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Evidence for the formation of maillardized insoluble dietary fiber in bread: A specific kind of dietary fiber in thermally processed food
Food Research International 55 (2014) 391–396
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Evidence for the formation of maillardized insoluble dietary fiber in bread: A specific kind of dietary fiber in thermally processed food J. Pérez-Jiménez 1, M.E. Díaz-Rubio 1, M. Mesías, F.J. Morales, F. Saura-Calixto ⁎ Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Madrid, Spain
a r t i c l e
i n f o
Article history: Received 2 September 2013 Accepted 20 November 2013 Keywords: Dietary fiber Maillard reaction products Polyphenols Maillardized dietary fiber
a b s t r a c t Bakery products are rich in insoluble dietary fiber (IDF), and Maillard reaction products (MRP), which are commonly studied as two independent constituents. The aim of this work was to elucidate whether IDF contains MRP as an intrinsic constituent, as well as to check the possible contribution of polyphenols to MRP in bread. It was found that MRP contribute to the increase in IDF from wheat flour to bread; indeed, 8% of furosine (a common indicator of MR) is associated with IDF. Also, ferulic acid and (−)-epicatechin got incorporated to MRP in a model system. This work provides evidence for the existence of a complex DF-protein-MRP-polyphenols, named maillardized IDF, in bakery products, a term applicable to other thermally processed food. Moreover, the present results strengthen the concept that MRP are formed not only from reducing sugars and amino acids, but also from phenolic compounds, including phenolic acids and flavonoids. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Dietary fiber (DF) is a key food component that consists of a wide variety of chemical structures. Since the first definition of DF, which considered that its constituents were exclusively non-starch polysaccharides (NSP) and lignin resistant to hydrolysis by human digestive enzymes (Southgate, 1977; Trowell, 1976), the concept has evolved to include other food components, such as oligosaccharides, resistant starch and others (AACC, 2001; Codex Alimentarius, 2009). Moreover, much work has emphasized that an important fraction of dietary polyphenols are constituents of DF (Goñi, Díaz-Rubio, Pérez-Jiménez, & Saura-Calixto, 2009; Saura-Calixto, Goni, Mañas, & Abia, 1991). All the constituents of DF have in common that, after ingestion, they share the same metabolic fate as those included in the original definition of DF: they reach the colon intact, where they may be subjected to extensive transformation by the microbiota. The Maillard reaction (MR) is one of the most studied chemical reactions in food. It is a non-enzymatic process that takes place during thermal processing and during storage in foodstuffs that contain reducing sugars as well as certain amino acids. The many chemical structures that are produced during the different stages of the MR are known as Abbreviations: DF, dietary fiber; fm, fresh matter; GAE, gallic acid equivalents; HMF, hydroxymethylfurfural; IDF, insoluble dietary fiber; MR, Maillard reaction; MRP, Maillard reaction products; NSP, non-starch polysaccharides; SDF, soluble dietary fiber; TDF, total dietary fiber. ⁎ Corresponding author at: Dep. Metabolism and Nutrition, ICTAN-CSIC, Jose Antonio Novais, 10, 28040 Madrid, Spain. Tel.: +34 91 549 23 00; fax: +34 91 549 36 27. E-mail addresses: [email protected] (J. Pérez-Jiménez), [email protected] (M.E. Díaz-Rubio), [email protected] (M. Mesías), [email protected] (F.J. Morales), [email protected] (F. Saura-Calixto). 1 J. P.-J. and M.E. D.-R. contributed equally to this work. 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.11.031
Maillard reaction products (MRP) and include Amadori compounds, hydroxymethylfurfural (HMF), furfural, aldehydes, aldols and melanoidins, among other compounds (Nursten, 2005). MRP are widely studied, either from a sensorial and/or technological point of view (Martins, Jongen, & Van Boekel, 2000; Newton, Fairbanks, Golding, Andrewes, & Gerrard, 2012), from a toxicological one (Lee & Shibamoto, 2002) or for their nutritional value (Moreira, Nunes, Domingues, & Coimbra, 2012); but they are commonly considered independently from DF. Therefore, DF approach does not take into account MRP as intrinsic constituents that may affect their properties and, similarly, MRP approach ignores that a substantial part of melanoidins is indeed insoluble dietary fiber (IDF). In this context, it was recently shown that MRP contribute significantly to soluble dietary fiber (SDF) content in roast coffee (Gniechwitz, Reichardt, Blaut, Steinhart, & Bunzel, 2007; Silván, Morales, & Saura-Calixto, 2010); one of the most common examples of a thermally treated food in whose production the MR takes place. This finding gave rise to the concept of maillardized DF (Silván et al., 2010), which refers to the MRP that are associated with polysaccharides, polyphenols and lignin in certain processed food. Indeed, due to these strong interactions, traditional determinations of SDF would also include a fraction of MRP. In the same way as the original concept of DF proved inadequate, since it did not include polyphenols or MRP as key constituents and it did not use physiological conditions for its determination (Goñi et al., 2009), so the possible presence of phenolic compounds in MRP has been scarcely addressed. While it has been reported that MRP in coffee (particularly melanoidins formed in the final stage of the MR) include phenolic compounds such as chlorogenic acid and caffeic acid (Bekedam et al., 2008; Gniechwitz et al., 2008; Moreira et al., 2012; Nunes & Coimbra, 2007, 2010) and some model studies have been
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carried out to evaluate the incorporation of certain polyphenols into MRP (Deshou, Christopher, Pranav, Sandeep, & Peterson, 2009), for most processed food it is commonly considered that MRP are generated exclusively from reducing sugars and amino acids, either free or as constituents of peptides or proteins. In contrast with beverages such as coffee, processed solid foodstuffs do not only include SDF, but also IDF. However, the possible application of the term maillardized DF to IDF has not yet been explored. In this study, bread was chosen as a case study, in order to elucidate whether a fraction of MRP are constituents of IDF. The research is challenging, since baking is a complex process inducing physical, chemical and biochemical changes in the cereal matrix, such as volume expansion, evaporation of water and the formation of a porous structure, denaturation of proteins, starch gelatinization, crust formation, the development of a desirable taste and pleasant flavors and browning. The presence of polyphenols as intrinsic constituents of MRP in bread was also explored. 2. Materials and methods 2.1. Chemicals and samples Gallic acid, ferulic acid, (−)-epicatechin, glucose, glycine, HMF, furfural, pancreatin, α-amylase and amyloglucosidase were obtained from Sigma Chemical (MO, USA). Pepsin, methanol, formic acid, acetonitrile, hydrochloric acid, sodium heptane sulfonate, sulfuric acid, ethylenediaminetetraacetic acid, potassium hexacyanoferrate and zinc sulfate were purchased from Merck (Darmstadt, Germany). FolinCiocalteu reagent was acquired from Panreac (Castellar del Vallés, Spain). Furosine was obtained from Neosystem Laboratories (Strasbourg, France). All the reagents used were of analytical grade. The following samples were provided by Ruipan S.A. (Leganés, Spain), all of them belonging to the same baking process: wheat flour, French bread (subjected to baking at 190–220 °C for 35 min) and toasted bread (subjected to a second baking at 280 °C for 9 min). For analysis, the French bread was freeze-dried and milled to a particle size of 0.5 mm. A fraction of the bread crust was removed from French bread for separate analysis. 2.2. Determinations 2.2.1. Dietary fiber DF was determined in wheat flour, French bread, toasted bread and bread crust according to the indigestible fraction method (Goñi et al., 2009). Samples (300 mg), after being solved in phosphate buffer, were incubated with pepsin (0.2 mL of a 300 mg/mL solution in 0.08 mol/L HCl–KCl buffer, pH 1.5, 40 °C, 1 h), pancreatin (1 mL of a 5 mg/mL solution in 0.1 mol/L phosphate buffer, pH 7.5, 37 °C, 6 h) and α-amylase (1 mL of a 120 mg/mL solution in 0.1 mol/L tris–maleate buffer, pH 6.9, 37 °C, 16 h). The samples were centrifuged (15 min, 3000 g) and the supernatants removed. The residues were washed twice with 5 mL of distilled water, and all the supernatants were combined. Each supernatant was incubated with 100 μL of amyloglucosidase for 45 min at 60 °C before being transferred to dialysis tubes (12,000–14,000 molecular weight cutoff, Visking dialysis tubing; Medicell International Ltd., London, U.K.) and dialyzed against water for 48 h at 25 °C to eliminate the digested compounds. The product of all these treatments was therefore a residue after enzymatic treatments, corresponding to IDF, and a supernatant from the enzymatic treatments later subjected to dialysis, corresponding to SDF. In the SDF, soluble NSP were hydrolyzed with 1 M sulfuric acid at 100 °C for 90 min and spectrophotometrically quantitated after alkalinization with NaOH (3.9 mol/L) and reaction with dinitrosalicylic acid (Englyst & Cummings, 1988). For the IDF, the residue was weighed to determine the IDF content of the sample gravimetrically, and it was divided into different fractions to
