Effect of the mixture of oleaster (E. angustifolia L.) and black cumin (Nigella sativa) flours as functional compounds on the quality characteristics of toast bread

Abstract The present study was conducted to evaluate the addition of a mixture of oleaster (OL; E. angustifolia L.) and black cumin (BC; Nigella sativa) flours on the quality characteristics of toast bread. The concentration of OL and BC mixture (1:1 w/w ratio) was 0 (T1), 1.5% (T2), 2% (T3), and 2.5% (T4) of total flour content. The bread samples containing the mixture of OL and BC flours had more protein content (8.49%–9.65%) than the control (6.81%; p < .05). The highest phenolic compounds and DPPH free radical scavenging capacity were observed in T4 and T3 samples, respectively. The OL and BC flours decreased the brightness, yellowness, and chroma and increased the redness compared to the control. The mixed flour concentration influenced the bread's hardness and chewiness. Adding OL and BC flours increased the calcium content in the bread (467.65–600.41 ppm) compared to the control (363.9 ppm; p < .05). The OL and BC flour mixture created a more compact texture in the bread samples. In the sensory evaluation, there was not a significant difference between the overall acceptability of the bread containing the mixture of OL and BC flours and the control (p > .05). Finally, it is recommended to utilize a mixture of 2% of OL and BC flours in toast bread to improve its nutritional properties.

strated that oleaster flour increased the total sugar and ash, and decreased the protein content in lavash bread (Iranian flatbread). Osman et al. (2015) added BC flour to flatbread and observed an increase in protein, fat, ash, and fiber contents. According to Al-Ansi et al. (2019), the total phenol content, antioxidant activity, protein, fat, and ash increased in biscuits containing BC flour. To the best of our knowledge, the mixture of oleaster and BC flours has not been used in toast bread formulation. The present study aims to evaluate the effect of the simultaneous addition of oleaster and BC flours on the quality characteristics and nutritional properties of toast bread.

| Materials
The ingredients used to prepare the bread samples included wheat flour (82% extraction rate), sugar, milk, baker's yeast, improving agent (DATEM), salt, vegetable oil, oleaster, and BC. The improving agent was purchased from Sahar Co. and other ingredients were purchased from a local store. Oleaster (fruit and crust) and BC were ground and passed through a 60-mesh sieve after removing the impurities and separating the seed from the oleaster.
All the chemicals purchased were of analytical grade (Merck).
DPPH was obtained from Sigma-Aldrich.

| Preparation of toast bread
To produce the bread samples, wheat flour and powdered ingredients were first mixed with a mixer according to Table 1. Then, vegetable oil, milk, and a mixture of OL and BC flours were added.
After mixing the ingredients, the dough mass was rested for 10 min in a proofer (Morshed Gohar) at 35°C and a relative humidity of 90%. Then, the dough was divided into pieces of about 650 g, poured into one-third of the toast molds, and placed in the fermentation chamber for 30 min for final fermentation. Baking was performed in a rotary oven (Morshed Gohar) at 150-160°C for about 40 min. After cooling, the bread samples were sliced and packed in polyethylene bags (Hadidi et al., 2021). Soxhlet (AOAC 935.38) and Kjeldahl (AOAC 950.36) methods were used for fat and protein measurement, respectively (AOAC, 2006).

| Analysis of samples
The moisture (AOAC 925.10) and ash content (AOAC 923.03) were determined by the gravimetric method (AOAC, 2006). Iron and calcium were measured according to Carocho et al. (2020) by atomic absorption spectrophotometry (AAS8020-YOUNG LIN). All analyses were carried out for flours (wheat, oleaster, and BC) and bread samples in triplicate. The pH of bread was measured with a sevenCOMPACT-METTLER Swiss pH meter according to AACC 02-52.01, and the specific volume was evaluated by the AACCapproved method 10-05.01 (AACC, 2000) in triplicate.

| Determination of the total phenolic content (TPC)
Dried bread sample (3 g) was mixed with 30 mL of 80% ethanol on a magnetic stirrer (SCI FINETECH) for 30 min. Then, the contents were centrifuged at 1792 × g for 15 min (Pars Azma). The supernatant (20 mL) was mixed with 5 mL of hexane, stirred for 15 min, and TA B L E 1 Toast bread formulation.

