Antimicrobial potential and phyto-physio-chemical characterization of brans from wheat, oat, and rice

2.1 Collection of raw materials

Raw materials were procured from the local markets of Dera Ismail khan. Materials comprised of wheat flour, Sugar, yeast, salt, stabilizers, shortenings, preservatives; wheat, oat and rice bran; and garlic.

2.2 Bread preparation

Bread preparation was done according to procedures defined in AACC (2000).

2.3 Proximate analysis of flour

Proximate analysis of flour viz. Moisture, fat, ash, iron, Zn, protein, crude fiber and caloric value of flour was analyzed by the methods of AACC (2000).

2.4 Dough rheological studies

The rheological properties of dough with various proposed treatments were assessed using Brabender Farinograph and Amylograph methods following the protocol established by the American Association of Cereal Chemists (AACC, 2000). The findings of Brabender Farinograph analysis were in compliance with ICC standard No.115/1, where a 300 g flour dough with a 14% mixture was combined with 500 BU water to achieve optimal dough consistency. The following attributes were evaluated: dough development time, dough stability, degree of dough softening, and water absorption (%). The studies of Brabender Amylograph were conducted in accordance with ICC standard No.126/1, 16, where a mixture of 14% distilled water and 80 g flour was homogenized with a glass stick and then transferred to the Amylograph dish at 25 °C. The temperature was then increased by 1.5 °C per minute until the desired viscosity was achieved.

2.5 Chemical analysis of bread

For an hour the bread was cooled in an ambient temperature and then put on instrumental measurements and sensory tests. Slices of 12.5 mm width were made from one part of bread, mechanically by a bread slicer. Tests of proximate constituents via mineral content, protein, crude fat, antioxidant activity, phenolic content, water activity, colorimetric analysis and texture profile analyses (TPA) were done using central slice of bread by the methods of AACC (2000). After cooling for one-hour in open air, bread was preceded by sensory tests and instrumental measurements. By a bread slicer, bread was cut mechanically into slices of 12.5 mm thickness. The mid slice of bread was used for the analytical assessment of proximate composition, viz. minerals content, protein, crude fat, antioxidant activity, phenolic content, water activity, colorimetric analysis and texture profile analyses (TPA) by the methods of AACC (2000).

2.6 Chemical attributes

Analysis of moisture, Ash, crude protein, Crude fats, and fiber was made by the procedures as depicted in AOAC (2005).

2.7 Moisture determination

Samples moisture was determined by methods of AOAC (2005) No. 925.10. Moisture of samples was determined by AOAC methods (2005) No. 925.10.

2.8 Procedure

Washed dishes with moisture were taken and afterward gauged. Two grams of the test said something about every dish and covered the top. The dishes were set open in a hot air boiler for 130 °C for 60 min (one hr. was viewed as when temperature reach to 130 °C). Samples were taken off from broiler and covered with top and placed in desiccators for cooling approximately 25 min. Afterward gauged the sample. Following equation was utilized to register the level of dampness. $$\mathrm{M}\mathrm{o}\mathrm{i}\mathrm{s}\mathrm{t}\mathrm{u}\mathrm{r}\mathrm{e}(\%)=\frac{\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{t}-\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{w}\mathrm{t}}{\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{w}\mathrm{t}}\times 100$$

2.9 Ash determination

First, a known weight of the sample was placed into a crucible and heated in a muffle furnace at a high temperature until all organic matter was completely burned away. The resulting residue, or ash, was then cooled in a desiccator and weighed. The percentage of ash content in the sample was calculated as the weight of the ash divided by the weight of the original sample, multiplied by 100. The AOAC method No. 923.03 is a standard method used for the determination of ash content in food and agricultural products, and is widely accepted for its accuracy and precision.

2.10 Determination of crude fat

To analyze crude fat content in the sample, the n-hexane extraction method was used in a Buchi extraction system following the guidelines of the AOAC (2005) method No. 203.06. The procedure involves mixing the sample with n-hexane to extract the fat, followed by filtration and evaporation to remove the solvent. The remaining material is then weighed to determine the crude fat content of the sample. This method is widely used for the determination of fat content in food and agricultural products.

