In vitro bioaccessibility and bioavailability of quercetin from the quercetin-fortified bread products with reduced glycemic potential

Food Chemistry 286 (2019) 629–635

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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Research Article

In vitro bioaccessibility and bioavailability of quercetin from the quercetinfortified bread products with reduced glycemic potential Jing Lina, Lexin Melina Teoa, Lai Peng Leonga, 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

ARTICLE INFO

ABSTRACT

Keywords: Quercetin Bread In vitro digestion Glycemic potential Bioaccessibility Bioavailability

Quercetin, a plant-extracted flavonol, is a potent inhibitor of carbohydrate digestive enzymes. This study aimed to examine: (1) the extent to which quercetin altered the starch digestion and the glycemic potential of the fortified western baked bread as well as oriental steamed bread, (2) the matrix effects of bread on the potential bioaccessibility and bioavailability of quercetin. The starch digestion of bread was quantitatively evaluated by determination of starch digestion rate (k) via a developed mathematical model. Results showed a significant reduction of k for the 1.5, 3, and 6% quercetin-fortified baked and steamed bread compared to the controls by up to 18.3%. The predicted GIs of the baked bread samples with quercetin were significantly reduced in comparison to control bread. Moreover, the potential bioaccessibility of quercetin incorporated in bread products was significantly higher than that of quercetin supplement probably as a result of the protective effects from food matrix.

1. Introduction Bread is a staple food that has been widely consumed all over the world. As a carbohydrate-rich food product, bread contains a large amount of rapidly digestible starch, often leading to a high glycemic index (GI). Diets higher in GI account for 10–33% higher risk of type 2 diabetes (T2D) among 3 large US cohorts (Bhupathiraju et al., 2014). Low dietary GI have been associated with a reduced risk of some chronic diseases, such as diabetes and stroke, in the epidemiologic studies (Barclay et al., 2008). It is of great interest to reformulate bread products to reduce their glycemic potentials. One of the promising strategies is fortification of natural plant extracts rich in flavonoids that could influence the starch digestibility. For example, incorporation of anthocyanins-rich black rice extract or catechin-rich green tea extract has reduced the digestibility of bread (Goh et al., 2015; Sui, Zhang, & Zhou, 2016). Quercetin is one of the most common flavonols rich in onion, kale and broccoli (Miean & Mohamed, 2001). As it could inhibit the carbohydrate digestive enzymes including α-glucosidase and α-amylase (Tadera, Minami, Takamatsu, & Matsuoka, 2006), quercetin may retard starch digestion in food. Incorporation of quercetin in bread is probably an effective way to develop low GI food products. Besides the inhibitory effect on digestive enzymes, one of the other well-known health

benefits of quercetin is its strong antioxidant capacity (Iacopini, Baldi, Storchi, & Sebastiani, 2008). Through its interactions with reactive oxygen species (ROS), quercetin could significantly reduce in vivo oxidative stress and the associated damages (Nabavi, Nabavi, Eslami, & Moghaddam, 2012). When it comes to the beneficial effect of a compound, one of the main concerns is its bioavailability. Highly variable bioavailability of quercetin has been reported as this is strongly related to its solubility in the vehicle for administration (Guo & Bruno, 2015). Generally, the low water solubility of pure quercetin supplement largely limits its absorption in the bodies (Guo & Bruno, 2015). Food matrices can act as the efficient delivery systems for quercetin and affect its absorption. Egert et al. (2012) found significantly higher bioavailability of quercetin from the quercetin-enriched cereal bars as compared to quercetin powder capsules as a result of the positive influences from the food components and the relatively uniform distribution of quercetin. In this context, incorporation of quercetin in bread has high potential to be a win-win strategy. It was hypothesized that the glycemic potential of bread could be lowered by quercetin while the bread matrix could improve the absorption of quercetin. This study aimed to investigate the digestive profiles of western oven-baked bread and oriental steamed bead fortified with quercetin. These two types of bread are usually prepared using different

⁎ 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.foodchem.2019.01.199 Received 3 August 2018; Received in revised form 30 December 2018; Accepted 31 January 2019 Available online 07 February 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

Food Chemistry 286 (2019) 629–635

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ingredients (e.g. high protein flour for baked bread and low to medium protein flour for steamed bread) and different processing methods (e.g. baking at 200 °C and steaming at 100 °C with saturated relative humidity) (Lau, Soong, Zhou, & Henry, 2015). The impact of quercetin on the digestion of these two bread systems might thus vary. The extent to which quercetin affected bread starch digestion and further altered the GI and glycemic load (GL) values of the fortified bread products was determined. The feasibility of utilizing bread matrix to enhance the absorption of quercetin was scientifically evaluated through an in vitro digestion model.

