Effects of microwave treatment of durum wheat kernels on quality characteristics of flour and pasta

Food Chemistry 283 (2019) 454–461

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Effects of microwave treatment of durum wheat kernels on quality characteristics of flour and pasta

T

Lucia Padalino, Matteo Alessandro Del Nobile , Barbara la Gatta, Mariacinzia Rutigliano, Aldo Di Luccia, Amalia Conte ⁎

University of Foggia, Department of Agricultural Sciences, Food and Environment, Via Napoli – 25, 71122 Foggia, Italy

ARTICLE INFO

ABSTRACT

Keywords: Microwave treatment Gluten network Size exclusion-HPLC and electrophoresis analysis Rheological properties Cooking quality

The influence of microwave treatment of hydrated durum wheat kernels of two different cultivars (cv Aureo and Sfinge), on wholemeal flour and pasta quality was addressed. Size exclusion-HPLC and electrophoresis analysis were used to investigate changes in the gluten network arrangement as affected by the microwave treatment. Rheological properties of dough, cooking quality and sensory properties of pasta were also assessed. Results suggested that the microwave treatment on hydrated durum wheat kernels blocks gluten protein conformation through SeS bonds formation and the free -SH are no longer able to create a strong network during pasta processing, due to the conformational changes. Rheological study of dough confirmed that the modifications induced by microwave treatment greatly affected pasta making characteristics of wheat flour, with significant negative consequences on product quality, especially for pasta cooking quality. Pasta from treated durum wheat showed low sensory quality, mainly due to high bulkiness and adhesiveness.

1. Introduction The gluten proteins contained in wheat flour are crucial during bread and pasta making processes because they confer viscosity and elasticity to the dough. Numerous studies have shown that gluten proteins are also the major causing agents for wheat-dependent immune-mediated disorders. The unique structural features of gluten proteins are long repetitive amino acid sequences rich in glutamine and proline. These sections are involved in most wheat sensitivities, such as as coeliac disease, wheat allergy and non-coeliac gluten sensitivity (Scherf, Koehler, & Wieser, 2016). The cereals generally used by the majority of industrial producers of gluten-free products are rice and maize (Rosell, Barro, Sousa, & Mena, 2014). Nevertheless, these types of flour lack proteins and other important nutrients, such as dietary fibre (Pellegrini & Agostoni, 2015). For these reasons, specific processing techniques and ingredients and/or additives (starches, hydrocolloids and gums) are necessary to improve the textural properties and the nutritional characteristics of gluten-free products (Marti, Seetharaman, & Pagani, 2010; Padalino, Mastromatteo & De Vita, et al., 2013, 2015). Although many advances have been made, most food exhibits low nutritional quality, poor palatability and taste and high cost (Arendt, O’Brien, Schober, Gormley, & Gallagher, 2002). Several studies are currently devoted to preparing pasta and baked

⁎

goods made from modified wheat flours in order to eliminate or reduce the immune toxicity of gluten proteins (detoxification process) (Gianfrani et al., 2007; Rizzello et al., 2007). Recently, a new and innovative method to detoxify gluten proteins from cereal grains has been developed (Lamacchia, Di Luccia, & Gianfrani, 2015). This innovation is usually referred to as “gluten friendly” and relies on the application of microwave treatment to hydrated wheat kernels before milling, reaching a high temperature for a short time and inducing structural changes in gluten proteins (Lamacchia, Landriscina, & D’Agnello, 2016). However, detoxification of gluten by the “gluten friendly” method has been questioned by Gianfrani et al. (2017), who demonstrated that, while the microwave treatment of kernels affected protein conformation with a clear reduction of R5-immunoreactivity, the gluten toxicity persisted after in vitro digestion of flour from treated kernels. These results confirmed the doubts of Leszczynska, Lacka, Szemraj, Lukamowicz, and Zegota (2003) who, investigating the effects of microwave treatment (MWT) on gliadins, suggested that while this treatment lead to conformational and chemical changes in gliadin structure and their immunoreactivity, the microwave heating does not eliminate their allergic properties. Despite these findings, MWT is subject to debate and no information are yet available concerning the technological properties of treated flours for making pasta. In reality, from a biochemical and functional point of view, some

Corresponding author. E-mail address: [email protected] (M.A. Del Nobile).

https://doi.org/10.1016/j.foodchem.2019.01.027 Received 9 October 2017; Received in revised form 21 November 2018; Accepted 3 January 2019 Available online 14 January 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.

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untreated (CTRL) and microwave treated (MWT) samples, were extracted using 10 ml of an extraction buffer containing Tris-HCl 0.0625 mol/L pH 6.8, SDS 2 g/100 g, Glycerol 10 ml/100 ml (v/v) and DTT 1,5 g/100 ml. Samples were stirred with the extraction buffer for 3 h and were then centrifuged at 13000g for 15 min at 25 °C. The supernatants, containing the ‘total proteins’, were carefully removed and stored at −20 °C until the use. Protein fractions (gliadins and glutenins) were extracted according to Wieser and Zimmermann (1998). Protein quantification was determined using the 2D Quant-Kit™ from GE Healthcare, according to supplier’s instructions. Each extract was analyzed in duplicate. To separate the extracted proteins, SDS-PAGE was performed on a 12% gel under reducing conditions with dithiotreitol (DTT) 5 g/100 g (w/v), using a horizontal electrophoresis system Hoefer SE 600, (GE Healthcare, Milan, Italy). SDS-PAGE analysis was carried out at 25 mA for 3 h at room temperature. The gels were stained with 0.25% w/v Coomassie Brilliant Blue (CBB) overnight. Image analysis was carried out using Quantity One software (Bio-Rad, Hercules, CA), calculating the trace quantity (Intensity × mm) of each electrophoretic pattern and indicating the results as percentage values.

changes have been recorded on the properties of the gluten proteins after microwave treatment. Grundas, Warchalewski, Dolińska, and Gralik (2008) and Yalcin, Sakiyan, Sumnu, Celik, and Koksel (2008) found that these changes in protein properties appeared unfavourable to wheat quality, particularly in terms of textural properties of microwave-baked products. These authors also concluded that more detailed studies would be needed to investigate the effects of the microwave treatment on functional properties of wheat gluten. Therefore, the aim of this research was to study the impact of microwave treatment on the structure of the gluten protein network, cooking and sensory quality of durum wheat spaghetti, thus contributing to the discussion, enhancing the knowledge about the protein arrangement for producing pasta Specifically, size exclusion liquid chromatography (SE-HPLC) and electrophoretic analysis (SDS-PAGE) were used to determine the changes on polymeric protein distribution in durum wheat flour and in spaghetti samples, as affected by the microwave treatment. 2. Materials and methods 2.1. Raw materials

2.5. Size exclusion-HPLC analysis

Two cultivars of durum wheat grain (cv Aureo and Sfinge) were provided by the C.R.A. (Agricultural Research Council, Foggia, Italy). The wholemeal flour was obtained with a stone mill (Mod MB250 Partisani).

