Effects of toasting on the carbohydrate profile and antioxidant properties of chickpea (Cicer arietinum L.) flour added to durum wheat pasta

Effects of toasting on the carbohydrate profile and antioxidant properties of chickpea (Cicer arietinum L.) flour added to durum wheat pasta

Food Chemistry 131 (2012) 1140–1148 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/food...

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Food Chemistry 131 (2012) 1140–1148

Contents lists available at SciVerse ScienceDirect

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

Effects of toasting on the carbohydrate profile and antioxidant properties of chickpea (Cicer arietinum L.) flour added to durum wheat pasta Clara Fares ⇑, Valeria Menga CRA, Centre for Cereal Research, S.S. 16, Km 675, 71100 Foggia, Italy

a r t i c l e

i n f o

Article history: Received 14 January 2011 Received in revised form 1 August 2011 Accepted 20 September 2011 Available online 2 October 2011 Keywords: Chickpea Toasting process Phenolic acids Carbohydrates Trolox equivalent antioxidant capacity Total phenolic content Pasta processing Cooking

a b s t r a c t The effects of the toasting process on the carbohydrate profile and antioxidant properties of chickpea flour were studied, along with the cooking behaviour, and antioxidant and nutritional properties of pasta enriched with the chickpea flour. The toasting process increased the resistant starch, insoluble dietary fibre and antioxidant properties of the flour. Addition of chickpea flour (raw and toasted) to durum wheat semolina changed the carbohydrate profile in the uncooked and cooked enriched pasta, especially with the toasted chickpea, and worsened the overall quality of the pasta. The increase in total phenolic content and total free phenolic acid content in the uncooked pasta was due to positive effects of addition of the chickpea flours, while the increase in the bound phenolics fraction in the cooked pasta was from the durum wheat, which was crucial for its high concentrations of ferulic acid. The increase in the free fraction of the Trolox equivalent antioxidant capacity in cooked pasta was consistent with the addition of chickpea. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Pasta is a traditional food in Italy, and it is also very popular in several other countries around the World. Pasta is a cheap food that is easy to prepare and is well accepted by all age groups. Moreover, in more recent years, it has become recognised as a healthy food, with a low fat content, no cholesterol and a low glycaemic index (Cleary & Brennan, 2006). It can also be used to carry nutraceutical substances (Chillo, Laverse, Falcone, Protopapa, & Del Nobile, 2008), and it has antioxidant activity (Fares, Platani, Baiano, & Menga, 2010). The low content of lysine and threonine in pasta (Baroni, 1988), which represent two of the eight essential amino acids, can be counterbalanced by the inclusion of legumes. Indeed, legumes are themselves low in fat, and rich in protein, complex carbohydrates and minerals (Geil & Anderson, 1994). As such, legumes can be viewed as a simple system to obtain several goals: to improve the nutritional value of durum wheat pasta, to enhance the cultivation of typical crops of the Mediterranean area, and to increase the market supply of new food for consumers. Several studies have indicated the possibility of adding legume flour to durum wheat semolina in the preparation of pasta, even if the results remain controversial, relating to the effects of such additions on the organoleptic properties of pasta. Wood (2009) showed that chickpea-fortified spaghetti has lower cooking losses ⇑ Corresponding author. Tel.: +39 0881 742972; fax: +39 0881 713150. E-mail address: [email protected] (C. Fares). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.09.080

and less stickiness than the control durum wheat spaghetti, while Gallegos-Infante et al. (2010) showed that the quality of spaghetti that contains flour of the Mexican common bean decreases as a function of the bean-flour substitution, while the total phenols content (TPC) increases. Zhao, Manthey, Chang, Hou, and Yuan (2005) incorporated 5–30%, of different pulse flours, into spaghetti and showed that the firmness and colour intensity of the spaghetti increased, while the overall quality decreased. Sabanis, Makri, and Doxastakis (2006) studied the effects of chickpea flour inclusion in durum wheat lasagne at up to 50% and showed that the nutritional value of the pasta increased with the level of fortification, although the cooking characteristics deteriorated at the higher levels of substitution. Epidemiological studies have indicated that legume consumption is inversely associated with the risk of coronary heart disease (Bazzano et al., 2001), type II diabetes mellitus (Villegas et al., 2008) and obesity (Rizkalla, Bellisle, & Slama, 2002). Furthermore, legume consumption can result in lower LDL cholesterol and higher HDL cholesterol levels (Bazzano, Tees, & Nguyen, 2008). For all of these reasons, legumes are an ideal complement to cereals in a healthy diet, even if their consumption has decreased over recent years, due to the perception that the cooking of legumes is time consuming. Legume seeds generally promote a slow post-prandial blood glucose increase (García-Alonso, Goñi, & Saura-Calixto, 1998), due to the presence of a high portion of non-digestible carbohydrates, including portions of resistant starch and fibre (Björck, Liljeberg, &

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Östman, 2000). They also contain a rich variety of phytochemicals, including natural antioxidants and bioactive carbohydrates (Amarowicz & Pegg, 2008; Kalogeropoulos et al., 2010). Thus there have been studies into the fortifying of durum wheat pasta by the addition of raw materials of vegetable origin, to increase the protein nutritional value, and the dietary fibre and mineral contents (Chillo et al., 2008; Tudorica˘, Kuri, & Brennan, 2002). Among the legumes, chickpea does not have a prominent ‘beany’ flavour, so also for this reason it is a preferred ingredient for the improvement of the protein nutritional value of durum wheat. Goni and Valentın-Gamazo (2003) showed that pasta with 25% chickpea flour has a significantly lower glycaemic index and a higher mineral content than durum wheat pasta. Recently, several studies have shown that the thermal processing of legumes (i.e. soaking, cooking and dehydration) can produce changes in both their carbohydrate composition (Aguilera et al., 2009; Hoover & Ratnayake, 2002; Martin-Cabrejas et al., 2006) and their antioxidant properties (Han & Baik, 2008; Kalogeropoulos et al., 2010). Gallegos-Infante et al. (2010) showed that the TPC and the furosine content increased in spaghetti enriched with the flour of processed Mexican common beans (cooked and dried), with respect to durum wheat pasta. To our knowledge, there are no studies that have investigated the effects of the toasting process on chickpea seeds and on the fortification of durum wheat pasta with toasted chickpea flour. Therefore, the aims of the present study were to determine: (i) the effects of the toasting process on the carbohydrate profile and antioxidant properties of chickpea flour; and (ii) the effects of the addition of chickpea flours to durum wheat semolina on the cooking behaviour and antioxidant and nutritional properties of the enriched pasta. 2. Materials and methods