Sugar 10
Improving agent (DATEM) 1.12 Bakery yeast 4.8 Salt 2 Vegetable oil 1.99 Milk 64 The mixture of oleaster (OL) and black cumin (BC) flours 1.5-2.5 then centrifuged for 20 min. The obtained supernatant, which contained hexane and fat, was gently separated by a syringe. The remaining solution was used to measure the concentration of phenolic compounds and evaluate the antioxidant activity (for each sample with three replicates). The total phenol content was determined according to the Folin-Ciocalteu method (Bourekoua et al., 2018). One milliliter of each extracted solution was mixed with 0.25 mL of Folin-Ciocalteu reagent and 10 mL of distilled water on a magnetic stirrer for 5 min. Then, 2 mL of sodium carbonate 7.5% (w/v) was added to the mixture and incubated in the dark for 60 min. The absorbance was measured using a Ultraviolet-Visible spectrophotometer (SU-6100-Philler Scientific) at 750 nm. The result was expressed as mg GAE/g d.m.

| Antioxidant activity
The antioxidant activity of samples was determined using DPPH free radical scavenging method in triplicate (Piechowiak et al., 2020). The extracted solution (0.1 mL) containing phenolic compounds (from the total phenolic determination step) was mixed with 3.9 mL of 0.1 mM DPPH and incubated in the dark for 30 min at room temperature. Then, the absorbance was measured by a Ultraviolet-Visible spectrophotometer (SU-6100-Philler Scientific) at 510 nm.

| Texture analysis
The texture profile analysis of bread crumb samples was determined using a texture analyzer (CT3 10K). The test was performed on the first, fourth, and sixth days after baking. The center of the bread was sliced into 2 × 2 × 2 cm cubes and compressed to 40% of the initial height at a testing speed of 1 mm/s with a 3.0 s of delay between the first and second compressions. Hardness, springiness, cohesiveness, and chewiness were measured on six repetitions (Bourekoua et al., 2018).
where ΔL, Δa, and Δb are the lightness, redness, and yellowness differences of samples, respectively (2) C * = √ a * 2 + b * 2 TA B L E 2 Physiochemical composition and antioxidant characteristics of oleaster flour (OL), black cumin flour (BC), and wheat flour (WF). Note: Results are presented as a mean value ± SD, the same superscript letters are not significant in the same column (p > .05).

| Microstructure
The microstructure was evaluated using scanning electron microscopy (SEM; MIRA3-TESCAN). After drying with a freeze dryer (Christ alpha 1-2 D plus), the bread sample was placed on a metal stand and covered with gold under vacuum by a desk sputter coater.

| Sensory evaluation
Sensory evaluation was performed by 30 panelists (15 women and 15 men) using a nine-point hedonic scale for taste, odor, after-taste, color, texture, and overall acceptance. Bread samples with a threedigit code were randomly provided to the panelists. The evaluation was done from strong dislike to extremely like, which were scored from 1 to 9, respectively (Mikulec et al., 2020).

| Statistical analysis
The statistical analysis of the results was conducted using MINITAB16 software. The data were analyzed using a one-way analysis of variance. Means were compared using Tukey's test with a significance level of p < .05.

| Flour characteristics
The physicochemical and antioxidant characteristics of the three flours used in this study are represented in Table 2

| Physicochemical properties of breads
The physicochemical properties of bread samples are shown in Table 3. There was no significant difference in moisture content (p < .05). Despite the high amount of fiber in OL (Öztürk et al., 2018) and In the current study, OL and BC flours increased the ash content (Table 3;  BC increased the ash content of biscuits and bread, respectively. BC flour contains various minerals, including iron, manganese, copper, magnesium, and zinc (Mamun & Absar, 2018). Oleaster also contains calcium, magnesium, potassium, iron, and manganese (Çakmakçı et al., 2015). The results of the specific volume (3.86-4.34 cm 3 /g) in Table 3 demonstrate no significant difference (p > .05) among samples.