Proteins from the CTRL and MWT samples (flours and milled spaghetti) were extracted following the two-step extraction procedure (Gupta, Khan, & MacRitchie, 1993). In the first step, the SDS-extractable proteins (proteins soluble in sodium dodecyl sulphate – SDS) were extracted; the second extract contains the SDS-unextractable proteins (proteins soluble only after sonication). SE-HPLC was performed using a liquid chromatograph Agilent 1100 Series system (Santa Clara, CA, USA) equipped with a Phenomenex Biosep SEC-S4000 column (300 × 7.8 mm, Phenomenex, Torrence, CA, USA). Each sample (20 μl) was injected into the column and the eluted proteins were monitored at 214 nm. The mobile phase was 50% acetonitrile containing 0.1% trifluoroacetic acid, with a flow rate of 0.7 ml/min. The SE-HPLC column was calibrated using protein standards with a range of molecular weights (KDa) as follows: Vitamin B12 (1.35), Myoglobin (17.0), ovalbumin (44.0), γ- globulin (158.0) and thyroglobulin (6 7 0). The percentage of unextractable polymeric proteins (UPP) was calculated as described by Kuktaite, Larsson, and Johansson (2004). The percentage of large unextractable polymeric protein (large UPP) was calculated as [peak 1 area (unextractable)/peak 1 area (total)] × 100. Peak 1 (total) refers to the sum of peak 1 (extractable) and peak 1 (unextractable) area. The percentage of total unextractable polymeric protein (total UPP) was calculated as [peak 1 + 2 area (unextractable)/peak 1 + 2 area (total)] × 100. Peak 1 + 2 (total) refers to the sum of the peak 1 + 2 (extractable) and peak 1 + 2 (unextractable) area.

2.2. Microwave treatment Durum wheat kernels underwent microwave treatment according to Gianfrani et al. (2017). Briefly, samples (150 g for each cultivar) were hydrated for 1 h (moisture 19–21%), dried at room temperature with the aid of ventilation (10 min) (moisture 17–20%) to remove the excess of exterior water and then put in a microwave oven (Whirpool, Italy) at 600 W for 5 min (100–110 °C). The humidity of the kernel, before and after the treatment, was measured with a halogen thermal balance MB120 (OHAUS Europe GmbH, Greifensee, Switzerland). After microwave treatment, the wheat kernels (moisture 10.5%) were cooled at ambient temperature for 24 h and then grounded. 2.3. Spaghetti preparation Spaghetti were made using the same operating conditions for both cultivars: wholemeal flour was mixed with water in a rotary shaft mixer (Namad, Rome, Italy) at 25 °C for 20 min so as to obtain a dough with 32 g/100 g moisture content. The dough was extruded with a 60VR extruder (Namad, Rome, Italy). The extrusion pressure was about 3.4 bar, whereas the temperature of the spaghetti after the extrusion was about 27–28 °C. The extruder was equipped with a screw (30 cm in length, 5.5 cm in diameter), which ended with a bronze die (diameter hole of 1.70 mm). The screw speed was 50 rpm. Subsequently, the pasta was dried in a dryer (SG600; Namad). The process conditions applied were the following: in the first step (external drying) the just-extruded spaghetti samples were subjected to drying for 20 min at 60 °C and 65% moisture in order to avoid the so-called “stuck” (the product sticking to itself). The second step (wrapping), carried out for 130 min at 90 °C and 79% moisture, was conducted to give consistency and shape to the dough; at this stage the extraction of moisture must be pushed. The third step (drying), at 150 min at 75 °C and 78% moisture, mainly aimed to remove the water inside the spaghetti. The fourth step for 160 min at 45 °C and 63% moisture and the fifth step for 1040 min at 50 °C and 50% moisture were used for cooling spaghetti (Padalino, Mastromatteo, & Lecce, et al., 2013).

2.6. Determination of crude proteins Nitrogen content of flours, milled spaghetti and residues obtained after the extraction of the unextractable polymeric proteins, for both control and MWT samples, was estimated by Kjeldhal method and converted to protein by using a factor of 5.70. The analyses were carried out by an automatic digestion unit and through an automatic distillation and titration system (VELP Scientifica Srl, Usmate, MonzaBrianza – Italy). Three determinations for each sample were performed. 2.7. Determination of thiol and disulphide groups The protein disulfide and sulphydryl content in the flours and pasta samples was estimated by a colorimetric determination of free SH groups, using a solid phase assay NTSB2−, according to the method of Chan and Wassermann (1993). Three measurements for each sample were performed.

2.4. SDS-PAGE analysis Total proteins from flours and milled spaghetti (1 g), of both 455

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A

B

250

250

150

75

x-

7

y-

8

HMW-GS MW -G

150

HMW-GS MW G

100

x-

6

y-

8

100

75

50

50

37

37

25

25

20

20

S

1

2

3

4

cv Sfinge

5 cv Aureo

6

8

7

9

1

cv Sfinge

CTRL

1

1

S

cv Aureo

1

2

3

4

cv Sfinge

5 cv Aureo

CTRL

MWT

6

7

8

9

1

cv Sfinge

1

1

cv Aureo

MWT

Fig. 1. SDS-PAGE of Flours (A) and Pasta (B) samples. Samples were ordered according to the cultivars (Sfinge and Aureo) and in control (CTRL) and microwave treated (MWT) samples. In the two panels, the lanes 1 and 4 show total proteins of CTRL samples; lanes 2 and 5 gliadins of CTRL samples; lanes 3 and 6 glutenins of CTRL samples; lanes 7 and 10 show total proteins of MWT samples; lanes 8 and 11 gliadins of MWT samples; lanes 9 and 12 glutenins of MWT samples.

