IJFS#60_DURAZZO_bozza


	
  

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PAPER 
 
 
 
 
 

TOTAL POLYPHENOL CONTENT AND 
ANTIOXIDANT PROPERTIES OF SOLINA (TRITICUM 

AESTIVUM L.) AND DERIVATIVES THEREOF 
 
 
 

A. DURAZZO*, G. CASALE, V. MELINI, G. MAIANI and R. ACQUISTUCCI 
Consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria - Centro di ricerca per gli alimenti e la 

nutrizione (CREA-AN), Via Ardeatina 546, 00178 Roma, Italy 
*Corresponding author. Tel.: +39 0651494499; fax: +39 0651494550 

E-mail address: alessandra.durazzo@crea.gov.it 
 
 
 
 
 
 

ABSTRACT 
 
This study aims to characterize grains and derivatives of Solina, an Italian traditional 
winter soft wheat (Triticum aestivum L.). Total polyphenol content and antioxidant 
properties were investigated in grains, whole flours and bread, both in aqueous-organic 
extracts and residues. 
Results showed the important contribution of hydrolysable polyphenols, isolated in the 
residue of the aqueous-organic extract, to antioxidant properties (over 90% in grains and 
derivatives), and the key role played by milling and baking in antioxidant properties. 
Moreover, the analysis of potentially bioactive antioxidants remaining in the residues 
showed to be required for a comprehensive determination of antioxidant capacity in 
cereals. The study highlights that Solina represents a valuable Italian traditional wheat 
cultivar with proved antioxidant properties. 
 
 
 
 
 

Keywords: antioxidants, Total Polyphenol Content (TPC), traditional foods, soft wheat Solina, whole wheat 
flours, bread 

 



	
  

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1. INTRODUCTION 
 
Nowadays the traditional character of foodstuffs is a value-adding characteristic which 
contributes to defining quality thereof and is also often associated with positive health 
benefits. Traditional foods, in fact, not only play an important role in the cultural and 
nutritional food patterns of every country because their cultivation or preparation have 
been passed down from generation to generation, and/or because they have been 
consumed locally for years and become part of daily life in communities; they are often 
considered healthy and wholesome and are also often associated with positive health 
benefits because of their chemical profile. 
Solina is a traditional Italian winter soft wheat (Triticum aestivum L.), typical of the 
province of L’Aquila, in the Abruzzo region, within the National Park of Gran Sasso. Its 
cultivation in Abruzzo dates back to 1500 a.C. and there is evidence of a strong connection 
between this variety and the life of people from Abruzzo who keep appreciating it for the 
sensory properties it provides to bakery products. It is grown in mountainous areas and 
shows a high adaptability to marginal areas, as well as resistance to cold. That ancient 
cultivar was, in fact, defined by last century agronomists “Wheat hybernum”. Solina also 
shows a high stability in yields, and according to agronomists it is also compatible with 
organic farming methods because it does not require a large amount of nitrogen; moreover 
it is able to tolerate and compete strongly with weeds for its size and tillering capacity 
(PORFIRI et al., 2001). 
In this study, grains of the Solina cultivar, whole flours and bread samples thereof were 
examined in order to evaluate the phenolic components and the antioxidant properties of 
this traditional crop. 
Some technological and chemical parameters of grains were also considered, so as to 
identify a comprehensive portrait of this Italian wheat cultivar. 
According to literature, grains, in particular whole grains, provide a wide range of 
nutrients and phytochemicals that work in synergy to maintain human health (LIU, 2007; 
OKARTER and LIU, 2010). Whole grain consumption has been associated with reduced 
risk of chronic diseases, such as cardiovascular diseases and cancer (SCHATZKIN et al., 
2007; MELLEN et al., 2008; AUNE et al., 2013). These beneficial properties have been 
rediscovered by consumers and producers (ARVOLA et al., 2007). 
As the content of bioactive molecules is affected by genetic and growing conditions in 
grains as well as by processing conditions in foods (SLAVIN et al., 2000), samples of Solina 
grains, flours and bread formulated with this soft wheat cultivar were studied. 
 