Disulfide bonds from sulfur amino acids (methionine and cysteine)
of flour improve the gluten quality and help to the better leavening of bread dough (Tao et al., 2018). The average content of sulfur amino acids in BC, OL, and wheat flour has been reported 0.5, 0.83, and 2.83 mg/g, respectively (Artikova et al., 2020;Kabir et al., 2019;Litwinek et al., 2013). In the present study, the results of specific volume indicate that probably the sulfur amino acids were not able to strengthen the gluten network enough to TA B L E 3 Physicochemical properties of bread samples. increase the specific volume significantly. According to the study conducted by Sarraf et al. (2017), there was no significant difference in the volume of donuts adding OL flour up to 3%, and utilizing more amounts of OL flour decreased the volume. There is also a report about a decrease in the specific volume of glutenfree bread by partial substitution of rice flour with BC and sesame flours in higher amounts than concentrations used in the present study (Al-Subhi, 2014). Hence, using low flour concentrations (below 3%) in the present study did not cause a significant difference in the specific volume of bread samples.
The OL and BC significantly increased the protein content of bread samples (p < .05). According to Nezamdoost-sani et al. (2018), the protein decreased as the OL flour increased in lavash bread.
In the present study, a higher amount of protein in BC flour compared to that of OL flour (Table 2)  An increase in the percentage of OL and BC flours caused a significant decrease in iron and a significant increase in calcium (p < .05; Table 3). The samples containing 1% and more OL and BC flours had lower iron content. It seems that it is due to the lower iron content of OL flour than wheat flour (Table 2). An increase in the calcium content of bread from 363.9 mg/kg in control to 467.65-600.41 mg/ kg (in T2-T4) was observed with the increase in the concentrations of OL and BC flours. The amount of calcium in OL (526.8 mg/kg) and BC flours (5149.90 mg/kg) was much higher than the wheat flour (0.1 mg/kg; Table 2). BC flour contains high amounts of calcium (Mamun & Absar, 2018). The calcium in BC flour in the present study was nine times more than that of OL flour. Hence, an increase in the calcium content of toast bread was more related to the presence of BC flour. Figure 1 depicts the total phenolic content of bread samples.

| Total phenolic compounds
As the OL and BC content increased, the TPC in toast bread in-

| Antioxidant activity
The highest antioxidant activity (DPPH scavenging capacity) was ob-

| Texture profile analysis
The results of the texture analysis are shown in Note: T 1 : Control, T 2 : 0.75% OL flour + 0.75% BC flour, T 3 : 1% OL flour + 1% BC flour, T 4 : 1.25% OL + 1.25% BC flour. Results are presented as a mean value ± SD, the different superscript letters are significant in the same column (p < .05).

TA B L E 4 Changes in texture
parameters of breads containing a mixture of oleaster and BC flours during storage. ing parameters on the hardness of its texture. The bread with a smaller volume has a more compact texture and is harder (Mikulec et al., 2019). In this study, the hardness of each bread sample increased significantly during 6 days of storage due to staling (p < .05). On the sixth day, the bread samples containing 1.5% and 2.5% OL and BC flours had higher hardness than the control.

TA B L E 5 Color values of breads containing a mixture of oleaster and BC flours.
However, no significant difference was observed between the control (2.96 N) and bread containing a 2% BC and OL mixture (3.45 N).
Partial substitution of wheat with a gluten-free flour especially in high amounts increases the hardness by diluting the gluten net-  Table 4).

| Color
The results of the effect of using OL and BC in bread formulation are shown in Table 5 is probably due to the low concentrations of OL and BC. If ∆E is less than 1, the color difference between the sample and the control cannot be seen by the eye. In values between 1 and 3, the existing color difference will not be easily seen, but if it is higher than 3, the color difference will be remarkable (Mikulec et al., 2019). The chroma value which indicates the color intensity was significantly lower in samples containing OL and BC flours (p < .05). It seems that it is related to a significant decrease in the yellowness compared to the control. Çakmakçı et al. (2015) also declared that the addition of OL flour to ice cream reduced the chroma.

| Microstructure
The microstructures of the control (A) and T3 sample (B) are shown in

| Sensory evaluation
The sensory evaluation results in Figure 4 indicate no significant difference between the evaluated characteristics (p > .05), except for color. The color score decreased significantly by increasing the amount of OL and BC flours (p < .05). These results were in accordance with the sensory evaluation results of Nezamdoost-sani et al. (2018) and Osman et al. (2015). Since OL has a specific astringency and BC may also have a specific aftertaste, we also evaluated the aftertaste in the sensory analysis. As shown in Figure 4, the aftertaste of none of the OL and BC-containing samples was significantly different from the control. The reason seems to be the addition of relatively small amounts of OL and BC flour in the formulation of toast bread. Besides, it should be noted that the overall acceptability of OL-and BC-containing bread samples was satisfying.

| CON CLUS ION
Based on the obtained results, developing a functional toast bread with suitable physicochemical characteristics is possible.
Incorporating the combination of oleaster and BC flours improved the nutritional quality of bread. The amount of protein, calcium, and antioxidant properties increased significantly, particularly in T3 and T4 samples. The sensory evaluation results were acceptable, and the proper concentration for the simultaneous use of oleaster and BC flour was 2% in toast bread.

ACK N OWLED G M ENTS
We acknowledge from cereal research and pilot plant center of Tehran Medical Sciences of Islamic Azad University, Tehran, Iran for providing the bread production and analysis facilities.

FU N D I N G I N FO R M ATI O N
This research received no specific grant from any funding source.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no conflict of interest in this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

E TH I C S S TATEM ENT
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.