2.3.2. HPLC analysis The samples obtained after filtration by a Phenex 0.45 μm PTFE syringe filter were analyzed using a reserved-phase C18 Sunfire column (250 mm × 4.6 mm/5 μm; Waters, Wexford, Ireland) at 30 °C on a Shimadzu HPLC system (Shimadzu, Japan) coupled with a diode array detector (DAD). The sample injection volume was 20 μL. The flow rate was 1 mL/min. Two mobile phases (A: 1% acetic acid; B: 100% acetonitrile) were used in the ratio of 6:4. Detection was performed at 374 nm for 10 min and the quantification of quercetin was carried out by external calibrations. 2.4. In vitro digestibility study

2. Materials and methods

2.4.1. In vitro digestion of bread samples The in vitro digestion was performed as described by Minekus et al. (2014), with some modification according to Goh et al. (2015) and Sui et al. (2016). The preparation of Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF) followed the protocol reported by Minekus et al. (2014). Oral phase. Bread crumb without crust was blended to obtain fine crumb samples. Five grams of bread crumb sample were mixed with 4 mL of α-amylase in SSF buffer (75 U/mL in the final mixture) and 1 mL of 7.5 mM CaCl2 solution. The mixture was subject to a 20 s vortex and then incubated for 2 min with a magnetic stirring rod on a 15-place magnetic stirrer (MIXdrive 15, 2mag AG, Germany) seated in a 37 °C circulating water bath. Gastric phase. Samples obtained after the oral phase were mixed with 8 mL of pepsin in SGF buffer (2000 U/mL in the final mixture), 5 μL of 0.3 M CaCl2 solution, 1.345 mL of water, as well as 0.65 mL of 1 M HCl to reach pH 3.0. The mixture was then incubated with stirring at 37 °C for 2 h. Intestinal phase. Gastric chyme was mixed with 16 mL of pancreatin in SIF buffer (100 U/mL for trypsin activity in the final mixture), 0.18 g of bile extract (10 mM in the final mixture), 80 μL of amyloglucosidase (21 U/mL in the final mixture), 40 μL of 0.3 M CaCl2 and 3.48 mL of water. To reach pH 7.0, 0.4 mL of 1 M NaOH was added. The resultant mixture was then transferred into a dialysis tube (cut-off size 14 kDa) and dialyzed for 6 h at 37 °C in 200 mL of PBS buffer (phosphate buffered saline, pH 7.4).

2.1. Materials The raw materials for breadmaking were purchased from the local supermarket: baked bread flour containing 13.1% of protein (Prima, Singapore), steamed bread flour containing 7.9% of protein (Gin Hin Lee, Singapore), instant dry yeast (Saccharomyces cerevisiae, S.I. Lesaffre, France), vegetable shortening (Bake King, Gin Hin Lee, Singapore), sugar (NTUC Fairprice, Singapore) and salt (Pagoda, Siem Trading, Singapore). The food grade quercetin (98% of purity) was obtained from Hunan Kang Biotech Co., Ltd, China. α-Amylase from porcine pancreas (A3176), pepsin from porcine gastric mucosa (P7000), pancreatin from porcine pancreas (P7545), amyloglucosidase from Aspergillus niger (A7095), porcine bile extract (B8631), dialysis tubing cellulose membrane (D9777), sodium potassium tartrate tetrahydrate and 3,5-dinitrosalicyclic acid (DNS) were obtained from Sigma-Aldrich (St Louis, MO, USA). 2.2. Bread sample preparation Baked and steamed bread samples were made following the protocol reported in our previous studies (Lin & Zhou, 2018; Lin, Tan, Leong, & Zhou, 2018). Two hundred grams of flour were mixed with quercetin at the addition levels of 1.5, 3, and 6%, to produce quercetin-fortified flour. For baked bread, the quercetin-fortified flour was mixed with 8 g sugar, 2.4 g salt, 6 g shortening, 2 g instant dry yeast and 120 g water for 6 min using a KitchenAid mixer (5KPM50, St. Joseph, Michigan, USA), while for the steamed bread, the quercetin-fortified flour was mixed with 2 g sugar, 2 g salt, 2 g instant dry yeast and 110 g water for 5 min. The dough for both types of bread was allowed to rest under ambient conditions for 10 min, and then divided and molded into 50 ± 1 g small pieces. The dough pieces of baked bread were proofed at 40 °C under 85% relative humidity for 70 min and subsequently baked at 200 °C for 8 min, while the dough pieces of steamed bread were proofed for 45 min instead, and then steamed for 10 min. All the bread samples were cooled to room temperature for further analysis.