2.11 Crude protein determination

The procedure for determining crude protein content involved the use of an Auto Kjeldahl analyzer, following the AOAC (2005) method No. 46.10. The raw materials were subjected to digestion with concentrated sulfuric acid and a catalyst. The resulting ammonium sulfate was then distilled and the ammonia produced was absorbed in a boric acid solution. The amount of nitrogen present in the sample was then calculated using titration and conversion factors. The crude protein content was determined by multiplying the amount of nitrogen by a conversion factor of 6.25.

2.12 Crude fiber determination

To determine the crude fiber content of the sample, the AOAC (2005) method No.926.09 was used. First, a 2-gram sample was taken and placed in a crucible. Then, the crucible was heated over a low flame for 1 h to remove any moisture. After cooling, the sample was treated with 1.25% H₂SO₄ and heated in a boiling water bath for 30 min. It was then filtered through a Gooch crucible with the help of suction. The residue was washed with boiling water, then with ethanol and finally with ether. The crucible was dried at 130 °C for 1 h and then weighed. The residue obtained was then treated with 1.25% NaOH solution and heated in a boiling water bath for 30 min. After cooling, it was filtered through the Gooch crucible and washed with boiling water, ethanol, and ether as before. The crucible was dried at 130 °C for 1 h and then weighed. The difference between the weights of the crucible before and after the two treatments was used to calculate the crude fiber content of the sample. The weight of the residue obtained after the second treatment was subtracted from the weight of the residue obtained after the first treatment, and the difference was multiplied by a correction factor of 0.69. The result was then expressed as a percentage of the original sample weight.

2.13 Shelf-life evaluation

Mold growth on the crumb and crust of breads was assessed using a modified method based on Delacy et al. (1993) through visual observation and counting. Additionally, several analyses were conducted on the bread samples, including antioxidant activity measured by the DPPH method, water activity, color analysis, phenolic content, texture profile analysis (TPA), and sensory evaluation. These analyses were conducted following the protocol established by the American Association of Cereal Chemists (AACC, 2000).

2.14 Determination of antioxidant activity

To conduct hydrophilic ORAC testing, pure mixes were dissolved in a 50 + 50 (v/v) CH3)2CO water blend and diluted with 75 mM potassium phosphate buffer (pH 7.4) for analysis. For plant ingredients, 0.5 g of the powder was weighed and mixed with 20 mL CH3)2CO water (50 + 50, v/v) extraction solvent. The mixture was shaken at 400 rpm on an orbital shaker at 4 °C for 1 h. The extracts were centrifuged at 14,000 rpm for 15 min, and the supernatant was diluted with support solution for analysis. For liquid samples, a 20 mL aliquot was centrifuged for 15 min, and the supernatant was diluted with support solution for analysis. Blood plasma or serum samples were diluted 100 to 200-fold with pH 7.4 phosphate buffer before analysis. To determine the non-protein fraction, protein was removed from plasma using 0.5 N perchloric acid (1 + 1, v/v), and the samples were centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was taken as the serum non-protein fraction and diluted with pH 7.4 phosphate buffer for analysis (see Table 1).

2.15 Determination of phenolic content

To estimate the total phenolic content in samples, the Folin and Ciocalteu's phenol reagent (Folin-C reagent) was used. The samples were first extracted with water using sonication, and then the dried extracts were treated with the Folin-C reagent. The resultant colorimetric reaction was measured at 765 nm and compared with a standard curve produced with gallic acid standard solutions. The results were developed and compared using the Standard Method Performance Requirement established by the Stakeholder Panel on Dietary Supplements (AOAC SMPR 2015.009).

2.16 Statistical analysis

Statistical analysis of data for given parameters was done by using the Analysis of Variance (ANOVA) technique and the Least Significance Difference (LSD) to compare the means according to Steel and Torrie (1980) using the Statistix version 8.1.

