± 27d,C 1328 ± 19b,B 1135 ± 33c,B 939 ± 19d,B 771 ± 27e,C 574 ± 31a,A 464 ± 24b,A 162 ± 8c,C 119 ± 4c,C

1445 ± 41a,A 1420 ± 86a,A 1381 ± 73ab,A 1194 ± 64b,A 1523 ± 78b,A 1475 ± 74b,A 1303 ± 70bc,A 1168 ± 63c,A 590 ± 29a,A 467 ± 28b,A 389 ± 23c,A 245 ± 13d,A

1452 ± 13a,A 1407 ± 66a,A 1377 ± 78a,A 944 ± 27b,B 1489 ± 76b,A 1263 ± 66c,B 1168 ± 65 cd,A 981 ± 85d,B 592 ± 24a,A 464 ± 27b,A 391 ± 11c,A 168 ± 16d,B

0.1% 0.5% 1.0%

ND ND ND

ND ND ND

ND ND ND

ND ND ND

2.0%

ND

ND

ND

1524 ± 116 12936 ± 792 21587 ± 2108 2107,63 43065 ± 939

Compounds resulting from the Maillard reaction 2-Methylpyrazine 578 ± 17a

2,6-Dimethylpyrazine

125 ± 1a

2-Ethylpyrazine

153 ± 4a

2-Vinylpyrazine

48.4 ± 2.2a

2-Ethyl-6-methylpyrazine

49.1 ± 2.5a

2-Ethyl-3-methylpyrazine

76.5 ± 1.3a

5-Methyl-2-vinylpyrazine

59.5 ± 3.1a

Thermal decarboxylation product of FA 4-Vinylguaiacol ND

ND

Values are mean ± SD of three replicates. Sample codes: CB: control model bread; CB + Q: model bread with quercetin; CB + C: model bread with (+)-catechin; CB + CA: model bread with caffeic acid; CB + FA: model bread with ferulic acid; CB + GA: model bread with gallic acid. Results with different lowercase letters (comparing the same phenolic compounds at different concentrations), and different uppercase letters (comparing the same concentrations but different phenolic compounds; regular font for 0.1%, oblique font for 0.5%, bold font for 1.0%, and underlined font for 2.0%) are significantly different (p < 0.05). ND is not detected.

balsamic note (Table 1). It can be seen that flavonoids and caffeic acid suppressed the generation of these two components, while ferulic acid and gallic acid significantly increased their levels (Table 1). When ferulic acid was added at 2.0% to the model bread, 2-pentylfuran and benzyl alcohol increased by 97% and 196%, while the addition of gallic

Qhairul Izzreen et al., 2016), 2-heptanone is rather less important in the final bread aroma. There are other odor-active components that can result from Maillard reactions or lipid oxidation, including 2-pentylfuran with a flavor of green beans and raw nuts, and benzyl alcohol with its fruity, 6

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Fig. 4. Effect of phenolic compounds on the concentration (µg/kg) of lipid oxidation products in bread crust. Sample codes: CB: control model bread; CB + Q: model bread with quercetin; CB + C: model bread with (+)-catechin; CB + CA: model bread with caffeic acid; CB + FA: model bread with ferulic acid; CB + GA: model bread with gallic acid. Results with different lowercase letters (comparing the same phenolic compounds at different concentrations), and different uppercase letters (comparing the same concentrations but different phenolic compounds; regular font for 0.1%, oblique font for 0.5%, bold font for 1.0%, and underlined font for 2.0%) are significantly different (p < 0.05).

in this study has a heterogeneous nature, consisting of separate phases, each with a predominant specific polymer—a gluten-rich phase and an arabinoxylan/soluble protein-rich phase. It thus seems that the solubility of phenols plays a subsidiary role. Pekkarinen, Stöckmann, Schwarz, Heinonen, and Hopia (1999) reported that, in bulk oil, the order of phenolic acid activity inhibiting hydroperoxide formation is as follows: caffeic acid and sinapic acid, followed by α-tocopherol, ferulic acid, and vanillic acid; in an emulsion system, the order is different: αtocopherol, than sinapic acid, ferulic acid, vanillic acid, and caffeic acid. Hence, the partitioning behavior of phenolic compounds between the lipid and aqueous phases, depending on the polarity of the compound, seems to play an important role in the formation of lipid oxidation products in bread.