2.4.2. Reducing sugars released An aliquot of dialysate (0.5 mL) was withdrawn at 0, 5, 10, 15, 30, 45, 60, 75, 90, 105, 120, 150, 180, 240, 300 and 360 min in the intestinal phase. The reducing sugar (RS) released in the dialysate at each time point was measured according to the method described by Miller (1959). In brief, 0.5 mL of DNS reagent reacted with the dialysate samples in boiling water for 10 min. The absorbance of the mixture was determined spectrophotometrically at 540 nm (UVMini 1240, Shimadzu, Kyoto, Japan) when it was cooled to the room temperature. RS was calculated and expressed as the glucose equivalent (GE, mg/mL) using a glucose calibration curve.

2.3. Quantification of quercetin

2.4.3. Determination of potentially bioaccessible and bioavailable quercetin The potentially bioaccessible fraction of quercetin was evaluated as the total amount of quercetin released from the bread matrix and present in the digestion solution. The potentially bioavailable quercetin was measured as the amount of quercetin passing through the dialysis tube, ready to be absorbed in vitro. The digesta in the dialysis tube obtained after the intestinal phase was centrifuged at 17,387g for 10 min and the supernatant (1 mL) was mixed with methanol (1.5 mL) for quercetin quantification by HPLC. The dialysate was first concentrated at 40 °C by a vacuum rotary evaporator. The condensed dialysate was mixed with methanol in a ratio of 2:3 to quantify the amount of quercetin by HPLC. To better understand the matrix effect of bread on quercetin profile during digestion, control baked and steamed bread samples were spiked

2.3.1. Quercetin extraction To determine the amount of quercetin in the bread before digestion, the crumb of the bread samples were lyophilized, blended into powder form, and sieved (Mesh 45) for extraction. The fine bread powders (0.5 g) were weighed and mixed homogenously with 8 mL of 60% methanol in 15 mL centrifuge tubes using a vortex. Subsequently, the tubes were incubated at 60 °C for 5 min in a water bath, followed by 10 min shaking at 300 rpm in an orbital shaker. Centrifugation at 3233g (Eppendorf 5810R, Thermo Fisher Scientific, MA, USA) for 5 min was applied to obtain liquid extract and solid fraction. The solid fraction was re-extracted by heating and shaking for a total of 4 cycles. The combined liquid extract was made up to 50 mL with 60% methanol for HPLC analysis. 630

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with quercetin extracts to make their quercetin contents equivalent to those left in the corresponding bake and steamed bread products with 3% fortification (i.e. matched 3%); these samples were prepared also for the in vitro digestion.

bread was the reference food for GI, namely GIbread (GI value for white bread = 100). To estimate GIglucose of samples with glucose as the reference (i.e. GI value for glucose = 100), the generated GIbread of each sample was multiplied by 0.7 (Wolever et al., 2008). The estimated GL was calculated based on a 50 g bread portion according to Eq. (5) considering the TAC content of each sample.