3.1 Proximate analysis of raw material

As shown in the Table 2, the three brans have varying levels of moisture content, ash content, fiber content, fat content, protein content, zinc content, and iron content. Wheat bran has the highest protein content (10.1%), while rice bran has the highest fat content (14.72%). Oat bran has the highest fiber content (3.54%), while rice bran has the highest ash content (7.50%). Zinc content is highest in wheat bran (7.30 mg/kg), while oat bran has the highest iron content (13.76 mg/kg). These differences in nutrient composition could affect their potential as sources of functional food ingredients or as antimicrobial agents.

3.2 Moisture content (%)

The moisture content of the samples varied significantly across the treatments. Treatment T0 had the highest moisture content with a value of 25.87%, which was used as the control. The moisture content in the other treatments was lower than that of T0, with treatment T4 showing the most significant reduction in moisture by 16.17%. Treatment T1 also had a significant reduction in moisture by 6.40% compared to T0. Treatment T2 had a reduction in moisture by 9.64%, T3 by 13.66%, T5 by 2.77%, and T6 by 7.54%. The LSD (0.05) value of 0.1935 indicates that the difference in moisture content between any two treatments that are greater than this value can be considered statistically significant. Overall, the results show that the treatments had a significant effect on reducing moisture content compared to the control (T0), with T4 showing the most significant reduction. The moisture present in the dough made showed its nutritional value. Higher the moisture in dough lowers the nutrition (fat, starch, etc.) as supported by the research of Bagheri and Mehdi (2011).

3.3 Ash content (%)

Wheat flour 60%+ Rice bran 40% showed maximum ash contents (0.97%) nearly to wheat flour 60%+ wheat bran 40% (0.94%) as compared to control and others. Ash content gradually increased from 0.66 to 0.94% with an increase in concentration of wheat from 20 to 40% bran. Similar results were found in rice where ash contents increased from 0.68 to 0.97% which increased the concentration of rice bran from 20 to 40%. Also same in the case of oat, ash contents decreased from 0.70to 0.84% which increased its concentration 20–40%. According to proximate analysis wheat, rice and oat have almost similar ash contents 0.62% which were increased in wheat, oat and rice to 0.94%, 0.84% and 0.97%, respectively consistent with the results of Mishra (2017).

3.4 Fat content (%)

The initial value of Fat content in T0 was 4.66, which is considered as 0% change. Treatment T1 had the highest Fat content value of 4.87, showing an increase of 4.50% compared to T0. Treatment T2 showed the least increase of 0.64% compared to T0, with a Fat content value of 4.69. Treatment T3 had a 2.79% increase compared to T0, with a Fat content value of 4.79, while Treatment T4 had a 2.57% increase with a value of 4.78. Treatment T5 showed a 4.92% increase compared to T0, with a Fat content value of 4.89. Finally, Treatment T6 showed the highest percentage increase of 6.85% compared to T0, with a Fat content value of 4.98.

3.5 Fiber content (%)

Maximum fiber contents were recorded in combination of wheat flour 60%+ wheat bran 40% which was 1.92%. While minimum wasfound in T₃ (0.94%) other than control. Results showed similar behaviour for all combinations. In case of wheat,fiber contents increased from 1.07 to 1.92 with increased in wheat bran concentration 20–40% respectively. Similar results were found for oat and rice which increased from 0.94 to 1.36 and 0.99–1.77 respectively with increase in their bran concentration from 20 to 40%. But according to their proximate analysis, fiber contents increased in 30 and 40% of all cereal bran. These results are consistent with the research of Ndala et al. (2019).

3.6 Protein content (%)

The statistical analysis showed significant behaviours among treatments means given in the Table 3. Maximum crude protein found in Wheat flour 60%+ oat bran 40% with 11.94 while minimum was found in Wheat flour 80%+ wheat bran 20% having 10.03% other than control. All the combinations showed different protein values while increasing in the recipe. In case of wheat bran, protein contents increased from 10.03 to 10.63 from 20–40% concentration. While in case of oat it increased from 10.62 to 11.94 with increased in concentration from 20 to 40%. For rice bran, 20% and 40% have the value of 10.87 and 11.44% respectively.