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pump to remove the humidity without increasing the temperature (30 °C; 760 mm Hg), and then ground with a Tecator1093 Cyclotec (1 mm screen). Determination of total dietary fibre was based on Method 991.43 (AOAC, 1997), as proposed by Prosky, Asp, Schweizer, De Vries, and Furda (1992). The resistant starch was determined with Megazyme Resistant Starch enzymatic assay kits (Megazyme International Ireland Ltd., Bray Business Park, Ireland). Protein content (%N  5.70) was determined using the UNI 10 274 method. All of the determinations were performed in triplicate and expressed according to dry matter.

2.4. Extraction of phytochemicals (free and bound) The free and bound phenolics were extracted as triplicate samples, according to a method described previously (Fares et al., 2010). Briefly, 1 g of sample was homogenised in 7 ml methanol/ 10% acetic acid (85:15; v/v). The sample extract was ultrasonicated for 30 min and made up to a volume of 10 ml with distilled water. After mixing, 1 ml was filtered for HPLC analysis of the free phenolic acids. Next, 12 ml distilled water and 5 ml 10 M NaOH were added, with the tubes sealed and stirred overnight at 20 °C, using a magnetic stirrer. The solution was then adjusted to pH 2, with HCl (37%), and the liberated phenolics were extracted three times with 15 ml cold diethyl ether/ethyl acetate (1:1; v/v). The organic layers were combined, evaporated to dryness, dissolved in 1.5 ml methanol, filtered, and analysed. After alkaline hydrolysis, acid hydrolysis was performed by adding 2.5 ml concentrated HCl to the tubes, with an incubation in a water bath, at 85 °C, for 30 min. The samples were then cooled, and further sample handling was performed in the same way as following the alkaline hydrolysis.

2.1. Chickpea flour preparation The chickpea sample (cv Sultano) used in the present study was cultivated in the field of the Centre for Cereal Research in Foggia (Italy) during the 2008–2009 season. Two types of flour (about 5 kg) were prepared from the chickpea: from the straight milling of the cleaned seeds, and from the cleaned and oven-toasted seeds (105 °C for 3 h). All of the chickpea samples were milled with granite stone milling (300 mm diameter; model Osttiroler Getreidemühle, from Colombini Sergio s.a.s Abbiategrasso, Milan, Italy). Commercial durum wheat semolina (from Molino Tamma srl, Foggia, Italy) was blended with 5%, 10%, 15% and 20% (w/w) of each type of chickpea flour. 2.2. Pasta production The pasta was prepared by mixing 100% semolina (control) and semolina containing 5%, 10%, 15% and 20% (w/w) of each type of chickpea flour, with 30% tap water (v/w), to obtain a total dough water content of 43–44%. The dough was processed into spaghetti (1.70 ± 0.03 mm diameter) using a laboratory press (NAMAD, Rome, Italy), with a 2-kg capacity. The dough extrusion conditions were: temperature, 50 ± 5 °C; pressure, 70 ± 10 atm; and vacuum, 700 mm Hg. The extruded spaghetti samples were dried in a laboratory pasta drier (NAMAD, Rome, Italy), using a low temperature drying procedure (18 h, at about 50 °C). Nine kinds of spaghetti were produced. 2.3. Proximate analysis The analyses were performed on the semolina and chickpea flours, and the uncooked and cooked spaghetti. Samples of cooked spaghetti (20 g) were dried in an oven equipped with a vacuum

2.5. Determination of phenolic acids and total phenols content The samples were run on an Agilent 1100 HPLC System (Hewlett–Packard, Germany) equipped with a diode array detector (Agilent, Waldbronn, Germany), according to the method proposed by Kim, Tsao, Yang, and Cui (2006). Phenolic acid separation was carried out with a Zorbax SB-C18 column (250 mm  4.6 mm  5.0 lm; Agilent, USA), with the temperature of the column oven set at 35 °C. Gradient elution was used, with a mobile phase of acetonitrile (solution A) and 2% acetic acid (solution B), as follows: 0 min, 100% B; 30 min, 85% B; 50 min, 50% B; 55 min, 0% B; 60 min, 100% B; 60–70 min, isocratic elution with 100% B. The flow rate of the mobile phase was 1 ml/min, and the injection volume was 10 ll. The wavelengths used for the quantification of the phenolic acids were 280 and 320 nm. All of the quantifications were based on peak areas of phenolic acid standards (Sigma–Aldrich, Milan, Italy) of: p-hydroxybenzoic, vanillic, caffeic syringic, vanillin, pcoumaric and ferulic acids. The data for the free, alkaline and acid hydrolysed phenolic acids were summed to get the total phenolic acids content (TPAC free and bound). The TPC of the free and bound extracts of all of the samples were determined according to the procedure of Singleton and Rossi (1965). Briefly, 200 ll extract was added to 1.5 ml, 10-fold diluted (distilled water), Folin–Ciocalteau reagent. The mixture was allowed to equilibrate for 5 min, and was then neutralised with sodium carbonate. After an incubation at room temperature for 90 min, the absorbances of the mixtures were measured at 725 nm, with methanol used as the blank. Ferulic acid was used as the standard, and the data are expressed as lg ferulic acid equivalents/g sample. Analyses were performed in triplicate for each extract, and the data reported according to dry matter. The moisture content was determined by drying at 130 °C, for 3 h, in an oven.