2.5. Quercetin recovery from digesta The recovery of quercetin from the digesta was calculated for 3% fortified and 3% spiked samples according to Eq. (1)

Quercetin recovery (%) = [1

(

minitial-mdigestate minitial

)] × 100%

GL =

3. Results and discussion

The bread starch digestion curve in this experiment followed a firstorder reaction. To evaluate the extent to which the different fortification levels of quercetin altered the digestive profile of the bread samples, the starch digestion curves were modeled according to Eq. (2), as proposed by Goñi, Garcia-Alonso, and Saura-Calixto (1997).

3.1. Retention levels of quercetin in bread The content (%) of retained quercetin in bread crumb after baking and steaming is shown in Fig. 1. There was significantly higher average retention level of quercetin in steamed bread (76.8%) compared to baked bread (71.3%). As reported by Rohn, Buchner, Driemel, Rauser, and Kroh (2007), the amount of quercetin remained constant under roasting at 180 °C for 60 min, showing a thermal-resistant property of quercetin. Here, the relatively lower retention level of quercetin in bread after baking (200 °C) and steaming (100 °C) might be ascribed to the unextractable portion of quercetin produced by the interactions between quercetin and wheat protein. The formation of polyphenolprotein complexes, involving hydrogen bonding and/or hydrophobic interactions, affected the extractability of polyphenols from a bread matrix (Sivam, Sun-Waterhouse, Perera, & Waterhouse, 2013). The higher protein content of the baked bread flour (13.1%) presumably accounted for the lower extractable quercetin amount in the baked bread, while the steamed bread flour contained only 7.9% of proteins. Moreover, the quercetin-starch interactions causing the formation of resistant starch (Zhang, Yang, Li, & Gao, 2011) and the quercetindietary fibre interactions leading to the reduced antioxidant activity (by 17%) of quercetin (Sun-Waterhouse, Smith, O’Connor, & Melton, 2008) could probably lead to the loss of free quercetin in the fortified-bread

(2)

kt )

where Ct is the RS concentration (GE, mg/mL) at time t and C∞ is the equilibrium concentration of RS; k is the rate constant of starch digestion (min−1) and t is time (min). Linear-least-squares regression of Eq. (2) was performed to obtain the k and C∞ values by minimizing the root mean square error (RMSE) based on the Eq. (3) using Matlab software (version 8.4.0.150421 R2014b, the MathWorks Inc., Massachusetts, USA).

RMSE =

n i=0

(CtMod, i

CtExp, i)2

n

(3)

where CtMod, i is modeled value; CtExp, i is experimental value; n is the number of experimental value. The RMSE, along with R2 in Eq. (4), was used to examine the quality of the model established.

R2 = 1

DFxRMSE 2 SST

(5)

The experiments were all conducted in triplicate. SPSS 20.0 software (IBM Corporation, Chicago, IL, USA) was employ to perform oneway analysis of variance (ANOVA) with Duncan test at p < 0.05 to evaluate significant differences within groups.

2.6. Mathematical modeling

e

100

2.8. Statistical analysis

(1)

where minitial is the amount of quercetin from bread samples after baking/steaming before digestion, mdigestate is the detectable amount of quercetin from the digesta

Ct = C (1

GIglucose × TAC

(4)

where DF is the degrees of freedom and SST is the total sum of squares. 2.7. Prediction of GI and GL 2.7.1. Evaluation of total available carbohydrates (TAC) The TAC of each bread sample was measured using a Megazyme assay kit (K-ACHDF 08/16, Megazyme, Ireland) following the study of Lau et al. (2015). Results were expressed as mg TAC per 5 g fresh bread for all the bread samples. 2.7.2. Estimation of GI and GL values Assessment on the in vitro hydrolysis reactions was conducted to obtain GI and GL following the protocol established by Wolter, Hager, Zannini, and Arendt (2014). The amount of RS in the dialysate, as the hydrolyzed TAC, was converted from glucose to starch equivalent (g) by the conversion factor of 0.9 (Goñi et al., 1997). Take into account the TAC content (g) in a 5 g sample, the hydrolysis percentage of TAC for each sample was calculated and plotted against time (t). The area under the hydrolysis curve (AUC) till 180 min for each sample was determined. A hydrolysis index (HI) was calculated by dividing AUC of each bread sample by the AUC of the reference, the control white bread (baked). The estimated GI was obtained based on the equation GI = 0.549 HI + 39.71, developed by Goñi et al. (1997). Here, white