2.8. Determination of dough rheological properties

2.10. Sensory analysis

Dough rheological investigations were performed by Mixolab (Chopin, Tripette et Renaud, Paris, France), which simultaneously determinates dough characteristics during process at a constant temperature (30 °C). Required amount of wholemeal flour for analysis was calculated by Mixolab software, according to input values of flour moisture, as well as water absorption. All the measurements were performed using the Mixolab “Chopin S” protocol whose parameters are: mixing speed 80 rpm; dough weight 75 g; temperature 30 °C; total analysis time 30 min. In particular, from the Mixolab curves, water absorption (percentage of water required to yield dough consistency of 1.1 Nm ± 0.05), dough development time (DDT, time to reach maximum consistency), stability (time during dough consistency is at 1.1 Nm) and amplitude (band width of the curve at the maximum consistency) were determined. Three measurements for each sample were performed.

Cooked and uncooked samples were examined by a panel of 15 trained tasters (seven men and eight women, aged between 28 and 45 years) in order to evaluate the sensory attributes. The panellists have several years of experience in sensory evaluation but they were further experienced in the products and terminology (ISO 11036, 7304). For sensory analysis, the panelists were asked to indicate color, homogeneity and resistance to breaking of uncooked spaghetti and elasticity, firmness, bulkiness, adhesiveness, color, odor and taste on cooked samples. For the evaluation, a nine-point scale was adopted, where 1 and 9 represented the lowest and the highest intensity of a particular attribute, respectively. On the basis of the above-mentioned attributes, the same panel evaluated overall acceptability of each pasta sample using a verbal nine-point scale, and the rating was converted to numerical scores, where 1 = dislike extremely, and 9 = like extremely. Pasta products with an overall acceptability mean score above 5 were considered as acceptable.

2.9. Cooking quality

2.11. Statistical analysis

The optimal cooking time (OCT) of pasta and the cooking loss (the amount of solid substance lost in the cooking water), were both evaluated according to the Approved Methods of the American Association of Cereal Chemistry (2007). The swelling index and the water absorption of cooked pasta (grams of water per gram of dry pasta) were determined according to the procedure described by Padalino, Mastromatteo and Lecce et al. (2013). Hardness (mean maximum force, N) and adhesiveness (mean negative area, Nmm) were determined by a Texture Analyzer (Zwick Roell Italia S.r.l., Genova, Italia model Z010) equipped with a stainless-steel cylinder probe (2 cm diameter), according to Padalino, Mastromatteo and Lecce et al. (2013). For each test, three spaghetti strands (40 mm length) cooked at OCT were put side by side on the lower plate, and the superior plate was moved down onto the spaghetti surface. Six measurements for each spaghetti sample were performed. Trial specifications were as follows: preload of 0.3 N; load cell of 1 kN; percentage deformation of 25%; crosshead speed constant of 0.25 mm s−1.

Experimental data on pasta characteristics were compared by oneway analysis of variance (ANOVA). A Duncan’s multiple range test, with the option of homogeneous groups (P < 0.05), was carried out to determine significant differences between samples. STATISTICA 7.1 for Windows (StatSoft, Inc, Tulsa, OK, USA) was used. 3. Results and discussion 3.1. Assessment of protein composition and polymeric protein distribution of flours and pasta The protein composition and the polymeric protein distribution after the microwave treatment were investigated by electrophoretic and chromatographic techniques. The electrophoretic patterns of ‘total proteins’ and protein fractions from flours (Panel A) and pasta (Panel B) 456

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under reducing conditions were shown in Fig. 1. To better understand the differences in gluten protein composition, as a consequence of the microwave treatment, the Figure shows the electrophoretic patterns of the gliadins and glutenins fractions, loaded with the same amount of protein (20 μg). The Fig. 1 (Panel A) shows the electophoretic patterns of control (CTRL) and microwave treated (MWT) flour samples (cv Sfinge and Aureo). This figure highlights the differences in the HMW-GS expression of the two flours, with cv Sfinge appearing to have the GluB1 HMW-GS 7+8 allele, while cv Aureo the Glu-B1 HMW-GS 6+8 allele, according to Visioli et al. (2018). The microwave treatment did not seem to affect the protein composition of either of the flours and pasta samples, although the differences among samples were clear from the image analysis of the electrophoretic patterns. Microwave treatment caused a decrease in the trace quantity of total proteins and gliadins patterns in the MWT flours sample (Fig. 1, Panel A). In the patterns of total proteins this decrease was 4% for cv Sfinge and 20% for cv Aureo, this latter value was also due to a lower number of detected bands in the MWT sample (21 vs 17). For the gliadins patterns the decrease was 15% for both the cultivars. The glutenin fraction did not markedly change, as the trace quantity values showed just a slight increase (2%, cv Sfinge and 5%, cv Aureo). As expected, the image analysis results showed a decrease in the protein solubility for the control samples, passing from flour to the pasta, as a consequence of pasta processing, which led to the formation of large polymeric protein aggregates (Lamacchia et al., 2007; la Gatta, Rutigliano, & Rusco et al., 2017). Differently, protein solubility did not significantly change in the microwave treated pasta samples compared to the respective flours. From the electrophoretic analysis, the difference between the two cultivars (cv Sfinge and Aureo), as related to their technological and pasting properties, were linked to their different expression of HMW-GS (High Molecular Weight – Glutenin Subunit). Samples from cv Aureo showed the typical configurations of Bx6 and By8, which have been suggested to have a correlation with the good technological properties of the flours (Gianibelli, Larroque, MacRitchie & Wrigley, 2001; Visioli et al., 2018), like those also found in our study in the case of cv Aureo. Other studies have dealt with the difference between the allelic configuration of HMW-GS 6+8 and 7+8, confirming the better quality of the 6+8 allelic expression than that of 7+8 (Brites & Carrillo, 2001; De Santis et al., 2017). Based on the electrophoretic pattern analysis, microwave treatment did not affect the gluten protein composition (Yalcin et al., 2008), but it did affect the building of a protein network already in the kernels, which in turn affected the protein solubility and the formation of a proper gluten network during pasta processing. The chromatographic data confirmed the electrophoretic results, indicating once again, that the microwave treatment reduced the solubility of gluten proteins by 52.8% for cv Sfinge and by 61.8% for cv Aureo flours, as indicated in Table 1, by the Δ values. The lower Δ values obtained for the pasta samples (39.1% for cv Sfinge and 47% for cv Aureo) were due to the denaturation of gluten proteins through pasta processing, which in any case reduced their solubility (Delcour et al., 2012). Comparing the total area of the chromatographic peaks, on the extractability index, we observed significant differences (P < 0.01), except for the areas of MWT samples. Actually, there was no statistical difference among MWT flours and the corresponding treated pasta total areas. It was also clear how the microwave treatment nullified the differences, in terms of extractability, between the two MWT flours, unlike the CTRL samples. In the case of CTRL samples, as already shown by the electrophoretic analysis, the extractability of the total proteins decreased with the transformation from flour to pasta (Delcour et al., 2012; la Gatta, Rutigliano, & Rusco, et al., 2017), whereas in the case of MWT samples we noted only a slight and not significant increase. The different arrangement of protein network in the CTRL and MWT pasta samples was assessed by the ratio between the sum of polymeric and monomeric proteins (TPP/TMP). In fact, we observed that the TMP prevailed in the MWT pasta samples, with ratios lower than the respective CTRL pasta samples (0.48 for cv Sfinge and 0.52 for cv Aureo,

