Antioxidant Properties of Experimental Wholegrain Pastas Made With Different Cereals

Pasta represents an identifying ingredient of traditional healthy dishes, in particular in Mediterranean areas. Traditional pasta is made with durum wheat semolina only. However, pasta manufactured with different cereals has become available on the market and its consumption is rapidly increasing. Five experimental dry pastas, manufactured with the same process by adding 60% wholegrain flours of different cereals (wheat, oat, rye, barley and rice) to the same durum wheat semolina, were supplied by Pavan s.p.a. (Galliera Veneta, PD, Italy). For each product, aqueous-organic extracts and their residues were studied. Their antioxidant properties were evaluated by FRAP (Ferric Reducing Antioxidant Power) assay and Total Polyphenols Content (TPC) was determined by the Folin Ciocalteau method. For cooked experimental wholegrain pastas made with different cereals, FRAP values ranged from 3.26 ± 0.08 μmol/g d.w. to 19.52 ± 1.28 μmol/g d.w. in aqueous-organic extracts and from 17.91 ± 2.83 μmol/g d.w. to 87.83 ± 5.06 μmol/g d.w. in residues. In both raw and cooked products the lowest FRAP values were found for semolina/60% whole rice pasta. The barley pasta has interesting antioxidant properties and this result was matched by the TPC one. Practical Application The interest in more nutritious and potentially functional foods has prompted the cereal industry towards new formulations of wholegrain products. The functional properties of minor cereals have been poorly investigated. Our results show that wholegrain pastas made with different cereals are rich in antioxidants, in particular whole barley based pasta, providing a scientific basis for the development of grain based functional foods. Then the study of free and bond bioactive compounds could be crucial to describe the potential of grains, with the aim of better understanding potential health benefits of wholegrain based food consumption.

Five experimental dry pastas, manufactured with the same process by adding 60% wholegrain flours of different cereals (wheat, oat, rye, barley and rice) to the same durum wheat semolina, were supplied by Pavan s.p.a. (Galliera Veneta, PD, Italy).
For each product, aqueous-organic extracts and their residues were studied. Their antioxidant properties were evaluated by FRAP (Ferric Reducing Antioxidant Power) assay and Total Polyphenols Content (TPC) was determined by the Folin Ciocalteau method.
For cooked experimental wholegrain pastas made with different cereals, FRAP values ranged from 3.26 ± 0.08 µmol/g d.w. to 19.52 ± 1.28 µmol/g d.w. in aqueous-organic extracts and from 17.91 ± 2.83 µmol/g d.w. to 87.83 ± 5.06 µmol/g d.w. in residues. In both raw and cooked products the lowest FRAP values were found for semolina/60% whole rice pasta.
The barley pasta has interesting antioxidant properties and this result was matched by the TPC one.

Practical Application
The interest in more nutritious and potentially functional foods has prompted the cereal industry towards new formulations of wholegrain products. The functional properties of minor cereals have been poorly investigated. Our results show that wholegrain pastas made with different cereals are rich in antioxidants, in particular whole barley based pasta, providing a scientific basis for the development of grain based functional foods.

Introduction
Pasta represents an identifying ingredient of traditional healthy dishes, in particular in Mediterranean areas (Krishnan & Prabhasankar, 2012).
A lot of scientific evidences have recognized the pasta's healthy place in the diet for its nutritional and beneficial properties (Augustin et al., 2001;Wolever et al., 2008).
In this work aqueous-organic extracts (extractable polyphenols) and potentially bioactive polyphenols that remain in the residues (non-extractable polyphenols) of experimental wholegrain pastas made with different cereals were investigated in the raw and in the cooked state.

Samples and Sample Preparation
Five experimental dry pastas, manufactured with the same process by adding 60% wholegrain flours of different cereals (wheat, oat, rye, barley and rice) to the same durum wheat semolina, were supplied by Pavan s.p.a. (Galliera Veneta, PD, Italy).
Preliminary tests were carried out by Pavan for determining the maximum percentage of non-semolina flour to use in the manufacturing of acceptable pasta as well as the best shape. All Pavan's pastas had the same shape (macaroni), average thickness (between 1.14 and 1.22 mm), optimum cooking time (between 8 and 9 min).
Raw pasta products were ground in a refrigerated mill (Janke and Kunkel, Ika Labortechnik, Germany) and the flours were sieved until a granulometry of 0.5 mm.
Wholegrain pastas were cooked following the manufacturer's directions in unsalted and distilled water, frozen, lyophilized and milled.

Chemicals and Reagents
All reagents and solvents used were of analytical or HPLC grade; all chemicals were purchased from Sigma-Aldrich Srl (Milan, Italy), Fluka Chemicals (Madrid, Spain), Carlo Erba (Milan, Italy). Double-distilled water (Millipore, Milan, Italy) was used throughout the study.

Sample Extraction
Extractable polyphenols (aqueous-organic extracts) and non-extractable polyphenols (residues) were extracted, isolated and studied as described by Durazzo et al. (2012). In particular in residues, among non-extractable polyphenols, hydrolysable polyphenols (comprising hydrolysable tannins, phenolic acids and hydroxycinnamic acids that are released from the food matrix by strong acidic hydrolysis) were determined. The antioxidant properties and the Total Polyphenol Content (TPC) were determined in both aqueous-organic extracts and their residues.