Fig. 1. Retention level of quercetin in baked and steamed bread before digestion. For the same type of bread, data values denoted by different lowercase letters are statistically different (p < 0.05) across the fortification levels of quercetin. At the same level of quercetin, data values denoted by different uppercase letters are statistically different (p < 0.05) between baked and steamed bread. 631

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Fig. 2. (a) The digestion trajectories of 0, 1.5, 3, 6% quercetin-fortified baked bread (colorful dots) and the corresponding developed mathematical models (dashline); (b), (c), (d), (e) validation plots for modeled data against experimental data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

samples. The food matrix could alter the profile of incorporated quercetin to a great extent. Zhang, Chen, and Wang (2014) reported an 88% loss of quercetin in cookies after 10 min baking at 200 °C, which resulted from the transformation of quercetin to other compounds in a low-moisture and lipid-rich food system. Compared to the cookies, bread was shown as a better carrier for quercetin fortification.

3, and 6% quercetin-fortified baked bread was reduced by 7.1, 8.6, and 12.8%, respectively, while for steamed bread, this reduction was 6.1, 8.2, and 9.5%, accordingly. These results indicated that the inhibition of bread starch digestion was enhanced with the increase of quercetin amount in both bread systems. One possible reason was the inhibitory effect of quercetin on α-amylase. A stable complex was formed as quercetin bound to α-amylase, which allowed the inhibition of αamylase in a competitive manner (Li, Gao, Shan, Bian, & Zhao, 2009). The conjugated structure of flavonol compounds, 4-keto group and the C2]C3 double bond, accounted for a stable binding of flavonols to the active site of α-amylase (Lo Piparo et al., 2008). Quercetin possesses a relatively higher inhibitory effect on pancreatin- or α-amylase-catalyzed starch digestion, compared to a cyanidin-catechin conjugate (vignacyanidin), due to its stronger hydrophobicity (Hirota & Takahama, 2017). Moreover, the hydrophobic binding between quercetin and starch could suppress the starch digestibility (Takahama, Yamauchi, & Hirota, 2013).

3.2. In vitro starch digestibility of bread Figs. 2(a) and 3(a) show a dose-dependent inhibition by quercetin on the release of RS during intestinal digestion of the baked and steamed bread, respectively. The transit of gastric chyme through the whole small intestine takes 3–4 h (DeSesso & Jacobson, 2001). As early as 60 min of dialysis, the concentrations of RS for the bread samples with quercetin were significantly lower than that of the control for both types of bread by up to 14.3% (Table S1). At the end of 240 min of dialysis, compared to the control bread, the concentration of RS for 1.5,

Fig. 3. (a) The digestion trajectories of 0, 1.5, 3, 6% quercetin-fortified steamed bread (colorful dots) and the corresponding developed mathematical models (dashline); (b), (c), (d), (e) validation plots for modeled data against experimental data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 632

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Table 1 Mathematical modelling parameters and regressed rate constants (k, min−1). Control (0%)

1.5% a,A

3.0% b,A

6.0% b,A

Baked bread

k C∞ R2 RMSE

0.5058 ± 0.0268 10.64 ± 0.39a,A 0.9979 0.1318

0.4545 ± 0.0269 10.57 ± 0.17a,A 0.9981 0.12

0.4228 ± 0.0165 10.43 ± 1.49a,A 0.9965 0.1395

0.4133 ± 0.0077b,A 10.31 ± 1.49a,A 0.9957 0.0174

Steamed bread

k C∞ R2 RMSE

0.4887 ± 0.0147a,A 10.65 ± 0.06a,A 0.9976 0.1355

0.4838 ± 0.0264ab,A 10.60 ± 0.30a,A 0.9979 0.1242

0.4598 ± 0.0359ab,A 10.44 ± 0.37a,A 0.9980 0.1175

0.4487 ± 0.0134b,A 10.28 ± 0.06a,A 0.9976 0.1291

Values are mean with standard deviation. Values with different superscripts in lowercase alphabets in the same row are significantly different (p < 0.05). Values with different superscripts in uppercase alphabets are significantly different across bread types (p < 0.05).