compared with 0.92 and 0.85, respectively). This result highlighted how the microwave treatment affected the arrangement of the gluten network and the formation of large polymeric protein. Polymeric protein formation has been described as one of the most important events triggered during industrial pasta making in order to achieve high quality pasta. The formation of a proper gluten network is necessary to have good cooking performance, avoiding the loss of material and the worsening of sensory properties (Bruneel, Pareyt, Brijs, & Delcour, 2010). The temperatures reached in pasta production cause protein disulfide cross-linking by oxidation of glutenin free sulfhydryl groups (Lagrain, Brijs, Veraverbeke, & Delcour, 2005; Lagrain, Thewissen, Brijs, & Delcour, 2008) as well as the formation of other covalent and non-covalent bonds, leading to large protein polymers (Lamacchia et al., 2007; Gerrard, 2002). The degree of polymerization can be measured by the amount of the unextractable protein in dilute sodium dodecyl sulphate (SDS) solution (Kuktaite et al., 2004; Lagrain et al., 2005), and the formation of large and insoluble protein aggregates is correlated to the good technological properties of the pasta product (la Gatta, Rutigliano, & Rusco et al., 2017; Bruneel et al., 2010). 3.2. Total unextractable polymeric protein, large unextractable polymeric protein and content of thiols and disulphide groups in flour and pasta To better understand the differences in the proportion of polymeric protein, which led to a different arrangement of the gluten network, as a consequence of the microwave treatment, we considered two indexes: the percentages of the total unextractable polymeric protein (tUPP) and the large unextractable polymeric protein (lUPP), which were shown in Fig. 2 (panel A, B, C and D) as histograms. As expected, both tUPP and lUPP increased in the transformation from flour to pasta and all the MWT samples exhibited lower values than the CTRL samples. The flour samples (panel A and B) did not exhibit significant differences between tUPP and lUPP percentages, both for CTRL and MWT samples, while the pasta samples (panel C and D) showed significant differences (P < 0.05) only between the lUPP values of CTRL and MWT samples. The MWT samples from cv Aureo showed a higher percentage of tUPP than lUPP and while not statistically different among the flour samples, the difference became significant (P < 0.05) in pasta samples. These two indexes, which were used to better describe the polymeric protein proportion in the control and treated samples, took into account the formation of polymeric aggregates (tUPP) and the weight that the largest polymeric protein aggregates (peak 1) had on the total polymeric protein (lUPP). The formation of large protein aggregates is an event promoted by the processing, which was evaluated by large unextractable polymeric protein index (lUPP), that, in turn, was correlated to the good technological performance of the pasta (la Gatta, Rutigliano, & Padalino, et al., 2017). From our results, it was evident how the processing lead to the formation of large unextractable polymeric aggregates, the values of lUPP, for both the cultivars, being higher than tUPP and respective flours. The formation of gluten network is the result of the self-assembling machinery of gluten proteins (Kuktaite et al., 2011) and of the re-assembling of them, as a consequence of processing, following a hierarchical organization of the supramolecular structure (la Gatta et al., 2017a), through the promotion of covalent and non-covalent bonds. The application of microwave treatment on hydrated kernels changed this mechanism by modifying protein conformation, thereby blocking gluten proteins in a pre-arranged structure, which was not able to be re-assembled into a larger structure, being in a cross-linked conformation and entailing no difference in the extractability between flours and pasta. Actually, tUPP and lUPP values increased in the passage from MWT flours to MWT pasta samples, but their values were lower than those of CTRL samples, suggesting the formation of aggregates which, probably, involved the smaller polymeric proteins, and eventually, the monomeric proteins, as also suggested by the TPP/TMP ratio (Table 1). 457

Food Chemistry 283 (2019) 454–461

1.23 0.48

100180.02 ± 3279.2b,d 100%

0.71

164470.65 ± 11553.4c 100% 64290.63 (39.09%) 0.92

24506.00 ± 2462.46 24.46% 57904.8 ± 1268.27 57.80% 18680.42 ± 2229.3711.36% 9712.82 ± 644.799.70%

Δ TPP/TMP

mAu⋅s Total area

mAu⋅s SMP

mAu⋅s LMP

mAu⋅s SPP

LPP: large polymeric proteins; SPP: small polymeric proteins; LMP: large monomeric proteins; SMP: small monomeric proteins; TPP: total polymeric proteins (LPP + SPP); TMP: total monomeric proteins (LMP + SMP). a-d Means in the same row followed by different superscript letters differ significantly (P < 0.01) referred to Sfinge samples (SF). A-D Means in the same row followed by different superscript letters differ significantly (P < 0.01) referred to Aureo samples (AU). Δ: referred to the difference between total area of CTRL and MWT samples, expressed both in absolute value and in percentage.