acid increased their levels by 24% and 269%, respectively. Similarly, Zhao et al. (2017) found that the addition of xylooligosaccharides at 50 mg/mL increased benzyl alcohol formation by 132% in dough model. Definitely, a concentration-dependent relationship in lipid oxidation products formation appeared. In this study, the addition of caffeic acid, quercetin, and (+)-catechin suppressed the generation of aliphatic aldehydes formed from lipid oxidation, while ferulic and gallic acid addition conversely increased the generation of these compounds. According to Rice-Evans, Miller, Bolwell, Bramley, and Pridham (1995), antioxidant activity is dependent on the number and arrangement of hydroxyl groups, the extent of structural conjugation, and the presence of electron-donating and electron-withdrawing substituents in the ring structure. However, some polyphenols may exhibit pro-oxidative properties, depending on the test system and their concentration (Rice-Evans et al., 1995). Maurya and Devasagayam (2010) found that the hydroxycinnamic acids have good antioxidant potentials at lower concentrations and begin to show pro-oxidant behavior at higher concentrations, probably on account of their reducing property. Moreover, to study the antioxidant and pro-oxidant nature of phenolic compounds, it is necessary to consider their solubility. Gallic acid has the highest solubility of the phenolic acids because of the higher number of hydroxyl groups, which leads to increased association interactions with water; this is followed by caffeic acid and ferulic acid (Mota, Queimada, Pinho, & Macedo, 2008). Additionally, the aqueous solubility of gallic acid was found to be significantly higher than that of (+)-catechin (12.6 g/L vs. 2.26 g/L at 298.75 K), while the solubility of quercetin was very low, being < 0.01 g/L at 20 °C (Srinivas, King, Howard, & Monrad, 2010). However, it should be kept in mind that the model system used

3.4. Effects of phenolic compounds on the formation of volatiles generated via fermentation In the blank bread crust, four volatile compounds from fermentation were detected: 2-methyl-1-propanol, 3-methyl-1-butanol, phenylethyl alcohol, and ethyl acetate (Fig. 5). The three alcohols were generated by the Ehrlich pathway, where α-keto acids are converted into fusel aldehydes, and further reduced to their fusel alcohols (Pico et al., 2015). The ethyl acetate arises from a reaction inside the yeast cell, catalyzed by acetyltransferases, between acetyl-CoA derivatives of fatty acids and alcohols (Birch et al., 2013). When antioxidants were added to the model bread, the generation of fermentation products was suppressed in a dose-dependent manner (Fig. 5). Ferulic acid was the most effective antioxidant at reducing the level of pleasant components derived from fermentation (∼42%), followed by caffeic acid (∼26%), 7

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Fig. 5. Effect of phenolic compounds on the concentration (µg/kg) of volatile compounds resulting from fermentation in the bread crust. Sample codes: CB: control model bread; CB + Q: model bread with quercetin; CB + C: model bread with (+)-catechin; CB + CA: model bread with caffeic acid; CB + FA: model bread with ferulic acid; CB + GA: model bread with gallic acid. Results with different lowercase letters (comparing the same phenolic compounds at different concentrations), and different uppercase letters (comparing the same concentrations but different phenolic compounds; regular font for 0.1%, oblique font for 0.5%, bold font for 1.0%, and underlined font for 2.0%) are significantly different (p < 0.05).