Our results are consistent with an early finding in which the starch digestibility of the bean paste fortified with onion skin, containing quercetin as one of the main phenolics, was significantly decreased by up to 22% because of the formation of phenolic-starch complexes (Świeca & Gawlik-Dziki, 2015). Goh et al. (2015) also found that baked and steamed bread enriched with 2% green tea catechin released significantly less reducing sugar during the pancreatic digestion.

Table 2 Values of GIbread, GIglucose, and GL.

3.3. Mathematical modeling of starch digestion Even though significantly lower RS concentrations of bread samples containing quercetin in comparison to the control were observed during digestion, evidences to discriminate the digestion profiles were not quantitative enough. Mathematical modeling of digestion profile was thus developed to provide digestion rate constant i.e. the k value in Eq. (1) and the equilibrium concentration C∞ of RS for all the samples. As detailed in Table 1, the k value was decreased as the quercetin level increased from 0% to 6%. The maximum reduction was observed to be 18.3% and 8.2% for baked and steamed bread, respectively, at the highest fortification level (6%). The lower the k value, the slower the starch digestion rate was. No significant difference was observed for C∞ between bread samples with and without quercetin. The modeled digestion profiles of all bread samples were plotted along with the experimental data as shown in Figs. 2(a) and 3(a). The small values of RMSE in Table 1, indicating a small difference between the modeled and experimental data, suggested a good quality of the established model. Supportively, as plotted in Figs. 2(b–e) and 3(b–e), the data points were evenly distributed around the 45° line, indicating a high agreement between the modeled and experimental result. The high values of R2 further confirmed the adequacy of the model to describe the digestion profiles of all bread samples. This result is in accordance with the study of Sui et al. (2016) who reported a 20.5% reduction of the regressed digestion coefficient of the bread enriched with 4% anthocyanin rich black rice extract in comparison to the control bread.

GIbread

GIglucose 70.0 67.3 66.6 65.6

± ± ± ±

0.8a 1.3cd 1.4de 0.3e

16.2 15.3 14.5 14.2

± ± ± ±

0.2d 0.3e 0.3f 0.1f

69.0 68.8 68.7 68.0

± ± ± ±

0.9ab 0.6abc 1.0abc 0.3bcd

17.5 17.1 16.7 16.1

± ± ± ±

0.2a 0.2b 0.3c 0.1d

Baked Bread

Control (0%) 1.5% 3% 6%

100a 96.1 ± 1.9cd 95.1 ± 2.0de 93.7 ± 0.5e

Steamed Bread

Control (0%) 1.5% 3% 6%

98.6 98.3 98.2 97.2

± ± ± ±

1.3ab 0.9abc 1.5abc 0.4bcd

GL

Values with different superscripts in lowercase alphabets within the same column across two bread types are significantly different (p < 0.05).

Quercetin-protein complexes were proven to be formed through the incorporation of quercetin-containing onion skin into bread system (Swieca, Gawlik-Dziki, Dziki, Baraniak, & Czyz, 2013), which might also negatively influence the starch digestion and GI values of bread products. As shown in Table 2, the maximum reduction of GIbread in both bread systems was observed at 6% of quercetin fortification; at this level of fortification, the GIbread value of baked bread was significantly lower than that of the steamed bread. As relatively lower protein and lipid contents present in the steamed bread samples, less accessibility of these ingredients towards interactions with quercetin was expected. The starch digestibility of steamed bread was thus probably less susceptible to be altered by quercetin fortification. Previously, steamed bread has been reported to be a “healthier” alternative to baked bread due to its lower in vitro starch digestibility and lower in vivo GI values (Lau et al., 2015). In this case, fortified with 3% quercetin, the baked bread had relatively lower estimated GI values compared to the respective steamed bread. Other than quercetin, fortifying bread products with varying levels of polyphenol-rich extracts such as green tea extract (0.4–2%), grape seed extract (0.2–1.6%), baobab fruit extract (1.88–3.13%), black rice extract (1–4%) and resveratrol (0.22–3.75%) has successfully reduced the starch digestibility as evaluated in different in vitro digestion systems, indicating the high potential of these approaches to reduce the in vivo glycemic response (Coe & Ryan, 2016; Goh et al., 2015; Sui et al., 2016). However, the green tea extract (0.4%) and the baobab fruit extract (1.88%) showed no effect on the glycemic response to the fortified bread samples in the human study involving 13 health participants (Coe & Ryan, 2016), while the impacts of other ingredients have not been examined in vivo. These conflicting results suggested the necessity of human study to validate the effect of quercetin on the glycemic response to the bread fortified with optimal doses (1.5%) as determined in this study.