3.7 Zinc contents

Maximum zinc found in Wheat flour 60%+ rice bran 40% with 8.98 while minimum was found in Wheat flour 80%+ oat bran 20% having 2.85% values. All the combinations showed variations in zinc values while increasing the bran concentration in the recipe. In case of wheat, zinc contents increased from 3.70 to 7.15% with increase its concentration from 20 to 40%. Similar behaviour was found in rice, where it increased from 5.94 to 8.98% with increased the level from 20 to 40%. But for oat bran, an increase of 2.85% to 4.56% was seen by increasing its percentage from 20 to 40%.

3.8 Iron contents

The statistical analysis showed significant behaviours among treatments means given in the Table 3. Maximum iron found in Wheat flour 60%+ rice bran 40% with 31.87 while minimum was found in Wheat flour 80%+ oat bran 20% having 22.31 zinc content. All the combinations showed variations in zinc values while increasing the bran concentration in the recipe. Continuous increase in zinc values from 24.32 to 27.70 in wheat and 22.31–23.99 in oat with increase the concentration from 20 to 40% of bran respectively. Likewise, for rice, 26.84% was observed in 20% bran and 31.87 at 40% bran addition as shown by the research of Mishra (2017).

3.9 Bread and dough quality

3.10 Stability

Compared to the control T0, treatments T1 to T5 showed a significant decrease in stability values ranging from −85.27% to −94.57%. Treatment T2 exhibited the lowest stability value with a −94.57% decrease, indicating the least stable among all the treatments. Treatment T6, on the other hand, showed a relatively smaller decrease in stability value of −42.25% compared to the other treatments. It is worth noting that treatment T4 had a stability value of only 1.50c, indicating that it is less stable compared to the control T0. Data revealed that variation is present in the stability as compared to control treatment.

3.11 Moisture

Treatments T1 to T6 did not result in any significant increase or decrease in moisture content compared to the control treatment T0, with only treatment T5 showing a slight increase of 1.55%. However, treatments T1 and T6 showed a decrease in moisture content of 0.77% and 5.04%, respectively.

4.1 L Value

The statistical analysis revealed a significant effect for the treatment on the L value of bread crumbs. L value in control was 65.2 that were reduced to 64.7 by incorporation of 20%wheatbranandto55.4by40%bran. Addition of oat bran and rice bran from 20 to 40% reduced the L Value from 65.4 to 52.3 and 64.9 to 53.0, respectively.

4.2 a* Value

Effect of treatment on a* value was found to be significant as measured statistically. Control bread showed a* value of 1.9 that increased to 3.4 by adding 20% wheat bran and to 5.9 by 40% wheat bran. Supplementation with oat bran increased a* value from 3.1 to 6.9 by 20% and 40%, respectively. Incorporation of rice bran from 20 to 40% increased the * value from 3.2 to 6.7.

4.3 b* Value

Addition of wheat, oat and rice bran in different percentages significantly affected the b* value of bread samples. b* value increased from 21.3 to 24.6 by supplementation of 20% and 40% wheat bran respectively. Addition of oat and rice bran increased the b* value from 21.5 to 24.9 and 21.6 to 25.4 respectively (Table 5).

4.4 Mould count

The mould count on bread crumb and crust was analysed during storage period of 96 h. It was revealed from statistical analysis that treatments had non-significant effect on mould count on bread crumb whilst storage period showed momentous rise in mould count. The mould count ranged from 1.41 to 2.15 (log CFU/g) by adding 20 to 40% wheat bran. It increased to 3.21 and3.87 with the addition of 20% and 40% wheat bran respectively. Likewise, addition of oat and rice bran gave mould count around 2.89, 2.83 (log CFU/g) and 2.58, 1.75 (log CFU/g) for 20 to 40% bran addition, respectively. It increased momentously during storage period of 96 h (Table 4) Likewise mould growth on bread crust increased with increase in storage interval.