Bread is one of the most important sources of dietary fibres, micronutrients, proteins, and vitamins. There- fore, it is considered effective, when fortified with sui- table fibre fractions, in treating various diseases, such as obesity, cardiovascular disease, type 2 diabetes, etc [1]. There is a growing demand for bread in the whole world, especially in developing countries such as Egypt. At the same time, consumers increasingly prefer functional foods that contain ingredients providing health benefits beyond basic nutrition [2, 3].

The whole grain of wheat consists of germ, endo- sperm, and bran. Milling results in a dramatic loss of healthy biochemical molecules, such as antiradical con- stituents, fibre, vitamins, and minerals, causing cardio- vascular and other types of disease [4]. Only endosperm, which contains a significant amount of carbohydrates, remains after milling. However, cereal products prepared from whole grain are not as popular as those from refined flour due to reduced quality and degraded sensory prop- erties caused by the presence of bran. The detrimental ef- fects of bran can be decreased by various methods such

as hydration, fermentation, and size reduction [5–7]. Bran is the main by-product of milling. It is a valuable and in- expensive source of dietary fibre that contains approxi- mately 27% of total carbohydrates, 14% of protein, and 5% of minerals [8, 9]. The chemical composition of wheat bran depends on wheat variety, environmental condi- tions, etc. Therefore, the source of bran is a critical factor for the quality of wheat grain products [10].

The world production of rice bran reaches 29.3 mil- lion tons annually [11]. Introducing defatted rice bran in wheat flour is a useful method of increasing lysine, protein, and fibre contents [12]. A high protein content (11–17%), excellent nutritional value, and a considerable amount of fibre (20–27%) make rice bran a good source of bread fortification [13, 14]. The addition of rice bran at a concentration of 15–30% did not change the physico- chemical properties of bread [15].

According to the Food and Agriculture Organisation (FAO), Egypt produces 117,113 tons of barley grains per annum on an area of about 87,752 ha [16]. It was repor- ted by Anderson et al. that barley contains a significant amount of β-glucan which helps to reduce low density li-

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poprotein (LDL) and total cholesterol in serum of both humans and animals [17–19]. A study into the effect of barley bran on bread quality found that 15% was the best amount of barley bran for bread fortification that ensured high quality and health benefits [20].

Texture, volume, and appearance of bread are im- portant quality criteria for consumers. However, taste and aroma play a dramatic role for both producers and consumers. Approximately 300 volatile compounds are identified in bread that fall into several classes such as alcohols, esters, aldehydes, etc. They result from vari- ous interactions between the type and concentration of ingredients during processing, yeast activity during fer- mentation, and fermentation conditions (time, tempera- ture, etc.) [21, 22].

The current study aims to compare the effects of sub- stituting wheat flour with various brans on the chemical composition, as well as antioxidant and volatile com- pounds of Egyptian Fino bread.

STUDY OBJECTS AND METHODS

Materials. Wheat flour (WF) (72% extraction) and wheat bran (WB) were obtained from the North Cairo Flour Mills Company (Egypt). Rice bran (RB) was ob- tained from a local milling factory (Zifta, Egypt). Barley bran (BB) was obtained from a pilot plant at the National Research Centre (Dokki, Egypt). All chemicals used in this study are of analytical grade.

Methods.

Milling. Barley grains (Giza, 136) were manually cleaned, tempered to 14% moisture content, milled using a Quadrumat Junior flour mill (Model MLV-202, Swit- zerland), and sieved to obtain flour and bran.

Stabilizing rice bran. The bran was immediately stabilized using oven heating (at 110°C for 10 minutes). Immediately subsequent to heating, the sample was re- moved from the oven and cooled to room temperature (25°C). The stabilized rice bran was milled into flour. The flour was screened through a 30-mesh sieve, supple- mented with wheat flour, and stored under freezing con- ditions.

Making Fino bread. Different Fino bread blends were prepared by using WF (72% extraction) and the studied bran at a concentration of 10%, 20%, and 30%. Active dry yeast (1.5%), NaCl (1.5%), sugar (2%), shor- tening (1%), bread improver (1%), and water (an amount required to reach 500 Brabender Units of consistency) were added to each sample in the pilot plant at the Na- tional Research Centre (NRC) in Dokki, Egypt. Fino bread was made according to Hussein et al. in an elec- tric oven (Mondial Formi, 4T 40/60, Italy) [23]. Firstly, yeast was dissolved in warm water (35°C) and added to the dry ingredients and the shortening; then the mix- ture was kneaded. The dough was fermented at 30°C for 30 min in a fermentation cabinet under 80–85% relative humidity. Then it was divided into 80 g pieces that were placed in the trays and proofed under the same condi- tions for 45 min. The dough loaves were baked at 325°C for 10–15 min after steaming for 10 sec. To enhance