determine resistant protein (see Section 2.2.2), indicators of MR (see Section 2.2.3) and antioxidant compounds (see Section 2.2.4). Total dietary fiber (TDF) was determined as the sum of SDF and IDF. 2.2.2. Resistant protein Resistant protein was determined in the whole samples (wheat flour, French bread, toasted bread and bread crust) and in the corresponding IDF using an automated nitrogen analyzer (FP-2000; Dumas Leco Corp., St. Joseph, MI), after calibration of the instrument with EDTA. Protein was calculated as N × 6.25. 2.2.3. Indicators of the Maillard reaction Indicators of the MR were measured in the whole samples (wheat flour and toasted bread) and in their corresponding IDF. Furosine: Furosine was determined by ion-pairing chromatography as described by Delgado et al. (Delgado, Corzo, Santa-Maria, Jimeno, & Olano, 1992) and modified by Delgado-Andrade et al. (DelgadoAndrade, Rufián-Henares, & Morales, 2007). A finely ground sample (30 mg) was hydrolyzed with 4 mL of 7.95 mol/L HCl at 110 °C for 23 h in a Pyrex screw-cap vial with PTFE-faced septa, and sealed under nitrogen. The hydrolyzate was aerated and cooled to room temperature, and subsequently centrifuged at 4000 g for 10 min. A sample of 0.5 mL of the supernatant was applied to a Sep-Pak C18 cartridge (Millipore, Billerica, MA, USA) pre-wetted with 5 mL of methanol and 10 mL of deionized water; it was then eluted with 3 mL of 3 mol/L HCl. An aliquot (1 mL) was dried with a speed-vac concentrator. The sample was dissolved in 1 mL of a mixture of water, acetonitrile and formic acid (95:5:0.2) and filtered (0.45 μm). The analysis was conducted with a Shimadzu HPLC system (Kyoto, Japan) equipped with an LC20AD pump, an SIL-10ADvp autosampler, a CTO-10ASVP oven, and a DAD (SPD-M20A). The chromatographic separations were performed in an Excel-ODS analytical column (250 mm × 4.6 mm, 5 μm particle size, Tecknokroma, Barcelona, Spain) at 32 °C. The mobile phase was prepared with 5 mmol/L sodium heptane sulfonate, including 20% of acetonitrile and 0.2% of formic acid. The elution was isocratic and the flow rate was 1.0 mL/min. The injection volume was 20 μL and detection was performed at 280 nm. Furosine was quantified by the external standard method; the calibration curve was established from a stock solution (1.2 mg/mL of furosine) in the range 1–10 mg/L. HMF and furfural. HMF and furfural were determined as described by Rufián-Henares et al. (Rufián-Henares, Delgado-Andrade, & Morales, 2006). Briefly, a finely ground sample (500 mg) was extracted with 5 mL of deionized water. The tube was shaken vigorously for 1 min and clarified with 0.25 mL each of potassium ferrocyanide, 15% w/v, and zinc acetate, 30% w/v, solutions. The resulting mixture was centrifuged at 4500 g for 10 min at 4 °C. The supernatant was collected in a 10 mL volumetric flask and two further extractions were performed using 2 mL of deionized water. The supernatants were pooled and filtered. The sample (20 μL) was analyzed by reverse-phase HPLC in a Shimadzu HPLC system as described above. The mobile phase was a mixture of acetonitrile in water (5% v/v) delivered at a flow rate of 1 mL/min under isocratic conditions through the analytical column (Mediterranea-SeaC18, 250 mm × 4.0 mm, 5 μm particle size, Teknokroma, Barcelona, Spain) and thermostated at 32 °C. The UV detector was set at 280 nm and 20 μL of the extract was injected. HMF and furfural were quantified using the external standard method within the range 0.1–20 mg/L and 1–10 mg/L respectively. The limit of quantitation was set as 0.05 mg/kg. Each sample was analyzed in duplicate and the mean of the two measurements is reported. 2.2.4. Antioxidant compounds Three different fractions of antioxidant compounds were determined in wheat flour, toasted bread and bread crust: - Extractable antioxidants. 0.5 g of sample was placed in a capped centrifuge tube; 20 mL of acidic methanol/water (50:50 v/v,
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acidified to pH = 2 with HCl 2 mol/L) was added and the tube was thoroughly shaken at room temperature for 1 h. The tube was centrifuged at 2500 g for 10 min and the supernatant was recovered. Twenty mL of acetone/water (70:30, v/v) was added to the residue, and shaking and centrifugation were repeated. The two extracts obtained for each replicate were combined and used to determine the content of extractable antioxidants (Pérez-Jiménez et al., 2008). - Non-extractable antioxidants. The residues from obtaining the extractable antioxidants were mixed with 20 mL of methanol and 2 mL of concentrated sulfuric acid. The samples were placed in a water bath with constant shaking at 85 °C for 20 h and then centrifuged (2500 g for 10 min) and the supernatants recovered, a procedure previously reported to release high molecular weight polyphenols and/or associated with the food matrix (Hartzfeld, Forkner, Hunter, & Hagerman, 2002; Pérez-Jiménez et al., 2008). After two washings with distilled water, the final volume was taken up to 50 mL (Hartzfeld et al., 2002). These hydrolyzates were used to determine the content of non-extractable antioxidants. - Non-extractable antioxidants associated with IDF. A portion of IDF was subjected to the same hydrolysis procedure as that used for obtaining non-extractable antioxidants, and the corresponding hydrolyzates were used to determine non-extractable antioxidants associated with IDF (Tabernero, Serrano, & SauraCalixto, 2007). The antioxidant content was determined in these extracts/hydrolyzates by the Folin–Ciocalteu procedure (Singleton, Orthofer, & Lamuela-Raventós, 1998). Samples (0.5 mL) were mixed with 0.5 mL of Folin-Ciocalteu reagent and swirled. After 3 min, 10 mL of sodium carbonate solution (75 g/L) was added and mixed. Additional distilled water was mixed thoroughly by inverting the tubes several times. After 1 h, the absorbance at 750 nm was recorded. The results were expressed as mg gallic acid equivalents (GAE)/kg.
2.2.5. Generation of melanoidin standard with and without polyphenols A melanoidin standard was generated as previously reported (Ames et al., 2000), but including also polyphenols, in order to determine their possible incorporation. Briefly, 25 mL of 0.05 mol/L solutions of both glucose and glycine in distilled water were prepared, including phenolic compounds in different proportions in relation to glycine (0.85–8.5– 85 mg of glycine per mg of ferulic acid or (−)-epicatechin). A control without phenolic compounds was also prepared. The solution was freeze-dried and the resulting solid was placed in an oven preheated to 125 °C, for 2 h. Of the resulting powder, 1.25 g was dissolved in 20 mL of distilled water and the mixture was filtered through Whatman No 4 filter paper. After two washings of the residue with 5 mL of distilled water, the washings and the original filtrate were combined and the absorbance of this solution was measured at 360, 420, 460 and 520 nm, according to the different stages of MR (Ames et al., 2000; Fogliano, Maria Monti, Musella, Randazzo, & Ritieni, 1999). The solution was then transferred to dialysis tubes (12,000–14,000 molecular weight cutoff) and dialyzed against water for 48 h at 25 °C. The Folin–Ciocalteu assay was then applied to the dialyzates, according to the procedure described in Section 2.2.4. The proportion of glycine and the polyphenols in the original mixture was calculated from that in white bread (about 85 mg of glycine per mg of ferulic acid). To evaluate possible dose–response relationships, ten-fold and one hundred-fold concentrations of the polyphenol were also tested. Absorbance values measured before dialysis and after dialysis (as Folin assay) were expressed in relation to the value of the solution obtained in the absence of polyphenols, which was considered as a value of 100.
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2.3. Statistics All the analyses were carried out in triplicate, and results are expressed as mean values ± standard deviation as fresh matter (fm) basis. Statistical significant differences between two samples were determined by Student's t test and between more samples by one-way ANOVA and Tukey's multiple-range test to compare means (p b 0.05), with the SPSS 19.0 statistical package for Windows software (SPSS, Chicago, IL, USA). 3. Results and Discussion 3.1. DF analysis considering the possible presence of MRP The present study explores whether MRP are ignored constituents of IDF in thermally processed food. First of all, SDF and IDF—including resistant protein—were determined in wheat flour, French bread (one of the most characteristic examples of a food that undergoes the MR), bread crust (where these compounds are concentrated) and toasted bread (where a second thermal process takes place, thereby increasing MRP), according to a methodology that includes resistant starch (Goñi et al., 2009), a key constituent of DF in may cereal products. The corresponding results are shown in Table 1. The transformation from wheat flour to bread significantly reduced TDF, which is probably due to an increase in the digestibility of resistant starch, as reported for instance for boiled potato (García-Alonso & Goñi, 2000). Regarding the different bread samples, significant differences in TDF content were found, according to the sequence: bread crust N toasted bread N French bread. This was mainly due to a significant increase in IDF, which also caused a significant decrease in the ratio SDF:IDF, from 0.45 in bread to 0.15 in bread crust. The increase in IDF in the samples with a higher MRP concentration (i.e., bread crust and toasted bread) is partly due to the higher content in those samples of resistant protein, but this does not completely explain the difference. Therefore, it may be that the higher IDF content in those samples in which the MR is more developed is due to the contribution to IDF of MRP. This is also supported by the observation that, while IDF content in bread crumb may be estimated to be about 4.3% (considering that IDF in bread was 5.06% and bread crumb is 77% of bread weight), IDF content in toasted bread, which is derived mostly from bread crumb, was 7.5%—this increase would be mainly due to the contribution of MRP to IDF. Therefore, common analysis of DF in thermally processed foodstuffs in fact includes a fraction of MRP, although this has not previously been considered, according to the traditional view of DF as NSP and lignin. Moreover, MRP would contribute to the health-related properties of these foodstuffs, even if they have a chemical structure quite different from those of the compounds originally considered as constituents of DF, since all of them will share the same metabolic fate after ingestion, reaching the colon nearly intact. Indeed, several in vitro studies have Table 1 Dietary fiber content in wheat flour, French bread, toasted bread and bread crust. Wheat flour Soluble dietary fiber 2.27 ± 0.14a (g/100 g fm) 17.87 ± 0.67a Insoluble dietary fiber (g/100 g fm) Resistant protein 2.09 ± 0.11a (mg/100 g fm) Total dietary fiber 20.14 ± 0.768a (g/100 g fm) Soluble/insoluble 0.11 ± 0.008a dietary fiber
French bread Toasted bread 2.28 ± 0.13a
Bread crust
2.69 ± 0.42b
1.57 ± 0.13c
5.06 ± 0.50b 7.51 ± 0.93c
10.21 ± 0.08d
1.54 ± 0.10b 1.80 ± 0.10b
2.22 ± 0.12a
7.34 ± 0.52b 10.2 ± 1.02c
11.78 ± 0.15c
0.45 ± 0.05b 0.39 ± 0.07b
0.15 ± 0.01a
Different superscript letters indicate significant differences (p b 0.05) between the samples. fm, fresh matter.