0.52

109472.5 ± 3257.5B,D 100%

10493.37 ± 1843.459.59%

61435.02 ± 1705.8856.12%

9897.27 ± 1352.66 9.04% 27646.82 ± 1660.4825.25%

25406.72 ± 2975.74 12.30% 69516.32 ± 1908.16 33.64% 85938.02 ± 1578.56 41.59% 25770.77 ± 3865.34 12.47% 206631.85 ± 8697.9C 100% 97159.35 (47%) 0.85 22984.17 ± 3579.09 23.46% 31095.77 ± 538.41 31.74% 37910.97 ± 2235.82 38.70% 5980.47 ± 939.56 6.10% 97971.03 ± 5912.2B 100%

71276.23 ± 2800.79 27.80% 74772.23 ± 5049.05 29.17% 85046.77 ± 2070.85 33.17% 25275.80 ± 2404.04 9.86% 256371.03 ± 3371.2A 100% 158400.00 (61.79%) 1.32 8055.47 ± 1377.11 8.04%

23308.7 ± 3256.33 14.17% 55675.12 ± 2027.30 33.85% 66.806.4 ± 4304.90 40.62%

14010.60 ± 400.67 14.32% 26735.90 ± 658,83 27.33% 50151.97 ± 1035.02 51.28% 6910.77 ± 911.03 7.07% 97809.23 ± 776.9b 100%

60557.37 ± 3429.45 29.21% 49747.77 ± 2096,07 23.99% 74473.73 ± 4325,21 35.92% 22568.63 ± 920.58 10.88% 207347.50 ± 6447.9a 100% 109538.27 (52.83%) 1.14 mAu⋅s LPP

SF-MWT SF-CTRL

Table 1 Total area of the SE-HPLC peaks of flour and pasta samples.

Pasta SF-CTRL

Pasta SF-MWT

AU-CTRL

AU-MWT

Pasta AU-CTRL

Pasta AU-MWT

L. Padalino et al.

In order to verify the influence of -SH/-SS- interchange reactions, we determined the content of free thiols and disulfide groups (nmol/mg prot), as well as SS/SH ratio, as reported in Table 2. Control flour samples showed a similar content in titrable free thiols and a high content of disulfide groups. In the case of CTRL pasta samples of cv Aureo, we observed the expected SH → SS interchange reaction, passing from flour to pasta (Delcour et al., 2012), with the increase in the ratio SS/SH (from 2.23 to 2.48). Surprisingly, in the case of CTRL pasta sample of cv Sfinge, we observed a more consistent increase in the titrable free thiols than in disulfide groups, that was translated into a lower-SS/-SH-ratio (2.61 and 1.57 respectively), suggesting a not proper formation of the gluten network, which was also confirmed by the worse technological properties of this pasta as compared with that obtained from cv Aureo, according to results on dough properties, cooking quality and sensory properties. As concerns MWT samples, the disulphide groups content was not statistically different in the passage from flour to pasta, unlike the free thiols content (P < 0.05), resulting in a decrease of the ratio SS/SH, (from 1.72 to 1.38 for MWT samples from cv Sfinge and from 1.97 to 1.55 for MWT samples from cv Aureo). These results were due to the extrusion conditions (Mei & Lee, 1997) and confirmed that in the MWT sample the microwave treatment, giving rise to protein conformational changes (Gianfrani et al., 2017), blocked gluten proteins through an earlier SeS bond formation, thus leading the free -SH to be no more able to create a strong network during pasta processing. From a technological point of view, the microwave treatment transformed gluten macromolecules from a thermoplastic polymer, which can be shaped under appropriate conditions, to a cross-linked polymer, which can be no longer be shaped into new conformations. Finally, we explored the potential presence of an insoluble protein residue with an insoluble in the structure pellets obtained after the extraction of the unextractable polymeric protein fractions, through the application of the Kjeldahl method. A protein residue was found and quantified as reported in Table S1 (Supplementary material). Two observations were highlighted: the former was the weight of the residue in MWT samples, which resulted higher than 2 g (the amount initially weighed), and probably, this was ascribable to the starch gelatinization during the microwave treatment; the latter was the significant higher percentage (P < 0.05) of unextracted crude proteins (%UCP) in MWT as compared with CTRL samples, with the highest value obtained in the pasta from cv Aureo (Table 2). All these results are summarized in Fig. S1 (Supplementary material), where the total area reported in Table 1 was correlated to the unextracted crude protein percentages (%UCP) (Table S1, Supplementary material). The CTRL samples showed that cv Aureo reached higher values than cv Sfinge, both for the total area and %UCP. It was interesting to note that passing from flour to pasta the extractability of the Sfinge and Aureo samples decreased in terms of total area and increased in terms of %UCP. This highlighted the formation of large insoluble protein aggregates, as a consequence of pasta processing (la Gatta et al., 2017; Lamacchia et al., 2007). In the case of microwave treated samples, we found no differences between the two flours, both for the total area values and %UCP. No significant changes were observed in the passage from flours to pasta for total area values and % UCP. However, a slight increase in the percentage of unextracted crude proteins was observed only for cv Aureo, due to the intense conformational modifications that make proteins insoluble. This finding would justify the lower value of lUPP in the MWT pasta sample from cv Aureo compared to cv Sfinge. 3.3. Dough rheological properties and pasta cooking quality Results from the rheological test highlighted that the microwave treatment of hydrated kernels greatly affected properties of both wholemeal flours. Water absorption, dough development time, stability, softening and amplitude derived from Mixolab are represented in 458

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Fig. 2. Total Un-extractable Polymeric Proteins (tUPP□) and Large Un-extractable Polymeric Proteins (lUPP□) in flour (Top) and pasta samples (bottom).