 
2. MATERIALS AND METHODS 
 
2.1. Sampling 
 
Analysis were performed on grain, whole wheat flour and bread samples. Grains were 
supplied by three farms located in three different spots of the same area: Castelvecchio 
Subequo, Capestrano and Introdacqua. According to sample location they were labeled as 
Solina 1, Solina 2 and Solina 3, collected respectively at Castelvecchio Subequo, 
Capestrano and Introdacqua. 
The grains of each collected sample were divided in two aliquots. An aliquot was milled 
by a water cooled mill (Janke & Kunkel IKA Labortechnik, Staufen, Germany) and stored 
at 4°C until chemical analyses were performed. An aliquot was tempered for 24 h to 15.5% 
moisture content, then milled in an experimental mill MLU-202 Bühler (Switzerland) 



	
  

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equipped with three breaks and three reduction rolls and six screens to obtain whole mill 
flours. 
Solina bread was also studied. Five loaves of bread (four long loaves and one round loaf), 
produced according to a traditional bread making process, were purchased in bakeries 
located in the same area of grain collection. They were coded as reported in Table 1. 
 
 
Table 1. Code, description and some chemical parameters of bread samples*. 
 

Code Description  Composition  

  Moisture (%) Protein (%) Ash (%) 

A Bread long loaf (900 g) 9.9 11.5 1.90 

B Bread long loaf (1660 g) 7.8 11.2 2.74 

C Bread round loaf (900 g) 10.8 12.5 2.96 

D Bread long loaf (810 g) 11.0 11.6 2.65 

E Bread long loaf (1700 g) 10.4 11.6 1.81 

 
*Data are expressed as mean of replicate measurements (n=3); differences within measurements were in the 
range reported in the method. 
 
 
2.2. Chemicals and standards 
 
Common reagents and standards were purchased from Sigma–Aldrich Srl (Milan, Italy), 
Extrasynthèse (Genay, France), Carlo Erba (Milan, Italy) and BDH Laboratory Supplies 
(Poole, UK) and their purity degree was chosen according to the analysis to be performed. 
Double-distilled water (Millipore, Milan, Italy) was used throughout the study. 
 
2.3 General chemical analysis 
 
Grain quality was determined after removing impurities, i.e., broken grains, heat-
damaged grains, shriveled grains, vegetable impurities, vitreous grains, on 100 g seeds by 
visual evaluation. Test weight (TW), thousand kernel weight (1,000 KW) and kernel 
hardness (KH) were assessed. TW was determined by a Shopper chondrometer equipped 
with 250 mL cylinder. 1,000 KW was determined by counting and weighing 1,000 kernels, 
while kernel hardness (KH), diameter and moisture of grains were determined by the 
SKCS 4100 apparatus (Perten Instruments, Sweden) according to the standard method 
AACC 55-31 (2003). 
Total proteins were determined by Kjeldahl method according to ICC standard method 
No 105/2  (2003) using 5.70 as specific conversion factor. Ash content was determined on 
the inorganic residue remaining after the incineration of the sample in a muffle furnace at 
the temperature of 900°C according to the ICC method No 104/1 (2003), while moisture 
was determined according to ICC standard method No 110/1 (2003). Moisture is reported 
as g/100 g fresh weight  (f. w.), while both protein and ash content are reported as g/100 g 
dry matter (d. m.). 
 
 
 



	
  

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2.4. Sample extraction for evaluation of Total Polyphenol Content (TPC) 
and antioxidant properties 
 
Total polyphenols were extracted as described by DURAZZO et al. (2013), with some 
modifications. Extractable polyphenols were determined on aqueous-organic extracts, 
while non extractable polyphenols were determined on solid residues of aqueous-organic 
extraction. In details, some non-extractable polyphenols, i.e. hydrolysable polyphenols, 
were isolated and determined following a specific and suitable acid hydrolysis as reported 
below. 
 
Extractable polyphenols 
 
About 3.0 g, 3.5 g, 4.5 g of grains, flours and bread, respectively, were placed in a test tube 
and added with 20 mL of acid methanol/water (50:50 v/v, pH= 2). Tubes were swirled at 
room temperature for 3 min, then mildly shaken for 1 h in a water bath at room 
temperature as well. Tubes were centrifuged at 2500 x g for 10 min, and the supernatant 
was recovered. Then, 20 mL acetone/water (70:30, v/v) were added to residue and the 
extraction repeated as reported above. Methanolic and acetonic extracts were combined 
and centrifuged at 2800 x g for 15 min. The resulting mixture was then used for the 
determination of total polyphenol content and antioxidant properties. 
 