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shown that melanoidins with different origins, including those from bread crust, promote the growth of different microbial species, such as Bifidobacteria strains (Borrelli & Fogliano, 2005; Corzo-Martínez, Hernandez-Hernandez, Villamiel, Rastall, & Moreno, 2013; Dell'Aquila, Ames, Gibson, & Wynne, 2003), but they considered MRP as compounds independent from DF. 3.2. Analysis of indicators of the MR as possible constituents of DF MR, caramelization and development of the gluten network are the most important chemical events that occur during the manufacture of bakery products and they are indeed related, since gluten is a key constituent of bread melanoidins (Dell'Aquila et al., 2003; Fogliano et al., 1999). To corroborate that a fraction of MRP in bread are constituents of IDF, we decided to analyze MRP in French bread IDF. Since determining all potential MRP would require a complicated combination of analytical techniques (Silván, van de Lagemaat, Olano, & del Castillo, 2006), some common indicators of the MR were selected instead: furosine, HMF and furfural. Furosine (N-2-furoylmethyl-L-lysine) is an amino acid derivative formed during the acid hydrolysis of the Amadori products (N-substituted 1-amino-1-deoxy-2-ketose) which is generated in the early stages of the MR. In contrast, HMF and furfural are classical indicators of the advanced stage of both the MR and caramelization. They were measured in wheat flour and toasted bread, and in the corresponding IDF, to observe the evolution of their content during the baking process from the first material to the last one (Table 2). The results show the expected increase in these indicators in toasted bread as compared with wheat flour. More interestingly, nearly 8% of total furosine and 1% of total HMF in toasted bread were found in IDF; i.e., they were also constituents of IDF. Although melanoidins have been widely characterized in different bakery products (Fogliano & Morales, 2011; González-Mateo, González-SanJosé, & Muñiz, 2009), indicators of MR in IDF from these products had not been determined. The present results are a confirmation of the presence of MRP in IDF, therefore extending the previously defined concept of maillardized SDF (Silván et al., 2010) to maillardized IDF. MRP would be therefore minor constituents of IDF. Interestingly, a recent work also reported the presence of melanoidins as constituents of IDF, in that case in a commercial DF from potato (Langner et al., 2013), showing that the concept of maillardized IDF would be extensible to other solid thermally processed food different from bakery products. 3.3. Antioxidant content analysis The selected indicators of the MR found in IDF of toasted bread were not able to explain the entire increase in IDF in toasted bread as compared with untoasted bread (Table 1). Moreover, due to the previously mentioned problems in measuring other MRP, total antioxidant content as well as antioxidants associated with IDF were evaluated by Folin Table 2 Furosine, furfural and hydroxymethylfurfural content in wheat flour and toasted bread (mg/kg fm).
Furosine Total Associated with IDF
Wheat flour
Toasted bread
n.d. n.d.
203 ± 20⁎⁎ 15 ± 1⁎⁎
assay in samples corresponding to the different stages of the baking process, as an alternative method for determining the contribution of MRP to DF. The use of this approach to estimate MRP associated with IDF is based on the fact that MRP have previously been described as antioxidants (Bekedam et al., 2008; Michalska, Amigo-Benavent, Zielinski, & del Castillo, 2008; Morales & Babbel, 2002; Moreira et al., 2012; Wang, Qian, & Yao, 2011). As shown in Table 3, toasted bread and bread crust had a significantly higher content of non-extractable antioxidants (structures with high molecular weight and/or associated with DF) than wheat flour, while there was no significant variation between the three samples in the content of extractable antioxidants (structures of low molecular weight). Therefore, while the thermal process did not generate new condensations or associations between DF and those antioxidants already present in the sample (extractable antioxidants), it did originate new antioxidant compounds, MRP, thereby confirming some previous results (Pérez-Jiménez & Saura-Calixto, 2005). Moreover, the increase in nonextractable antioxidants was higher in toasted bread than in bread crust, since the second thermal process involved in toasted bread increases the formation of MRP. Furthermore, antioxidants associated with IDF were specifically measured by Folin assay (Table 3). A significant increase in nonextractable antioxidants associated with IDF was observed in toasted bread and bread crust as compared with wheat flour. Although Folin assay measures all reducing compounds present in a sample, this increase associated with IDF during baking, concomitant with the increase in MRP content during the same process, postulates MRP as candidates to be responsible for it. Therefore, this association again suggests the contribution of MRP to IDF and to the overall antioxidant capacity of processed food. These results show that both polyphenols (present in wheat flour and in bread samples) and antioxidant MRP (present in bread samples) are associated with DF, and generate a complex structure. Indeed, while MRP are commonly considered to be formed exclusively from reducing sugars and amino acids, these results suggest that polyphenols may also be constituents of MRP as part of this complex structure with DF, what was later specifically tested (see Section 3.4). Considering the amount of non-extractable antioxidants associated with IDF in wheat flour (117.3 mg/100 g fm) and that in toasted bread (158.8 mg/100 g fm), this implies that toasted bread contains about 40 mg/100 g fm (expressed as GAE) of MRP associated with IDF. This is much more than the sum of the selected indicators of the MR—furosine and HMF (1.5 mg/100 g fm)—and would therefore correspond to other MRP. A way to solve the problem of the analysis of MRP may be to determine total antioxidants as an indirect estimation of MRP content. Another interesting aspect is that, while in wheat flour nearly all the non-extractable antioxidants were associated with IDF, as previously described for some raw foodstuffs (Goñi et al., 2009), in toasted bread and bread crust only about half of the non-extractable antioxidants were associated with IDF. Nevertheless, the other half of nonextractable antioxidants, due to their high molecular weight, will also have a similar metabolic fate to that of DF and reach the colon intact,
Table 3 Total, extractable and non-extractable antioxidants content in wheat flour, toasted bread and bread crust (mg GAE/100 g fm). Wheat flour Extractable antioxidants
Furfural Total Associated with IDF
n.d. n.d.
11.1 ± 0.2⁎⁎ n.d.
Hydroxymethylfurfural Total Associated with IDF
0.20 ± 0.06 0.14 ± 0.04
119.5 ± 3.1⁎⁎ 0.60 ± 0.10⁎⁎
fm, fresh matter; IDF, insoluble dietary fiber. ⁎⁎ p b 0.001.
Non-extractable antioxidants Total Associated with IDF Total antioxidants
271.5 ± 29.0a
119.08 ± 5.8a 117.3 ± 12.5a 390.58 ± 29.57a
Toasted bread
Bread crust
234.0 ± 24.1a
230.9 ± 26.1a
279.5 ± 19.9b 158.8 ± 14.4b 513.5 ± 30.61b
232.2 ± 0.01c 142.1 ± 11.9b 463.1 ± 26.1c
Different superscript letters indicate significant differences (p b 0.05) between the samples. fm, fresh matter; GAE, gallic acid equivalents; IDF, insoluble dietary fiber.
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even if they are not structurally associated with DF (Saura-Calixto et al., 2010).
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Table 5 Absorbance values in Folin assay of a melanoidin standard generated in the presence or absence of some polyphenols. Values were normalized in relation to those obtained without polyphenols, considered as 100.