the browning process of protein bread, the dough pie-

ces were brushed with melted margarine prior to baking. The baked loaves were cooled down at room tempera- ture for 60 min. Weight, volume, and specific volume of the bread samples were determined as described by [24]. Analytical  methods.  Moisture,  protein,  fat,  ash, and  fibre  of  raw  materials  and  Fino  bread  were  de- termined  according  to  AOAC  Official  Methods  of Analysis  International,  while  carbohydrates  were  cal- culated by difference as in Tadrus’s study [25, 26]. In- dividual elements (Ca, P, K, Na, Fe, Mn, and Cu) in all the  samples  were  determined  according  to  Chapman and  Pratt  [27].  Changes  in  Hunter  colour  parameters (L, a & b) of raw materials and Fino bread were followed up using a Tristimulus Colour Analyzer (Hunter, Lab

Scan XE, Reston, Virginia) with a standard white tile.

Bread freshness. The freshness of the bread samples was tested at day 0, 3 and 7 of storage at room tempera- ture by alkaline water retention capacity (AWRC) ac- cording to the method described by Hussein et al. [28].

Sensory properties. The Fino bread samples were evaluated for taste (20), aroma (20), mouth feel (10), crumb texture (15), crumb colour (10), break & shred (10), crust colour (10), and symmetry shape (5) accor- ding to the method described in [24].

Total phenolics extraction. Ten grams of powdered bread was extracted with 75 ml 100% methanol at 25°C for 24 hours along with stirring followed by filtration using Whatman no.1 filter paper. The residues were re-extracted twice as described above. The combined methanolic extracts were evaporated at 40°C under va- cuum until dry.

Total phenolics determination. The concentration of phenolic compounds in the bread samples was esti- mated with the Folin–Ciocalteu  reagent  according  to the method described by Singleton and Rossi [29]. One millilitre of a sample (5 mg) was mixed with 1 ml of the Folin-Ciocalteu’s phenol reagent. After 3 min, 1 ml of saturated sodium carbonate (20%) solution was added to the mixture and adjusted to 10 ml with distilled wa- ter. The reaction was kept in the dark for 90 min, after which the absorbance was read at 765 nm. Gallic acid was used to construct the standard curve (8–80 μg/ml). The results were expressed as mg of GAEs (gallic acid equivalents/g).

Determination of free radical scavenging activity. The antioxidant activity of the methanol extracts was determined by the DPPH radical scavenging method as described by Woldegiorgis et al. [30]. A 0.004% solu- tion of the DPPH radical solution in methanol was pre- pared and then 2 ml of the DPPH solution was mixed with 1 ml of various concentrations (0.1–0.5 mg/ml) of the extracts in methanol. Finally, the samples were in- cubated for 30 min in the dark at room temperature. The scavenging capacity was read spectrophotomet- rically by monitoring the decrease  in  absorbance  at 517 nm. The inhibition of free radical DPPH in percent (I %) was then calculated.

The scavenging activity, %, was calculated using the

following formula:

% Inhibition = [(A

treatment

/=A

control

)] × 100.

mass spectral search programme (Version 2.0; National

Institute of Standards & Technology) for library search

β-Carotene-linoleate  bleaching  assay.  The  anti-

oxidant activity of the methanol extract with various concentrations (0.1–0.5 mg/ml) was assayed according to the β-Carotene-linoleate bleaching method deve- loped by Velioglu et al. [31]. 0.2 mg of β-Carotene (in 1 ml chloroform), 0.02 ml of linoleic acid, and 0.2 ml of Tween were transferred into a round bottom flask. The mixture was then added to 0.2 mg of methanolic extract prepared for the  β-carotene-linoleate  bleaching  assay or 0.2 ml of standard methanol (as a control). Chloro- form was removed at room temperature under vacuum at reduced pressure using a rotary evaporator. Following evaporation, 50 ml of distilled water was added to the mixture and then shaken vigorously to form an emul- sion. Two millilitres aliquots of the emulsion was taken in test tubes and immediately placed in a water bath at 50°C. The absorbance was measured at 470 nm by a UV-Vis Shimadzu (UV-1601, PC) spectrophotometer. Readings of all the samples were performed immediately (t = 0 min) and after 120 minutes of incubation. The an- tioxidant activity (%) was evaluated in terms of β-caro- tene bleaching inhibition using as follows:

% Inhibition = [(A – C )/(C – C )] × 100,

t           t          0           t

where A and C are the absorbance values measured for

and identification, as well as software (EZChrom Elite; GL Sciences Inc.) for the quantification of identified to- tal ion peak areas. For the static headspace, 3 grams of the whole bread samples were encapsulated in a glass vial container (22 ml). The analytical conditions of the headspace included a sample weight of 3 g, a sample temperature of 80°C, an injection temperature of 160°C, an injection duration of 36 sec, a needle temperature of 80°C, and a transferring temperature of 160°C. High-pu- rity helium carrier gas (1.2 ml/min) was employed for gas chromatography. The column was held at 50°C for 3 min, with the temperature programmed to 220°C at a heating rate of 4°C/min, and then held at 220°C for

15 min. The analytical conditions of the mass spec- trometer included  an interface  temperature  of 220°C, a transferring temperature of 160°C, and an ion source temperature of 230°C. The ionization energy of  the mass spectrometer was 70 eV and a scan cycle time was 0.5 ms (33–40 m/z).

Compounds identification. The linear retention in- dex (RI) values for unknowns were determined based on retention time data obtained by analyzing a series of

normal alkanes (C6–C22). Volatile components were po- sitively identified by matching their RI values and mass spectra with those of standards, also run under identical

chromatographic conditions in the laboratory (Adams,

t                   t

the test sample and control, respectively, after 120 min

2007).

is the absorbance values for the con-

Statistical analysis. The obtained results were eva-

trol measured at zero time during incubation. All the ex- periments were carried out in triplicate.

Ascorbic acid was used as a standard, and the ex- tract-free mixture was used as a control.

Volatile compounds analysis. Flavour compounds were identified by GC/MS analyses. The instruments in- cluded a static headspace (Agilent 7890 GC coupled to a 5977 MS detector) with a column (DB-5; J&W Scientific Inc.) of 60 m in length, 0.25 mm in inside diameter, and

0.25 µm in membrane thickness. We also used a mass spectrometer (Automass SUN-200S; JEOL Ltd.) with a

luated statistically using analysis of variance as reported by Mc-Clave and Benson [32].

RESULTS AND DISCUSSION

Chemical composition of wheat flour and bran. The proximate  composition  of  the  brans  under  study is presented in Table 1. The protein content of diffe- rent brans ranged from 8.49 to 14.16%. The fat content varied from 2.16 to 8.12% in BB and RB, respective- ly. Rice bran exhibited the most concentrated source of dietary fibre  (36.18%) among  all cereal  brans. Barley

Table 1. Chemical composition of brans, %

Note: WF (72%) is wheat flour extract 72%; WB is wheat bran; and RB is rice bran; BB: barley bran

bran  had  the  maximum  value  for  total  carbohydrates (67.35%). Maximum ash content (14.59%) was observed

Table 2. Physical properties of bread with various amounts of brans

in  wheat  bran.  Wheat  flour  (72%)  contained  85.06%       

of  carbohydrates.  Similar  results  were  reported  by

Sample   Weight, g         Volume, cm   Specific volume, cm3/g

O. Ozdestan et al. [33, 34].

Mineral content. According to Table 1, the brans under study were superior in sodium, potassium, phos- phorus, calcium, iron, zinc, and copper, compared to wheat flour. These data agree with those found by Faria et al. who reported that Ca and P contents were 63.3 and 979 mg/100 g in rice bran and 65 and 979 mg/100 g in stabilized rice bran, respectively [35]. Also, the results in Table 1 showed an increased amount of K and P in rice bran, compared to the other raw materials.

Colour attributes. Colour plays an important role in the consumer’s choice of foods, especially bakery pro- ducts. The colour parameters of the brans, as well as wheat flour 72%, were evaluated using a Hunter laborato- ry colourimeter (Table 1). The bran samples were darker than WF. The same trend was observed with yellowness (a*): it was higher for different brans, compared to wheat flour. The obtained results are in good agreement with Kim et al. And Ramy et al.: the presence of bran produ- ces darker bakery products [36, 37]. Therefore, we should control its concentration or use suitable additives to re- duce this browning.

 Control    70.5 ± 0.12      290 ± 1.65     4.11 ± 0.19                      

WB concentration,%:

10            73.2 ± 0.15     280 ± 1.20     3.83 ± 0.19

20            77.5 ± 0.17     265 ± 1.35     3.42 ± 0.32

30            82.0 ± 0.21     250 ± 1.42     3.05 ± 0.39

RB concentration, %:

10            74.2 ± 0.22     270 ± 2.6       3.64 ± 0.19

20            78.5 ± 0.15     250 ± 1.35     3.18 ± 0.32

30            84.0 ± 0.11     230 ± 2.15     2.74 ± 0.39

gradually decreased in all the bread samples, compared to the control, with its values ranging from 220 (for 30% of BB) to 280 cm3 (for 10% of WB).