Table 3. These results are all consistent with the hypothesis that microwave treatment freezing gluten macromolecules in its native form, impedes the formation of a strong gluten network. Indeed, the water absorbed in the case of treated sample is higher than the CTRL. This is because the formation of a strong gluten network reduces starch granule hydration, thus reducing the amount of absorbed water. Data listed in Table 3 also show that the dough development time is lower in the case of treated samples, whereas the dough stability time for the same sample is shorter. Indeed, a strong gluten network takes more time to develop and it remains stable for longer time. Similar results were also found by Susanna and Prabhasankar (2011) who stated that microwave treatment caused a decline of semolina rheological properties, making it unsuitable for wheat-based products. Rheological studies also suggest that modifications induced by microwave treatment greatly affect pasting characteristics of wheat flour, as better explained in the subsequent paragraph dealing with pasta quality. Data on the cooking quality of spaghetti samples are also shown in

Table 3. As one can be inferred from data, CTRL samples from cv Aureo recorded slightly better values if compared to CTRL spaghetti from cv Sfinge, even though the differences are not statistically significant. Concerning the MWT and the corresponding control samples, data suggest that the microwave treatment reduced pasta cooking quality. This is true for both cultivars. Indeed, the cooking losses of treated samples are higher than that of the CTRL samples. This is consistent with the fact that in the case of MWT samples weaker gluten network is formed. Indeed, the formation of weaker gluten network facilitates the diffusion of amylose towards the spaghetti surface and then in the boiling water, thus increasing the cooking loss. The swelling index and the water absorption values recorded for the MWT samples are higher than those of the corresponding controls. These data also agree with the fact that a weaker gluten network is formed when the hydrated kernels are microwave-treated prior to the milling process. A weaker gluten network allows starch granules to expand and absorb more water. As one would expect, the value of adhesiveness is strictly correlated to

Table 2 Effect of MWT and pasta processing on contents in free thiol and disulfide groups. Flour SF-CTRL Free Thiol (μmol/mg prot) Disulfide group (μmol/mg prot) Disulfide/Free Thiol

a

Flour SF-MWT b

Pasta SF-CTRL b,c

36.92 ± 6.50 64.12 ± 9.96 65.42 ± 6.59 96.22 ± 4.65a 110.38 ± 6.97b 102.70 ± 3.24c 2.61 1.72 1.57

Flour SF-MWT d

Flour AU-MWT Flour AU-CTRL Pasta AU-MWT A

B

Pasta AU-MWT

79.31 ± 8.13 39.80 ± 7.29 49.02 ± 6.05 39.43 ± 8.44 62.83 ± 4.88C 109.72 ± 7.86b,d 88.87 ± 1.98A 96.94 ± 7.38B 97.74 ± 7.95B,C 97.29 ± 7.68B,C,D 1.38 2.23 1.97 2.48 1.55

Values are micromoles per milligram of sample. a–d Means in the same row followed by different superscript letters differ significantly (P < 0.05).-referred to Sfinge samples (SF). A–D Means in the same row followed by different superscript letters differ significantly (P < 0.05).-referred to Aureo samples (AU).

459

A,B

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Table 3 Dough rheological properties and pasta cooking quality. Dough SF-CTRL Water Absorption (%) Dough Development time (min) Stability (min) Amplitude (Nm) Cooking Loss (%) Swelling Index (g water per g dry Spaghetti) Water Absorption (%) Adhesiveness (Nmm) Hardness (N) a-b

65.9 1.53 6.57 0.10 – – – – –

± ± ± ±

a

0.02 0.03a 0.02a 0.00a

Dough SF-MWT 63.2 1.05 4.00 0.09 – –

± ± ± ±

b

Dough AU-CTRL a

0.01 0.02b 0.04b 0.01a

– – –

Dough AU-MWT

72.2 ± 0.02 1.60 ± 0.05a 13.96 ± 0.06a 0.11 ± 0.01a – –

77.3 1.35 6.97 0.09 – –

– – –

– – –

± ± ± ±

b

0.01 0.06b 0.05b 0.00a

Pasta SF-CTRL

Pasta SF-MWT

Pasta AU-CTRL

Pasta AU-MWT

– – – – 6.55 ± 0.02b 1.80 ± 0.06b

– – – – 7.78 ± 0.36a 1.93 ± 0.04a

– – – – 5.46 ± 0.47b 1.69 ± 0.02b

– – – – 8.43 ± 0.03a 1.84 ± 0.02a

136 ± 0.62b 0.75 ± 0.02b 6.40 ± 0.37a

144 ± 0.70a 1.25 ± 0.04a 5.13 ± 0.20b

126 ± 3.11b 0.64 ± 0.04b 8.73 ± 0.45a

138 ± 1.57a 1.04 ± 0.05a 6.51 ± 0.56b

Means in the same row followed by different superscript letters differ significantly (P < 0.05). Mean ± standard error for three replicates.

cooking loss. In the case of the hardness, the MWT samples showed lower values if compared to CTRL samples. In this case, the weak gluten network is the main responsible factor for a less cohesive structure. Indeed, looser gluten promotes an increase of water absorbed during cooking, which in turn reduces the hardness values. All the evidences reported beforehand are consistent with the hypothesis that microwave treatment compromises the gluten network, which in turn is the main cause of the cooking quality worsening. As reported before, the microwave treatment freezes the gluten macromolecules in the conformation they have in the kernels (gluten denaturation). Therefore, denatured gluten macromolecules can no longer form a strong gluten network during dough-making (i.e., the extrusion step). Generally, protein denaturation is promoted after the gluten network is formed; generally, high drying temperatures are used to promote SeS bond formation, which in turn makes the gluten network stronger. If gluten macromolecules are cross-linked via SeS bond formation before the gluten network is formed, gluten can no longer form a strong network.

resistance to break attributes. Regarding this latter one, it is strictly related to the formation of weak gluten network in the MWT samples. Concerning the change in color, this may be related to the high temperature reached during the microwave treatment. The spaghetti samples from treated wholemeal flour showed color worsening (bright brown) compared to the intense brown color of the CTRL samples. Regarding sensory quality of cooked spaghetti, data in terms of elasticity, firmness, adhesiveness and bulkiness show that the CTRL samples from cv Aureo are slightly better than CTRL pasta from cv Sfinge, even though the differences are not statistically significant. Furthermore, these experimental findings are in agreement with previous reports on spaghetti cooking quality and wholemeal flour quality. As regards the differences between treated and control samples, there is always a strong worsening of the sensory attributes of the MWT samples. It can be related to the fact that microwave treatment, freezing the gluten macromolecules in the conformation they do have in the seed via SeS bond formation, does not allow the formation of a strong gluten network, which in turn causes a reduction in spaghetti structure. In the case of the worsening of the attributes of odor, color and taste, they may be related to the high temperature experienced by hydrated seeds during the microwave treatment. While semolina and pasta are generally not considered for their aromatic properties, some data suggest that volatile compounds are present in the cereal flour and have an effect on product aroma (Bredie et al., 2002; Parker, Hassell, Mottram, & Guy, 2000). In summary, as consequence of microwave treatment, the overall quality of treated samples is much lower than that of the corresponding control pasta. It is also worth noting that its value is much lower than the threshold (score = 5), accounting for a score of about 3, which makes the treated samples unsuitable for commercial distribution.

