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Role of quercetin in the physicochemical properties, antioxidant and antiglycation activities of bread
Journal of Functional Foods 40 (2018) 299–306
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Role of quercetin in the physicochemical properties, antioxidant and antiglycation activities of bread Jing Lina, Weibiao Zhoua,b, a b
T
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Food Science & Technology Programme, c/o Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore National University of Singapore (Suzhou) Research Institute, 377 Linquan Street, Suzhou Industrial Park, Jiangsu 215123, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords: Quercetin Bread Antioxidant capacity Antiglycation activity
As a natural glycation inhibitor, quercetin was incorporated into bread to develop antiglycative functional food. Quercetin added in the wheat bread flour at 0.05, 0.1, and 0.2% caused a loss of dough elasticity with lower resistance and higher extensibility. It further altered the quality of bread in terms of deceasing bread volume and increasing bread hardness. The antioxidant potential of the bread with quercetin was enhanced in a dose-dependent manner. The antiglycation capacity was assessed according to the ability of the bread to inhibit the formation of advanced glycation endproducts (AGEs) in vitro. Results showed that bread with 0.2% quercetin addition was able to inhibit 46–52% of total AGEs formed during protein glycation. Overall, the results support quercetin as a functional food ingredient in bread system, offering consumers a higher intake of antioxidant and a lower load of AGEs.
1. Introduction Using natural compounds as glycation inhibitors to reduce the intake of advanced glycation end-products (AGEs) from diet is a promising way to decrease the risk of diabetic complications. Among these compounds, quercetin is one of the most efficient antiglycative agents (Wu, Hsieh, Wang, & Chen, 2009). Quercetin is a flavonol comprising of 15 carbon atoms, with two aromatic rings connected by a three-carbon bridge. The main structure of benzopyran-4-one makes it a hydrophobic compound. The rich food sources of quercetin are onions, berries, and apples. It exhibits multiple biological activities such as antioxidative, cardioprotective, and hypoglycemic effects that are related to the protein glycation process (Erlund, 2004; Wu & Yen, 2005). Protein glycation starts from the reactions between reducing sugars and amino groups in proteins. Diverse intermediates are produced during this process, including AGEs. The accumulation of AGEs in the body contributes to the development of many chronic diseases, especially diabetic complications (Peng, Ma, Chen, & Wang, 2011). Besides, reactive carbonyl species such as methylglyoxal (MGO) are also generated along the same process. MGO can bind to the amino groups of proteins and further produce AGEs. Such reactions can result in the undesired modification of proteins and cause protein dysfunction. The formation of AGEs occurs both in vivo through normal metabolism and in vitro from food sources. Dietary AGEs (dAGEs) produced by Maillard reaction is the major external source of exposure to human
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bodies. These compounds can enter the in vivo circulation of AGEs binding to the cellular receptors and cause abnormal metabolism such as the production of inflammatory cytokines, further contributing to the diabetic complications (Chinchansure, Korwar, Kulkarni, & Joshi, 2015). Meanwhile, reactive oxygen species (ROS) are largely generated during glycation, causing high oxidative stress in vivo. Different mechanisms have been reported to inhibit the glycation process (Peng et al., 2011). As for quercetin, it has been shown to inhibit protein glycation by its free radical scavenging activity to combat the oxidative stress and its ability to trap MGO (Li, Zheng, Sang, & Lv, 2014; Zhang, Chen, & Wang, 2014). Despite the well-known antiglycation activity of quercetin, most of the previous investigations were conducted with plant extract rich in quercetin or with quercetin standard (Kim, Lee, Yokozawa, Sakata, & Lee, 2011; Li et al., 2014; Wu et al., 2009). Only few studies have tried to explore the in vitro antiglycation behavior of quercetin in fortified food systems (Szawara-Nowak, Koutsidis, Wiczkowski, & Zieliński, 2014; Zhang et al., 2014). Still, these researches did not focus on just quercetin but also other polyphenols. For example, Szawara-Nowak et al. (2014) reported that buckwheat enhanced wheat bread rich in both rutin and quercetin reduced in vitro AGEs generation, and a high correlation between AGEs inhibition and the quercetin content (r = 0.92) as well as the rutin content (r = 0.86) was found. As there were more than one effective ingredient, it’s hard to distinguish the AGEs inhibitory effect of quercetin itself. It is thus necessary to
Corresponding author at: Food Science & Technology Programme, c/o Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore. E-mail address: [email protected] (W. Zhou).
https://doi.org/10.1016/j.jff.2017.11.018 Received 20 August 2017; Received in revised form 2 November 2017; Accepted 13 November 2017 Available online 22 December 2017 1756-4646/ © 2017 Elsevier Ltd. All rights reserved.
Journal of Functional Foods 40 (2018) 299–306
J. Lin, W. Zhou
2.4. Measurement of bread quality attributes
elucidate the antiglycation function of quercetin in practical food products in more detail and specifically. In this research, we aimed to develop quercetin-fortified baked bread as a functional food that helps consumers to lower the risk of diabetic complications. We used bread as a carrier for quercetin fortification because bread is a popular staple food widely consumed around the world. The tasty bread crust formed through Maillard reaction between proteins and sugars during baking process is a key attraction to consumers; however, it is also a rich source of dietary AGEs, posing a potential risk to human health, especially to the diabetic patients. Quercetin may exert its anti-glycation effect on both the AGEs in the body and the dAGEs in bread matrix. On the other hand, the quality of quercetin-fortified bread would largely affect the consumer’s acceptance. Therefore, it is also of great interest to examine the effect of quercetin fortification on dough development and the final bread quality attributes. In this study, the rheological properties of quercetin-fortified baked bread dough and the bread quality were evaluated. The antioxidant capacity of the bread with quercetin was examined. Bovine serum albumin (BSA) and glucose (GLU) in vitro model system was employed to investigate the potent therapeutic values of this functional bread. The knowledge gained from this research would provide some useful insights to the potentials of quercetin-containing functional food.
Specific volume, texture, colour, moisture content, and pH value were measured, as they are important indicators to assess the overall quality of quercetin-fortified bread. Bread volume was measured using a Volscan profiler (VSP 600, Stable Micro System Ltd., Surrey, U.K.). The specific volume was obtained through dividing bread volume (cm3) by bread weight (g). Texture analysis of the bread was performed using a texture analyser (TAXT2i, Stable Micro System, Surry, U.K.) with a 20 mm diameter cylindrical probe. A bread slice of 2 cm thick cut from the central part of the bread was compressed up to 40% of its original thickness using a double compression cycle at a cross head speed of 2 mm/s. Hardness, springiness, cohesiveness, chewiness and resilience of the bread crumb were quantified. The measurements of three chromatic coordinates L∗, a∗, and b∗ of bread crust and crumb individually were performed using a colourimeter (CM-3500d Spectrophotometer, Konica Minolta, Japan). The coordinate L∗ represents the lightness of colour (L∗ = 0 points to black and L∗ = 100 points to white); a∗ indicates the colour between red and green (negative values yield green, and positive values yield red); b∗ expresses the colour between yellow and blue (negative values suggest blue, and positive values suggest yellow). The moisture content was evaluated according to the study of Sui et al. (2015). Bread samples (2 g) were heated at 100 °C in on oven until constant weight, and the moisture content was expressed as the percentage change in the weight of the sample. As for the pH value measurement, 5 g of bread sample was agitated in 50 ml of deionized water for 30 min and the supernatant layer obtained was tested by a pH meter (Metrohm 744 pH meter, Switzerland).
2. Materials and methods 2.1. Chemicals Quercetin (food grade) was obtained from Xi’an Dowell Bio-tech Co. Ltd., China with 98% of purity. Wheat bread flour (Prima brand, protein content 13.1%), instant dry yeast (Saccharomyces cerevisiae, S.I. Lesaffre, France), salt (Fairprice Cooperative Ltd., Singapore), sugar (Fairprice Cooperative Ltd., Singapore), and vegetable shortening (Bake King, Gim Hin Lee Ltd., Singapore) were purchased from a local supermarket. DPPH (2,2-diphenyl-1- picrylhydrazyl), ABTS (2,2′-azinobis (3-ethylbenzothiazo- line-6-sulphonic acid)), potassium persulfate, trolox (6-hydroxy-2,5,7,8-tetramethylchloromane-2-carboxylic acid), gallic acid (3,4,5-Trihydroxybenzoic acid), Folin-Ciocalteu reagent, GLU, BSA, MGO (40% aqueous solution), and aminoguanidine (AG), were purchased from Sigma–Aldrich (Sigma–Aldrich, St Louis, MO, USA).
2.5. Extraction of quercetin Bread crumb and crust were separated, frozen for 24 h, and freezedried. The dry maters were ground into fine bread powders. Bread powder samples (2 g) were extracted with methanol (10 ml) under 30 min shaking at 300 rpm using an orbital shaker (IKA VXR basic Vibrax, Staufen, Germany). Liquid extracts were obtained after centrifugation at 3233g for 5 min and combined together for concentration at 40 °C by a vacuum rotary evaporator after 3 rounds of extraction where the maximum extractability of quercetin was achieved at around 92% and 77% for bread crumb and crust respectively. The same extraction process was conducted on the control bread powder spiked with quercetin to examine the recovery rate of the extraction process, which was found to be above 96%.
2.2. Farinograph and extensograph tests Wheat bread flour fortified with various levels of quercetin (0, 0.05, 0.10, and 0.20%) was loaded on a Farinograph-E equipped with a S50 mixer and sigma blades (Brabender, Duisburg, Germany). Farinograph test was conducted according to the constant flour weight procedure of AACC Method 54–21 (AACC, 2000). In the extensograph test, freshly prepared quercetin-fortified dough samples were examined using an Extensograph-E (Brabender, Duisburg, Germany) following the AACC Method 54–10 (AACC, 2000).
2.6. Quantification of quercetin using HPLC/DAD Quercetin was quantified by a Shimadzu High Performance Liquid Chromatography (HPLC) system equipped with a diode array detector (DAD) (Shimadzu, Kyoto, Japan) using a C18 reserved-phase column (250 × 4.6 mm, Sunfire, Waters, Wexford, Ireland). The flow rate was 1 ml/min and the oven temperature was 30 °C. Mobile phase A (1% acetic acid in DI water) and B (100% acetonitrile) were applied according to the ratio of 6:4. Detection was performed at 257 nm. Quantification of quercetin was accomplished by the external calibration ranging from 0.025 to 0.250 mg/ml.