Determination of Total Polyphenol Content (TPC)
The Total Polyphenol Content (TPC) was determined using the Folin-Ciocalteau method previously described by Singleton, Orthofer, and Lamuela-Raventos, 1999. Briefly, the appropriate dilutions of extracts were oxidized with Folin-Ciocalteau reagent, and the reaction mixture was neutralized with sodium carbonate. The absorbance of the resulting blue color was measured at 760 nm against a blank after 2h of reaction at room temperature. Gallic acid was used as the standard.

Evaluation of Antioxidant Properties
Antioxidant properties have been determined by the FRAP (Ferric Reducing Antioxidant Power) assay. The www.ccsenet.org/jfr Journal of Food Research Vol. 3, No. 4;2014 method was performed as described by Benzie and Strain (1996) and Pulido, Bravo, and Saura-Calixto, (2000) using a Tecan Sunrise® plate reader spectrophotometer. The method is based on the reduction of the yellow Fe 3+ -TPTZ (2,4,6-tripyridyl-s-triazine) complex to the blue ferrous complex at low pH. The absorbance was recorded at 595 nm after 30 min incubation at 37 °C.

Statistical Analysis
The data presented are the means of three separate extractions and determinations. Results were expressed as mean ± standard deviation (SD). Differences among groups using a significance level of P < 0.05 were performed by one-way analysis of variance (ANOVA) using the Statistica for Windows statistical package (release 4.5; StatSoft Inc., Vigonza PD, Italy). Significant differences between raw and cooked products have been evaluated by Student t test.

Results
Figures 1A-1B show TPC values (mg/100g d.w.) and FRAP values (µmol/g d.w.) in aqueous-organic extracts and residues of raw, experimental, wholegrain pastas made with different cereals. Figure 1A. TPC values in raw, experimental, wholegrain pastas made with different cereals * *mean ± S.D.; Anova, Tukey HSD Test; for each fraction, means followed by different letters are significantly different (P < 0.05). Figure 1B. FRAP values in raw, experimental, wholegrain pastas made with different cereals * *mean ± S.D.; Anova, Tukey HSD Test; for each fraction, means followed by different letters are significantly different (P < 0.05).  (2011) have obtained spaghetti enriched in bioactive compounds using barley coarse fraction as an ingredient.
In addition it is interesting to underline that in all selected products the hydrolysable polyphenols represent a quantitatively important fraction of polyphenols: the hydrolysable polyphenols content is major than the extractable polyphenols one in all samples.
In particular in raw products the hydrolysable polyphenols contribution to antioxidant properties ranged from 75% in whole barley based pasta to 94% in whole rice based pasta, whereas in cooked pasta ranged from 81% to 92%.
In Figure 2A-2B TPC values (mg/100g d.w.) and Frap values (µmol/g d.w.) in aqueous-organic extracts and residues of cooked, experimental, wholegrain pastas made with different cereals were reported. Anese, Nicoli, Massimi, and Lerici (1999) have shown that temperature, time and moisture conditions of the drying process have a significant impact of antioxidant properties of pasta. Slavin (2003) has observed that bioactive compounds produced during processing contribute to the antioxidant power of the grain products. Several researches have shown that non-enzymatic browning reactions such as the Maillard reaction could generate antioxidant molecules (Manzocco, Calligaris, Mastrocola, Nicoli, & Lerici, 2001;Amarowicz, 2009 Figure 2B). In both raw and cooked products the lowest FRAP values were found for semolina/60% whole rice pasta. In addition, in rice based pasta, after cooking, a decrease of FRAP value has been reported in residue (P < 0.001), even if significant increases in TPC values have been reported in aqueous-organic extract (P < 0.01) and in residue (P < 0.01). Comparing raw and cooked products, a significant increase in FRAP values of aqueous-organic extract (P < 0.05) and residue (P < 0.01) as well as in TPC of residue (P < 0.001) has been reported for semolina/60% whole wheat pasta. Fares, Platani, Baiano, and Menga (2010) have shown that boiling water improve the extraction of bound phenolics from the food matrix in wheat pasta enriched with debranning fractions of wheat. On the contrary, Hirawan, Ser, Arntfield, and Beta (2010) have found that the total phenols content reduce by 40% in both regular and whole wheat brands after cooking.
From our results, after cooking, also for barley based pasta significant increases in FRAP value (P < 0.01) and in TPC (P < 0.001) were observed in residue.
For semolina/60% whole oat pasta a different trend was reported: significant decreases in FRAP value (P < 0.001) of residue and in TPC (P < 0.05) of aqueous-organic extract were observed.
Then cooked rye based pasta has higher FRAP value (P < 0.05) and lower TPC (P < 0.05) in aqueous-organic extract than raw product, whereas in residue after cooking an increase in TPC (P < 0.001) has been reported.

Conclusions
In this research our results have shown the antioxidant characteristics of wholegrain pastas made with different cereals, barley added pasta in particular. Our results confirm that the effect of cooking depends on the nature of food matrix. These results can provide a scientific basis for the development of wholegrain based functional foods.