Dark RKB was collected from the Bandar Bazar market of Sylhet, Bangladesh. At first, insect or physically damaged beans were removed visually. Sorted beans were treated by the sun-drying process to lower the moisture content. Then the dried beans were grounded from a local grinder house. The RKB powder was sieved and kept in an airtight container for further analyzing process.

RKB powder was firstly mixed with market wheat flour at various level of 0% (control), 10%, 20%, and 30%. Then 60 g paste made from the mixture of 504 g of powdered sugar, 30 g of milk powder, 252 g refined oil and 12 g of sodium bi-carbonate was added to mixed flour. 34 ml of distilled water was also put into each mixture. Thereafter, the mixture was rubbed well to mix properly and avoid ball formation. The dough was given a shape of biscuit by hand with a thickness of 3.5 mm, and baked in oven at 160˚C for 20 min. Then the biscuits were cooled for 30 min and packed in airtight food grade plastic bags for further analysis.

Chemical composition and functional properties of RKB and proximate compositions of biscuits after the incorporation of bean powder were analyzed. Protein content was measured according to the Official methods of Analysis [6], where factor 6.25 was used for the conversion of nitrogen to protein. The method described by [7] was used for the determination of fat content. Moisture and ash content were determined using Approved Methods of the American Association of Cereal Chemists [8]. Difference method by [9] was used to determine the total carbohydrate content. Energy value was calculated by multiplying carbohydrate, protein, and fat by their factorial values as follows:

Energy value ( kcal / 100   g ) = [ ( carbohydrate % × 4 ) + ( protein % × 4 ) + ( crude fat % × 9 ) ]

Swelling capacity was determined according to the method described by [10] with some modifications. Water absorption capacity and oil holding capacity were measured using the modified method reported by [11]. And bulk density was determined following the method of [12].

Folin–Ciocalteu method [13] was followed to determine total phenolic contents using UV visible spectrophotometer. 1.0 mL aliquot sample was mixed to 1.5 mL of deionized water and 0.5 mL of 0.1 M Folin–Ciocalteu reagent, and were mixed methodically. After 1 min, 1.0 mL of 20% sodium carbonate solution was added and the mixture was again stirred thoroughly. The control for spectrometry analysis contained all chemical reagents excluding the sample. The sample was kept in incubation at 37˚C for 30 min and the absorbance was taken at 750 nm. The amount of phenolic contents in the sample was calculated using the standard calibration curve derived from known concentrations of gallic acid and the total phenolics were estimated as gallic acid equivalent (GAE).

Calculation of total polyphenol content:

y = 0.692 x + 0.344

where, y = absorbance found by the sample tested and; x = concentration found from the standard curve.

Antioxidant activity of bean powder and biscuit samples were measured at five different concentrations (0.016 mg/mL, 0.08 mg/mL, 0.4 mg/mL, 2 mg/mL and 10 mg/mL).

Our study followed the method described by [14] to assess the DPPH scavenging effects of samples. Briefy, 2.0 mL aliquot of the test sample (in methanol) and 2.0 mL of 0.16 mM DPPH methanolic solution were mixed and vortexed for 1 min, and then left to stay at room temperature for 30 min in the dark. The absorbance was taken at 517 nm. The ability to scavenge the DPPH radical was calculated using the following equation:

Scavenging effect (%) = [1 − (A sample − A sample blank/A control)] × 100

where, A control is the absorbance of the control (DPPH solution without sample); A sample is the absorbance of the test sample (DPPH solution plus test sample) and A sample blank is an absorbance of the sample only (sample without DPPH solution). Ascorbic acid was used as positive control.

All the data were presented in this study as mean value with standard deviation (SD). A significant difference among samples was measured using Analysis of variance (p < 0.05). To analyze the data SPSS-17 statistical software (SPSS Inc., Chicago, IL, USA) was used.

The nutritional composition of RKB powder is shown in Table 1. The moisture content of bean powder was 8.38%. It has been found that bean powder contains a high amount of protein (26.25%) and good amount of ash (3.27%), but very low amount of fat (2.40%). In addition, moisture and carbohydrate content of bean powder were 59.70% and 8.38%, respectively. The results were similar to the study [15] and [16].

*Values are mean ± standard deviation of three replicates.

The functional properties such as water absorption capacity, oil absorption capacity, swelling capacity and bulk density were analyzed shown in Table 2. The water absorption capacity and oil holding capacity of bean powder was 4.97 g/g and 4.50 g/g, respectively, which is similar to the study by [16]. The factors affecting water absorption capacity and oil holding capacity is protein content in food products [12], because protein has both hydrophilic and hydrophobic properties to interact with water and oil in food. The absorption or retention of water or oil of a flour has the ability to improve texture and mouth feel, and enhance the flavor [17]. The study indicated that swelling power of RKB is 9.6 g/g that was in accordance with similar findings [16]. High swelling power indicates that ingredients can be added to enhance the quality of baked goods. However the bulk density of the bean powder was found as 0.74 g/ml, where [18] reported the bulk density of RKB as 0.41 g/ml.