2.3. Bread sample preparation Bread making process was modified following the study of Sui, Yap, and Zhou (2015). Quercetin was firstly mixed with 1 kg wheat bread flour at the addition levels of 0, 0.05, 0.10, and 0.20%, which was further mixed with 10 g instant dry yeast, 12 g salt, 30 g shortening, 40 g sugar, and 600 g water for 5 min to form bread dough in a mixer. The dough was rested for 10 min at 20 °C followed by molding into small pieces (50 g each) and proofing in a proofer (Climatic chamberKBF, Binder, Germany) at 40 °C and 85% relative humidity condition. The baking process was conducted at 200 °C in an oven (Eurofours, France) for 8 min. The bread samples are cooled down to room temperature for 1 h after baking.
2.7. Antioxidant capacity analysis: ABTS assay The ABTS assay was performed according the method of Sui, Dong, and Zhou (2014) with some modifications. ABTS radical cation (ABTS+) was prepared by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate and being left in the dark for 12–16 h before use. The ABTS+ solution was diluted with methanol to an absorbance of 0.70 ± 0.05 at 734 nm. Bread extract or trolox (0.1 ml) was mixed with the diluted ABTS+ solution (1 ml) for 7 min. The absorbance at 734 nm was then measured using a UV–vis 300
Journal of Functional Foods 40 (2018) 299–306
J. Lin, W. Zhou
Table 1 Farinograph and extensograph analysis of bread flour fortified with different levels of quercetin. Formulation
Water absorption (%)
Control 0.05% 0.10% 0.20%
63.73 63.67 63.70 63.63
± ± ± ±
0.35a 0.40a 0.00a 0.15a
Development time (min) 7.77 8.53 9.27 9.63
± ± ± ±
0.12c 0.35b 0.45a 0.51a
Resistance [BU]
Extensibility [mm]
1220.00 ± 33.18a 970.67 ± 10.12d 1026.00 ± 32.92c 1124.00 ± 16.00b
111.00 121.67 131.67 123.33
± ± ± ±
4.00b 4.73ab 12.42a 2.31ab
Ratio number 12.90 10.60 10.20 11.47
± ± ± ±
0.95a 0.70b 1.22b 0.50b
Values in the same column followed by different superscript letters are significantly different (p < .05).
spectrophotometer (Shimadzu UVmini-1240, Tokyo, Japan). The antioxidant capacity was expressed as mg trolox equivalent per 100 g dried bread sample.
Inhibition(%) = {1−[(fluorescence of solution with bread extract
2.8. Antioxidant capacity analysis: DPPH assay
IC50 of the bread extract or AG solution were calculated by the Probit Regression Model using the Statistical Product and Service Solutions (SPSS) computer software (SPSS 21, IBM Corporation, New York, USA). The measurement of dietary AGEs in bread was conducted following the method described by Zhang et al. (2014). In brief, the fluorescent AGEs in 0.1 ml bread extract were indicated at the excitation wavelength of 355 nm and emission wavelength of 405 nm by the microplate reader.
− intrinsic fluorescence of bread extract) /fluorescence of solution without bread extract]} × 100
The DPPH assay was conducted based on the method of Sui, Bary, and Zhou (2016). The bread extract or trolox (0.1 ml) and 60 µM methanol DPPH% solution (3.9 ml) were mixed and incubated in the dark for 2 h. The absorbance at 515 nm was measured by the spectrophotometer. The antioxidant capacity was expressed as mg trolox equivalent per 100 g dried bread sample. 2.9. Total phenolic content analysis
2.11. Statistical analysis The total phenolic content (TPC) was determined as described by Sui et al. (2015). Bread liquid extract (0.1 ml) was mixed with FolinCiocalteau reagent (0.1 ml) and deionised water (1.58 ml). The mixture solution was incubated for 6 min, followed by adding 2 M of sodium carbonate (0.3 ml). After incubation in the dark for 2 h, the absorbance of the mixture was measured at 765 nm by the spectrophotometer. The TPC of the samples was expressed in gallic acid equivalent per 100 g dried bread sample.
All experiments were conducted in triplicate batches with three individual samples from each batch. The mean values of the analysis were reported as the final results with standard deviation (n = 9). Oneway ANOVA was used for statistical analysis adopting Duncan’s multiple range test on SPSS to determine the presence of any significant difference (p < .05) between samples. Pearson’s correlation was also conducted using SPSS.
2.10. Antiglycation capacity examination
3. Results and discussion
In the BSA-GLU system, the assay was performed according to the method described by Szawara-Nowak et al. (2014), with a slight modification based on the study of Izabela Sadowska-Bartosz, Galiniak, and Bartosz (2014). BSA (10 mg/ml) was mixed with GLU (0.5 M) in phosphate buffer (0.1 M, pH 7.4) as the test solution. Sodium azide (0.2 mg/ml) was added to achieve an aseptic condition. The test solution (2 ml) was incubated at 55 °C for 40 h with or without a bread extract (1 ml) in which the final concentration of quercetin ranging from 0.00 to 0.25 g/ml was selected with high inhibitory effect. The fluorescence intensity of the solution was measured at the characteristic excitation and emission wavelengths of 325/440, 330/415, 325/434 and 365/480 nm to detect the levels of AGEs and protein oxidation markers, dityrosine, N-formylkynurenine and kynurenine, respectively, by a microplate reader (Victor X4 Multilabel Plate Reader, PerkinElmer, USA). In the BSA-MGO system, the assay was based on the method of Wu and Yen (2005) with slight modification. BSA (1 mg/ml) was dissolved in phosphate buffer (0.1 M, pH 7.4) and mixed with MGO (5 mM) and sodium azide (0.2 mg/ml) to obtain a test solution. Bread extraction (1 ml) was added into the test solution (2 ml), followed by the incubation at 37 °C for 6 days. The sample fluorescence was examined at the excitation and emission wavelengths of 340 and 420 nm respectively. The positive control for both of the assays was AG at the concentrations of 1–25 mM. IC50 value, the specific concentration at which 50% of glycation was inhibited, was used to compare the anti-glycation effect between the commercial inhibitor AG with the quercetin-fortified bread. The percentage inhibition of total AGEs, oxidatively modified proteins formation, and AGEs induced by MGO was calculated based on the following equation:
3.1. Farinograph and extensograph analysis Effects of quercetin on the rheological properties of the dough are shown in Table 1. As expected, no significant difference of water absorption was observed between the control and the quercetin-fortified dough. The low water solubility of quercetin makes it not to compete for water with other constituents in the dough during mixing process. However, the development time of the dough with quercetin extended up to 19% compared to the control dough (from 7.77 to 9.63 min). The longer dough development time might be attributed to the slower hydration rate of protein that impaired the aggregation behaviour of high molecular weight (HMW) protein in flour (Rosell, Santos, & Collar, 2006). Quercetin could interact with gluten, negatively affecting the protein hydration and further causing a longer dough development time. Besides, the long development time of dough can be associated with the loss of its elasticity based on the dough structure alteration (Miś, Grundas, Dziki, & Laskowski, 2012). In fact, a typical interchain bonding namely disulfide bonds (SS) mainly accounts for the dough stability via the SS-SH interchange reactions (Sivam, Sun-Waterhouse, Quek, & Perera, 2010). Quercetin, as a reducing agent, is involved in these reactions, leading to lower amount of SS as a consequence of a weaker protein network. The development time was thus prolonged with impeded dough elasticity. From the extensograph results present in Table 1, all of the treated dough possessed significantly lower resistance compared to the control dough. This indicated a weaker gas retention capacity and possible disintegration of the gluten network during dough proofing. However, close bonds can also be formed between the gluten matrix and the additives that are strong enough to counteract the weakening impact of 301
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Table 2 Specific volume of and texture analysis of the bread fortified with different levels of quercetin. Sample
Specific Volume (cm3/g)
Control 0.05% 0.10% 0.20%
3.73 3.61 3.55 3.37
± ± ± ±
0.03a 0.02b 0.05b 0.03c
Hardness (N) 218.52 229.55 256.83 270.59
± ± ± ±
13.97b 31.46b 23.43a 12.85a
Springiness 1.01 1.01 0.97 0.97
± ± ± ±
0.02a 0.01a 0.00b 0.01b
Cohesiveness 0.86 0.85 0.84 0.85
± ± ± ±
0.01a 0.01a 0.01a 0.01a
Chewiness (N) 180.46 193.83 208.32 235.45
± ± ± ±
8.13c 30.04bc 11.27b 19.74a
Resilience 0.53 0.53 0.52 0.53
± ± ± ±
0.02a 0.02a 0.01a 0.00a
Values in the same column followed by different superscript letters are significantly different (p < .05).
from Zhang et al. (2014) that quercetin-enriched cookies obtained darker colour with reduced lightness. The b∗ value of quercetin-fortified bread crumb was significantly higher than that of the control by up to 45%, indicating the accumulation of yellow colour. The a∗ value dropped from −0.09 to −2.04 at 0.05% quercetin addition and progressively increased to -1.32 at the 0.2% addition, which coincided with the increasing b∗ value showing a shift from green to yellow. The colour of bread crumb seemed to be dramatically influenced by the original yellowish colour of quercetin powder. However, in contrast to the distinct alteration of bread crumb in colour, bread crust showed no significant change. This is in agreement with the result from Gómez, Ronda, Blanco, Caballero, and Apesteguía (2003) that in spite of the influence from colourful food additives, the characterized browning induced by Maillard reaction and caramelization mainly accounted for bread crust’s colour. In addition, the moisture content of the quercetin incorporated bread crumb and crust did not exhibit significant difference from the control (Table 3). Nonetheless, it was much lower in the curst than in the crumb due to more rapid water evaporation on bread surface during baking. Moverover, as quercetin is unstable under alkaline condition, the pH value of final food product is crucial for its retention. The results in Table 3 show that the pH value of the bread crumb and crust were statistically the same within the range of 5.52–5.61, which made bread a feasible carrier to produce quercetin-contaning functional food.
SS reduction on dough structure (Miś et al., 2012). Quercetin can hydrophobically interact with glutenins to form tight bonds and thus interfere in the formation of dough structure. As a consequence, the resistance of the quercetin-fortified dough firstly decreased at the lowest dosage (0.05%) and then increased with incremental quercetin addition, but the overall values were still lower than that of the control dough. Moreover, significant increasing of extensibility was observed in the dough with quercetin. The extensibility can be enhanced when the SS amount is reduced and the solubility of glutenin increases (Wieser, 2007). In the presence of quercetin, the ratio number of dough (reflecting the dough strength) was statistically lower than the control dough. This revealed a more delicate dough structure. Our result is in line with the study of Han and Koh (2011) who reported that the addition of phenolic acids hindered the rheological properties of dough by reducing the HMW proteins.