3.4. Polyphenols as constituents of MRP
Polyphenol tested
mg polyphenol: mg glycine
Absorbance
In order to confirm the previous results that suggest that polyphenols, MRP and indigestible carbohydrates together form the complex of maillardized DF in bread, a melanoidin standard was produced, from glucose and glycine. It was generated both in the presence and in the absence of ferulic acid, as an example of a phenolic acid and (−)-epicatechin, as an example of a flavonoid; the two main classes of phenolic compounds. After the thermal reaction and washings to remove the fraction of the phenolic compounds not incorporated into the melanoidins, absorbance was measured at 360, 420, 460 and 520 nm, corresponding to the different chemical species generated in the MR. As can be seen in Table 4, at all the wavelengths and concentrations tested, the absorbance value increased in the presence of ferulic acid, indicating its incorporation into MRP. A previous work reported the formation of two adducts ferulic acid-MRP in another model system (Deshou et al., 2009). At 420, 460 and 520 nm, the absorbance increased significantly when the polyphenol concentration increased ten-fold, but not when the increase was a hundred-fold; indicating saturation had been reached and no more polyphenol molecules were incorporated into the new compounds. In the case of (−)-epicatechin, the lowest concentration tested increased significantly the absorbance value at 360 nm, while the middle and the highest concentrations increased the absorbance values at all the wavelengths tested; showing the incorporation of this flavonoid, as itself or as a derived product, into MRP. Similarly, when the Folin assay was applied to the melanoidin standard obtained after dialysis (Table 5), all the concentrations tested of both ferulic acid and (−)-epicatechin showed a significant increase in absorbance, as compared with the control, and a dose–response relationship was observed for (−)-epicatechin. The present results, based on the proportion of ferulic acid and glycine found in wheat bread, show that ferulic acid may be incorporated into the melanoidins in other processed foodstuffs and not only in coffee. Moreover, we also observed for the first time that a flavonoid (i.e., (−)-epicatechin) is also incorporated into the melanoidins, despite some studies reported that this compound behave as an inhibitor of some stages of MR (Totlani & Peterson, 2007). Indeed, due to its chemical structure, (−)-epicatechin, with five hydroxyl moieties, is more prone to participate in this reaction than ferulic acid, with only one hydroxyl group. Further studies are needed to confirm this and, in particular, the exact linkages by which polyphenols are constituents of MRP, and for which only some preliminary data are available (Deshou et al., 2009; Moreira et al., 2012). Also, in a food, which is a much more complex matrix than the model system used in this assay, new interactions derived from other potential sites of reaction would appear. Anyway, the
– Ferulic acid
– 1:85 1:8.5 1:0.85 1:85 1:8.5 1:0.85
100 139 143 179 137 190 323
Table 4 Absorbance values of soluble melanoidins standard generated in the presence or absence of some polyphenols. Values were normalized in relation to those obtained without polyphenols, considered as 100. Polyphenol tested
mg polyphenol: mg glycine
360 nm
– Ferulic acid
– 1:85 1:8.5 1:0.85 1:85 1:8.5 1:0.85
100 140 130 127 139 156 169
(−)-Epicatechin
± ± ± ± ± ± ±
5a 19b 22b 23b 2b 10b 20b
420 nm
100 140 183 204 85 127 125
± ± ± ± ± ± ±
4a 5b 12b 10c 16a 13b 15b
460 nm
100 143 197 220 89 131 261
± ± ± ± ± ± ±
5a 5b 13c 11c 11a 32b 31c
520 nm
100 145 206 220 86 147 157
± ± ± ± ± ± ±
6a 7b 14c 13c 8a 18b 19b
Different superscript letters indicate significant differences (p b 0.05) between the solution with polyphenols and that without them for a same wavelength.
(−)-Epicatechin
± ± ± ± ± ± ±
4a 3b 23b 3c 4b 23c 8d
Different superscript letters indicate significant differences (p b 0.05) between the solution with polyphenols and that without them.
preliminary results provided in this work indicate that maillardized IDF is a complex comprising indigestible carbohydrates, MRP and polyphenols. 4. Conclusions The present work provides evidence of the existence of a complex constituted by indigestible carbohydrates-protein-MRP-polyphenols, named maillardized IDF in bread, chosen as a case study of solid thermally processed food. Regarding DF approach, this strengthens the idea that DF includes a wide variety of chemical structures with similar behavior throughout the digestive tube, i.e., reaching nearly intact the colon where they may be extensively fermented by the microbiota. And in MR approach, these findings should encourage the already ongoing research on these compounds not only as those originated from reducing sugars and amino acids, but as much more complex structures, including phenolic compounds that confer them antioxidant properties. Overall, these results indicate that some health effects that have been attributed exclusively to certain MRP, such as melanoidins, would in fact be due to the whole indigestible complex of maillardized IDF. Future studies should focus on the characterization of this complex in other thermally processed foodstuffs, the different chemical linkages between its different constituents and, in particular, its potential health effects. Acknowledgments This research has been supported by the Spanish Ministry of Science and Innovation (AGL2011-27741). M.E. D.-R. and J. P.-J. acknowledge the CSIC and the Spanish Ministry of Science and Innovation for granting them JAE-Doc and Juan de la Cierva postdoctoral contracts, respectively. M.L. García-González is thanked for her technical assistance. References AACC (2001). The definition of dietary fiber. Report of the Dietary Fiber Definition Committee to the Board of Directors of the American Association of Cereal Chemists. Cereal foods world, 46, 112–116. Ames, J. M., Caemmerer, B., Vélisek, J., Cejpek, K., Obretenov, C., & Cioroi, M. (2000). The nature of melanoidins and their investigation. In J. M. Ames (Ed.), Melanoidins in food and health (pp. 13–29). Luxembourg: European Communities. Bekedam, E. K., Schols, H. A., Cämmerer, B., Kroh, L. W., Van Boekel, M.A. J. S., & Smit, G. (2008). Electron spin resonance (ESR) studies on the formation of roasting-induced antioxidative structures in coffee brews at different degrees of roast. Journal of Agricultural and Food Chemistry, 56, 4597–4604. Borrelli, R. C., & Fogliano, V. (2005). Bread crust melanoidins as potential prebiotic ingredients. Molecular Nutrition and Food Research, 49, 673–678. Codex Alimentarius (2009). Report of the 31st session of the Codex committee on nutrition and foods for special dietary uses. ALINORM 10/33/26 (Düsseldorf, Germany) Available at www.codexalimentarius.org (acccessed on August 2013). Corzo-Martínez, M., Hernandez-Hernandez, O., Villamiel, M., Rastall, R. A., & Moreno, F. J. (2013). In vitro bifidogenic effect of Maillard-type milk protein-galactose conjugates on the human intestinal microbiota. International Dairy Journal, 31, 127–131. Delgado, T., Corzo, N., Santa-Maria, G., Jimeno, M. L., & Olano, A. (1992). Determination of furosine in milk samples by ion-pair reversed phase liquid chromatography. Chromatographia, 33, 374–376.
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Delgado-Andrade, C., Rufián-Henares, J. A., & Morales, F. J. (2007). Lysine availability is diminished in commercial fibre-enriched breakfast cereals. Food Chemistry, 100, 725–731. Dell'Aquila, C., Ames, J. M., Gibson, G. R., & Wynne, A. G. (2003). Fermentation of heated gluten systems by gut microflora. European Food Research and Technology, 217, 382–386. Deshou, J., Christopher, C., Pranav, M., Sandeep, K., & Peterson, D.G. (2009). Identification of hydroxycinnamic acid — Maillard reaction products in low-moisture baking model systems. Journal of Agricultural and Food Chemistry, 57, 9932–9943. Englyst, H. N., & Cummings, J. H. (1988). Improved method for measurement of dietary fiber as non-starch polysaccharides in plant foods. Journal of the Association of Official Analytical Chemists, 71, 808–814. Fogliano, V., Maria Monti, S., Musella, T., Randazzo, G., & Ritieni, A. (1999). Formation of coloured Maillard reaction products in a gluten-glucose model system. Food Chemistry, 66, 293–299. Fogliano, V., & Morales, F. J. (2011). Estimation of dietary intake of melanoidins from coffee and bread. Food and Function, 2, 117–123. García-Alonso, A., & Goñi, I. (2000). Effect of processing on potato starch: In vitro availability and glycaemic index. Starch/Staerke, 52, 81–84. Gniechwitz, D., Reichardt, N., Blaut, M., Steinhart, H., & Bunzel, M. (2007). Dietary fiber from coffee beverage: Degradation by human fecal microbiota. Journal of Agricultural and Food Chemistry, 55, 6989–6996. Gniechwitz, D., Reichardt, N., Ralph, J., Blaut, M., Steinhart, H., & Bunzel, M. (2008). Isolation and characterisation of a coffee melanoidin fraction. Journal of the Science of Food and Agriculture, 88, 2153–2160. Goñi, I., Díaz-Rubio, M. E., Pérez-Jiménez, J., & Saura-Calixto, F. (2009). Towards an updated methodology for measurement of dietary fiber, including associated polyphenols, in food and beverages. Food Research International, 42, 840–846. González-Mateo, S., González-SanJosé, M. L., & Muñiz, P. (2009). Presence of Maillard products in Spanish muffins and evaluation of colour and antioxidant potential. Food and Chemical Toxicology, 47, 2798–2805. Hartzfeld, P. W., Forkner, R., Hunter, M.D., & Hagerman, A. E. (2002). Determination of hydrolyzable tannins (gallotannins and ellagitannins) after reaction with potassium iodate. Journal of Agricultural and Food Chemistry, 50, 1785–1790. Langner, E., Nunes, F. M., Pozarowski, P., Kandefer-Szerszeń, M., Pierzynowski, S. G., & Rzeski, W. (2013). Melanoidins isolated from heated potato fiber (Potex) affect human colon cancer cells growth via modulation of cell cycle and proliferation regulatory proteins. Food and Chemical Toxicology, 57, 246–255. Lee, K. G., & Shibamoto, T. (2002). Toxicology and antioxidant activities of non-enzymatic browning reaction products: Review. Food Reviews International, 18, 151–175. Martins, S. I. F. S., Jongen, W. M. F., & Van Boekel, M.A. J. S. (2000). A review of Maillard reaction in food and implications to kinetic modelling. Trends in Food Science and Technology, 11, 364–373. Michalska, A., Amigo-Benavent, M., Zielinski, H., & del Castillo, M.D. (2008). Effect of bread making on formation of Maillard reaction products contributing to the overall antioxidant activity of rye bread. Journal of Cereal Science, 48, 123–132. Morales, F. J., & Babbel, M. B. (2002). Antiradical efficiency of Maillard reaction mixtures in a hydrophilic media. Journal of Agricultural and Food Chemistry, 50, 2788–2792. Moreira, A. S. P., Nunes, F. M., Domingues, M. R., & Coimbra, M.A. (2012). Coffee melanoidins: Structures, mechanisms of formation and potential health impacts. Food and Function, 3, 903–915.