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2.6. Trolox equivalent antioxidant capacity The Trolox equivalent antioxidant capacity (TEAC) was determined according to the procedure of Re et al. (1999), with some modifications: 2,2-azinobis-[3-ethylbenzothiazoline-6-sulphonic acid] (ABTS) was dissolved in water to 7 mM. The ABTS radical cation (ABTS+) was produced by the reaction of the ABTS stock solution with potassium persulphate (final concentration, 2.45 mM), for 12–16 h, in the dark and at room temperature before use. The ABTS+ solution was diluted with ethanol, to an absorbance of 0.70 ± 0.02, at 734 nm. The absorbance was read after exactly 1 min, and up to 6 min after the addition of 20 ll of the two kinds of extracts to 2 ml diluted ABTS+. All of the assays were conducted in triplicate. The TEAC of the extracts were calculated, using a Trolox standard curve, on the basis of the percentage inhibition of absorbance at 734 nm, and the data are expressed in lmol Trolox/g dry matter. 2.7. Cooking characteristics of the pasta To assess the pasta cooking quality, spaghetti (100 g) was cooked in 1 l of boiling tap water and the optimal cooking time was taken to be when, after squeezing the spaghetti between two glass plates, the white core from the strands disappeared. The total organic matter (TOM) was determined by the standard method of D’Egidio et al. (1981), and it represents the amount of surface material released from the cooked pasta into the washing water, after rinsing. TOM values > 2.1 g/100 g correspond to low quality pasta, between 2.1 and 1.4 g/100 g correspond to good quality pasta, and <1.4 g/100 g correspond to very good quality pasta (D’Egidio & Nardi, 1996). Analyses were performed in triplicate. 2.8. Statistical analysis Standard ANOVA procedures (one factor randomised, complete block design) was applied to the dataset with the statistical package STATISTICA (StatSoft Italia Srl, Vers.7.1, 2005). Means were separated by least significant difference tests at P = 0.05 probability level. 3. Results and discussion 3.1. Effects of the toasting process on the carbohydrate profile and antioxidant properties of chickpea flour The proximal composition of the raw chickpea flour (Table 1) for total protein (23.4%) and fibre content (16.91% and 6.4% for IDF and SDF, respectively) was in agreement with that described in recent studies, in which samples were grown in environments similar to the conditions in the present study (Mediterranean basin) (Aguilera et al., 2009; Kalogeropoulos et al., 2010). However, some differences were seen in the composition of the soluble fibre fraction, which in the present study was 27.5% of the total dietary fibre (TDF) in raw chickpea flour, while in the study of Aguilera et al. (2009) it represented only 4%. As also shown in Table 1, upon toasting of the chickpeas, the protein content increased (by 11.7%), as well as the resistant starch (by 3-fold) and insoluble dietary fibre (IDF; by 17%), while there were slight reductions in the available starch and soluble fibre. Our data are consistent with those of MartinCabrejas et al. (2006), who found a general increase in the IDF due to soluble sugars (glucose and arabinose) and Klason lignin residues, with the latter ascribed to the formation of new insoluble products (Maillard components), as an effect of the heat treatment. On the other hand, our data also show that products related to the Maillard reaction are formed, as seen by increases in the TPC and TEAC of the toasted chickpea flour.

Table 1 Proximal composition and antioxidant properties of the chickpea flours. Property

Protein (%dm) Available starch (%dm) Resistant starch (%dm) Insoluble dietary fibre (%dm) Soluble dietary fibre (%dm) TPC (lg FAE/g free phenolic extracts) TPC (lg FAE/g bound phenolic extracts) TPAC (lg/g free phenolic extracts) TPAC (lg/g bound phenolic extracts) TEAC (lmol Trolox/g free phenolic extracts) TEAC (lmol Trolox/g bound phenolic extracts)

Chickpea flour From raw chickpea

From toasted chickpea

23.40b 53.30a 1.20b 16.91b 6.40a 813b 48b

26.50a 51.40b 3.60a 20.51a 5.97b 1025a 63a

18.75b 24.31b 11.87b

21.41a 35.33a 16.81a

4.61ns

5.64ns

Data are means of three determinations. Different letters in the same row indicate statistical differences by LSD test (p < 0.05). Abbreviations: TPC = total phenolic content; TPAC = total phenolic acids content; TEAC = Trolox equivalent antioxidant capacity; dm = dry matter.

The antioxidant properties of the chickpea flours are also shown in Table 1. It is interesting to note that the TPC and TEAC values were always higher in the free fraction, as compared to the bound fraction, regardless of the treatment (raw and toasted), in agreement with the literature (Han & Baik, 2008). To our knowledge this study is the first to document the qualitative differences in TPAC of the free and bound fractions of chickpea flour. Despite being lower than the TPAC bound (24.31 and 35.33 lg/g for raw and toasted, respectively), the TPAC free (18.75 and 21.41 lg/g for raw and toasted, respectively) contained approximately 85% of all of the ferulic acid (Table 2) of the flour, and thus explained the higher activity observed in the free extract, with respect to the bound. Also of note, although the amounts of ferulic acid measured in the free fractions of the chickpea flours (3.55 and 3.01 lg/g for raw and toasted, respectively) was significantly higher in raw chickpea flour, the increased antioxidant activity (16.81 lmol Trolox/g) and the TPC (1025 lg FAE/g) measured in the toasted chickpea flour must be ascribed to other products that can improve the antioxidant activity and that formed as a result of the toasting (Amadori and Heyn’s products between reducing sugars and proteins) (Manzocco, Calligaris, Mastrocola, Vicoli, & Lerici, 2001). Goni & Valentın-Gamazo, (2003) have already shown that durum wheat pasta enriched with 25% chickpea flour produced a significant reduction in the glycaemic index, which suggested benefits of the inclusion of chickpea flour as an ingredient in pasta production. Our study has demonstrated that when toasted chickpea flours are added to pasta, they enhanced the health properties of the pasta, as shown by the increases in the resistant starch and the TDF. These changes might be responsible, in turn, for the reducing effects on the post-prandial blood glucose response observed by Goni & Valentın-Gamazo, (2003), thus providing a wider range of low-glycaemic-index food for consumers. 3.2. Processing effects on the nutritional properties and cooking behaviour of chickpea-enriched pasta Table 3 shows the values of the protein, starch (available and resistant) and TDF of the semolina and the uncooked and cooked pasta samples enriched with different percentages of the chickpea flour. The protein content in semolina (from 11.11% of control to 13.73% with 20% of toasted chickpea flour), uncooked pasta (from 11.77% of control to 14.43% with 20% of toasted chickpea flour) and cooked pasta (from 11.30% of control to 13.45% with 20% of