3.2. Bread quality attributes examination The specific volume of bread, shown in Table 2, decreased successively from 3.73 cm3/g (control bread) to 3.37 cm3/g (0.2% quercetinfortified bread). Quercetin might impair the rheological properties of dough at the expense of a small bread volume. The result is consistent to the literature reports that bread volume decreased with the incorporation of catechin, anthocyanin, and other polyphenols from fruit extracts (Sui, Zhang, & Zhou, 2016; Sun-Waterhouse et al., 2011; Wang, Zhou, Yu, & Chow, 2006). Besides, the characterization of bread texture in Table 2 shows that the hardness and chewiness of the 0.05–0.2% quercetin-fortified bread were significantly higher than that of the control bread by up to 23%. However, the springiness only showed statistical difference with more than 0.1% quercetin addition; no significant difference was found in the cohesiveness and resilience among all the quercetin-fortified bread and the control. Hardness is a measurement of peak force required to compress the sample, while chewiness represents a quantitative estimation of energy needed to disintegrate bread structure (Ribotta, Pérez, Añón, & León, 2010). Springiness indicates the capacity of sample to spring back after a deformation due to the compression. Harder texture implied the formation of denser bread crumb, corresponding to the smaller specific volume of quercetin-fortified bread. This was due to the smaller amount of SS bonds caused by the reducing capacity of quercetin, thus impairing the bread crumb’s elasticity and weakening the bread matrix. Our observation is in agreement with the results of Sui et al. (2016) as in their studies the bread incorporating black rice extract (up to 4%) showed increased hardness texture and decreased bread volume. Another recent study about the impact of incorporated quinoa leaves on bread quality also showed a similar trend: as the amount of quinoa leaves increased from 1 to 5%, bread volume decreased and crumb texture became harder and chewier (Swieca, Seczyk, Gawlik-Dziki, & Dziki, 2014). Regarding the effect of quercetin on the colourimetric profile of bread, a significant decrease in bread crumb’s lightness by up to 16% is shown in Table 3. The result suggested that the colour of bread crumb became dull and slightly grayish as the brightness was impeded after the fortification of quercetin. This is in agreement with the observations
3.3. Antioxidant capacity and total phenolic content analysis After the baking process, the average quercetin content remaining in the bread crumb at 0.05, 0.10, and 0.20% additions was 0.35, 0.77, and 1.42 mg/g Dry Weight (DW), respectively; meanwhile, that in the bread crust was 0.27, 0.60, and 1.20 mg/g DW, respectively. The smaller amount of quercetin in the crust was due to the much higher temperature in the crust layer than the crumb during baking (data not shown), leading to more quercetin degradation. As shown in Table 4, a stepwise fortification of quercetin in bread resulted in a corresponding significant increase in the antioxidant activity from 2.9 to 285.8 mg trolox/100 g DW examined by ABTS assay and from 8.4 to 298.5 mg trolox/100 g DW by DPPH assay. The total phenolic content of the quercetin-fortified bread also increased from 76.3 to 259.2 mg gallic acid/100 g DW. Moreover, in spite of less quercetin in the bread crust, its antioxidant activity was significantly higher than the crumb in both the control condition and 0.5% quercetin addition. The differences were assumed to stem from the antioxidant properties of the Maillard reaction products (MRPs) formed in bread crust. Besides, the thermal degradation products of quercetin also possess radical scavenging properties, such as protocatechuic acid (Buchner, Krumbein, Rohn, & Kroh, 2006). Similarly, the bread crust also exhibited significantly higher total phenolic content than the crumb. It is well understood that the antioxidant profiles of a polyphenol-fortified baked product could arise from various sources including the intrinsic polyphenols in wheat flour, the incorporated phenolic compounds, the intermediate or thermal degradation products of the phenolics, and the polyphenol-starch/protein complex (Sivam et al., 2010). 302
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Table 3 The colourimetric profiles (L*, a*, and b*), moisture content (MC%), and the pH value of the bread with different levels of quercetin. Sample
L* a* b* MC% pH
Crumb
Crust
Control
0.05%
0.10%
0.20%
Control
0.05%
0.10%
0.20%
76.72 ± 0.56a −0.09 ± 0.20d 16.05 ± 0.46d 41.02 ± 0.85a 5.52 ± 0.02a
72.43 ± 1.33b −2.04 ± 0.19a 23.34 ± 0.65c 41.69 ± 1.33a 5.49 ± 0.02a
67.89 ± 0.42c −1.76 ± 0.01b 27.09 ± 0.12b 42.15 ± 2.01a 5.61 ± 0.04a
64.29 ± 0.59d −1.32 ± 0.06c 29.39 ± 0.43a 40.48 ± 1.54a 5.54 ± 0.01a
50.42 ± 2.13a 17.05 ± 0.29a 32.17 ± 1.99a 22.51 ± 1.49a 5.39 ± 0.06a
49.99 ± 3.14a 16.87 ± 0.09a 31.84 ± 2.34a 21.87 ± 0.59a 5.41 ± 0.05a
50.64 ± 1.81a 17.23 ± 0.20a 33.48 ± 1.98a 22.46 ± 1.65a 5.32 ± 0.03a
49.17 ± 2.36a 16.79 ± 0.59a 32.98 ± 1.75a 20.59 ± 1.89a 5.36 ± 0.01a
Values in the same row followed by different superscript letters are significantly different (p < .05).
depleted quercetin by dAGEs would be. Therefore, the inhibition difference between the bread crust and crumb tended to be smaller and the inhibition effect reached statistically the same extent as the quercetin addition level was above 0.2%. As the AGEs inhibition ability of quercetin may not just come through a single pathway, we also investigated its AGEs inhibition effects in the BSA-MGO system, as shown in Fig. 1(B). It is noteworthy that the enhanced inhibition of MGO-induced AGEs by the bread extract with increasing quercetin amount was consistent with that in the BSAGLU system. Similarly, no significantly increased inhibition on AGEs was observed in this system when the quercetin concentration of the bread crumb and crust extracts was beyond 0.16 and 0.15 mg/ml, respectively. As known, the reactions between MGO and the amino/ sulfhydryl group of amino acid residues such as arginine, lysine and cysteine, lead to the formation of AGEs. Quercetin can directly deplete MGO (Li et al., 2014), and this reduces the protein-modifying reactions to inhibit AGEs formation.
3.4. Antiglycation capacity analysis Glycation process involves three main stages: first, an unstable and highly reversible Schiff base is produced, prone to oxidation with free radicals generation; then, it is followed by a transition of the Schiff base into stable Amadori products through isomerization; lastly, the Amadori adducts, namely ketosamines, undergo further rearrangement, dehydration and other complicate reactions to form reactive dicarbonyl intermediates like MGO and irreversible AGEs such as carboxymethyllysine (CML) (Peng et al., 2011). In this study, two methods were used to examine the anti-glycation effect of the quercetin-fortified bread: (1) the BSA-GLU model, targeting at the total AGEs produced by protein glycation; (2) the BSA-MGO model, focusing on the AGEs prompted by the main precursor, MGO.
3.4.1. Inhibition of AGEs formation The inhibitory effect of bread extracts on the in vitro AGEs formation using the BSA-GLU system is shown in Fig. 1(A). Below a certain quercetin concentration (0.16 and 0.15 mg/ml respectively), both quercetin-fortified bread crumb and crust extracts behaved in a dosedependent manner on the reduction of total AGEs produced by protein glycation from 23.4% to 59.0% and from 12.3% to 56.2%, respectively. Previous studies have shown that in terms of free radical scavenging capacity, antioxidant activity is the major action for polyphenols to suppress AGEs generation (Navarro, Fiore, Fogliano, & Morales, 2015; Zhang, Hu, Chen, & Wang, 2014). Supportively, our results showed a significant correlation between the antioxidant activity in terms of free radical scavenging capacity and the inhibition of total AGEs generation (r = 0.916, p < .05 for DPPH assay and r = 0.957, p < .05 for ABTS assay). It indicated high possibility of the free radical scavenging activity to dominate the mechanism of AGEs inhibition by quercetinfortified bread. The results also showed that below 0.1% quercetin addition, quercetin-fortified bread crumb extract (0.11 mg/ml) had significantly higher inhibitory effect (up to 47%) than quercetin-fortified bread crust extract (0.098 mg/ml). It might ascribe to the higher level of inherent AGEs (i.e. dAGEs) produced in the bread crust than that in the bread crumb. Discussion in detail would be conducted later in Section 3.4.4. In brief, the more the quercetin available in bread, the smaller ratio of
3.4.2. Oxidatively-modified proteins inhibition analysis Combination of glycation and oxidation as glycoxidation also contributes to the formation of AGEs. Due to the difficulty to detect the unstable intermediates during oxidation, the relatively stable end products were detected to reflect the extent of protein oxidative damage. Dityrosine, N-formylkynurenine and kynurenine were used to be the indices of oxidant-induced protein crosslinking (Sadowska-Bartosz, Galiniak, Skolimowski, Stefaniuk, & Bartosz, 2015; Shiba et al., 2008). The inhibitory effect of quercetin-fortified bread on these oxidative markers are displayed in Fig. 1(C), (D), and (E), respectively. Quercetinfortified bread extracts reduced the amount of all the three markers dose-dependently. Dityrosine is an indicator of crosslinking formation between two tyrosines by hydroxyle radicals under oxidative stress (Leeuwenburgh, Hansen, Holloszy, & Heinecke, 1999). Our results supported quercetinfortified bread as a plausible inhibitor to reduce such crosslinking formed during protein glycation and therefore helping maintain the normal function of proteins. Besides, N-formylkynurenine, as a main product of tryptophan modification with exposure to ROS, was also inhibited by the quercetin-fortified bread extract, following the same pattern as that of dityrosine. Nevertheless, kynurenine was inhibited to
Table 4 Antioxidant capacity and total phenolics content of the bread with different levels of quercetin. Sample
Control 0.05% 0.10% 0.20%
ABTS (mg Trolox/100 g DW)
DPPH (mg Trolox/100 g DW)
TPC (mg Gallic acid/100 g DW)
Crumb
Crust
Crumb
Crust
Crumb
Crust
2.9 ± 1.8d,B 55.5 ± 4.7c,B 155.1 ± 20.8b,A 285.8 ± 14.2a,A
22.0 ± 9.5d.A 94.8 ± 5.1c,A 155.5 ± 24.8b,A 290.8 ± 15.9a,A
8.4 ± 1.2d,B 62.9 ± 1.7c,B 153.8 ± 3.1b,B 298.5 ± 14.5a,A
20.5 ± 0.7d.A 91.6 ± 3.9c,A 159.8 ± 2.0b,A 295.2 ± 16.4a,A
63.9 ± 5.3d,B 97.2 ± 2.0c,B 151.0 ± 2.9b,B 233.4 ± 1.3a,B
76.3 ± 5.5d,A 123.3 ± 2.4c,A 168.4 ± 3.2b,A 259.2 ± 12.6a,A
Within each column, data values denoted by different lowercase letters are statistically different (p < .05) across the quercetin percentage. Within each row, for the same test (i.e. ABTS, DPPH or TPC), data values denoted by different uppercase letters are statistically different (p < .05) between bread crumb and crust.