Newton, A. E., Fairbanks, A. J., Golding, M., Andrewes, P., & Gerrard, J. A. (2012). The role of the Maillard reaction in the formation of flavour compounds in dairy products — Not only a deleterious reaction but also a rich source of flavour compounds. Food and Function, 3, 1231–1241. Nunes, F. M., & Coimbra, M.A. (2007). Melanoidins from coffee infusions. Fractionation, chemical characterization, and effect of the degree of roast. Journal of Agricultural and Food Chemistry, 55, 3967–3977. Nunes, F. M., & Coimbra, M.A. (2010). Role of hydroxycinnamates in coffee melanoidin formation. Phytochemistry Reviews, 9, 171–185. Nursten, H. (2005). The Maillard reaction: Chemistry, biochemistry and implications. Cambridge: Royal Society of Chemistry. Pérez-Jiménez, J., Arranz, S., Tabernero, M., Díaz- Rubio, M. E., Serrano, J., Goñi, I., et al. (2008). Updated methodology to determine antioxidant capacity in plant foods, oils and beverages: Extraction, measurement and expression of results. Food Research International, 41, 274–285. Pérez-Jiménez, J., & Saura-Calixto, F. (2005). Literature data may underestimate the actual antioxidant capacity of cereals. Journal of Agricultural and Food Chemistry, 53, 5036–5040. Rufián-Henares, J. A., Delgado-Andrade, C., & Morales, F. J. (2006). Analysis of heat-damage indices in breakfast cereals: Influence of composition. Journal of Cereal Science, 43, 63–69. Saura-Calixto, F., Goni, I., Mañas, E., & Abia, R. (1991). Klason lignin, condensed tannins and resistant protein as dietary fibre constituents: Determination in grape pomaces. Food Chemistry, 39, 299–309. Saura-Calixto, F., Pérez-Jiménez, J., Touriño, S., Serrano, J., Fuguet, E., Torres, J. L., et al. (2010). Proanthocyanidin metabolites associated with dietary fibre from in vitro colonic fermentation and proanthocyanidin metabolites in human plasma. Molecular Nutrition and Food Research, 54, 939–946. Silván, J. M., Morales, F. J., & Saura-Calixto, F. (2010). Conceptual study on maillardized dietary fiber in coffee. Journal of Agricultural and Food Chemistry, 58, 12244–12249. Silván, J. M., van de Lagemaat, J., Olano, A., & del Castillo, M.D. (2006). Analysis and biological properties of amino acid derivates formed by Maillard reaction in foods. Journal of Pharmaceutical and Biomedical Analysis, 41, 1543–1551. Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1998). Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods in Enzymology, 299, 152–178. Southgate, D. A. T. (1977). The definition and analysis of dietary fiber. Nutrition Reviews, 35, 31–37. Tabernero, M., Serrano, J., & Saura-Calixto, F. (2007). Dietary fiber intake in two European diets with high (Copenhagen, Denmark) and low (Murcia, Spain) colorectal cancer incidence. Journal of Agricultural and Food Chemistry, 55, 9443–9449. Totlani, V. M., & Peterson, D.G. (2007). Influence of epicatechin reactions on the mechanisms of maillard product formation in low moisture model systems. Journal of Agricultural and Food Chemistry, 55, 414–420. Trowell, H. (1976). Definition of dietary fiber and hypotheses that it is a protective factor in certain diseases. American Journal of Clinical Nutrition, 29, 417–427. Wang, H. Y., Qian, H., & Yao, W. R. (2011). Melanoidins produced by the Maillard reaction: Structure and biological activity. Food Chemistry, 128, 573–584.
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Evidence for the formation of maillardized insoluble dietary fiber in bread: A specific kind of dietary fiber in thermally processed food J. Pérez-Jiménez 1, M.E. Díaz-Rubio 1, M. Mesías, F.J. Morales, F. Saura-Calixto ⁎ Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), Madrid, Spain
a r t i c l e
i n f o
Article history: Received 2 September 2013 Accepted 20 November 2013 Keywords: Dietary fiber Maillard reaction products Polyphenols Maillardized dietary fiber
a b s t r a c t Bakery products are rich in insoluble dietary fiber (IDF), and Maillard reaction products (MRP), which are commonly studied as two independent constituents. The aim of this work was to elucidate whether IDF contains MRP as an intrinsic constituent, as well as to check the possible contribution of polyphenols to MRP in bread. It was found that MRP contribute to the increase in IDF from wheat flour to bread; indeed, 8% of furosine (a common indicator of MR) is associated with IDF. Also, ferulic acid and (−)-epicatechin got incorporated to MRP in a model system. This work provides evidence for the existence of a complex DF-protein-MRP-polyphenols, named maillardized IDF, in bakery products, a term applicable to other thermally processed food. Moreover, the present results strengthen the concept that MRP are formed not only from reducing sugars and amino acids, but also from phenolic compounds, including phenolic acids and flavonoids. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Dietary fiber (DF) is a key food component that consists of a wide variety of chemical structures. Since the first definition of DF, which considered that its constituents were exclusively non-starch polysaccharides (NSP) and lignin resistant to hydrolysis by human digestive enzymes (Southgate, 1977; Trowell, 1976), the concept has evolved to include other food components, such as oligosaccharides, resistant starch and others (AACC, 2001; Codex Alimentarius, 2009). Moreover, much work has emphasized that an important fraction of dietary polyphenols are constituents of DF (Goñi, Díaz-Rubio, Pérez-Jiménez, & Saura-Calixto, 2009; Saura-Calixto, Goni, Mañas, & Abia, 1991). All the constituents of DF have in common that, after ingestion, they share the same metabolic fate as those included in the original definition of DF: they reach the colon intact, where they may be subjected to extensive transformation by the microbiota. The Maillard reaction (MR) is one of the most studied chemical reactions in food. It is a non-enzymatic process that takes place during thermal processing and during storage in foodstuffs that contain reducing sugars as well as certain amino acids. The many chemical structures that are produced during the different stages of the MR are known as Abbreviations: DF, dietary fiber; fm, fresh matter; GAE, gallic acid equivalents; HMF, hydroxymethylfurfural; IDF, insoluble dietary fiber; MR, Maillard reaction; MRP, Maillard reaction products; NSP, non-starch polysaccharides; SDF, soluble dietary fiber; TDF, total dietary fiber. ⁎ Corresponding author at: Dep. Metabolism and Nutrition, ICTAN-CSIC, Jose Antonio Novais, 10, 28040 Madrid, Spain. Tel.: +34 91 549 23 00; fax: +34 91 549 36 27. E-mail addresses: [email protected] (J. Pérez-Jiménez), [email protected] (M.E. Díaz-Rubio), [email protected] (M. Mesías), [email protected] (F.J. Morales), [email protected] (F. Saura-Calixto). 1 J. P.-J. and M.E. D.-R. contributed equally to this work. 0963-9969/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2013.11.031
Maillard reaction products (MRP) and include Amadori compounds, hydroxymethylfurfural (HMF), furfural, aldehydes, aldols and melanoidins, among other compounds (Nursten, 2005). MRP are widely studied, either from a sensorial and/or technological point of view (Martins, Jongen, & Van Boekel, 2000; Newton, Fairbanks, Golding, Andrewes, & Gerrard, 2012), from a toxicological one (Lee & Shibamoto, 2002) or for their nutritional value (Moreira, Nunes, Domingues, & Coimbra, 2012); but they are commonly considered independently from DF. Therefore, DF approach does not take into account MRP as intrinsic constituents that may affect their properties and, similarly, MRP approach ignores that a substantial part of melanoidins is indeed insoluble dietary fiber (IDF). In this context, it was recently shown that MRP contribute significantly to soluble dietary fiber (SDF) content in roast coffee (Gniechwitz, Reichardt, Blaut, Steinhart, & Bunzel, 2007; Silván, Morales, & Saura-Calixto, 2010); one of the most common examples of a thermally treated food in whose production the MR takes place. This finding gave rise to the concept of maillardized DF (Silván et al., 2010), which refers to the MRP that are associated with polysaccharides, polyphenols and lignin in certain processed food. Indeed, due to these strong interactions, traditional determinations of SDF would also include a fraction of MRP. In the same way as the original concept of DF proved inadequate, since it did not include polyphenols or MRP as key constituents and it did not use physiological conditions for its determination (Goñi et al., 2009), so the possible presence of phenolic compounds in MRP has been scarcely addressed. While it has been reported that MRP in coffee (particularly melanoidins formed in the final stage of the MR) include phenolic compounds such as chlorogenic acid and caffeic acid (Bekedam et al., 2008; Gniechwitz et al., 2008; Moreira et al., 2012; Nunes & Coimbra, 2007, 2010) and some model studies have been