Hydrolysable polyphenols 
 
The residue left after the above described extraction was dried in a ventilated oven at 
25°C. 250 mg, 300 mg, 400 mg of grain, flour and bread residue, respectively, were mixed 
with 20 mL of methanol and 2 mL of sulfuric acid (18 M). Samples were gently stirred for 1 
min and shaked at 85°C for 20 h in a water bath. 
Samples were then centrifuged (2500 x g for 10 min), and the supernatant was recovered. 
After two washings with minimum volume of distilled water and re-centrifuging as 
necessary, the final volume was taken up to 50 mL. The tube was centrifuged at 2800 x g 
for 20 min and the resulting supernatant was used for the determination of total 
polyphenol content and antioxidant properties. 
 
2.5. Determination of Total Polyphenol Content (TPC) 
 
The TPC was determined using the Folin-Ciocalteau procedure (SINGLETON et al., 1999). 
Briefly, appropriate dilutions of extracts were oxidised with Folin-Ciocalteau reagent, and 
the reaction was neutralised with sodium carbonate. The absorbance of the resulting blue 
colour solution was measured at 760 nm against an appropriate blank after 2 h of reaction 
at room temperature at dark. Gallic acid was used as standard. 
 
2.6. Antioxidant properties evaluation 
 
The determination of antioxidant properties was carried out by FRAP (Ferric Reducing 
Antioxidant Power) assay according to methods of BENZIE and STRAIN (1996) and 
PULIDO et al. (2000) and by using a Tecan Sunrise® plate reader spectrophotometer. The 
method is based on the reduction of Fe3+-TPTZ (2,4,6-tripyridyl-s-triazine) complex to 
ferrous at low pH. 
 
 
 



	
  

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2.7. Statistical Analysis 
 
All analyses were performed in triplicate. Data are presented as mean±standard deviation 
(s.d.). 
Data obtained by Official Methods are presented as mean. Statistica for Windows 
(Statistical package; release 4.5; StatSoft Inc., Vigonza PD, Italy) was used to perform One-
way Analysis of Variance (ANOVA). Significant differences between grain and flours of 
the same origin were evaluated by the Student’s t test. 
 
 
3. RESULTS AND DISCUSSIONS 
 
The inspection of grain impurities in the samples is an important control procedure 
necessary to verifying wheat quality and it is a preliminary step prior to the milling phase. 
The percentage of ripe grains were higher than 95% in all samples. This confirms the good 
quality of analysed grains. 
The values obtained for the main physical parameters (kernel hardness, test weight, 
thousand kernel weight, diameter) and the chemical composition (moisture, protein, ash) 
of grain samples are reported in Table 2. As concerns physical parameters, no significant 
differences were found among grain samples. The values obtained for TW (74.1 - 80.2 
Kg/hL), 1,000 KW (>43 g) and grain diameter  (2.11-2.47 mm) show that kernels are 
wholesome, sound, not sprout damaged. 
 
 
Table 2. Physical and chemical parameters of grains*. 
 

Sample  KH TW 1,000 KW Diameter Moisture Protein Ash 

  (kg/hL) (g) (mm) (g/100 g) (g/100 g d.m.) 
(g/100 g 

d.m.) 
SOLINA 1 19.70 74.1 44.6 2.47 12.4 13.1 2.04 

SOLINA 2 16.69 80.2 54.4 2.46 12.6 11.9 1.75 

SOLINA 3 33.70 79.0 43.4 2.11 12.4 13.7 2.00 

 
*Data are expressed as mean of replicate measurements (n=3); differences within measurements were in the 
range reported in the method. 
 
 
As regards kernel hardness, the data reported in Table 2 confirm our samples belong to 
the “soft” species and their variation may depend on the different growing locations. 
According to POMERANZ and WILLIAMS (1990), in fact, the main factors that affect the 
grain hardness are growing location (soil type, elevation, planting type, irrigation, 
fertilizers and cultivation practice), growing season (precipitation and temperature during 
maturation and post ripening), storage conditions, protein content, moisture and kernel 
size. Regarding the main chemical parameters, significant differences were observed: 
Solina 2 showed the lowest protein and ash content. Those data show, therefore, that 
environmental and growing factors not only influence hardness, but also chemical 
parameters such as protein and ash content. 
In Table 3, total polyphenol content (TPC) and antioxidant properties (FRAP) are reported 
for grains and whole wheat flours. Values refer to aqueous-organic extracts and residues. 
In grains, TPC values ranged between 165.57 and 183.75 mg/100g d.m. in aqueous-organic 