the activity of baker’s yeast, while Zhang and Kashket (1998) demonstrated the inhibitory activity of tannins against salivary amylase. It is therefore possible that the activity of bakers’ yeast in bread dough could be partially depressed by phenolic compounds and that the activities of amylases might be restricted, leading to inadequate maltose for the yeast’s activity, and thus less pleasant flavor generation. Poinot et al. (2008) reported that baker’s yeast plays a role in generating pleasant flavors through the Ehrlich pathway, but that if there is no external addition of yeast, the major aroma impact compounds comes from lipid oxidation. In fact, it is possible that when yeast is inhibited by phenolic compounds, oxygen is more available and is used by lipoxygenase to generate aldehydes from the oxidation of lipids. This was seen in particular for ferulic acid, where strong inhibition of the most important fermentation aromas simultaneously enhanced the generation of compounds from lipid oxidation. In addition, significant negative correlations (r = −0.993 and r = −0.992; p < 0.05) were obtained between the generation of fermentation and lipid oxidation products in the presence of ferulic acid and gallic acid. However, the bread model is a multiphase system, much more complex than the model system, and so translating the results is not straightforward, and further investigation of the effects of phenolic compounds on baker’s yeast activity and amylase activity as well as volatile profile is necessary. The high level of 4-vinylguaiacol (from 1524 to 43065 µg/kg) in the bread crust with the addition of ferulic acid is noteworthy (Table 1). This component has a low odor threshold value, so even a trace amount produces a specific medicinal, phenolic, or smoky flavor. 4-Vinylguaiacol originates from ferulic acid decarboxylation, facilitated by the p-phenol hydroxyl group, which is reported to exhibit antioxidant activity comparable to that of α-tocopherol (Terpinc et al., 2011). According to Terpinc et al. (2011), 4-vinylguaiacol showed weaker

(+)-catechin (∼20%), and quercetin and gallic acid (∼18%). 2-Methyl-1-propanol with its malty note and 3-methyl-1-butanol with its balsamic note are characteristic volatile compounds of bread crust (Jensen et al., 2011). Due to its much lower OT value, 3-methyl-1butanol (OT value 4 µg/L) is expected to be sensorially more important than 2-methyl-1-propanol (OT value 6505 µg/L) (Nor Qhairul Izzreen et al., 2016). When ferulic acid was added at the highest treatment level to wheat bread, the formation of the pleasant flavors were suppressed by about 95% (Fig. 5 a,b). The addition of the remaining two phenolic acids at the highest concentration decreased the formation of those alcohols by about 46% (caffeic acid) and 37% (gallic acid), while the addition of flavonoids suppressed their level by about 24%. According to Birch et al. (2013), the level of ethyl acetate increases with increasing yeast concentration and decreasing fermentation temperature. Generally, higher concentrations of this ester in bread crust aroma are of interest, since it possesses a pleasant, sweet, fruity aroma (Birch et al., 2013). At the highest concentration (2.0%), the order of reactivity of the phenolic compounds for suppressing ethyl acetate formation was ferulic acid (51%), followed by caffeic acid (36%), gallic acid (17%) and quercetin (17%), and (+)-catechin (16%) (Fig. 5 d). There are also some aldehydes that can result from Maillard reactions or the Ehrlich pathway during fermentation, including 3-methylbutanal and 2-methylbutanal (Table 1). These components are responsible for crust aroma of fresh bread, due to their low odor threshold value (Jensen et al., 2011). When phenolic compounds were added at the highest concentration, caffeic acid exhibited the strongest suppression activity towards those pleasant aldehydes (52%), followed by gallic acid and (+)-catechin (∼40%), quercetin (30%), and ferulic acid (27%). Turchetti et al. (2005) found that plant extracts are able to inhibit 8

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towards flavor profile modification and acrylamide mitigation in heterogeneous systems like bread, more than differences in radicalscavenging activities do. Many phenolic compounds with significant health benefits are characterized by bitterness and astringency, thus the instrumental analysis needs to be complemented with further sensory study.