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carried out to evaluate the incorporation of certain polyphenols into MRP (Deshou, Christopher, Pranav, Sandeep, & Peterson, 2009), for most processed food it is commonly considered that MRP are generated exclusively from reducing sugars and amino acids, either free or as constituents of peptides or proteins. In contrast with beverages such as coffee, processed solid foodstuffs do not only include SDF, but also IDF. However, the possible application of the term maillardized DF to IDF has not yet been explored. In this study, bread was chosen as a case study, in order to elucidate whether a fraction of MRP are constituents of IDF. The research is challenging, since baking is a complex process inducing physical, chemical and biochemical changes in the cereal matrix, such as volume expansion, evaporation of water and the formation of a porous structure, denaturation of proteins, starch gelatinization, crust formation, the development of a desirable taste and pleasant flavors and browning. The presence of polyphenols as intrinsic constituents of MRP in bread was also explored. 2. Materials and methods 2.1. Chemicals and samples Gallic acid, ferulic acid, (−)-epicatechin, glucose, glycine, HMF, furfural, pancreatin, α-amylase and amyloglucosidase were obtained from Sigma Chemical (MO, USA). Pepsin, methanol, formic acid, acetonitrile, hydrochloric acid, sodium heptane sulfonate, sulfuric acid, ethylenediaminetetraacetic acid, potassium hexacyanoferrate and zinc sulfate were purchased from Merck (Darmstadt, Germany). FolinCiocalteu reagent was acquired from Panreac (Castellar del Vallés, Spain). Furosine was obtained from Neosystem Laboratories (Strasbourg, France). All the reagents used were of analytical grade. The following samples were provided by Ruipan S.A. (Leganés, Spain), all of them belonging to the same baking process: wheat flour, French bread (subjected to baking at 190–220 °C for 35 min) and toasted bread (subjected to a second baking at 280 °C for 9 min). For analysis, the French bread was freeze-dried and milled to a particle size of 0.5 mm. A fraction of the bread crust was removed from French bread for separate analysis. 2.2. Determinations 2.2.1. Dietary fiber DF was determined in wheat flour, French bread, toasted bread and bread crust according to the indigestible fraction method (Goñi et al., 2009). Samples (300 mg), after being solved in phosphate buffer, were incubated with pepsin (0.2 mL of a 300 mg/mL solution in 0.08 mol/L HCl–KCl buffer, pH 1.5, 40 °C, 1 h), pancreatin (1 mL of a 5 mg/mL solution in 0.1 mol/L phosphate buffer, pH 7.5, 37 °C, 6 h) and α-amylase (1 mL of a 120 mg/mL solution in 0.1 mol/L tris–maleate buffer, pH 6.9, 37 °C, 16 h). The samples were centrifuged (15 min, 3000 g) and the supernatants removed. The residues were washed twice with 5 mL of distilled water, and all the supernatants were combined. Each supernatant was incubated with 100 μL of amyloglucosidase for 45 min at 60 °C before being transferred to dialysis tubes (12,000–14,000 molecular weight cutoff, Visking dialysis tubing; Medicell International Ltd., London, U.K.) and dialyzed against water for 48 h at 25 °C to eliminate the digested compounds. The product of all these treatments was therefore a residue after enzymatic treatments, corresponding to IDF, and a supernatant from the enzymatic treatments later subjected to dialysis, corresponding to SDF. In the SDF, soluble NSP were hydrolyzed with 1 M sulfuric acid at 100 °C for 90 min and spectrophotometrically quantitated after alkalinization with NaOH (3.9 mol/L) and reaction with dinitrosalicylic acid (Englyst & Cummings, 1988). For the IDF, the residue was weighed to determine the IDF content of the sample gravimetrically, and it was divided into different fractions to
determine resistant protein (see Section 2.2.2), indicators of MR (see Section 2.2.3) and antioxidant compounds (see Section 2.2.4). Total dietary fiber (TDF) was determined as the sum of SDF and IDF. 2.2.2. Resistant protein Resistant protein was determined in the whole samples (wheat flour, French bread, toasted bread and bread crust) and in the corresponding IDF using an automated nitrogen analyzer (FP-2000; Dumas Leco Corp., St. Joseph, MI), after calibration of the instrument with EDTA. Protein was calculated as N × 6.25. 2.2.3. Indicators of the Maillard reaction Indicators of the MR were measured in the whole samples (wheat flour and toasted bread) and in their corresponding IDF. Furosine: Furosine was determined by ion-pairing chromatography as described by Delgado et al. (Delgado, Corzo, Santa-Maria, Jimeno, & Olano, 1992) and modified by Delgado-Andrade et al. (DelgadoAndrade, Rufián-Henares, & Morales, 2007). A finely ground sample (30 mg) was hydrolyzed with 4 mL of 7.95 mol/L HCl at 110 °C for 23 h in a Pyrex screw-cap vial with PTFE-faced septa, and sealed under nitrogen. The hydrolyzate was aerated and cooled to room temperature, and subsequently centrifuged at 4000 g for 10 min. A sample of 0.5 mL of the supernatant was applied to a Sep-Pak C18 cartridge (Millipore, Billerica, MA, USA) pre-wetted with 5 mL of methanol and 10 mL of deionized water; it was then eluted with 3 mL of 3 mol/L HCl. An aliquot (1 mL) was dried with a speed-vac concentrator. The sample was dissolved in 1 mL of a mixture of water, acetonitrile and formic acid (95:5:0.2) and filtered (0.45 μm). The analysis was conducted with a Shimadzu HPLC system (Kyoto, Japan) equipped with an LC20AD pump, an SIL-10ADvp autosampler, a CTO-10ASVP oven, and a DAD (SPD-M20A). The chromatographic separations were performed in an Excel-ODS analytical column (250 mm × 4.6 mm, 5 μm particle size, Tecknokroma, Barcelona, Spain) at 32 °C. The mobile phase was prepared with 5 mmol/L sodium heptane sulfonate, including 20% of acetonitrile and 0.2% of formic acid. The elution was isocratic and the flow rate was 1.0 mL/min. The injection volume was 20 μL and detection was performed at 280 nm. Furosine was quantified by the external standard method; the calibration curve was established from a stock solution (1.2 mg/mL of furosine) in the range 1–10 mg/L. HMF and furfural. HMF and furfural were determined as described by Rufián-Henares et al. (Rufián-Henares, Delgado-Andrade, & Morales, 2006). Briefly, a finely ground sample (500 mg) was extracted with 5 mL of deionized water. The tube was shaken vigorously for 1 min and clarified with 0.25 mL each of potassium ferrocyanide, 15% w/v, and zinc acetate, 30% w/v, solutions. The resulting mixture was centrifuged at 4500 g for 10 min at 4 °C. The supernatant was collected in a 10 mL volumetric flask and two further extractions were performed using 2 mL of deionized water. The supernatants were pooled and filtered. The sample (20 μL) was analyzed by reverse-phase HPLC in a Shimadzu HPLC system as described above. The mobile phase was a mixture of acetonitrile in water (5% v/v) delivered at a flow rate of 1 mL/min under isocratic conditions through the analytical column (Mediterranea-SeaC18, 250 mm × 4.0 mm, 5 μm particle size, Teknokroma, Barcelona, Spain) and thermostated at 32 °C. The UV detector was set at 280 nm and 20 μL of the extract was injected. HMF and furfural were quantified using the external standard method within the range 0.1–20 mg/L and 1–10 mg/L respectively. The limit of quantitation was set as 0.05 mg/kg. Each sample was analyzed in duplicate and the mean of the two measurements is reported. 2.2.4. Antioxidant compounds Three different fractions of antioxidant compounds were determined in wheat flour, toasted bread and bread crust: - Extractable antioxidants. 0.5 g of sample was placed in a capped centrifuge tube; 20 mL of acidic methanol/water (50:50 v/v,
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acidified to pH = 2 with HCl 2 mol/L) was added and the tube was thoroughly shaken at room temperature for 1 h. The tube was centrifuged at 2500 g for 10 min and the supernatant was recovered. Twenty mL of acetone/water (70:30, v/v) was added to the residue, and shaking and centrifugation were repeated. The two extracts obtained for each replicate were combined and used to determine the content of extractable antioxidants (Pérez-Jiménez et al., 2008). - Non-extractable antioxidants. The residues from obtaining the extractable antioxidants were mixed with 20 mL of methanol and 2 mL of concentrated sulfuric acid. The samples were placed in a water bath with constant shaking at 85 °C for 20 h and then centrifuged (2500 g for 10 min) and the supernatants recovered, a procedure previously reported to release high molecular weight polyphenols and/or associated with the food matrix (Hartzfeld, Forkner, Hunter, & Hagerman, 2002; Pérez-Jiménez et al., 2008). After two washings with distilled water, the final volume was taken up to 50 mL (Hartzfeld et al., 2002). These hydrolyzates were used to determine the content of non-extractable antioxidants. - Non-extractable antioxidants associated with IDF. A portion of IDF was subjected to the same hydrolysis procedure as that used for obtaining non-extractable antioxidants, and the corresponding hydrolyzates were used to determine non-extractable antioxidants associated with IDF (Tabernero, Serrano, & SauraCalixto, 2007). The antioxidant content was determined in these extracts/hydrolyzates by the Folin–Ciocalteu procedure (Singleton, Orthofer, & Lamuela-Raventós, 1998). Samples (0.5 mL) were mixed with 0.5 mL of Folin-Ciocalteu reagent and swirled. After 3 min, 10 mL of sodium carbonate solution (75 g/L) was added and mixed. Additional distilled water was mixed thoroughly by inverting the tubes several times. After 1 h, the absorbance at 750 nm was recorded. The results were expressed as mg gallic acid equivalents (GAE)/kg.