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C. Fares, V. Menga / Food Chemistry 131 (2012) 1140–1148 Table 2 Phenolic acids profile (free and bound extracts) of durum wheat semolina, uncooked and cooked pasta control, and chickpea flours (raw and toasted). Semolina

Uncooked pasta

Cooked pasta

Raw chickpea

Toasted chickpea

Free phenolic acid (lg/g dm) p-Hydroxybenzoic acid Vanillic acid Caffeic acid Syringic acid Vanillin p-Coumaric acid Ferulic acid

0 1.15 ± 0.00c nd nd nd 0.78 ± 0.17c 0.85 ± 0.16d

0.90 ± 0.01c 2.78 ± 0.04a nd nd nd 0.59 ± 0.06c 1.65 ± 0.06c

0 1.21 ± 0.05c nd nd nd 0.54 ± 0.00c 0.78 ± 0.00d

11.76 ± 0.84b 1.84 ± 0.08b nd nd nd 1.53 ± 0.02b 3.55 ± 0.08a

15.38 ± 1.04a 0.68 ± 0.03d nd nd nd 2.05 ± 0.10a 3.01 ± 0.08b

Bound phenolic acid (lg/g dm) p-Hydroxybenzoic acid Vanillic acid Caffeic acid Syringic acid Vanillin p-Coumaric acid Ferulic acid

1.33 ± 0.19d 1.75 ± 0.07ns 0.31 ± 0.03b 0.25 ± 0.05b 1.45 ± 0.11a 2.41 ± 0.08b 84.82 ± 1.69b

2.03 ± 0.06cd 2.10 ± 0.04ns 0.38 ± 0.04b 0.30 ± 0.04b 0.62 ± 0.16b 2.19 ± 0.12b 63.97 ± 0.96c

2.58 ± 0.22c 2.16 ± 0.03ns 1.11 ± 0.00a 0.73 ± 0.09a 0.49 ± 0.00b 2.91 ± 0.06a 101.74 ± 2.51a

20.97 ± 0.29b 1.65 ± 0.78ns nd nd nd 1.07 ± 0.15c 0.62 ± 0.09d

31.38 ± 0.23a 2.22 ± 0.33ns nd nd nd 1.01 ± 0.03c 0.72 ± 0.01d

Different letters in the same row for each sample correspond to significant differences by LSD test (P < 0.05). Values are mean of three determinations ± standard deviation; nd = not detectable.

Table 3 Cooking behaviour of pasta samples. Protein, available starch, resistant starch and total dietary fibre in semolina, uncooked and cooked pasta samples with the different percentages of substitution with chickpea flour (raw and toasted). Samples

Control

Raw chickpea 5%

g

Toasted chickpea

10% f

15% e

20% d

5%

10%

b

f

15% d

20% c

Semolina

Protein (% dm) Available starch (% dm) Resistant starch (% dm) TDF (% dm)

11.11 81.51a 0.65d 5.32f

11.75 71.45cd 1.46c 5.95e

12.21 70.85bc 1.66c 6.59d

12.65 69.01bd 1.62c 7.88bc

13.41 70.94bd 1.65c 7.65c

11.89 72.55b 2.10b 5.89e

12.45 71.27bc 2.25b 6.68d

12.89 70.65cd 2.25b 8.22b

13.73a 69.64d 3.29a 8.90a

Uncooked pasta

Protein (% dm) Available starch (% dm) Resistant starch (% dm) TDF (% dm)

11.77e 71.97b 0.61ab 4.68e

12.20d 73.68a 0.44ab 5.44d

12.55c 71.76b 0.74a 5.975d

13.29b 67.50cd 0.67ab 7.04c

13.88a 65.54e 0.75a 7.77b

12.53d 67.86c 0.20b 5.45d

13.16c 66.43de 0.46ab 5.845d

13.82b 63.03f 0.55ab 7.24bc

14.43a 68.39c 0.76a 8.41a

Cooked pasta

Protein (% dm) Available starch (% dm) Resistant starch (% dm) TDF (% dm) OCT (min) Weight after cooking (g) TOM (g)

11.30e 73.55a 1.45d 6.61f 10ns 260.5a 0.97a

11.84e 68.89b 1.53d 7.02ef 10ns 252b 1.85e

12.34d 67.79c 2.01c 7.44e 10ns 250.0b 1.95d

12.91c 66.82d 2.07c 8.16cd 10ns 249.0b 2.09c

13.41b 65.44c 2.41b 9.27ab 10ns 248.0b 2.84b

11.91d 64.23c 1.54d 7.64de 10ns 264a 1.66f

12.28c 67.38c 2.55ab 8.64bc 10ns 250.0b 2.12c

12.91b 65.24c 2.62a 8.92b 10ns 254b 2.95a

13.45a 63.44d 2.51ab 9.80a 10ns 238c 2.97a

Data are means of three determinations. Different letters in the same row indicate statistical differences by LSD test (p < 0.05). Abbreviations: dm = dry matter; ns = not significant; TDF = total dietary fibre; OCT = optimum cooking time; TOM = total organic matter.