	
  

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extracts, and between 1084.42 and 1325.32 mg/100g d.m. in residues, whereas the range 
for FRAP values was 6.34-6.60 !mol/g d.m. in aqueous-organic extracts and 85.06-109.12 
!mol/g d.m. in residues. TPC and FRAP values in aqueous-organic extracts showed no 
differences between the grain samples supplied by the three farmers. On the other hand, 
as to the residue, the TPC resulted significantly different in the grain samples and the 
highest value was obtained for Solina 1; the values for antioxidant properties in Solina 1 
were higher than those obtained for Solina 2 and Solina 3 that were, at their turn, 
comparable. As reported by several authors (ADOM et al., 2003; CARCEA et al., 2009) 
differences in the bioactive molecule distribution and antioxidant properties in wheat, as 
well as in foodstuffs in general, could be related to genetic factors, agronomic practices 
and environmental conditions. It is interesting to notice that in grains the FRAP values in 
residues are about 15 fold higher than in aqueous-organic extracts. This trend was also 
confirmed by the data obtained for TPC. The hydrolysable polyphenols, isolated in 
residue, belong to several classes of phenolic compounds, i.e. hydrolysable tannins, 
phenolic acids and hydroxycinnamic acids, and consist on ferulic acid, caffeic acid, sinapic 
acid, etc. These phenols are linked to carbohydrates and protein by covalent bonds, 
hydrogen bonds and/or hydrophobic interactions and they are released from food matrix 
after a strong acid hydrolysis (PÉREZ JIMÉNEZ and TORRES, 2011; PÉREZ-JIMÉNEZ et 
al., 2013). These compounds represent a significant fraction in some groups of foods, 
among which cereals and have appreciable antioxidant properties and possibly specific 
health properties. However, they have been generally underestimated and studies are still 
required to tackle this issue (SAURA-CALIXTO, 2012). In addition, recent studies have 
shown that non-extractable polyphenols significantly contribute to the beneficial 
properties associated to dietary fibre (VITAGLIONE et al., 2008; SAURA-CALIXTO, 2011). 
 
 
Table 3: TPC and FRAP values in aqueous-organic extracts and their residues in grains and flours*. 
 

Products Aqueous-organic extract (extractable polyphenols) 
 TPC (mg/100g d.m.) FRAP (μmol/g d.m.) 
 Grain Flour P valueΩ Grain Flour P valueΩ 

SOLINA 1 170.43±28.61 129.28±13.64a 0.01 6.60±0.47 2.75±0.17b 0.0001 
SOLINA 2 165.57±26.11 130.87±15.85a 0.001 6.34±0.27 2.35±0.14a 0.0001 
SOLINA 3 183.75±14.07 155.24±12.74b 0.01 6.47±0.60 2.31±0.07a 0.0001 

Mean 173.25±24.05 138.46±18.16 0.0001 6.47±0.46 2.48±0.24 0.0001 
 

Products Residue (hydrolysable polyphenols) 
 TPC (mg/100g d.m.) FRAP (μmol/g d.m.) 
 Grain Flour P valueΩ Grain Flour P valueΩ 

SOLINA 1 1325.32±57.99c 981.01±50.27b 0.0001 109.12±10.54b 114.25±3.24b n.s. 
SOLINA 2 1084.42±66.99a 811.65±38.57a 0.0001 85.06±9.90a 99.20±11.14a 0.01 
SOLINA 3 1198.73±37.49b 791.50±80.14a 0.0001 90.40±4.30a 118.29±4.26b 0.0001 

Mean 1202.82±114.39 866.82±104.77 0.0001 92.07±12.22 111.61±10.13 0.0001 
*mean ± s.d.; Anova, Tukey HSD Test: by column, means followed by different letters are significantly 
different (p < 0.05). 
" Student’s t test ; n.s.=not significant 
 
 



	
  