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Fig. 1. Inhibitory activity of quercetin-fortified bread extract on the formation of (A) total fluorescent AGEs produced by protein glycation process, (B) fluorescent AGEs induced by its precursor MGO (C) ditryrosine, (D) N-formylkynurenine, (E) kynurenine. Within the same curve, data values denoted by different lowercase letters are statistically different (p < .05) across the quercetin concentrations. At the same quercetin concentration, data values denoted by different uppercase letters are statistically different (p < .05) between bread crumb and crust.
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to scavenge free radicals and to trap the AGEs precursors, as the lipid content in bread was low. In general, the modification of AGE formation by inhibitors in food systems is mostly product dependent. This involves considerations of complicated food components in the matrix and the processing conditions including temperature, moisture content, as well as processing time (Poulsen et al., 2013; Srey et al., 2010).
Table 5 Concentration (mg/ml) of quercetin in the bread crumb and crust extract and concentration (mg/ml) of AG exerting the 50% inhibition of AGEs or oxidative damage markers. IC50-antiglycation activity
Bread Crumb extract
Bread Crust extract
AG
AGEs from protein glycation AGEs derived from MGO Dityrosine N-formylkynurenine Kynurenine
0.11 ± 0.03b
0.12 ± 0.02b
0.74 ± 0.22a
0.03a 0.02b 0.02b 0.32
0.17 ± 0.07a 0.12 ± 0.03b 0.13 ± 0.03b N.A.*
0.16 ± 0.16a 0.70 ± 0.18a 0.70 ± 0.16a N.A.
0.14 0.09 0.10 0.42
± ± ± ±
4. Conclusion In conclusion, quercetin fortification altered dough development, resulting in a slight loss of dough elasticity. Furthermore, it caused significant quality changes to bread, including smaller volume, harder texture, and green-yellowish appearance. The antioxidant capacity and total phenolic content of the bread were significantly enhanced by the quercetin fortification. Quercetin-fortified bread inhibited the in vitro glycation process and reduced AGEs formation, most probably by free radical scavenging and trapping reactive dicarbonyl intermediates. These results indicated that quercetin fortification might be a promising way to produce functional bread that helps consumers to prevent glycation-associated diseases.
Different superscript letters in the same row denote significant differences (p < .05). * N.A: Not applicable.
Acknowledgements Supports from the Agency for Science, Technology and Research (A∗STAR) Singapore through research grant BMRC 14/1/21/24/009 under Singapore-New Zealand Foods for Health Grant Call and Jiangsu Province under the Scientific Research Platform scheme BY2014139 are gratefully acknowledged. The first author also thanks the National University of Singapore (NUS) for financial support. References AACC (2000). Approved methods of the American association of cereal chemists. Methods, 54, 21. Buchner, N., Krumbein, A., Rohn, S., & Kroh, L. W. (2006). Effect of thermal processing on the flavonols rutin and quercetin. Rapid Communications in Mass Spectrometry, 20(21), 3229–3235. Chinchansure, A. A., Korwar, A. M., Kulkarni, M. J., & Joshi, S. P. (2015). Recent development of plant products with anti-glycation activity: A review. RSC Advances, 5(39), 31113–31138. Erlund, I. (2004). Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemiology. Nutrition Research, 24(10), 851–874. Gómez, M., Ronda, F., Blanco, C. A., Caballero, P. A., & Apesteguía, A. (2003). Effect of dietary fibre on dough rheology and bread quality. European Food Research and Technology, 216(1), 51–56. Han, H. M., & Koh, B. K. (2011). Effect of phenolic acids on the rheological properties and proteins of hard wheat flour dough and bread. Journal of the Science of Food and Agriculture, 91(13), 2495–2499. Kim, H. Y., Lee, J. M., Yokozawa, T., Sakata, K., & Lee, S. (2011). Protective activity of flavonoid and flavonoid glycosides against glucose-mediated protein damage. Food Chemistry, 126(3), 892–895. Leeuwenburgh, C., Hansen, P. A., Holloszy, J. O., & Heinecke, J. W. (1999). Hydroxyl radical generation during exercise increases mitochondrial protein oxidation and levels of urinary dityrosine. Free Radical Biology and Medicine, 27(1–2), 186–192. Li, X., Zheng, T., Sang, S., & Lv, L. (2014). Quercetin inhibits advanced glycation end product formation by trapping methylglyoxal and glyoxal. Journal of Agricultural Food Chemistry, 62(50), 12152–12158. Miś, A., Grundas, S., Dziki, D., & Laskowski, J. (2012). Use of farinograph measurements for predicting extensograph traits of bread dough enriched with carob fibre and oat wholemeal. Journal of Food Engineering, 108(1), 1–12. Navarro, M., Fiore, A., Fogliano, V., & Morales, F. J. (2015). Carbonyl trapping and antiglycative activities of olive oil mill wastewater. Food & Function, 6(2), 574–583. Peng, X., Ma, J., Chen, F., & Wang, M. (2011). Naturally occurring inhibitors against the formation of advanced glycation end-products. Food & Function, 2(6), 289–301. Poulsen, M. W., Hedegaard, R. V., Andersen, J. M., de Courten, B., Bügel, S., Nielsen, J., ... Dragsted, L. O. (2013). Advanced glycation endproducts in food and their effects on health. Food and Chemical Toxicology, 60, 10–37. Ribotta, P. D., Pérez, G. T., Añón, M. C., & León, A. E. (2010). Optimization of additive combination for improved soy–wheat bread quality. Food and Bioprocess Technology, 3(3), 395–405. Rosell, C. M., Santos, E., & Collar, C. (2006). Mixing properties of fibre-enriched wheat bread doughs: A response surface methodology study. European Food Research and Technology, 223(3), 333–340. Sadowska-Bartosz, I., Galiniak, S., & Bartosz, G. (2014). P78 - Polyphenols protect against protein glycoxidation. Free Radical Biology and Medicine, 75(Supplement 1), S47.
Fig. 2. Inhibitory activity of quercetin on the formation of dAGEs in bread. Within the same bread sample type (i.e. crumb or crust), columns denoted with different lowercase letters are statistically different (p < .05) across the quercetin addition levels. At the same quercetin addition level, columns denoted with different uppercase letters are statistically different (p < .05) between the bread crumb and crust extract.
a less extent. This is consistent with another study investigating the inhibition of oxidative protein modifications by nitroxides, in which weaker inhibition of kynurenine was also found due to the concurrent conversion of N-formylkynurenine into kynurenine. (Sadowska-Bartosz et al., 2015). 3.4.3. IC50-antiglycation activity analysis IC50-antiglycation activity of the bread is presented in Table 5. With a significantly smaller IC50 value, quercetin-fortified bread crumb and crust extracts (0.110 and 0.120 mg/ml, respectively) showed a stronger antiglycation effect compared to the positive control AG (0.744 mg/ml) in terms of the total AGEs formation. In contrast, no significant difference in the IC50 value between the bread crumb and crust extract (0.138 and 0.174 mg/ml, respectively) and AG (0.158 mg/ml) was observed in the BSA-MGO system. The IC50-antiglycation activity of quercetin-fortified bread showed its strong potential to be an efficient natural glycation inhibitor instead of AG. 3.4.4. Inhibition of dAGEs in baked bread As shown in Fig. 2, the inhibitory effect on dAGEs by both crumb and crust of the quercetin-fortified bread exhibited a linear increasing trend. The maximum inhibition (above 55%) was shown at the 0.2% quercetin addition based on flour weight. It is in line with an previous study in which 0.25% quercetin incorporated cookies showed around 80% inhibition on dAGEs by the suppression of lipid peroxidation (Zhang et al., 2014). However, rather than inhibiting lipid peroxidation, quercetin in bread exerted its antiglycation activity by its abilities 305
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N. (2011). Effects of added fruit polyphenols and pectin on the properties of finished breads revealed by HPLC/LC-MS and Size-Exclusion HPLC. Food Research International, 44(9), 3047–3056. Swieca, M., Seczyk, L., Gawlik-Dziki, U., & Dziki, D. (2014). Bread enriched with quinoa leaves - the influence of protein-phenolics interactions on the nutritional and antioxidant quality. Food Chemistry, 162, 54–62. Szawara-Nowak, D., Koutsidis, G., Wiczkowski, W., & Zieliński, H. (2014). Evaluation of the in vitro inhibitory effects of buckwheat enhanced wheat bread extracts on the formation of advanced glycation end-products (AGEs). LWT-Food Science and Technology, 58(2), 327–334. Wang, R., Zhou, W., Yu, H. H., & Chow, W. F. (2006). Effects of green tea extract on the quality of bread made from unfrozen and frozen dough processes. Journal of the Science of Food and Agriculture, 86(6), 857–864. Wieser, H. (2007). Chemistry of gluten proteins. Food Microbiology, 24(2), 115–119. Wu, J.-W., Hsieh, C.-L., Wang, H.-Y., & Chen, H.-Y. (2009). Inhibitory effects of guava (Psidium guajava L.) leaf extracts and its active compounds on the glycation process of protein. Food Chemistry, 113(1), 78–84. Wu, C.-H., & Yen, G.-C. (2005). Inhibitory effect of naturally occurring flavonoids on the formation of advanced glycation endproducts. Journal of Agricultural and Food Chemistry, 53(8), 3167–3173. Zhang, X., Chen, F., & Wang, M. (2014). Antioxidant and antiglycation activity of selected dietary polyphenols in a cookie model. Journal of Agricultural and Food Chemistry, 62(7), 1643–1648. Zhang, X., Hu, S., Chen, F., & Wang, M. (2014). Treatment of proteins with dietary polyphenols lowers the formation of AGEs and AGE-induced toxicity. Food & Function, 5(10), 2656–2661.