toasted chickpea flour) increased with the percentage of chickpea flour added, independent of the treatment (raw or toasted chickpeas). Therefore these increases were associated with the amounts of chickpea flour added. This trend is consistent with several other studies (Sabanis et al., 2006; Wood, 2009). As a result of the pastamaking process, there was a slight increase in the protein content, although this was lost after the cooking of the pasta, where the values were not different from those measured in the corresponding enriched semolina. As expected, the resistant starch of the durum wheat semolina (0.65%) was low (Fares et al., 2008), as the amount of native resistant starch is low. With the addition of the chickpea flour (raw and toasted), the resistant starch increased proportionally, with the greatest increases (to almost double) seen with the addition of the toasted chickpea flour to the durum wheat semolina (3.29%). Apart from the control, which did not show any differences between the semolina (0.65%) and the uncooked pasta (0.61%), the pasta process caused a sharp decrease in the resistant starch, which was proportional to the addition of the chickpea flour and stronger in the presence of the toasted chickpea flour: 5% raw chickpea from 1.46% (semolina) to 0.44% (uncooked pasta), 10%

raw chickpea from 1.66% (semolina) to 0.74% (uncooked pasta), 15% raw chickpea from 1.62% (semolina) to 0.67% (uncooked pasta), 20% raw chickpea from 1.65% (semolina) to 0.75% (uncooked pasta); 5% toasted chickpea from 2.10% (semolina) to 0.20% (uncooked pasta), 10% toasted chickpea from 2.25% (semolina) to 0.46% (uncooked pasta), 15% toasted chickpea from 2.25% (semolina) to 0.55% (uncooked pasta), 20% toasted chickpea from 3.29% (semolina) to 0.76% (uncooked pasta). The decrease here ranged from 95% to 75%, and the values for the uncooked pasta with different percentages of chickpea flour enrichment (raw and toasted) did not show any statistical differences. Therefore, we hypothesise that the decrease in the resistant starch in the chickpea-enriched pasta can be ascribed to the legume starch typology. Indeed, raw chickpea starch has large amounts of soluble carbohydrate, with respect to other legumes (Aguilera et al., 2009), and the soluble portions might have leached out from pasta during the drying process. Moreover, the heat treatment also altered the dietary fibre of the chickpeas, as reported by Martin-Cabrejas et al. (2006); indeed, they reported that some of the glucose can be released from the resistant starch included in the IDF fraction. Only the soluble portions of the carbohydrates included in dietary fibre and resistant

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Fig. 1. Total phenolic content of the free extracts (a) and bound extracts (b) in the control and enriched chickpea flour samples (as indicated; left, raw; right, toasted). Data are means of three determinations. Different letters indicate statistical differences by LSD test (p < 0.05) ± standard deviation.

starch are involved in the phenomenon of leaching, which occurs during the drying of the pasta when about 32% of the water has been removed. Therefore, we speculate that only the chickpea flour is involved in this loss. The main phenomenon responsible of this change was also involved in the slight decrease in the dietary fibre seen. For the non-resistant starch, there was a slight decrease with the addition of the chickpea flour, which was more noted in the presence of the toasted chickpea flour. As expected, compared to the control (4.68%), the TDF in the samples of uncooked pasta was consistent with the enrichment (from 5.44% to 7.77% for 5– 20% of raw chickpea flour added), and was more pronounced with the toasted chickpea flour (from 5.45% to 8.41%). After cooking, there were increases in the resistant starch, in both the control and enriched pasta, which were more pronounced in those with the toasted chickpea flour (at >10% enrichment). This increase in the resistant starch after cooking was also reported by Fares et al. (2008) in pasta samples of durum and emmer wheat. The increase in resistant starch after cooking, for all of the products analysed, is also in agreement with Rabe and Sievert (1992), who showed that during cooking, more amylose molecules reorganise to form the resistant starch when the pasta is cooled, as occurred in our samples. Moreover, chickpea has a lower starch content, with a higher proportion of amylose than for durum wheat (Wood, 2009), and this probably explains the further resistant starch increase in the chickpea-enriched pasta. The formation of resistant starch from material with almost no initial resistant starch occurs after heat processing, with the starch granule completely hydrated, and this is thought to depend on the amylose/amylopectin ratio, polymer chain length, lipid content and processing conditions, such temperature and water content (Eerlingen & Delcour, 1995; Haralampu, 2000). The TDF was positively affected by cooking (from 4.68% of control uncooked pasta to 6.61% cooked), as it showed significant increased values in all of the samples tested (from 7.02% of cooked pasta with 5% of raw chickpea flour to 9.80% of cooked pasta with 20% of toasted chickpea flour), in agreement with a previous study (Fares et al., 2008). The main phenomenon responsible for these