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In our study, the determined hydrolysable polyphenols (isolated in the residue of the 
aqueous-organic extract of grains) showed to account for 87-89% of total polyphenols and 
contribute to the total antioxidant properties for about 93%. These data match those 
obtained in the other studies on cereals (PEREZ-JIMENEZ and SAURA-CALIXTO, 2005; 
CHANRASEKARA and SHAHIDI, 2010). 
Regarding the effect of milling process on TPC content, whole flours exhibited a more 
significant reduction than in grains, both in the aqueous-organic extract and in the residue. 
Results showed that antioxidant properties also appear to be affected by the milling 
process: a decrease in FRAP values was, in fact, observed in aqueous-organic extract 
(mean value: 6.47±0.46 vs 2.48±0.24 !mol/g d.m.; P<0.0001), whereas an increase in FRAP 
values was reported in residues (mean value: 92.07±12.22 vs 111.61±10.13 !mol/g d.m.; 
P<0.0001). The increase of FRAP values in residue after milling process could be due to a 
better efficiency of extraction of bound compounds with antioxidant properties, related to 
improvement of solvent penetration for the enlargement of the particle surface. These data 
confirm the importance of milling process in retention of components contributing to 
antioxidant properties (DUODU, 2011). 
Nowadays, several investigations have been carried out to study the effects of cereal 
processing technologies, such as milling, baking, extrusion, etc., on antioxidant properties 
(LIYANA-PATHIRANA and SHAHIDI, 2007; RAGAEE et al., 2014). As a consequence, 
TPC (mg/100g d.m.) and FRAP (!mol/g d.m.) in aqueous-organic extracts and their 
residues were also studied in Solina bread samples, as reported in Table 4. It is worth 
mentioning that whole wheat bread represents a rich source of bioactive compounds as 
reported in literature (JENSEN et al., 2011; ABDEL-AAL and RABALSKI, 2013). 
 
 
Table 4. TPC and FRAP values in aqueous-organic extracts and their residues in bread*. 
 

Loaves TPC (mg/100g d.m.) FRAP (μmol/g d.m.) 

 Aqueous-organic extract Residue 
Aqueous-organic 

extract Residue 

Code A 63.13±6.03cd 832.91±46.21a 8.57±0.34d 106.21±16.96a 

Code B 49.44±6.98b 894.07±62.64a 3.93±0.17a 120.27±30.77ab 

Code C 52.87±6.93bc 1326.90±48.00b 6.14±0.34b 141.90±8.33b 

Code D 36.96±5.59a 1237.41±69.35b 4.14±0.37a 114.19±3.52a 

Code E 71.23±6.04d 877.73±88.77a 7.80±0.26c 126.60±7.80ab 
*mean ± s.d.; Anova, Tukey HSD Test: by column, means followed by different letters are significantly 
different (p < 0.05). 
 
 
In bread samples, as it was observed in raw materials, the major contribution to 
antioxidant properties was given by hydrolysable polyphenols (93-97%). Moreover, the 
comparison of the TPC and FRAP values of the residue in bread samples and the 
corresponding values in grains showed that a decrease of TPC, from grains to bread, 
corresponded to an increase of FRAP values. Several authors have observed that some 
antioxidants can be generated during non-enzymatic browning reactions, such as the 
Maillard reaction (MANZOCCO et al., 2001). 
 
 
 



	
  

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4. CONCLUSIONS 
 
Our results showed that in grains and derivatives thereof, hydrolysable polyphenols 
(isolated in the residue of the aqueous-organic extract) represent an important fraction, 
with appreciable antioxidant properties and possibly health properties. In particular, this 
research highlighted that the analysis of potentially bioactive antioxidants, remaining in 
the residues, is required for an adequate and comprehensive determination of antioxidant 
capacity in cereals. Results also pointed out the key role played by the milling and baking 
process in antioxidant properties. 
So, in conclusion, Solina wheat represents a valuable Italian traditional wheat cultivar 
with proved antioxidant properties. It also contributes to highlighting the benefits that 
traditional foodstuffs may have Europe-wide: they represent foods with an increased 
nutritional value and bring a strong contribution to land protection and conservation of 
biodiversity. 
 
 
ACKNOWLEDGEMENTS 
 
This work was undertaken within the project TERRAVITA, funded by the Italian Ministry of Agriculture, Food and 
Forestry Policies. The authors wish to thank Dr. D. Silveri, Dr. V. Galli and Mr. L. Bartoli for the technical assistance and 
Dr. Francesca Melini for the technical and linguistic assistance in the revision of this paper. 
 
 
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Paper Received February 13, 2015 Accepted June 20, 2015