3.4. Estimation of GI and GL The estimated GI and GL values for bread samples are summarized in Table 2. It is noteworthy that a dose-dependent reduction of GIbread, GIglucose, and GL values was observed for all the bread samples with quercetin as compared to the control in each of the two bread systems. On one hand, this might be ascribed to the formation of resistant starch. Quercetin was able to interact with starch hydrophobically, contributing to the generation of resistant starch (Hirota & Takahama, 2017). The addition of resistant starch helps to reduce the GI value of a food product (Fuentes-Zaragoza, Riquelme-Navarrete, Sánchez-Zapata, & Pérez-Álvarez, 2010). On the other hand, starch digestion is known to be hindered by the surrounding proteins and lipids present in the food matrix due to the reduced accessibility and binding of enzymes on the starch granule (Dhital, Warren, Butterworth, Ellis, & Gidley, 2017).

3.5. Potentially bioaccessible and bioavailable quercetin from digestion The potential bioaccessibility is an important index to evaluate the nutritional value of a component: the portion of an ingested component 633

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Table 3 Potentially bioaccessible and bioavailable quercetin from digestion. Bread type Baked bread

Steamed bread

Bioaccessible quercetin (mg/50 g fresh bread) a

Bioavailable quercetin (mg/50 g fresh bread) b

Bioaccessibility % a

Bioavailability %

1.5% 3.0% 6.0%

6.09 ± 1.75 6.29 ± 2.94a 6.61 ± 3.40a

1.43 ± 0.47 2.19 ± 0.54ab 2.33 ± 0.70a

1.55 ± 0.39 0.80 ± 0.36b 0.43 ± 0.22c

0.37 ± 0.13ab 0.28 ± 0.07bc 0.15 ± 0.05de

Matched 3%

–

–

0.36 ± 0.05*

0.19 ± 0.02*

1.5% 3.0% 6.0%

6.19 ± 0.80a 5.98 ± 1.20a 6.22 ± 0.59a

1.98 ± 0.63ab 1.93 ± 0.66ab 1.74 ± 0.55ab

1.41 ± 0.27a 0.72 ± 0.16bc 0.39 ± 0.04c

0.45 ± 0.13v 0.23 ± 0.08cd 0.11 ± 0.04e

Matched 3%

–

–

0.42 ± 0.13*

0.26 ± 0.01

a,b,c,d,e

Values with different superscripts in lowercase alphabets within the same column across two bread types are significantly different (p < 0.05). * Significant difference (p < 0.05) is observed as compared to the 3% quercetin-fortified baked or steamed bread.

solid bread matrix, a solid dispersion. Solid dispersions, offering a large effective surface area for poorly water-soluble compounds to be available for dissolution, could help to enhance the potential bioavailability of the compounds (Egert et al., 2012). Likewise, comparing the digestion profiles of quercetin supplement (130 mg) in capsules and the equivalent amount of quercetin incorporated in cereal bars, Egert et al. (2012) reported a 6 times higher maximum plasma level of quercetin from the quercetin-enriched cereal bar compared to that from the capsules. The protective effects of bread matrix on quercetin during digestion were examined by the determination of quercetin recovered from the digesta (Fig. S1). The recovery of quercetin from 3% matched samples was significantly lower than that from the 3% fortified samples by 20.4 and 12.0% in the baked and steamed bread system, respectively. This result demonstrated the quercetin loss during digestion was up to 15% in bread systems, while the loss of quercetin supplement spiked was almost double (up to 30%). In the neutral pH environment of intestinal phase, quercetin is relatively unstable and can be oxidized or degraded in the presence of digestive enzymes (Boyer, Brown, & Liu, 2005); this study also found that the recovery of quercetin during in vitro digestion varied widely based on the food matrix as the quercetin recovered from onion was 21.8% lower than that from its pure compound. The relatively higher recovery level of quercetin in bread systems showed protective effects of the bread matrix on the stability of quercetin during in vitro digestion, possibly due to the complex formed between quercetin and bread ingredients. Similarly, Lamothe, Azimy, Bazinet, Couillard, and Britten (2014) reported an significantly enhanced stability (30%) of green tea extract incorporated into dairy products during in vitro intestinal phase digestion.