observed increases in fibre is, the gelatinisation and retro-degradation of the starch during cooking, which changes part of it into resistant starch and leads to an increase in the TDF (Unlu & Faller, 1998; Vasanthan, Caosong, Yeung, & Li, 2002). For the cooking behaviour of the enriched pasta (Table 3), there were differences seen between the control and chickpea-enriched pasta. The substitution of semolina with the chickpea flours (raw and toasted) generally increased the total organic matter (TOM) and decreased the weight of the cooked pasta, while it had no influence on the cooking time. So, the overall quality of the chickpea-enriched pasta worsened, as indicated by the increase in the TOM, which represented the surface material released from the cooked spaghetti after the exhaustive rinsing (D’Egidio et al., 1982). The increase in the TOM is in agreement with the decrease in the weight as the percentage of chickpea flour increased. The addition of non-gluten flours in spaghetti production has been reported to dilute the gluten network of the semolina and to interrupt, and weaken, the overall structure of the spaghetti. Indeed, several reports have shown that partial substitution of durum wheat semolina with another material can result in negative changes (Gallegos-Infante et al., 2010; Sabanis et al., 2006; Zhao et al., 2005). 3.3. Processing effects on the antioxidant properties of chickpeaenriched pasta The antioxidant properties (TPC, TPAC and TEAC) of the samples were evaluated for the free and bound extracts. Table 2 gives the phenolic acid profiles of the durum wheat control (semolina, uncooked and cooked pasta) and the chickpea flours (raw and toasted), for the free and bound extracts. The qualitative and quantitative compositions of the phenolic acids in the durum wheat and the chickpea flours varied, and in particular, they highlight the absence of free p-hydroxybenzoic acid in durum wheat products, as compared to the chickpea flours (especially from the toasted chickpea with 15.38 lg/g), where free p-hydroxybenzoic acid was found. Although ferulic acid was found in trace amounts in the semolina (0.85 lg/g) and pasta controls (1.65 lg/g), these values

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2.48 ± 0.05c 1.45 ± 0.07e 7.21d 5.29 ± 0.01a 2.49 ± 0.04b 13.96a TPAC: Total phenolic acids content. Different letters in the same row for each sample correspond to significant differences by LSD test (P < 0.05). Values are mean of three determinations ± standard deviation.

2.34 ± 0.00c 2.78 ± 0.04a 7.42c 1.99 ± 0.01e 1.46 ± 0.08e 7.04e 3.25 ± 0.11b 2.00 ± 0.05c 8.35b 2.16 ± 0.00d 0.91 ± 0.08g 3.94h 1.34 ± 0.12g 1.24 ± 0.01f 5.86g 2.46 ± 0.02c 1.78 ± 0.01d 7.22d 1.42 ± 0.01g 1.02 ± 0.03g 3.77i 0.95 ± 0.05h 1.25 ± 0.04f 5.80g 1.02 ± 0.00h 0.91 ± 0.02g 3.21l Toasted chickpea p-Hydroxybenzoic acid Ferulic acid TPAC

1.70 ± 0.02f 1.57 ± 0.03e 6.36f

2.37 ± 0.07ef 1.25 ± 0.11cd 6.04e 6.77 ± 0.02a 2.16 ± 0.07a 12.51a 2.43 ± 0.22d 0.83 ± 0.05de 4.69g 2.04 ± 0.06eg 1.05 ± 0.07de 5.59f 5.12 ± 0.51b 1.89 ± 0.18ab 9.96b 1.28 ± 0.05hi 0.77 ± 0.25e 3.44i 1.52 ± 0.04gi 0.99 ± 0.00de 4.67g 4.14 ± 0.30c 1.64 ± 0.01bc 7.84c 1.81 ± 0.15fh 0.72 ± 0.26 e 4.65g 1.09 ± 0.07i 0.87 ± 0.12 de 4.21h 2.27 ± 0.09ef 1.25 ± 0.05cd 6.49d 0.96 ± 0.04i 0.71 ± 0.26e 3.22i Raw chickpea p-Hydroxybenzoic acid Ferulic acid TPAC

Uncooked pasta Semolina

20%

Cooked pasta Uncooked pasta Semolina

15%

Cooked pasta Semolina

Uncooked pasta 10%

Uncooked pasta

Cooked pasta 5%

Semolina

Free extract (lg/g dm)

Table 4 Phenolic acids profile (free extracts) in chickpea-enriched (raw and toasted) semolina and pasta samples.

tripled in the chickpea flour free extracts (3.55 and 3.01 lg/g for raw and toasted, respectively). In the bound fraction of the chickpea flours, p-hydroxybenzoic acid (20.97 and 31.38 lg/g for raw and toasted flour, respectively) was found in front of traces of ferulic acid (0.62 and 0.72 lg/g for raw and toasted flour, respectively); in contrast, in the durum wheat semolina and pasta (uncooked and cooked) there was little p-hydroxybenzoic acid, but large amounts of ferulic acid (84.82, 63.97 and 101.74 lg/g for semolina, uncooked and cooked pasta, respectively). Negligible amounts of other phenolic acids were detected in all of the samples. As expected, in the enriched semolina samples, the addition of the toasted chickpea flour affected the TPC of the free extract (Fig. 1a), and this increase paralleled the additions (from 548.9 lg FAE/g of control to 813.4 lg FAE/g of 20%). Instead, the addition of the various percentages of the raw chickpea flour did not affect the TPC of the free extract, and the values were lower than the control (the range varied from 460 lg FAE/g of 5% to 504.9 lg FAE/g of 20%). As a result of the pasta-making process, a significant decrease in the TPC was seen in the uncooked pasta from 100% durum wheat (from 548.9 lg FAE/g of semolina to 445.1 lg FAE/g of uncooked pasta). Indeed, it is well documented that during pasta processing the presence of oxygen, water and heat induces oxidative degradation of antioxidants (carotenoid pigments, polyphenols) (Borrelli, Troccoli, Di Fonzo, & Fares, 1999; Fares et al., 2008). The same trend was seen for the TPC of toasted chickpea pasta at different percentages of enrichment (from 442.6, 625.4, 609.3 and 813.4 lg FAE/g of enriched semolina at increasing level of chickpea flour substitution to 447.8, 492.0, 551.6 and 661.4 lg FAE/g of corresponding uncooked pasta); in contrast, the addition of the raw chickpea flour reversed the effects of this processing, and the pasta had higher values than the corresponding enriched semolina. The explanation for this behaviour becomes clear in the light of the qualitative/quantitative composition of the phenolic acids reported in Table 4. The pasta processing increased the TPAC (Table 4) in the free uncooked pasta, when compared with the corresponding enriched semolina, independent of the kind of chickpea flour used for the enrichment (e.g. from 3.22 to 6.49 lg/g and 3.21 to 6.36 lg/g for enriched sample with 5% raw and toasted chickpea, respectively). These data appear not to be in agreement with the data from an earlier study of Fares et al. (2010), in which the free fraction of phenolic acids was reduced during the pasta processing and cooking. This loss was entirely due to the drastic reduction in p-hydroxybenzoic acid, which was found to be abundant in the semolina. On the contrary, in the samples analysed in the present study, phydroxybenzoic acid was absent in both the durum wheat semolina and pasta (Table 2). Recently, Okarter, Liu, Sorrells, and Liu (2010) found no evidence of p-hydroxybenzoic acid in six different varieties of whole wheat, while Hung, Maeda, Miyatake, and Morita (2009) found significant quantities. So there is variability in the qualitative/quantitative composition of phenolic acids across different wheat varieties. Therefore, the increase in the free TPAC observed in the present study from semolina to pasta should be solely explained by the positive effects due to the inclusion of the chickpea flours, with their good supply of free phenolic acids. In the light of the data given in Table 4, the increase in the TPC (Fig. 2), seen as a result of the enrichment with the raw chickpea flour, was in agreement with the TPAC data. Indeed, with the enrichment of the uncooked pasta, with 5–20% raw chickpea flour, the amount of p-hydroxybenzoic acid (2.27, 4.14, 5.12 and 6.77 lg/g, respectively) was more than doubled, with respect to the enriched semolina (0.96, 1.81, 1.28 and 2.43 lg/g, respectively), while for the toasted chickpea flour in the uncooked pasta, the increases were lower for the additions of 5%, 10%, 15% and 20% toasted chickpea flour (57%, 57%, 66%