Sadowska-Bartosz, I., Galiniak, S., Skolimowski, J., Stefaniuk, I., & Bartosz, G. (2015). Nitroxides prevent protein glycoxidation in vitro. Free Radical Research, 49(2), 113–121. Shiba, Y., Kinoshita, T., Chuman, H., Taketani, Y., Takeda, E., Kato, Y., ... Terao, J. (2008). Flavonoids as substrates and inhibitors of myeloperoxidase: Molecular actions of aglycone and metabolites. Chemical Research in Toxicology, 21(8), 1600–1609. Sivam, A. S., Sun-Waterhouse, D., Quek, S., & Perera, C. O. (2010). Properties of bread dough with added fiber polysaccharides and phenolic antioxidants: A review. Journal of Food Science, 75(8), R163–R174. Srey, C., Hull, G. L. J., Connolly, L., Elliott, C. T., del Castillo, M. D., & Ames, J. M. (2010). Effect of Inhibitor Compounds on Nε-(Carboxymethyl)lysine (CML) and Nε(Carboxyethyl)lysine (CEL) Formation in Model Foods. Journal of Agricultural Food Chemistry, 58(22), 12036–12041. Sui, X., Bary, S., & Zhou, W. (2016). Changes in the colour, chemical stability and antioxidant capacity of thermally treated anthocyanin aqueous solution over storage. Food Chemistry, 192, 516–524. Sui, X., Dong, X., & Zhou, W. (2014). Combined effect of pH and high temperature on the stability and antioxidant capacity of two anthocyanins in aqueous solution. Food Chemistry, 163, 163–170. Sui, X., Yap, P. Y., & Zhou, W. (2015). Anthocyanins during baking: Their degradation kinetics and impacts on colour and antioxidant capacity of bread. Food and Bioprocess Technology, 8(5), 983–994. Sui, X., Zhang, Y., & Zhou, W. (2016). Bread fortified with anthocyanin-rich extract from black rice as nutraceutical sources: Its quality attributes and in vitro digestibility. Food Chemistry, 196, 910–916. Sun-Waterhouse, D., Sivam, A. S., Cooney, J., Zhou, J., Perera, C. O., & Waterhouse, G. I.
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Role of quercetin in the physicochemical properties, antioxidant and antiglycation activities of bread Jing Lina, Weibiao Zhoua,b, a b
T
⁎
Food Science & Technology Programme, c/o Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore National University of Singapore (Suzhou) Research Institute, 377 Linquan Street, Suzhou Industrial Park, Jiangsu 215123, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords: Quercetin Bread Antioxidant capacity Antiglycation activity
As a natural glycation inhibitor, quercetin was incorporated into bread to develop antiglycative functional food. Quercetin added in the wheat bread flour at 0.05, 0.1, and 0.2% caused a loss of dough elasticity with lower resistance and higher extensibility. It further altered the quality of bread in terms of deceasing bread volume and increasing bread hardness. The antioxidant potential of the bread with quercetin was enhanced in a dose-dependent manner. The antiglycation capacity was assessed according to the ability of the bread to inhibit the formation of advanced glycation endproducts (AGEs) in vitro. Results showed that bread with 0.2% quercetin addition was able to inhibit 46–52% of total AGEs formed during protein glycation. Overall, the results support quercetin as a functional food ingredient in bread system, offering consumers a higher intake of antioxidant and a lower load of AGEs.
1. Introduction Using natural compounds as glycation inhibitors to reduce the intake of advanced glycation end-products (AGEs) from diet is a promising way to decrease the risk of diabetic complications. Among these compounds, quercetin is one of the most efficient antiglycative agents (Wu, Hsieh, Wang, & Chen, 2009). Quercetin is a flavonol comprising of 15 carbon atoms, with two aromatic rings connected by a three-carbon bridge. The main structure of benzopyran-4-one makes it a hydrophobic compound. The rich food sources of quercetin are onions, berries, and apples. It exhibits multiple biological activities such as antioxidative, cardioprotective, and hypoglycemic effects that are related to the protein glycation process (Erlund, 2004; Wu & Yen, 2005). Protein glycation starts from the reactions between reducing sugars and amino groups in proteins. Diverse intermediates are produced during this process, including AGEs. The accumulation of AGEs in the body contributes to the development of many chronic diseases, especially diabetic complications (Peng, Ma, Chen, & Wang, 2011). Besides, reactive carbonyl species such as methylglyoxal (MGO) are also generated along the same process. MGO can bind to the amino groups of proteins and further produce AGEs. Such reactions can result in the undesired modification of proteins and cause protein dysfunction. The formation of AGEs occurs both in vivo through normal metabolism and in vitro from food sources. Dietary AGEs (dAGEs) produced by Maillard reaction is the major external source of exposure to human
⁎
bodies. These compounds can enter the in vivo circulation of AGEs binding to the cellular receptors and cause abnormal metabolism such as the production of inflammatory cytokines, further contributing to the diabetic complications (Chinchansure, Korwar, Kulkarni, & Joshi, 2015). Meanwhile, reactive oxygen species (ROS) are largely generated during glycation, causing high oxidative stress in vivo. Different mechanisms have been reported to inhibit the glycation process (Peng et al., 2011). As for quercetin, it has been shown to inhibit protein glycation by its free radical scavenging activity to combat the oxidative stress and its ability to trap MGO (Li, Zheng, Sang, & Lv, 2014; Zhang, Chen, & Wang, 2014). Despite the well-known antiglycation activity of quercetin, most of the previous investigations were conducted with plant extract rich in quercetin or with quercetin standard (Kim, Lee, Yokozawa, Sakata, & Lee, 2011; Li et al., 2014; Wu et al., 2009). Only few studies have tried to explore the in vitro antiglycation behavior of quercetin in fortified food systems (Szawara-Nowak, Koutsidis, Wiczkowski, & Zieliński, 2014; Zhang et al., 2014). Still, these researches did not focus on just quercetin but also other polyphenols. For example, Szawara-Nowak et al. (2014) reported that buckwheat enhanced wheat bread rich in both rutin and quercetin reduced in vitro AGEs generation, and a high correlation between AGEs inhibition and the quercetin content (r = 0.92) as well as the rutin content (r = 0.86) was found. As there were more than one effective ingredient, it’s hard to distinguish the AGEs inhibitory effect of quercetin itself. It is thus necessary to
Corresponding author at: Food Science & Technology Programme, c/o Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543, Singapore. E-mail address: [email protected] (W. Zhou).
https://doi.org/10.1016/j.jff.2017.11.018 Received 20 August 2017; Received in revised form 2 November 2017; Accepted 13 November 2017 Available online 22 December 2017 1756-4646/ © 2017 Elsevier Ltd. All rights reserved.
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2.4. Measurement of bread quality attributes
elucidate the antiglycation function of quercetin in practical food products in more detail and specifically. In this research, we aimed to develop quercetin-fortified baked bread as a functional food that helps consumers to lower the risk of diabetic complications. We used bread as a carrier for quercetin fortification because bread is a popular staple food widely consumed around the world. The tasty bread crust formed through Maillard reaction between proteins and sugars during baking process is a key attraction to consumers; however, it is also a rich source of dietary AGEs, posing a potential risk to human health, especially to the diabetic patients. Quercetin may exert its anti-glycation effect on both the AGEs in the body and the dAGEs in bread matrix. On the other hand, the quality of quercetin-fortified bread would largely affect the consumer’s acceptance. Therefore, it is also of great interest to examine the effect of quercetin fortification on dough development and the final bread quality attributes. In this study, the rheological properties of quercetin-fortified baked bread dough and the bread quality were evaluated. The antioxidant capacity of the bread with quercetin was examined. Bovine serum albumin (BSA) and glucose (GLU) in vitro model system was employed to investigate the potent therapeutic values of this functional bread. The knowledge gained from this research would provide some useful insights to the potentials of quercetin-containing functional food.
Specific volume, texture, colour, moisture content, and pH value were measured, as they are important indicators to assess the overall quality of quercetin-fortified bread. Bread volume was measured using a Volscan profiler (VSP 600, Stable Micro System Ltd., Surrey, U.K.). The specific volume was obtained through dividing bread volume (cm3) by bread weight (g). Texture analysis of the bread was performed using a texture analyser (TAXT2i, Stable Micro System, Surry, U.K.) with a 20 mm diameter cylindrical probe. A bread slice of 2 cm thick cut from the central part of the bread was compressed up to 40% of its original thickness using a double compression cycle at a cross head speed of 2 mm/s. Hardness, springiness, cohesiveness, chewiness and resilience of the bread crumb were quantified. The measurements of three chromatic coordinates L∗, a∗, and b∗ of bread crust and crumb individually were performed using a colourimeter (CM-3500d Spectrophotometer, Konica Minolta, Japan). The coordinate L∗ represents the lightness of colour (L∗ = 0 points to black and L∗ = 100 points to white); a∗ indicates the colour between red and green (negative values yield green, and positive values yield red); b∗ expresses the colour between yellow and blue (negative values suggest blue, and positive values suggest yellow). The moisture content was evaluated according to the study of Sui et al. (2015). Bread samples (2 g) were heated at 100 °C in on oven until constant weight, and the moisture content was expressed as the percentage change in the weight of the sample. As for the pH value measurement, 5 g of bread sample was agitated in 50 ml of deionized water for 30 min and the supernatant layer obtained was tested by a pH meter (Metrohm 744 pH meter, Switzerland).
2. Materials and methods 2.1. Chemicals Quercetin (food grade) was obtained from Xi’an Dowell Bio-tech Co. Ltd., China with 98% of purity. Wheat bread flour (Prima brand, protein content 13.1%), instant dry yeast (Saccharomyces cerevisiae, S.I. Lesaffre, France), salt (Fairprice Cooperative Ltd., Singapore), sugar (Fairprice Cooperative Ltd., Singapore), and vegetable shortening (Bake King, Gim Hin Lee Ltd., Singapore) were purchased from a local supermarket. DPPH (2,2-diphenyl-1- picrylhydrazyl), ABTS (2,2′-azinobis (3-ethylbenzothiazo- line-6-sulphonic acid)), potassium persulfate, trolox (6-hydroxy-2,5,7,8-tetramethylchloromane-2-carboxylic acid), gallic acid (3,4,5-Trihydroxybenzoic acid), Folin-Ciocalteu reagent, GLU, BSA, MGO (40% aqueous solution), and aminoguanidine (AG), were purchased from Sigma–Aldrich (Sigma–Aldrich, St Louis, MO, USA).