that is potentially available for absorption, is dependent on digestion and release from food matrix; considering the further absorption through intestinal wall, the potential bioavailability is pointed to the compound that is ready to reach the systematic circulation (Cardoso, Afonso, Lourenço, Costa, & Nunes, 2015; Etcheverry, Grusak, & Fleige, 2012). As the passive diffusion was one of the main ways for quercetin to be absorbed (Day, Gee, DuPont, Johnson, & Williamson, 2003), here, quercetin in the dialysate passing through the dialysis tube was assumed to be the in vitro bioavailable portion. As presented in Table 3, no significant difference was observed in the potentially bioaccessible amount of quercetin across all the bread samples with quercetin fortification. Therefore, it was not surprising to see that the potential bioaccessibility (%), as a percentage of the quercetin content in bread before digestion, was significantly decreased with increasing quercetin fortification level in both bread systems. Similarly, such decreasing trend was also observed in the potential bioavailability (%) for both baked and steamed bread. Results in Table 3 also show that at each fortification level, there was no significant difference of quercetin profiles between the baked and steamed bread during digestion. In vivo, a compound is ready to be transported across the intestinal epithelium into body circulation once this compound is released from its matrix in the digesta. In a static in vitro digestion model, without removal of the compound during digestion, the potential bioaccessibility may be underestimated when saturation of the compound in the aqueous phase occurs. Our results are in accordance with a previous study in which a does-dependently reduced the potential bioaccessibility and bioavailability of quercetin from the onion skin-enriched bread was observed with increasing addition levels; the absolute values, however, were not comparable to our results due to the different methods used for quercetin extraction and analysis (Gawlik-Dziki et al., 2013).

4. Conclusions

3.6. Matrix effects of bread on quercetin during digestion

Results of this study demonstrated that the incorporation of quercetin (1.5–6%) into bread systems could significantly reduce the starch digestion rate of the bread samples and lower their glycemic potentials by up to 18.3% and 6.3%, respectively, allowing for healthier food choices. Both baked and steamed bread were proven to be promising food carriers for delivering quercetin more efficiently compared to the pure quercetin supplement. Limitations of this study include (1) possibly underestimated potential bioaccessibility and bioavailability of quercetin due to the saturation of quercetin in the aqueous phase in a static in vitro digestion system and (2) the lack of identifying the quercetin-protein/starch complexes that were proposed to reduce the starch digestibility. In the future, animal studies or clinical trials are recommended to be conducted on these quercetin-fortified bread products to validate their in vivo health benefits.

To investigate the effect of bread matrix on digestion and absorption of quercetin, control baked bread and control steamed bread were spiked with an equivalent amount of quercetin that was left in the baked and steamed bread samples with 3% fortification before digestion (denoted as Matched 3%), respectively. Significantly lower potential bioaccessibility of quercetin (up to 55%) from the matched 3% sample was observed compared to that from the 3% fortified bread samples in both bread systems (Table 3). This result indicated a possible interaction between bread matrix and quercetin, enhancing the potential of quercetin to be absorbed. Quercetin could interact with wheat protein (gluten network) during breadmaking (Lin & Zhou, 2018), most probably via hydrogen bonding and hydrophobic interactions as a flavonoid (Sivam, Sun-Waterhouse, Perera, & Waterhouse, 2012). This result also indicated that not only did the food ingredients affect the absorption of quercetin, but also that the manufacturing process might play an important role in it. Intimate mixing of quercetin with bread ingredients led to a homogenous distribution of quercetin in the semi-

Conflicts of interest There are no conflicts to declare. 634

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Acknowledgements

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