3.4. Pasta sensory quality Sensory data obtained on MWT and corresponding CTRL samples of both cultivars are listed in Table 4. Concerning the uncooked spaghetti, overall quality data show that the CTRL from cv Aureo is slightly better than CTRL from cv Sfinge; however, the differences between these two samples are not statistically significant. The above-mentioned differences are due to slightly better color and resistance to breaking. These data are in agreement with what reported beforehand on spaghetti cooking quality and wholemeal flour quality. Concerning the differences between control and treated samples of both cultivars investigated in this work a worsening overall quality of uncooked spaghetti was recorded. This is due to a low score of both color and Table 4 Sensory properties of dried uncooked and cooked spaghetti samples. Uncooked Spaghetti

SF-CTRL

SF-MWT a

Color Break to resistance Overall Quality

6.75 ± 0.25 6.50 ± 0.20a 6.75 ± 0.27a

Cooked Spaghetti Elasticity Firmness Adhesiveness Bulkiness Color Odor Taste Overall Quality

5.75 6.50 6.50 6.75 6.50 7.25 7.25 6.50

a-b

± ± ± ± ± ± ± ±

0.25a 0.20a 0.27a 0.25a 0.27a 0.30a 0.25a 0.20a

AU-CTRL b

6.00 ± 0.20 4.75 ± 0.30b 5.00 ± 0.25b 3.05 4.05 3.25 3.00 4.20 3.30 3.25 3.00

± ± ± ± ± ± ± ±

0.35b 0.27b 0.32b 0.25b 0.20b 0.27b 0.25b 0.25b

7.25 ± 0.25 6.75 ± 0.20a 7.00 ± 0.27a 6.00 7.00 7.00 6.75 7.20 7.25 7.25 7.00

Means in the same row followed by different superscript letters differ significantly (P < 0.05).

460

AU-MWT a

± ± ± ± ± ± ± ±

0.25a 0.20a 0.27a 0.25a 0.27a 0.30a 0.25a 0.25a

6.50 ± 0.20b 5.02 ± 0.30b 5.00 ± 0.25b 3.25 4.25 3.00 3.20 4.50 3.25 3.50 3.25

± ± ± ± ± ± ± ±

0.25b 0.27b 0.25b 0.25b 0.20b 0.25b 0.27b 0.25b

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4. Conclusions

bread wheats. I. Effects of variation in the quantity and size distribution of polymeric protein. Journal of Cereal Science, 18, 23–41. Kuktaite, R., Larsson, H., & Johansson, E. (2004). Variation in protein composition of wheat flour and its relationship to dough mixing behaviour. Journal of Cereal Science, 40, 31–39. Kuktaite, R., Plivelic, T. S., Cerenius, Y., Hedenqvist, M. S., Gällstedt, M., Marttila, S., et al. (2011). Structure and morphology of wheat gluten films: From polymeric protein aggregates towards superstructure arrangements. Biomacromolecules, 12, 1438–1448. la Gatta, B., Rutigliano, M., Rusco, G., Petrella, G., & Di Luccia, A. (2017). Evidence for different supramolecular arrangements in pasta from durum wheat (Triticum durum) and einkorn (Triticum monococcum) flours. Journal of Cereal Science, 73, 76–83. la Gatta, B., Rutigliano, M., Padalino, L., Conte, A., Del Nobile, M. A., & Di Luccia, A. (2017). The role of hydration on the cooking quality of bran-enriched pasta. LWT – Food science and Technology, 84, 489–496. Lagrain, B., Brijs, K., Veraverbeke, W. S., & Delcour, J. A. (2005). The impact of heating and cooling on the physico-chemical properties of wheat gluten–water suspensions. Journal of Cereal Science, 42, 327–333. Lagrain, B., Thewissen, B. G., Brijs, K., & Delcour, J. A. (2008). Mechanism of gliadin–glutenin cross-linking during hydrothermal treatment. Food Chemistry, 107, 753–760. Lamacchia, C., Di Luccia, A., Baiano, A., Gambacorta, G., la Gatta, B., Pati, S., et al. (2007). Changes in pasta proteins induced by drying cycles and their relationship to cooking behaviour. Journal of Cereal Science, 46, 58–63. Lamacchia, C., Di Luccia, A., & Gianfrani, C. (2015). Method for the detoxification of gluten proteins from grains of cereals. Patent Cooperation Treaty. PCT/IB2013/ 000797. Lamacchia, C., Landriscina, L., & D’Agnello, P. (2016). Changes in wheat kernels proteins induced by microwave treatment. Food Chemistry, 197, 634–640. Leszczynska, J., Lacka, A., Szemraj, J., Lukamowicz, J., & Zegota, H. (2003). The effect of microwave treatment on the immunoreactivity of gliadin and wheat flour. European Food Research and Technology, 217, 387–391. Marti, A., Seetharaman, K., & Pagani, M. A. (2010). Rice-based pasta: A comparison between conventional pasta-making and extrusion-cooking. Journal of Cereal Science, 52, 404–409. Mei, L., & Lee, T. C. (1997). Relationship of the extrusion temperature and the solubility and disulfide bond distribution of wheat proteins. Journal of Agricultural and Food Chemistry, 45, 2711–2717. Padalino, L., Mastromatteo, M., De Vita, P., Ficco, D. B. M., & Del Nobile, M. A. (2013). Effects of hydrocolloids on chemical properties and cooking quality of gluten-free spaghetti. International Journal of Food Science and Technology, 48, 972–983. Padalino, L., Mastromatteo, M., Lecce, L., Spinelli, S., Contò, F., & Del Nobile, M. A. (2013). Chemical composition, sensory and cooking quality evaluation of durum wheat spaghetti enriched with pea flour. International Journal of Food Science and Technology, 49, 1544–1556. Padalino, L., Mastromatteo, M., Lecce, L., Spinelli, S., Conte, A., & Del Nobile, M. A. (2015). Optimization and characterization of gluten-free spaghetti enriched with chickpea flour. International Journal of Food Science and Nutrition, 66, 148–158. Parker, J. K., Hassell, G. M. E., Mottram, D. S., & Guy, R. C. E. (2000). Sensory and instrumental analysis of volatiles generated during extrusion cooking of oat flours. Journal of Agricultural and Food Chemistry, 48, 3497–3505. Pellegrini, N., & Agostoni, C. (2015). Nutritional aspects of gluten-free products. Journal of Scence Food Agriculture, 95, 2380–2385. Rizzello, C. G., de Angelis, M., di Cagno, R., Camarca, A., Silano, M., Losito, I., et al. (2007). Highly efficient gluten degradation by lactobacilli and fungal proteases during food processing: New perspectives for Celiac disease. Applied and Environmental Microbiology, 73, 4499–4507. Rosell, C. M., Barro, F., Sousa, C., & Mena, M. C. (2014). Cereals for developing glutenfree products and analytical tools for gluten detection. Journal of Cereal Science, 59, 354–364. Scherf, K. A., Koehler, P., & Wieser, H. (2016). Gluten and wheat sensitivities. An overview. Journal of Cereal Science, 67, 2–11. Susanna, S., & Prabhasankar, P. (2011). A comparative study of different bio-processing methods for reduction in wheat flour allergens. European Food Research Technology, 233, 999–1006. Visioli, G., Bonas, U., Dal Cortivo, C., Pasini, G., Marmiroli, N., Mosca, G., et al. (2018). Variations in yield and gluten proteins in durum wheat varieties under late-season foliar versus soil application of nitrogen fertilizer in a northern Mediterranean environment. Journal of the Science of Food and Agriculture, 98, 2360–2369. Wieser, H., & Zimmermann, G. (2000). Importance of amounts and proportions of high molecular weight subunits of glutenin for wheat quality. European Food Research and Technology, 210, 324–330. Yalcin, E., Sakiyan, O., Sumnu, G., Celik, S., & Koksel, H. (2008). Functional properties of microwave-treated wheat gluten. European Food Research and Technology, 227, 1411–1417.