Cooked pasta

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Fig. 2. Trolox equivalent antioxidant capacity (TEAC) of free (a) and bound (b) fractions of the control and chickpea-enriched cooked pasta samples. Data are means of three determinations. Different letters indicate statistical differences by LSD test (p < 0.05) ± standard deviation.

and 44%, respectively). Moreover, the ferulic acid also doubled in the different formulations, in agreement with the percentages of chickpea enrichment: with raw chickpea flour from 0.71 to 1.25 lg/g, from 0.72 to 1.64 lg/g, from 0.77 to 1.89 lg/g and from 0.83 to 2.16 lg/g for semolina and uncooked pasta, respectively; with toasted chickpea flour: from 0.91 to 1.57 lg/g, from 1.02 to 1.78 lg/g, from 0.91 to 2.0 lg/g and from 2.78 to 2.49 lg/g for semolina and uncooked pasta, respectively. After cooking, both the control and the enriched pasta samples showed reductions in the TPC and TPAC free, which were more evident in the raw-chickpea-enriched pasta. The contribution of the bound fraction to the TPC (Fig. 1b) observed in the enriched semolina samples (708.9, 692.5, 734.9 and 682 lg FAE/g for 5–20% raw chickpea flour, respectively; 658.9, 652, 687.2 and 752 lg FAE/g for 5–20% toasted chickpea flour, respectively), was not different from that in the durum wheat control (664.9 lg FAE/g), as in the chickpea flour most of the antioxidant compounds are concentrated in the free fraction (Han & Baik, 2008). The bound fraction of the TPC increased in the uncooked (767.2 lg FAE/g) and cooked control pasta (996.3 lg FAE/g). A similar trend was seen for the raw chickpea pasta samples, while a non-significant decrease was seen in the toasted chickpea pasta samples (from 1102.2 to 1054 lg FAE/g, from 1111.8 to 1054 lg FAE/g, from 1080.4 to 1050.8 lg FAE/g and from 943.9 to 901.9 lg FAE/g for uncooked and cooked pasta, respectively at 5– 20% chickpea flour). The same trend was seen for the TPAC, with the exception of the control (Table 2), which after showing a decline from the semolina (92.32 lg/g) to the uncooked pasta (71.59 lg/g), showed a sharp increase after the cooking (111.72 lg/g). The explanation of this behaviour can be revealed by observing the compositions of the phenolic acids in the semolina, the uncooked and cooked pasta, and the chickpea flours reported in Table 2. The profile of phenolic acids of the chickpeas (raw and toasted) differed greatly from the semolina and pasta controls, and defined the derived products (enriched pasta samples). The durum wheat semolina had a content of ferulic acid (84.82, 63.97 and 101.74 lg/g for semolina, uncooked and cooked pasta, respectively) that was a lot higher than that in the chickpea flour (0.62 and 0.72 lg/g for raw and toasted, respectively), but had little p-hydroxybenzoic acid (1.33, 2.03 and 2.58 lg/g for semolina, uncooked and cooked pasta, respectively), which was instead, well represented in the chickpea flours (20.97 lg/g for raw), and especially in that toasted (31.38 lg/g). So, the addition of the chickpea flours caused the increase in p-hydroxybenzoic acid, in both the semolina and the uncooked enriched pasta samples, which were greater in samples with the toasted chickpeas. After the cooking, the bound ferulic acid in the control sample (Table 2) was greatly increased (101.74 lg/g), and this trend also affected all of the enriched cooked products. The cooking, however, caused a generalised loss of p-hydroxybenzoic acid in all of the

samples (Table 5), which was stronger in the pasta made with the toasted chickpeas (from 8.88 to 4.40 lg/g, from 12.32 to 5.36 lg/g, from 16.14 to 6.99 lg/g and from 17.15 to 8.69 lg/g for 5–20%, respectively). Regarding the ferulic acid, a significant increase in the cooked pasta was seen for the raw chickpea addition (103.20, 98.95, 93.54 and 91.3 lg/g for 5–20% chickpea flour, respectively), as for the pasta control, while in the toasted-chickpea-enriched pasta samples, no increases were detected and the values were comparable, or slightly lower than, those found in the uncooked samples. The present study also confirmed that the boiling water during the cooking can enhance the extraction of bound phenolics from the food matrix, and can thereby increase the amount of bound phenolics during the chemical extraction and determination. This positive cooking effect was large for the bound ferulic acid of the durum wheat matrix, and it is consistent with a previous study by Fares et al. (2010). In summary, we observed that the presence of durum wheat caused the variability of the bound phenolic acids (ferulic acid), while the chickpea flour determined the variability of the free phenolic acids.