These results were in agreement with [39] that sub- stituted wheat flour with high concentrations of rice bran (20 and 30%), which decreased the loaf volume.

Bran effects on colour attributes. The colour measurements of different bread samples are shown in Table 3. The bread samples containing different pro- portions of bran had lower values of L, b, and a; more- over, the values decreased as the concentration of bran increased. All the fortified samples had slightly lower L values for crust than the control and therefore a slightly darker crumb colour was noticed.

Bran effects on sensory evaluation. The sensory characteristics of the Fino bread samples with different amounts of WB, RB, and BB are shown in Table 4. The results indicated that the addition of bran did not have a clear effect on the crust and crumb colour, whereas its effect on taste and smoothness was quite remarkable. All the changes, however, were in the acceptable range. The colour changes may be due a higher content of reducing sugars in bran, compared to wheat flour, and the Mail- lard reaction during the baking process. We can also

Table 3. Hunter colour parameters of Fino bread with various amounts of brans

Table 4. Sensory evaluation of bread with various amounts of brans

notice that increased concentrations of bran lead to a gradual decrease in hardness and smoothness, especial- ly in the bread samples containing 30% of WB, RB, and BB. The results also showed a significant effect of WB, RB, and BB on the aroma of bread, primarily due to the flavour compounds.

The bread samples with 30% of WB, RB, and BB were significantly harder than the others. This may be due to the dilution of gluten and the thickening of the walls surrounding air bubbles in the crumb [40, 41]. All the Fino  bread samples containing WB,  RB,  and BB showed an observed acceptability. Also, the addition of the brans changed the bread colour slightly and reduced the size of the holes, as confirmed by Sharma and Chau- han [39]. As can be noticed, there were no significant differences in break and shred and symmetry shape be- tween the Fino bread from WF (control) and the samples with 10% of WB. However, the samples fortified with bran manifested significant differences in taste, aroma, mouth feel, crumb texture and colour, and crust colour. As the bran level increased, the crust colour score de- creased.

Table 5. Changes in freshness of bread with various amounts of brans during storage

Sample                                       Storage time, days 0                         3              7

Control                 300a ± 0.11      295.3a ± 0.13    290.4a ± 0.09

WB amount, %:

10                       290b ± 0.19      285.6b ± 0.15    280.1b ± 0.13

20                       280c ± 0.22      275.3c ± 0.22    270.3c ± 0.19

30                       275cd ± 0.17     270.5d ± 0.19    263.5d ± 0.25

RB amount, %:

10                       295ab  ±0.15     286.2b ± 0.20    278.5b ± 0.30

20                       287bc ± 0.12     276.3c ± 0.16    269.8c ± 0.26

30                       280c ± 0.10      271.5d ± 0.14    262.7d ± 0.17

BB amount, %:

10                       292b ± 0.25      287.3b ± 0.23    278.5b ± 0.15

20                       285 c± 0.17      279.5c ± 0.19    269.4c ± 0.13

30                       278d ± 0.23      269.7d ± 0.15    263.3d ± 0.32

LSD at 0.05       5.9                   9.4                     8.9

Bran effects on staling. The changes that occur after baking can be defined as staling. They can be measured by the alkaline water retention capacity (AWRC) test. Increases in the AWRC showed the freshness of baked products [42]. Our results revealed a gradual increase in the staling rate for all the Fino bread samples during a prolonged storage time of about 7 days (Table 5). No dif- ferences were observed in the first 3 days, while 7 days of storage caused an increase in the staling rate for all the bread samples. It is clear that the Fino bread with WB, RB, and BB at concentrations of 10, 20, and 30% was fresher than the control under the same conditions due to its higher water retention capacity and a conse- quent improvement of its staling rate. This might be due to a higher content of fibres in bran-fortified bread com- pared to the control. The Fino bread samples with 10 or 20% of bran had a higher water retention capacity, com- pared to the control. Such an increase can be related to a higher hydrophilic nature of proteins. It was noticed that the bread fortified with 10 or 20% of bran showed a bet- ter consistency or high texture characteristics.

Bran effects on total phenolic content and antiox- idant activity. The total phenolic content (TFC) of the brans under study, as well as the bread samples fortified with different concentrations of bran, was determined by the Folin-Ciocalteu method (Fig. 1). It was clear that the addition of all the brans showed a remarkable increase in the total phenolic content of the bread samples, com-