antioxidant activity in a homogeneous polar medium than did the majority of hydroxycinnamic acids, but its activity in the emulsion system was significantly higher than ferulic acid, which was greater than sinapic acid, which in turn was greater than caffeic acid, as the presence of a methoxyl substituent in the monophenolic structure led to an increase in its reducing capacity and free radical-scavenging activity. When 4-vinylguaiacol was used in place of ferulic acid in the glucose–glycine reaction model, a similar pattern of volatile modification was observed as result of pericyclic reactions with Maillard intermediates (e.g., 3-deoxy-2-hexosulose) to generate phenolic Maillard adducts (Deshou, Christopher, Pranav, Sandeep, & Peterson, 2009). Thus, the high reactivity acting to suppress flavor formation and acrylamide level in the ferulic acid-formulated bread was probably due to the presence of both ferulic acid and its corresponding derivative. Hierarchical cluster analysis of the data on volatile compounds shows that three groups are observed (Fig. 2b). The model breads formulated with ferulic and gallic acid were characterized by higher levels of unpleasant lipid oxidation products, and flavonoids and caffeic acid constituted two distinct clusters. The control bread without addition of phenolic compounds formed a more distinct third cluster, a long distance from the other two clusters (by 80% cut-off point), indicating a significant difference. The sensory properties of phenolic compounds might be summarized with two main taste characteristics, bitterness and astringency. These are well known to elicit negative consumer reactions at high levels, but the intensity of these sensations can be modified by adding masking agents, or by modifying the concentrations and compositions of the polyphenols (Lesschaeve & Noble, 2005). Nevertheless, this study did not examine the effect of antioxidants on the sensory acceptance of the bread. It seems that a lower level of polyphenol addition might strike a good balance between their desirable effects and their undesirable sensory qualities. It is thus necessary to perform further studies, including a sensory analysis of samples using significantly lower levels of phenolic compounds. Another promising strategy for maximizing the bioactivity of wheat breads might be to enrich wheat flour with low-cost fruit by-products, instead of pure phenolic compounds. Such a strategy might allow them to work as functional ingredients, usefully reducing acrylamide-associated health risk and promoting sustainable competitive worldwide production. The attractiveness of such enrichment breads should definitely be investigated with respect to their sensory properties, in order to determine the maximum acceptable dose.