3.4. Antioxidant activity of pasta samples In the cooked pasta (Fig. 2), the antioxidant properties of the free fraction increased proportionally, and significantly, to the addition of the chickpea flours (5–20% raw: 1.40, 1.93, 3.14 and 3.88 lmol Trolox/g; 5–20% toasted: 4.01, 4.11, 4.83 and 5.33 lmol Trolox/g), with respect to the control pasta (2.37 lmol Trolox/g), which was more evident with the toasted chickpea flour. These data are consistent with the composition of the phenolic acids reported in Table 2, where the control pasta had limited amounts of free ferulic acid and p-hydroxybenzoic acid, while the chickpea flours contained large amounts of these. These data are also in agreement with the literature, that have confirmed the prevalence of antioxidant activity in the free fraction in chickpea flour (Han & Baik, 2008). In the opposite way, the antioxidant activity seen in the bound fraction decreased significantly with the addition of the chickpea flour (5–20% raw: 22.01, 21.80, 20.97 and 20.36 lmol Trolox/g; 5–20% toasted: 22.49, 19.39, 19.33 and 18.41 lmol Trolox/g), with respect to the control pasta (25.36 lmol Trolox/g). These data are again consistent with those observed in Tables 2 and 5, and the reductions in the enriched pasta samples were due to the dilution effects of the chickpea additions to the durum wheat semolina, with the consequent reduction in the ferulic acid content. This confirms that the antioxidant activity of the enriched pasta is due to the ferulic acid, that was supplied as ester-linked, to the cell walls (which was not measurable before cooking) of the durum wheat pasta matrix during the cooking, and that despite the heat, preserved its antioxidant properties measured in vitro.

80.34 ± 1.67cd 93.69 i 85.22 ± 1.85bc 108.27 e TPAC: Total phenolic acids content. Different letters in the same row for each sample correspond to significant differences by LSD test (P < 0.05). Values are mean of three determinations ± standard deviation.

81.20 ± 2.65c 97.25 h 95.68 ± 0.95a 118.38 a 74.44 ± 2.72de 88.48 l 86.20 ± 2.44bc 97.68 c 97.13 ± 0.87a 116.17 b 71.45 ± 0.56e 85.48 m 94.52 ± 1.42a 104.13 f 96.00 ± 0.36a 111.87 c 69.64 ± 1.12e 78.31 n

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4. Conclusions

89.62 ± 2.65b 108.93 d

8.69 ± 0.40de 17.15 ± 0.51a 8.88 ± 0.17de

Toasted chickpea p-Hydroxybenzoic acid Ferulic acid TPAC

4.59 ± 1.33g

4.40 ± 0.17g

9.90 ± 1.95d

12.32 ± 0.33c

5.36 ± 0.25fg

6.99 ± 0.06ef 16.14 ± 0.89ab 9.35 ± 0.26de

13.85 ± 0.04bc

91.32 ± 0.31de 111.32d 67.16 ± 2.65i 93.05l 98.95 ± 0.67bc 113.86 b 88.50 ± 0.91de 105.82 f 92.84 ± 2.55ce 102.09 g 86.86 ± 3.01eg 101.03 h 75.92 ± 2.66h 83.59 m

103.20 ± 0.88a 115.39 a

7.91 ± 0.87e 11.43 ± 0.08d 4.37 ± 0.18g 7.91 ± 0.37e

Raw chickpea p-Hydroxybenzoic acid Ferulic acid TPAC

3.03 ± 0.26h

5.22 ± 0.10g

93.54 ± 1.98cd 112.95 c 83.01 ± 3.62fg 105.90 f 94.31 ± 1.23cd 106.72 d

80.82 ± 0.64gh 94.89 i

13.04 ± 0.24c 19.94 ± 0.16a 16.39 ± 0.83b 6.46 ± 0.03f

8.62 ± 0.06e

Semolina Uncooked pasta

11.66 ± 0.06d

20%

Semolina Semolina

Uncooked pasta 10%

Semolina

Cooked pasta 5%

Bound extract (lg/ g dm)

Table 5 Phenolic acids profile (bound extracts) in chickpea-enriched (raw and toasted) semolina and pasta samples.

Cooked pasta

Cooked pasta Uncooked pasta 15%

Uncooked pasta

Cooked pasta

C. Fares, V. Menga / Food Chemistry 131 (2012) 1140–1148

These data indicate that the chickpea toasting treatment induces significant changes in the carbohydrate and antioxidant profiles of the resulting chickpea flours, while enhancing the overall nutritional profile. Our data show that the toasting of the chickpeas further reduces the calorie intake of chickpea flour (see the resistant starch and IDF increases and the reduction in available sugars, which in turn, might be responsible for the reduced effects on the post-prandial blood glucose response), and also improves the nutritional profile (TEAC), and might achieve a wider range of low glycaemic index food for consumers. To our knowledge, this type of information has not been reported to date. When toasted chickpea flours are added to pasta, they enhanced the health properties of the pasta, as shown by the increases in the resistant starch and the TDF. The protein content also increased, while the cooking quality was reduced. For the antioxidant properties (as the TPC, TPAC and TEAC) in the enriched pasta, the main effects were due to the increase in the free phenolics fraction as a result of the addition of the chickpea flours, while the increase observed in the bound fraction, depended on the contribution of the durum wheat semolina. This result is not surprising, and largely reflects the results of a previous study that we carried out (Fares et al., 2010), which showed that the boiling water can enhance the extraction of the bound phenolics from the food matrix, and can thereby increase the amount of the phenolics during the subsequent chemical extraction and determination.

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