2.5. Extraction of quercetin Bread crumb and crust were separated, frozen for 24 h, and freezedried. The dry maters were ground into fine bread powders. Bread powder samples (2 g) were extracted with methanol (10 ml) under 30 min shaking at 300 rpm using an orbital shaker (IKA VXR basic Vibrax, Staufen, Germany). Liquid extracts were obtained after centrifugation at 3233g for 5 min and combined together for concentration at 40 °C by a vacuum rotary evaporator after 3 rounds of extraction where the maximum extractability of quercetin was achieved at around 92% and 77% for bread crumb and crust respectively. The same extraction process was conducted on the control bread powder spiked with quercetin to examine the recovery rate of the extraction process, which was found to be above 96%.
2.2. Farinograph and extensograph tests Wheat bread flour fortified with various levels of quercetin (0, 0.05, 0.10, and 0.20%) was loaded on a Farinograph-E equipped with a S50 mixer and sigma blades (Brabender, Duisburg, Germany). Farinograph test was conducted according to the constant flour weight procedure of AACC Method 54–21 (AACC, 2000). In the extensograph test, freshly prepared quercetin-fortified dough samples were examined using an Extensograph-E (Brabender, Duisburg, Germany) following the AACC Method 54–10 (AACC, 2000).
2.6. Quantification of quercetin using HPLC/DAD Quercetin was quantified by a Shimadzu High Performance Liquid Chromatography (HPLC) system equipped with a diode array detector (DAD) (Shimadzu, Kyoto, Japan) using a C18 reserved-phase column (250 × 4.6 mm, Sunfire, Waters, Wexford, Ireland). The flow rate was 1 ml/min and the oven temperature was 30 °C. Mobile phase A (1% acetic acid in DI water) and B (100% acetonitrile) were applied according to the ratio of 6:4. Detection was performed at 257 nm. Quantification of quercetin was accomplished by the external calibration ranging from 0.025 to 0.250 mg/ml.
2.3. Bread sample preparation Bread making process was modified following the study of Sui, Yap, and Zhou (2015). Quercetin was firstly mixed with 1 kg wheat bread flour at the addition levels of 0, 0.05, 0.10, and 0.20%, which was further mixed with 10 g instant dry yeast, 12 g salt, 30 g shortening, 40 g sugar, and 600 g water for 5 min to form bread dough in a mixer. The dough was rested for 10 min at 20 °C followed by molding into small pieces (50 g each) and proofing in a proofer (Climatic chamberKBF, Binder, Germany) at 40 °C and 85% relative humidity condition. The baking process was conducted at 200 °C in an oven (Eurofours, France) for 8 min. The bread samples are cooled down to room temperature for 1 h after baking.
2.7. Antioxidant capacity analysis: ABTS assay The ABTS assay was performed according the method of Sui, Dong, and Zhou (2014) with some modifications. ABTS radical cation (ABTS+) was prepared by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate and being left in the dark for 12–16 h before use. The ABTS+ solution was diluted with methanol to an absorbance of 0.70 ± 0.05 at 734 nm. Bread extract or trolox (0.1 ml) was mixed with the diluted ABTS+ solution (1 ml) for 7 min. The absorbance at 734 nm was then measured using a UV–vis 300
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Table 1 Farinograph and extensograph analysis of bread flour fortified with different levels of quercetin. Formulation
Water absorption (%)
Control 0.05% 0.10% 0.20%
63.73 63.67 63.70 63.63
± ± ± ±
0.35a 0.40a 0.00a 0.15a
Development time (min) 7.77 8.53 9.27 9.63
± ± ± ±
0.12c 0.35b 0.45a 0.51a
Resistance [BU]
Extensibility [mm]
1220.00 ± 33.18a 970.67 ± 10.12d 1026.00 ± 32.92c 1124.00 ± 16.00b
111.00 121.67 131.67 123.33
± ± ± ±
4.00b 4.73ab 12.42a 2.31ab
Ratio number 12.90 10.60 10.20 11.47
± ± ± ±
0.95a 0.70b 1.22b 0.50b
Values in the same column followed by different superscript letters are significantly different (p < .05).
spectrophotometer (Shimadzu UVmini-1240, Tokyo, Japan). The antioxidant capacity was expressed as mg trolox equivalent per 100 g dried bread sample.
Inhibition(%) = {1−[(fluorescence of solution with bread extract
2.8. Antioxidant capacity analysis: DPPH assay
IC50 of the bread extract or AG solution were calculated by the Probit Regression Model using the Statistical Product and Service Solutions (SPSS) computer software (SPSS 21, IBM Corporation, New York, USA). The measurement of dietary AGEs in bread was conducted following the method described by Zhang et al. (2014). In brief, the fluorescent AGEs in 0.1 ml bread extract were indicated at the excitation wavelength of 355 nm and emission wavelength of 405 nm by the microplate reader.
− intrinsic fluorescence of bread extract) /fluorescence of solution without bread extract]} × 100
The DPPH assay was conducted based on the method of Sui, Bary, and Zhou (2016). The bread extract or trolox (0.1 ml) and 60 µM methanol DPPH% solution (3.9 ml) were mixed and incubated in the dark for 2 h. The absorbance at 515 nm was measured by the spectrophotometer. The antioxidant capacity was expressed as mg trolox equivalent per 100 g dried bread sample. 2.9. Total phenolic content analysis
2.11. Statistical analysis The total phenolic content (TPC) was determined as described by Sui et al. (2015). Bread liquid extract (0.1 ml) was mixed with FolinCiocalteau reagent (0.1 ml) and deionised water (1.58 ml). The mixture solution was incubated for 6 min, followed by adding 2 M of sodium carbonate (0.3 ml). After incubation in the dark for 2 h, the absorbance of the mixture was measured at 765 nm by the spectrophotometer. The TPC of the samples was expressed in gallic acid equivalent per 100 g dried bread sample.
All experiments were conducted in triplicate batches with three individual samples from each batch. The mean values of the analysis were reported as the final results with standard deviation (n = 9). Oneway ANOVA was used for statistical analysis adopting Duncan’s multiple range test on SPSS to determine the presence of any significant difference (p < .05) between samples. Pearson’s correlation was also conducted using SPSS.
2.10. Antiglycation capacity examination
3. Results and discussion
In the BSA-GLU system, the assay was performed according to the method described by Szawara-Nowak et al. (2014), with a slight modification based on the study of Izabela Sadowska-Bartosz, Galiniak, and Bartosz (2014). BSA (10 mg/ml) was mixed with GLU (0.5 M) in phosphate buffer (0.1 M, pH 7.4) as the test solution. Sodium azide (0.2 mg/ml) was added to achieve an aseptic condition. The test solution (2 ml) was incubated at 55 °C for 40 h with or without a bread extract (1 ml) in which the final concentration of quercetin ranging from 0.00 to 0.25 g/ml was selected with high inhibitory effect. The fluorescence intensity of the solution was measured at the characteristic excitation and emission wavelengths of 325/440, 330/415, 325/434 and 365/480 nm to detect the levels of AGEs and protein oxidation markers, dityrosine, N-formylkynurenine and kynurenine, respectively, by a microplate reader (Victor X4 Multilabel Plate Reader, PerkinElmer, USA). In the BSA-MGO system, the assay was based on the method of Wu and Yen (2005) with slight modification. BSA (1 mg/ml) was dissolved in phosphate buffer (0.1 M, pH 7.4) and mixed with MGO (5 mM) and sodium azide (0.2 mg/ml) to obtain a test solution. Bread extraction (1 ml) was added into the test solution (2 ml), followed by the incubation at 37 °C for 6 days. The sample fluorescence was examined at the excitation and emission wavelengths of 340 and 420 nm respectively. The positive control for both of the assays was AG at the concentrations of 1–25 mM. IC50 value, the specific concentration at which 50% of glycation was inhibited, was used to compare the anti-glycation effect between the commercial inhibitor AG with the quercetin-fortified bread. The percentage inhibition of total AGEs, oxidatively modified proteins formation, and AGEs induced by MGO was calculated based on the following equation:
3.1. Farinograph and extensograph analysis Effects of quercetin on the rheological properties of the dough are shown in Table 1. As expected, no significant difference of water absorption was observed between the control and the quercetin-fortified dough. The low water solubility of quercetin makes it not to compete for water with other constituents in the dough during mixing process. However, the development time of the dough with quercetin extended up to 19% compared to the control dough (from 7.77 to 9.63 min). The longer dough development time might be attributed to the slower hydration rate of protein that impaired the aggregation behaviour of high molecular weight (HMW) protein in flour (Rosell, Santos, & Collar, 2006). Quercetin could interact with gluten, negatively affecting the protein hydration and further causing a longer dough development time. Besides, the long development time of dough can be associated with the loss of its elasticity based on the dough structure alteration (Miś, Grundas, Dziki, & Laskowski, 2012). In fact, a typical interchain bonding namely disulfide bonds (SS) mainly accounts for the dough stability via the SS-SH interchange reactions (Sivam, Sun-Waterhouse, Quek, & Perera, 2010). Quercetin, as a reducing agent, is involved in these reactions, leading to lower amount of SS as a consequence of a weaker protein network. The development time was thus prolonged with impeded dough elasticity. From the extensograph results present in Table 1, all of the treated dough possessed significantly lower resistance compared to the control dough. This indicated a weaker gas retention capacity and possible disintegration of the gluten network during dough proofing. However, close bonds can also be formed between the gluten matrix and the additives that are strong enough to counteract the weakening impact of 301
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Table 2 Specific volume of and texture analysis of the bread fortified with different levels of quercetin. Sample
Specific Volume (cm3/g)
Control 0.05% 0.10% 0.20%
3.73 3.61 3.55 3.37
± ± ± ±
0.03a 0.02b 0.05b 0.03c
Hardness (N) 218.52 229.55 256.83 270.59
± ± ± ±
13.97b 31.46b 23.43a 12.85a
Springiness 1.01 1.01 0.97 0.97
± ± ± ±
0.02a 0.01a 0.00b 0.01b
Cohesiveness 0.86 0.85 0.84 0.85
± ± ± ±
0.01a 0.01a 0.01a 0.01a
Chewiness (N) 180.46 193.83 208.32 235.45
± ± ± ±
8.13c 30.04bc 11.27b 19.74a
Resilience 0.53 0.53 0.52 0.53
± ± ± ±
0.02a 0.02a 0.01a 0.00a
Values in the same column followed by different superscript letters are significantly different (p < .05).
from Zhang et al. (2014) that quercetin-enriched cookies obtained darker colour with reduced lightness. The b∗ value of quercetin-fortified bread crumb was significantly higher than that of the control by up to 45%, indicating the accumulation of yellow colour. The a∗ value dropped from −0.09 to −2.04 at 0.05% quercetin addition and progressively increased to -1.32 at the 0.2% addition, which coincided with the increasing b∗ value showing a shift from green to yellow. The colour of bread crumb seemed to be dramatically influenced by the original yellowish colour of quercetin powder. However, in contrast to the distinct alteration of bread crumb in colour, bread crust showed no significant change. This is in agreement with the result from Gómez, Ronda, Blanco, Caballero, and Apesteguía (2003) that in spite of the influence from colourful food additives, the characterized browning induced by Maillard reaction and caramelization mainly accounted for bread crust’s colour. In addition, the moisture content of the quercetin incorporated bread crumb and crust did not exhibit significant difference from the control (Table 3). Nonetheless, it was much lower in the curst than in the crumb due to more rapid water evaporation on bread surface during baking. Moverover, as quercetin is unstable under alkaline condition, the pH value of final food product is crucial for its retention. The results in Table 3 show that the pH value of the bread crumb and crust were statistically the same within the range of 5.52–5.61, which made bread a feasible carrier to produce quercetin-contaning functional food.