The results indicated that the microwave treatment affected the distribution of polymeric proteins and the formation of a proper gluten network, regardless of the functional properties of the cultivars used (cv Sfinge and Aureo), involving gluten proteins denaturation, mainly gliadins, in treated samples. Microwave treatment entailed the lock of the gluten proteins through an earlier SeS bond formation, the extrusion conditions caused the exposure of further free sulfhydryl groups and hence a not proper protein network organization during pasta processing was achieved. In addition, rheological study suggested that the modifications induced by microwave treatment greatly affected pasting characteristics of wheat flour. In reality, the MWT samples recorded considerable cooking loss, swelling index and water absorption when compared to the CTRL samples. With respect to the textural properties, treated samples were more adhesive and less hard, when compared to the untreated ones. Regarding the sensory quality there was a clear worsening of all the evaluated attributes when the kernels were microwave-treated. The overall quality of the MWT samples was much lower than the threshold, above all for elasticity, firmness, bulkiness and adhesiveness, making the pasta completely un-acceptable. Conflict of interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.01.027. References Approved Methods of the American Association of Cereal Chemistry (2007). Methods 6650. Saint Paul, MN. Arendt, E. K., O’Brien, C. M., Schober, T., Gormley, T. R., & Gallagher, E. (2002). Development of gluten-free cereal products. Farm Food, 12, 21–27. Bredie, W. L. P., Mottram, D. S., & Guy, R. C. E. (2002). Effect of temperature and pH on the generation of flavor volatiles in extrusion cooking of wheat flour. Journal of Agricultural and Food Chemistry, 50, 1118–1125. Brites, C., & Carrillo, J. M. (2001). Influence of high molecular weight (HMW) and low molecular weight (LMW) glutenin subunits controlled by Glu-1 and Glu-3 loci on durum wheat quality. Cereal Chemstry, 78, 59–63. Bruneel, C., Pareyt, B., Brijs, K., & Delcour, J. A. (2010). The impact of the protein network on the pasting and cooking properties of dry pasta products. Food Chemistry, 120, 371–378. Chan, K. Y., & Wassermann, B. P. (1993). Direct colorimetric assay of free thiol groups and disulfide bonds in suspensions of solubilized and particulate cereal proteins. Cereal Chemistry, 70, 22–26. De Santis, M. A., Giuliani, M. M., Giuzio, L., De Vita, P., Lovegrove, A., Shewry, P. R., & Flagella, Z. (2017). Differences in gluten protein composition between old and modern durum wheat genotypes in relation to 20th century breeding in Italy. European Journal of Agronomy, 87, 19–29. Delcour, J. A., Joye, I. J., Pareyt, B., Wilderjans, E., Brijs, K., & Lagrain, B. (2012). Wheat gluten functionality as a quality determinant in cereal-based food products. Annual Review of Food Science and Technology, 3, 469–492. Gerrard, J. A. (2002). Protein–protein crosslinking in food: Methods, consequences, applications. Trends in Food Science & Technology, 13, 391–399. Gianfrani, C., Mamone, G., la Gatta, B., Camarca, A., Di Stasio, L., Maurano, F., et al. (2017). Microwave-based treatments of wheat kernels do not abolish gluten epitopes implicated in celiac disease. Food and Chemical Toxicology, 101, 105–113. Gianfrani, C., Siciliano, R. A., Facchiano, A. M., Camarca, A., Mazzeo, M. F., Costantini, S., et al. (2007). Transamidation inhibits the intestinal immune response to gliadin in vitro. Gastroenterology, 133, 780–789. Gianibelli, M. C., Larroque, O. R., MacRitchie, F., & Wrigley, W. (2001). Biochemical, genetic, and molecular characterization of wheat glutenin and its component subunits. Cereal Chemistry, 78, 635–646. Grundas, S., Warchalewski, J. R., Dolińska, R., & Gralik, J. (2008). Influence of microwave heating on some physicochemical properties of wheat grain harvested in three consecutive years. Cereal Chemistry, 85, 224–229. Gupta, R. B., Khan, K., & MacRitchie, F. (1993). Biochemical basis of flour properties in

Further reading ISO International Organization for Standardization (2007). Sensory Analysis – General Guidance for the Design of Test Rooms. ISO 8589:2007, Geneva, Switzerland. Linsday, M. P., & Skerritt, J. H. (1999). The glutenin macropolymer of wheat flour dough: Structure-function perspectives. Trends in Food Science and Technology, 10, 247–253.

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