2.2.5. Generation of melanoidin standard with and without polyphenols A melanoidin standard was generated as previously reported (Ames et al., 2000), but including also polyphenols, in order to determine their possible incorporation. Briefly, 25 mL of 0.05 mol/L solutions of both glucose and glycine in distilled water were prepared, including phenolic compounds in different proportions in relation to glycine (0.85–8.5– 85 mg of glycine per mg of ferulic acid or (−)-epicatechin). A control without phenolic compounds was also prepared. The solution was freeze-dried and the resulting solid was placed in an oven preheated to 125 °C, for 2 h. Of the resulting powder, 1.25 g was dissolved in 20 mL of distilled water and the mixture was filtered through Whatman No 4 filter paper. After two washings of the residue with 5 mL of distilled water, the washings and the original filtrate were combined and the absorbance of this solution was measured at 360, 420, 460 and 520 nm, according to the different stages of MR (Ames et al., 2000; Fogliano, Maria Monti, Musella, Randazzo, & Ritieni, 1999). The solution was then transferred to dialysis tubes (12,000–14,000 molecular weight cutoff) and dialyzed against water for 48 h at 25 °C. The Folin–Ciocalteu assay was then applied to the dialyzates, according to the procedure described in Section 2.2.4. The proportion of glycine and the polyphenols in the original mixture was calculated from that in white bread (about 85 mg of glycine per mg of ferulic acid). To evaluate possible dose–response relationships, ten-fold and one hundred-fold concentrations of the polyphenol were also tested. Absorbance values measured before dialysis and after dialysis (as Folin assay) were expressed in relation to the value of the solution obtained in the absence of polyphenols, which was considered as a value of 100.
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2.3. Statistics All the analyses were carried out in triplicate, and results are expressed as mean values ± standard deviation as fresh matter (fm) basis. Statistical significant differences between two samples were determined by Student's t test and between more samples by one-way ANOVA and Tukey's multiple-range test to compare means (p b 0.05), with the SPSS 19.0 statistical package for Windows software (SPSS, Chicago, IL, USA). 3. Results and Discussion 3.1. DF analysis considering the possible presence of MRP The present study explores whether MRP are ignored constituents of IDF in thermally processed food. First of all, SDF and IDF—including resistant protein—were determined in wheat flour, French bread (one of the most characteristic examples of a food that undergoes the MR), bread crust (where these compounds are concentrated) and toasted bread (where a second thermal process takes place, thereby increasing MRP), according to a methodology that includes resistant starch (Goñi et al., 2009), a key constituent of DF in may cereal products. The corresponding results are shown in Table 1. The transformation from wheat flour to bread significantly reduced TDF, which is probably due to an increase in the digestibility of resistant starch, as reported for instance for boiled potato (García-Alonso & Goñi, 2000). Regarding the different bread samples, significant differences in TDF content were found, according to the sequence: bread crust N toasted bread N French bread. This was mainly due to a significant increase in IDF, which also caused a significant decrease in the ratio SDF:IDF, from 0.45 in bread to 0.15 in bread crust. The increase in IDF in the samples with a higher MRP concentration (i.e., bread crust and toasted bread) is partly due to the higher content in those samples of resistant protein, but this does not completely explain the difference. Therefore, it may be that the higher IDF content in those samples in which the MR is more developed is due to the contribution to IDF of MRP. This is also supported by the observation that, while IDF content in bread crumb may be estimated to be about 4.3% (considering that IDF in bread was 5.06% and bread crumb is 77% of bread weight), IDF content in toasted bread, which is derived mostly from bread crumb, was 7.5%—this increase would be mainly due to the contribution of MRP to IDF. Therefore, common analysis of DF in thermally processed foodstuffs in fact includes a fraction of MRP, although this has not previously been considered, according to the traditional view of DF as NSP and lignin. Moreover, MRP would contribute to the health-related properties of these foodstuffs, even if they have a chemical structure quite different from those of the compounds originally considered as constituents of DF, since all of them will share the same metabolic fate after ingestion, reaching the colon nearly intact. Indeed, several in vitro studies have Table 1 Dietary fiber content in wheat flour, French bread, toasted bread and bread crust. Wheat flour Soluble dietary fiber 2.27 ± 0.14a (g/100 g fm) 17.87 ± 0.67a Insoluble dietary fiber (g/100 g fm) Resistant protein 2.09 ± 0.11a (mg/100 g fm) Total dietary fiber 20.14 ± 0.768a (g/100 g fm) Soluble/insoluble 0.11 ± 0.008a dietary fiber
French bread Toasted bread 2.28 ± 0.13a
Bread crust
2.69 ± 0.42b
1.57 ± 0.13c
5.06 ± 0.50b 7.51 ± 0.93c
10.21 ± 0.08d
1.54 ± 0.10b 1.80 ± 0.10b
2.22 ± 0.12a
7.34 ± 0.52b 10.2 ± 1.02c
11.78 ± 0.15c
0.45 ± 0.05b 0.39 ± 0.07b
0.15 ± 0.01a
Different superscript letters indicate significant differences (p b 0.05) between the samples. fm, fresh matter.
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shown that melanoidins with different origins, including those from bread crust, promote the growth of different microbial species, such as Bifidobacteria strains (Borrelli & Fogliano, 2005; Corzo-Martínez, Hernandez-Hernandez, Villamiel, Rastall, & Moreno, 2013; Dell'Aquila, Ames, Gibson, & Wynne, 2003), but they considered MRP as compounds independent from DF. 3.2. Analysis of indicators of the MR as possible constituents of DF MR, caramelization and development of the gluten network are the most important chemical events that occur during the manufacture of bakery products and they are indeed related, since gluten is a key constituent of bread melanoidins (Dell'Aquila et al., 2003; Fogliano et al., 1999). To corroborate that a fraction of MRP in bread are constituents of IDF, we decided to analyze MRP in French bread IDF. Since determining all potential MRP would require a complicated combination of analytical techniques (Silván, van de Lagemaat, Olano, & del Castillo, 2006), some common indicators of the MR were selected instead: furosine, HMF and furfural. Furosine (N-2-furoylmethyl-L-lysine) is an amino acid derivative formed during the acid hydrolysis of the Amadori products (N-substituted 1-amino-1-deoxy-2-ketose) which is generated in the early stages of the MR. In contrast, HMF and furfural are classical indicators of the advanced stage of both the MR and caramelization. They were measured in wheat flour and toasted bread, and in the corresponding IDF, to observe the evolution of their content during the baking process from the first material to the last one (Table 2). The results show the expected increase in these indicators in toasted bread as compared with wheat flour. More interestingly, nearly 8% of total furosine and 1% of total HMF in toasted bread were found in IDF; i.e., they were also constituents of IDF. Although melanoidins have been widely characterized in different bakery products (Fogliano & Morales, 2011; González-Mateo, González-SanJosé, & Muñiz, 2009), indicators of MR in IDF from these products had not been determined. The present results are a confirmation of the presence of MRP in IDF, therefore extending the previously defined concept of maillardized SDF (Silván et al., 2010) to maillardized IDF. MRP would be therefore minor constituents of IDF. Interestingly, a recent work also reported the presence of melanoidins as constituents of IDF, in that case in a commercial DF from potato (Langner et al., 2013), showing that the concept of maillardized IDF would be extensible to other solid thermally processed food different from bakery products. 3.3. Antioxidant content analysis The selected indicators of the MR found in IDF of toasted bread were not able to explain the entire increase in IDF in toasted bread as compared with untoasted bread (Table 1). Moreover, due to the previously mentioned problems in measuring other MRP, total antioxidant content as well as antioxidants associated with IDF were evaluated by Folin Table 2 Furosine, furfural and hydroxymethylfurfural content in wheat flour and toasted bread (mg/kg fm).
Furosine Total Associated with IDF
Wheat flour
Toasted bread
n.d. n.d.
203 ± 20⁎⁎ 15 ± 1⁎⁎
assay in samples corresponding to the different stages of the baking process, as an alternative method for determining the contribution of MRP to DF. The use of this approach to estimate MRP associated with IDF is based on the fact that MRP have previously been described as antioxidants (Bekedam et al., 2008; Michalska, Amigo-Benavent, Zielinski, & del Castillo, 2008; Morales & Babbel, 2002; Moreira et al., 2012; Wang, Qian, & Yao, 2011). As shown in Table 3, toasted bread and bread crust had a significantly higher content of non-extractable antioxidants (structures with high molecular weight and/or associated with DF) than wheat flour, while there was no significant variation between the three samples in the content of extractable antioxidants (structures of low molecular weight). Therefore, while the thermal process did not generate new condensations or associations between DF and those antioxidants already present in the sample (extractable antioxidants), it did originate new antioxidant compounds, MRP, thereby confirming some previous results (Pérez-Jiménez & Saura-Calixto, 2005). Moreover, the increase in nonextractable antioxidants was higher in toasted bread than in bread crust, since the second thermal process involved in toasted bread increases the formation of MRP. Furthermore, antioxidants associated with IDF were specifically measured by Folin assay (Table 3). A significant increase in nonextractable antioxidants associated with IDF was observed in toasted bread and bread crust as compared with wheat flour. Although Folin assay measures all reducing compounds present in a sample, this increase associated with IDF during baking, concomitant with the increase in MRP content during the same process, postulates MRP as candidates to be responsible for it. Therefore, this association again suggests the contribution of MRP to IDF and to the overall antioxidant capacity of processed food. These results show that both polyphenols (present in wheat flour and in bread samples) and antioxidant MRP (present in bread samples) are associated with DF, and generate a complex structure. Indeed, while MRP are commonly considered to be formed exclusively from reducing sugars and amino acids, these results suggest that polyphenols may also be constituents of MRP as part of this complex structure with DF, what was later specifically tested (see Section 3.4). Considering the amount of non-extractable antioxidants associated with IDF in wheat flour (117.3 mg/100 g fm) and that in toasted bread (158.8 mg/100 g fm), this implies that toasted bread contains about 40 mg/100 g fm (expressed as GAE) of MRP associated with IDF. This is much more than the sum of the selected indicators of the MR—furosine and HMF (1.5 mg/100 g fm)—and would therefore correspond to other MRP. A way to solve the problem of the analysis of MRP may be to determine total antioxidants as an indirect estimation of MRP content. Another interesting aspect is that, while in wheat flour nearly all the non-extractable antioxidants were associated with IDF, as previously described for some raw foodstuffs (Goñi et al., 2009), in toasted bread and bread crust only about half of the non-extractable antioxidants were associated with IDF. Nevertheless, the other half of nonextractable antioxidants, due to their high molecular weight, will also have a similar metabolic fate to that of DF and reach the colon intact,
Table 3 Total, extractable and non-extractable antioxidants content in wheat flour, toasted bread and bread crust (mg GAE/100 g fm). Wheat flour Extractable antioxidants
Furfural Total Associated with IDF
n.d. n.d.