SS reduction on dough structure (Miś et al., 2012). Quercetin can hydrophobically interact with glutenins to form tight bonds and thus interfere in the formation of dough structure. As a consequence, the resistance of the quercetin-fortified dough firstly decreased at the lowest dosage (0.05%) and then increased with incremental quercetin addition, but the overall values were still lower than that of the control dough. Moreover, significant increasing of extensibility was observed in the dough with quercetin. The extensibility can be enhanced when the SS amount is reduced and the solubility of glutenin increases (Wieser, 2007). In the presence of quercetin, the ratio number of dough (reflecting the dough strength) was statistically lower than the control dough. This revealed a more delicate dough structure. Our result is in line with the study of Han and Koh (2011) who reported that the addition of phenolic acids hindered the rheological properties of dough by reducing the HMW proteins.
3.2. Bread quality attributes examination The specific volume of bread, shown in Table 2, decreased successively from 3.73 cm3/g (control bread) to 3.37 cm3/g (0.2% quercetinfortified bread). Quercetin might impair the rheological properties of dough at the expense of a small bread volume. The result is consistent to the literature reports that bread volume decreased with the incorporation of catechin, anthocyanin, and other polyphenols from fruit extracts (Sui, Zhang, & Zhou, 2016; Sun-Waterhouse et al., 2011; Wang, Zhou, Yu, & Chow, 2006). Besides, the characterization of bread texture in Table 2 shows that the hardness and chewiness of the 0.05–0.2% quercetin-fortified bread were significantly higher than that of the control bread by up to 23%. However, the springiness only showed statistical difference with more than 0.1% quercetin addition; no significant difference was found in the cohesiveness and resilience among all the quercetin-fortified bread and the control. Hardness is a measurement of peak force required to compress the sample, while chewiness represents a quantitative estimation of energy needed to disintegrate bread structure (Ribotta, Pérez, Añón, & León, 2010). Springiness indicates the capacity of sample to spring back after a deformation due to the compression. Harder texture implied the formation of denser bread crumb, corresponding to the smaller specific volume of quercetin-fortified bread. This was due to the smaller amount of SS bonds caused by the reducing capacity of quercetin, thus impairing the bread crumb’s elasticity and weakening the bread matrix. Our observation is in agreement with the results of Sui et al. (2016) as in their studies the bread incorporating black rice extract (up to 4%) showed increased hardness texture and decreased bread volume. Another recent study about the impact of incorporated quinoa leaves on bread quality also showed a similar trend: as the amount of quinoa leaves increased from 1 to 5%, bread volume decreased and crumb texture became harder and chewier (Swieca, Seczyk, Gawlik-Dziki, & Dziki, 2014). Regarding the effect of quercetin on the colourimetric profile of bread, a significant decrease in bread crumb’s lightness by up to 16% is shown in Table 3. The result suggested that the colour of bread crumb became dull and slightly grayish as the brightness was impeded after the fortification of quercetin. This is in agreement with the observations
3.3. Antioxidant capacity and total phenolic content analysis After the baking process, the average quercetin content remaining in the bread crumb at 0.05, 0.10, and 0.20% additions was 0.35, 0.77, and 1.42 mg/g Dry Weight (DW), respectively; meanwhile, that in the bread crust was 0.27, 0.60, and 1.20 mg/g DW, respectively. The smaller amount of quercetin in the crust was due to the much higher temperature in the crust layer than the crumb during baking (data not shown), leading to more quercetin degradation. As shown in Table 4, a stepwise fortification of quercetin in bread resulted in a corresponding significant increase in the antioxidant activity from 2.9 to 285.8 mg trolox/100 g DW examined by ABTS assay and from 8.4 to 298.5 mg trolox/100 g DW by DPPH assay. The total phenolic content of the quercetin-fortified bread also increased from 76.3 to 259.2 mg gallic acid/100 g DW. Moreover, in spite of less quercetin in the bread crust, its antioxidant activity was significantly higher than the crumb in both the control condition and 0.5% quercetin addition. The differences were assumed to stem from the antioxidant properties of the Maillard reaction products (MRPs) formed in bread crust. Besides, the thermal degradation products of quercetin also possess radical scavenging properties, such as protocatechuic acid (Buchner, Krumbein, Rohn, & Kroh, 2006). Similarly, the bread crust also exhibited significantly higher total phenolic content than the crumb. It is well understood that the antioxidant profiles of a polyphenol-fortified baked product could arise from various sources including the intrinsic polyphenols in wheat flour, the incorporated phenolic compounds, the intermediate or thermal degradation products of the phenolics, and the polyphenol-starch/protein complex (Sivam et al., 2010). 302
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Table 3 The colourimetric profiles (L*, a*, and b*), moisture content (MC%), and the pH value of the bread with different levels of quercetin. Sample
L* a* b* MC% pH
Crumb
Crust
Control
0.05%
0.10%
0.20%
Control
0.05%
0.10%
0.20%
76.72 ± 0.56a −0.09 ± 0.20d 16.05 ± 0.46d 41.02 ± 0.85a 5.52 ± 0.02a
72.43 ± 1.33b −2.04 ± 0.19a 23.34 ± 0.65c 41.69 ± 1.33a 5.49 ± 0.02a
67.89 ± 0.42c −1.76 ± 0.01b 27.09 ± 0.12b 42.15 ± 2.01a 5.61 ± 0.04a
64.29 ± 0.59d −1.32 ± 0.06c 29.39 ± 0.43a 40.48 ± 1.54a 5.54 ± 0.01a
50.42 ± 2.13a 17.05 ± 0.29a 32.17 ± 1.99a 22.51 ± 1.49a 5.39 ± 0.06a
49.99 ± 3.14a 16.87 ± 0.09a 31.84 ± 2.34a 21.87 ± 0.59a 5.41 ± 0.05a
50.64 ± 1.81a 17.23 ± 0.20a 33.48 ± 1.98a 22.46 ± 1.65a 5.32 ± 0.03a
49.17 ± 2.36a 16.79 ± 0.59a 32.98 ± 1.75a 20.59 ± 1.89a 5.36 ± 0.01a
Values in the same row followed by different superscript letters are significantly different (p < .05).
depleted quercetin by dAGEs would be. Therefore, the inhibition difference between the bread crust and crumb tended to be smaller and the inhibition effect reached statistically the same extent as the quercetin addition level was above 0.2%. As the AGEs inhibition ability of quercetin may not just come through a single pathway, we also investigated its AGEs inhibition effects in the BSA-MGO system, as shown in Fig. 1(B). It is noteworthy that the enhanced inhibition of MGO-induced AGEs by the bread extract with increasing quercetin amount was consistent with that in the BSAGLU system. Similarly, no significantly increased inhibition on AGEs was observed in this system when the quercetin concentration of the bread crumb and crust extracts was beyond 0.16 and 0.15 mg/ml, respectively. As known, the reactions between MGO and the amino/ sulfhydryl group of amino acid residues such as arginine, lysine and cysteine, lead to the formation of AGEs. Quercetin can directly deplete MGO (Li et al., 2014), and this reduces the protein-modifying reactions to inhibit AGEs formation.
3.4. Antiglycation capacity analysis Glycation process involves three main stages: first, an unstable and highly reversible Schiff base is produced, prone to oxidation with free radicals generation; then, it is followed by a transition of the Schiff base into stable Amadori products through isomerization; lastly, the Amadori adducts, namely ketosamines, undergo further rearrangement, dehydration and other complicate reactions to form reactive dicarbonyl intermediates like MGO and irreversible AGEs such as carboxymethyllysine (CML) (Peng et al., 2011). In this study, two methods were used to examine the anti-glycation effect of the quercetin-fortified bread: (1) the BSA-GLU model, targeting at the total AGEs produced by protein glycation; (2) the BSA-MGO model, focusing on the AGEs prompted by the main precursor, MGO.