Declaration of Competing Interest None declared. Acknowledgements This study was financially supported by the National Science Centre, Poland (Project No. 2013/09/B/NZ9/01626). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125008. References Açar, Ö.Ç., & Gökmen, V. (2009). Investigation of acrylamide formation on bakery products using a crust-like model. Molecular Nutrition and Food Research, 53(12), 1521–1525. Andrzejewski, D., Roach, J. A. G., Gay, M. L., & Musser, S. M. (2004). Analysis of coffee for the presence of acrylamide by LC-MS/MS. Journal of Agricultural and Food Chemistry, 52(7), 1996–2002. Bassama, J., Brat, P., Bohuon, P., Boulanger, R., & Günata, Z. (2010). Study of acrylamide mitigation in model system: Effect of pure phenolic compounds. Food Chemistry, 123(2), 558–562. Birch, A. N., Petersen, M. A., & Hansen, Å. S. (2013). The aroma profile of wheat bread crumb influenced by yeast concentration and fermentation temperature. LWT – Food Science and Technology, 50(2), 480–488. Cheng, K. W., Zeng, X., Yun, S. T., Wu, J. J., Liu, Z., Sze, K. H., ... Wang, M. (2009). Inhibitory mechanism of naringenin against carcinogenic acrylamide formation and nonenzymatic browning in maillard model reactions. Chemical Research in Toxicology, 22(8), 1483–1489. Deshou, J., Christopher, C., Pranav, M., Sandeep, K., & Peterson, D. G. (2009). Identification of hydroxycinnamic acid – Maillard reaction products in low-moisture baking model systems. Journal of Agricultural and Food Chemistry, 57(21), 9932–9943. Favreau-Farhadi, N., Pecukonis, L., & Barrett, A. (2015). The inhibition of Maillard browning by different concentrations of rosmarinic acid and epigallocatechin-3-gallate in model, bakery, and fruit systems. Journal of Food Science, 80(10), C2140–C2146. Fujiwara, Y., Kiyota, N., Tsurushima, K., Yoshitomi, M., Mera, K., Sakashita, N., ... Nagai, R. (2011). Natural compounds containing a catechol group enhance the formation of Nε-(carboxymethyl)lysine of the Maillard reaction. Free Radical Biology and Medicine, 50(7), 883–891. Guinet, R., & Godon, B. (1994). La panification française. Collection sciences et techniques agro-alimentaires. Tec & Doc-Lavoisier. Jensen, S., Oestdal, H., Skibsted, L. H., Larsen, E., & Thybo, A. K. (2011). Chemical changes in wheat pan bread during storage and how it affects the sensory perception of aroma, flavour, and taste. Journal of Cereal Science, 53(2), 259–268. Jin, C., Wu, X., & Zhang, Y. (2013). Relationship between antioxidants and acrylamide formation: A review. Food Research International, 51, 611–620. Kellard, B., Busfield, D. M., & Kinderlerer, J. L. (1985). Volatile off-flavour compounds in desiccated coconut. Journal of the Science of Food and Agriculture, 36(5), 415–420. Kotsiou, K., Tasioula-Margari, M., Capuano, E., & Fogliano, V. (2011). Effect of standard phenolic compounds and olive oil phenolic extracts on acrylamide formation in an emulsion system. Food Chemistry, 124(1), 242–247. Lesschaeve, I., & Noble, A. C. (2005). Polyphenols: Factors influencing their sensory properties and their effects on food and beverage preferences. The American Journal of Clinical Nutrition, 81(1 Suppl), 330S–335S. Martins, S. I. F. S., Marcelis, A. T. M., & Van Boekel, M. A. J. S. (2003). Kinetic modelling of Amadori N-(1-deoxy-D-fructos-1-yl)-glycine degradation pathways. Part I – Reaction mechanism. Carbohydrate Research, 338(16), 1651–1663. Maurya, D. K., & Devasagayam, T. P. A. (2010). Antioxidant and prooxidant nature of hydroxycinnamic acid derivatives ferulic and caffeic acids. Food and Chemical Toxicology, 48(12), 3369–3373. Mildner-Szkudlarz, S., Siger, A., Szwengiel, A., Przygoński, K., Wojtowicz, E., & ZawirskaWojtasiak, R. (2017). Phenolic compounds reduce formation of Nε-(carboxymethyl) lysine and pyrazines formed by Maillard reactions in a model bread system. Food Chemistry, 23, 175–184. Moskowitz, M. R., Bin, Q., Elias, R. J., & Peterson, D. G. (2012). Influence of endogenous ferulic acid in whole wheat flour on bread crust aroma. Journal of Agricultural and Food Chemistry, 60(45), 11245–11252. Mota, F. L., Queimada, A. J., Pinho, S. P., & Macedo, E. A. (2008). Aqueous solubility of

4. Conclusion Our results here demonstrate that gallic acid, ferulic acid, caffeic acid, (+)-catechin, and quercetin can reduce acrylamide levels in model bread matrices. Of all the phenolic compounds, regardless of concentration, ferulic acid showed the highest level of acrylamide inhibition; this is probably due to the presence of 4-vinylguaiacol, a degradation derivative that has shown strong antioxidant activity in heterogeneous systems. Although the phenolic compounds mitigated acrylamide formation in a bread system, this adversely affected the bread’s volatile profile. Due to the generation of high levels of unpleasant lipid oxidation products, ferulic and gallic acids are not good candidates for acrylamide mitigation. Among the additives studied here, the most promising seems to be (+)-catechin, as it exerted the most significant inhibitory effect on acrylamide formation at the lowest treatment level; it is thus possible that lower concentrations will continue to have the reducing effect without altering the generation of flavors. Most likely, the suppression of pleasant fermentation-type aromas by ferulic and gallic acids, and their promoting effect on the generation of lipid oxidation volatiles, was due to the inhibition of amylases and yeast activity, thus providing a sufficient amount of active oxygen for oxidation. It seems that differences in affinities for the nonpolar phase determine the antioxidant activity of phenolic compounds 9

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