11.1 ± 0.2⁎⁎ n.d.
Hydroxymethylfurfural Total Associated with IDF
0.20 ± 0.06 0.14 ± 0.04
119.5 ± 3.1⁎⁎ 0.60 ± 0.10⁎⁎
fm, fresh matter; IDF, insoluble dietary fiber. ⁎⁎ p b 0.001.
Non-extractable antioxidants Total Associated with IDF Total antioxidants
271.5 ± 29.0a
119.08 ± 5.8a 117.3 ± 12.5a 390.58 ± 29.57a
Toasted bread
Bread crust
234.0 ± 24.1a
230.9 ± 26.1a
279.5 ± 19.9b 158.8 ± 14.4b 513.5 ± 30.61b
232.2 ± 0.01c 142.1 ± 11.9b 463.1 ± 26.1c
Different superscript letters indicate significant differences (p b 0.05) between the samples. fm, fresh matter; GAE, gallic acid equivalents; IDF, insoluble dietary fiber.
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even if they are not structurally associated with DF (Saura-Calixto et al., 2010).
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Table 5 Absorbance values in Folin assay of a melanoidin standard generated in the presence or absence of some polyphenols. Values were normalized in relation to those obtained without polyphenols, considered as 100.
3.4. Polyphenols as constituents of MRP
Polyphenol tested
mg polyphenol: mg glycine
Absorbance
In order to confirm the previous results that suggest that polyphenols, MRP and indigestible carbohydrates together form the complex of maillardized DF in bread, a melanoidin standard was produced, from glucose and glycine. It was generated both in the presence and in the absence of ferulic acid, as an example of a phenolic acid and (−)-epicatechin, as an example of a flavonoid; the two main classes of phenolic compounds. After the thermal reaction and washings to remove the fraction of the phenolic compounds not incorporated into the melanoidins, absorbance was measured at 360, 420, 460 and 520 nm, corresponding to the different chemical species generated in the MR. As can be seen in Table 4, at all the wavelengths and concentrations tested, the absorbance value increased in the presence of ferulic acid, indicating its incorporation into MRP. A previous work reported the formation of two adducts ferulic acid-MRP in another model system (Deshou et al., 2009). At 420, 460 and 520 nm, the absorbance increased significantly when the polyphenol concentration increased ten-fold, but not when the increase was a hundred-fold; indicating saturation had been reached and no more polyphenol molecules were incorporated into the new compounds. In the case of (−)-epicatechin, the lowest concentration tested increased significantly the absorbance value at 360 nm, while the middle and the highest concentrations increased the absorbance values at all the wavelengths tested; showing the incorporation of this flavonoid, as itself or as a derived product, into MRP. Similarly, when the Folin assay was applied to the melanoidin standard obtained after dialysis (Table 5), all the concentrations tested of both ferulic acid and (−)-epicatechin showed a significant increase in absorbance, as compared with the control, and a dose–response relationship was observed for (−)-epicatechin. The present results, based on the proportion of ferulic acid and glycine found in wheat bread, show that ferulic acid may be incorporated into the melanoidins in other processed foodstuffs and not only in coffee. Moreover, we also observed for the first time that a flavonoid (i.e., (−)-epicatechin) is also incorporated into the melanoidins, despite some studies reported that this compound behave as an inhibitor of some stages of MR (Totlani & Peterson, 2007). Indeed, due to its chemical structure, (−)-epicatechin, with five hydroxyl moieties, is more prone to participate in this reaction than ferulic acid, with only one hydroxyl group. Further studies are needed to confirm this and, in particular, the exact linkages by which polyphenols are constituents of MRP, and for which only some preliminary data are available (Deshou et al., 2009; Moreira et al., 2012). Also, in a food, which is a much more complex matrix than the model system used in this assay, new interactions derived from other potential sites of reaction would appear. Anyway, the
– Ferulic acid
– 1:85 1:8.5 1:0.85 1:85 1:8.5 1:0.85
100 139 143 179 137 190 323
Table 4 Absorbance values of soluble melanoidins standard generated in the presence or absence of some polyphenols. Values were normalized in relation to those obtained without polyphenols, considered as 100. Polyphenol tested
mg polyphenol: mg glycine
360 nm
– Ferulic acid
– 1:85 1:8.5 1:0.85 1:85 1:8.5 1:0.85
100 140 130 127 139 156 169
(−)-Epicatechin
± ± ± ± ± ± ±
5a 19b 22b 23b 2b 10b 20b
420 nm
100 140 183 204 85 127 125
± ± ± ± ± ± ±
4a 5b 12b 10c 16a 13b 15b
460 nm
100 143 197 220 89 131 261
± ± ± ± ± ± ±
5a 5b 13c 11c 11a 32b 31c
520 nm
100 145 206 220 86 147 157
± ± ± ± ± ± ±
6a 7b 14c 13c 8a 18b 19b
Different superscript letters indicate significant differences (p b 0.05) between the solution with polyphenols and that without them for a same wavelength.
(−)-Epicatechin
± ± ± ± ± ± ±
4a 3b 23b 3c 4b 23c 8d
Different superscript letters indicate significant differences (p b 0.05) between the solution with polyphenols and that without them.
preliminary results provided in this work indicate that maillardized IDF is a complex comprising indigestible carbohydrates, MRP and polyphenols. 4. Conclusions The present work provides evidence of the existence of a complex constituted by indigestible carbohydrates-protein-MRP-polyphenols, named maillardized IDF in bread, chosen as a case study of solid thermally processed food. Regarding DF approach, this strengthens the idea that DF includes a wide variety of chemical structures with similar behavior throughout the digestive tube, i.e., reaching nearly intact the colon where they may be extensively fermented by the microbiota. And in MR approach, these findings should encourage the already ongoing research on these compounds not only as those originated from reducing sugars and amino acids, but as much more complex structures, including phenolic compounds that confer them antioxidant properties. Overall, these results indicate that some health effects that have been attributed exclusively to certain MRP, such as melanoidins, would in fact be due to the whole indigestible complex of maillardized IDF. Future studies should focus on the characterization of this complex in other thermally processed foodstuffs, the different chemical linkages between its different constituents and, in particular, its potential health effects. Acknowledgments This research has been supported by the Spanish Ministry of Science and Innovation (AGL2011-27741). M.E. D.-R. and J. P.-J. acknowledge the CSIC and the Spanish Ministry of Science and Innovation for granting them JAE-Doc and Juan de la Cierva postdoctoral contracts, respectively. M.L. García-González is thanked for her technical assistance. References AACC (2001). The definition of dietary fiber. Report of the Dietary Fiber Definition Committee to the Board of Directors of the American Association of Cereal Chemists. Cereal foods world, 46, 112–116. Ames, J. M., Caemmerer, B., Vélisek, J., Cejpek, K., Obretenov, C., & Cioroi, M. (2000). The nature of melanoidins and their investigation. In J. M. Ames (Ed.), Melanoidins in food and health (pp. 13–29). Luxembourg: European Communities. Bekedam, E. K., Schols, H. A., Cämmerer, B., Kroh, L. W., Van Boekel, M.A. J. S., & Smit, G. (2008). Electron spin resonance (ESR) studies on the formation of roasting-induced antioxidative structures in coffee brews at different degrees of roast. Journal of Agricultural and Food Chemistry, 56, 4597–4604. Borrelli, R. C., & Fogliano, V. (2005). Bread crust melanoidins as potential prebiotic ingredients. Molecular Nutrition and Food Research, 49, 673–678. Codex Alimentarius (2009). Report of the 31st session of the Codex committee on nutrition and foods for special dietary uses. ALINORM 10/33/26 (Düsseldorf, Germany) Available at www.codexalimentarius.org (acccessed on August 2013). Corzo-Martínez, M., Hernandez-Hernandez, O., Villamiel, M., Rastall, R. A., & Moreno, F. J. (2013). In vitro bifidogenic effect of Maillard-type milk protein-galactose conjugates on the human intestinal microbiota. International Dairy Journal, 31, 127–131. Delgado, T., Corzo, N., Santa-Maria, G., Jimeno, M. L., & Olano, A. (1992). Determination of furosine in milk samples by ion-pair reversed phase liquid chromatography. Chromatographia, 33, 374–376.
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