3.4.1. Inhibition of AGEs formation The inhibitory effect of bread extracts on the in vitro AGEs formation using the BSA-GLU system is shown in Fig. 1(A). Below a certain quercetin concentration (0.16 and 0.15 mg/ml respectively), both quercetin-fortified bread crumb and crust extracts behaved in a dosedependent manner on the reduction of total AGEs produced by protein glycation from 23.4% to 59.0% and from 12.3% to 56.2%, respectively. Previous studies have shown that in terms of free radical scavenging capacity, antioxidant activity is the major action for polyphenols to suppress AGEs generation (Navarro, Fiore, Fogliano, & Morales, 2015; Zhang, Hu, Chen, & Wang, 2014). Supportively, our results showed a significant correlation between the antioxidant activity in terms of free radical scavenging capacity and the inhibition of total AGEs generation (r = 0.916, p < .05 for DPPH assay and r = 0.957, p < .05 for ABTS assay). It indicated high possibility of the free radical scavenging activity to dominate the mechanism of AGEs inhibition by quercetinfortified bread. The results also showed that below 0.1% quercetin addition, quercetin-fortified bread crumb extract (0.11 mg/ml) had significantly higher inhibitory effect (up to 47%) than quercetin-fortified bread crust extract (0.098 mg/ml). It might ascribe to the higher level of inherent AGEs (i.e. dAGEs) produced in the bread crust than that in the bread crumb. Discussion in detail would be conducted later in Section 3.4.4. In brief, the more the quercetin available in bread, the smaller ratio of
3.4.2. Oxidatively-modified proteins inhibition analysis Combination of glycation and oxidation as glycoxidation also contributes to the formation of AGEs. Due to the difficulty to detect the unstable intermediates during oxidation, the relatively stable end products were detected to reflect the extent of protein oxidative damage. Dityrosine, N-formylkynurenine and kynurenine were used to be the indices of oxidant-induced protein crosslinking (Sadowska-Bartosz, Galiniak, Skolimowski, Stefaniuk, & Bartosz, 2015; Shiba et al., 2008). The inhibitory effect of quercetin-fortified bread on these oxidative markers are displayed in Fig. 1(C), (D), and (E), respectively. Quercetinfortified bread extracts reduced the amount of all the three markers dose-dependently. Dityrosine is an indicator of crosslinking formation between two tyrosines by hydroxyle radicals under oxidative stress (Leeuwenburgh, Hansen, Holloszy, & Heinecke, 1999). Our results supported quercetinfortified bread as a plausible inhibitor to reduce such crosslinking formed during protein glycation and therefore helping maintain the normal function of proteins. Besides, N-formylkynurenine, as a main product of tryptophan modification with exposure to ROS, was also inhibited by the quercetin-fortified bread extract, following the same pattern as that of dityrosine. Nevertheless, kynurenine was inhibited to
Table 4 Antioxidant capacity and total phenolics content of the bread with different levels of quercetin. Sample
Control 0.05% 0.10% 0.20%
ABTS (mg Trolox/100 g DW)
DPPH (mg Trolox/100 g DW)
TPC (mg Gallic acid/100 g DW)
Crumb
Crust
Crumb
Crust
Crumb
Crust
2.9 ± 1.8d,B 55.5 ± 4.7c,B 155.1 ± 20.8b,A 285.8 ± 14.2a,A
22.0 ± 9.5d.A 94.8 ± 5.1c,A 155.5 ± 24.8b,A 290.8 ± 15.9a,A
8.4 ± 1.2d,B 62.9 ± 1.7c,B 153.8 ± 3.1b,B 298.5 ± 14.5a,A
20.5 ± 0.7d.A 91.6 ± 3.9c,A 159.8 ± 2.0b,A 295.2 ± 16.4a,A
63.9 ± 5.3d,B 97.2 ± 2.0c,B 151.0 ± 2.9b,B 233.4 ± 1.3a,B
76.3 ± 5.5d,A 123.3 ± 2.4c,A 168.4 ± 3.2b,A 259.2 ± 12.6a,A
Within each column, data values denoted by different lowercase letters are statistically different (p < .05) across the quercetin percentage. Within each row, for the same test (i.e. ABTS, DPPH or TPC), data values denoted by different uppercase letters are statistically different (p < .05) between bread crumb and crust.
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Fig. 1. Inhibitory activity of quercetin-fortified bread extract on the formation of (A) total fluorescent AGEs produced by protein glycation process, (B) fluorescent AGEs induced by its precursor MGO (C) ditryrosine, (D) N-formylkynurenine, (E) kynurenine. Within the same curve, data values denoted by different lowercase letters are statistically different (p < .05) across the quercetin concentrations. At the same quercetin concentration, data values denoted by different uppercase letters are statistically different (p < .05) between bread crumb and crust.
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to scavenge free radicals and to trap the AGEs precursors, as the lipid content in bread was low. In general, the modification of AGE formation by inhibitors in food systems is mostly product dependent. This involves considerations of complicated food components in the matrix and the processing conditions including temperature, moisture content, as well as processing time (Poulsen et al., 2013; Srey et al., 2010).
Table 5 Concentration (mg/ml) of quercetin in the bread crumb and crust extract and concentration (mg/ml) of AG exerting the 50% inhibition of AGEs or oxidative damage markers. IC50-antiglycation activity
Bread Crumb extract
Bread Crust extract
AG
AGEs from protein glycation AGEs derived from MGO Dityrosine N-formylkynurenine Kynurenine
0.11 ± 0.03b
0.12 ± 0.02b
0.74 ± 0.22a
0.03a 0.02b 0.02b 0.32
0.17 ± 0.07a 0.12 ± 0.03b 0.13 ± 0.03b N.A.*
0.16 ± 0.16a 0.70 ± 0.18a 0.70 ± 0.16a N.A.
0.14 0.09 0.10 0.42
± ± ± ±
4. Conclusion In conclusion, quercetin fortification altered dough development, resulting in a slight loss of dough elasticity. Furthermore, it caused significant quality changes to bread, including smaller volume, harder texture, and green-yellowish appearance. The antioxidant capacity and total phenolic content of the bread were significantly enhanced by the quercetin fortification. Quercetin-fortified bread inhibited the in vitro glycation process and reduced AGEs formation, most probably by free radical scavenging and trapping reactive dicarbonyl intermediates. These results indicated that quercetin fortification might be a promising way to produce functional bread that helps consumers to prevent glycation-associated diseases.
Different superscript letters in the same row denote significant differences (p < .05). * N.A: Not applicable.
Acknowledgements Supports from the Agency for Science, Technology and Research (A∗STAR) Singapore through research grant BMRC 14/1/21/24/009 under Singapore-New Zealand Foods for Health Grant Call and Jiangsu Province under the Scientific Research Platform scheme BY2014139 are gratefully acknowledged. The first author also thanks the National University of Singapore (NUS) for financial support. References AACC (2000). Approved methods of the American association of cereal chemists. Methods, 54, 21. Buchner, N., Krumbein, A., Rohn, S., & Kroh, L. W. (2006). Effect of thermal processing on the flavonols rutin and quercetin. Rapid Communications in Mass Spectrometry, 20(21), 3229–3235. Chinchansure, A. A., Korwar, A. M., Kulkarni, M. J., & Joshi, S. P. (2015). Recent development of plant products with anti-glycation activity: A review. RSC Advances, 5(39), 31113–31138. Erlund, I. (2004). Review of the flavonoids quercetin, hesperetin, and naringenin. Dietary sources, bioactivities, bioavailability, and epidemiology. Nutrition Research, 24(10), 851–874. Gómez, M., Ronda, F., Blanco, C. A., Caballero, P. A., & Apesteguía, A. (2003). Effect of dietary fibre on dough rheology and bread quality. European Food Research and Technology, 216(1), 51–56. Han, H. M., & Koh, B. K. (2011). Effect of phenolic acids on the rheological properties and proteins of hard wheat flour dough and bread. Journal of the Science of Food and Agriculture, 91(13), 2495–2499. Kim, H. Y., Lee, J. M., Yokozawa, T., Sakata, K., & Lee, S. (2011). Protective activity of flavonoid and flavonoid glycosides against glucose-mediated protein damage. Food Chemistry, 126(3), 892–895. Leeuwenburgh, C., Hansen, P. A., Holloszy, J. O., & Heinecke, J. W. (1999). Hydroxyl radical generation during exercise increases mitochondrial protein oxidation and levels of urinary dityrosine. Free Radical Biology and Medicine, 27(1–2), 186–192. Li, X., Zheng, T., Sang, S., & Lv, L. (2014). Quercetin inhibits advanced glycation end product formation by trapping methylglyoxal and glyoxal. Journal of Agricultural Food Chemistry, 62(50), 12152–12158. Miś, A., Grundas, S., Dziki, D., & Laskowski, J. (2012). Use of farinograph measurements for predicting extensograph traits of bread dough enriched with carob fibre and oat wholemeal. Journal of Food Engineering, 108(1), 1–12. Navarro, M., Fiore, A., Fogliano, V., & Morales, F. J. (2015). Carbonyl trapping and antiglycative activities of olive oil mill wastewater. Food & Function, 6(2), 574–583. Peng, X., Ma, J., Chen, F., & Wang, M. (2011). Naturally occurring inhibitors against the formation of advanced glycation end-products. Food & Function, 2(6), 289–301. Poulsen, M. W., Hedegaard, R. V., Andersen, J. M., de Courten, B., Bügel, S., Nielsen, J., ... Dragsted, L. O. (2013). Advanced glycation endproducts in food and their effects on health. Food and Chemical Toxicology, 60, 10–37. Ribotta, P. D., Pérez, G. T., Añón, M. C., & León, A. E. (2010). Optimization of additive combination for improved soy–wheat bread quality. Food and Bioprocess Technology, 3(3), 395–405. Rosell, C. M., Santos, E., & Collar, C. (2006). Mixing properties of fibre-enriched wheat bread doughs: A response surface methodology study. European Food Research and Technology, 223(3), 333–340. Sadowska-Bartosz, I., Galiniak, S., & Bartosz, G. (2014). P78 - Polyphenols protect against protein glycoxidation. Free Radical Biology and Medicine, 75(Supplement 1), S47.
Fig. 2. Inhibitory activity of quercetin on the formation of dAGEs in bread. Within the same bread sample type (i.e. crumb or crust), columns denoted with different lowercase letters are statistically different (p < .05) across the quercetin addition levels. At the same quercetin addition level, columns denoted with different uppercase letters are statistically different (p < .05) between the bread crumb and crust extract.
a less extent. This is consistent with another study investigating the inhibition of oxidative protein modifications by nitroxides, in which weaker inhibition of kynurenine was also found due to the concurrent conversion of N-formylkynurenine into kynurenine. (Sadowska-Bartosz et al., 2015). 3.4.3. IC50-antiglycation activity analysis IC50-antiglycation activity of the bread is presented in Table 5. With a significantly smaller IC50 value, quercetin-fortified bread crumb and crust extracts (0.110 and 0.120 mg/ml, respectively) showed a stronger antiglycation effect compared to the positive control AG (0.744 mg/ml) in terms of the total AGEs formation. In contrast, no significant difference in the IC50 value between the bread crumb and crust extract (0.138 and 0.174 mg/ml, respectively) and AG (0.158 mg/ml) was observed in the BSA-MGO system. The IC50-antiglycation activity of quercetin-fortified bread showed its strong potential to be an efficient natural glycation inhibitor instead of AG. 3.4.4. Inhibition of dAGEs in baked bread As shown in Fig. 2, the inhibitory effect on dAGEs by both crumb and crust of the quercetin-fortified bread exhibited a linear increasing trend. The maximum inhibition (above 55%) was shown at the 0.2% quercetin addition based on flour weight. It is in line with an previous study in which 0.25% quercetin incorporated cookies showed around 80% inhibition on dAGEs by the suppression of lipid peroxidation (Zhang et al., 2014). However, rather than inhibiting lipid peroxidation, quercetin in bread exerted its antiglycation activity by its abilities 305
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