Nutritional compositions of biscuits partially replaced with bean flour at various levels (0, 10, 20, and 30) are presented in Table 3. The result shows that protein and ash content of control biscuits was 13.23% and 0.40%, respectively. When the level of RKB powder in the biscuits increased from 10% to 30%, the protein and ash content also increased significantly (p ≤ 0.05) ranged from 15.31% to 20.80% and from 0.80% to 1.90%, respectively. The similar trend was found in a study conducted by [19]. On the other hand, with increasing the level of RKB

*Values are mean ± standard deviation of three replicates.

*Values are mean ± standard deviation of three replicates.

from 0% to 30%, fat content did not change notably ranged from 12.23% - 10.80%, but carbohydrate contents reduced extensively from 72% - 63.89%, almost similar to the study by [19]. As a result, the energy value with the addition of kidney bean powder decreased significantly from 1620.73 to 1594.68 kJ/100g, while control biscuit possessed the highest energy (1628.92 kJ/100g). However, the moisture content did not show any significant changes with increasing the level of fortification.

The results from Table 4 showed that total phenolic content of RKB was found 14.14 mg GAE/g which is in the range of TPC (5.87 - 14.14 mg GAE/g) for the common beans reported by ( [20] [21] ). But another study [22] showed that the TPC of twelve Italian cultivars of P. vulgaris ranged from 1.17 - 4.40 mg GAE/g. It probably varied because of the extraction and determination method of phenolic contents and it also could be the regional variation of the beans species. The TPC of wheat flour biscuit was found as 10.31 mg GAE/g, which was similar to the study conducted by [23] who found that TPC of control was 11.37 mg GAE/g. Increasing the level of RKB powder from 10% - 30% also increased the TPC from 11.19 - 12.50 mg GAE/g, similar trend was found in the study reported by [24], where prickly pear peel and potato peel powder was incorporated with wheat flour.

Figure 1 shows DPPH-radical scavenging activity (DRSA) of raw RKB powder, control, 10%, 20%, 30% RKB fortified biscuits and a standard ascorbic acid at various concentration (from 0.016 - 10 mg/ml). The results reveal that ascorbic acid showed highest antioxidant activity ranged from 89.37% - 93.63% at various concentration ranged from 0.016 - 10 mg/ml, followed by raw RKB, control, 10%, 20%, and 30% supplementation. Raw bean powder showed significantly (p < 0.05) higher antioxidant activity than other fortified biscuits, which was reflected in the IC₅₀ value of 0.0210 mg/ml (Table 5). Another study by [3] found that at 0.4 mg/ml concentration DPH-1 antioxidant activity of RKB powder was

*Values are mean ± standard deviation. Values in column with different letter superscripts are significantly different at p ≤ 0.05.

*Values are mean ± standard deviation. Values in column with different letter superscripts are significantly different at p ≤ 0.05.

81.41%, where this study shows slightly different capacity (71.11%) because of extraction and determination method. Compared to the ascorbic acid, the samples showed significantly lower (p < 0.05) DPPH-radical scavenging activity. Loss of antioxidant activity was observed during baking process. Polyphenol compounds are liable for showing antioxidant activity [25]. So for leaching out of polyphenols, antioxidant activity might be decreased significantly (p < 0.05). Antioxidant activity increased with the increasing concentration of bean powder, similar study was found in another study [26] for cocoa powder incorporation in biscuits.

The cakes were evaluated for their diameter, thickness, weight and spread ratio after and before the incorporation of RKB powder shown in Table 6. The control cake contained the highest spread ratio (8.92) and the spread ratio decreased from 8.25 - 7.86 with the increase of fortification level from 10% - 30%. The decrease in spread ratio was observed for the dilution of gluten and less availability of water for gluten hydration reported by [27]. The decrease in the spread ratio was due to the decrease in the diameter (from 5.21 - 5.20 cm) and increase in the thickness of the cake (from 0.53 - 0.57 cm). Similar results had been reported for biscuits with cocoa powder [26]. On the other hand, the weight of the RKB fortified biscuits increased with the fortification ranged from 4.25 - 6.03 g.

The biscuits added with 10%, 20% and 30% bean powder were subjected to sensory analysis shown in Figure 2. Control biscuits got satisfactory score in color (6.27), aroma (6.45) taste (6.27) and overall acceptability (6.18), but 10% supplementation superseded the control in color by 6.64, aroma by 6.47, taste by 6.89, and overall acceptability by 6.34. The formulation with 20% and 30% kidney bean did not get satisfactory score in sensory evaluation. Color and aroma depends on reducing sugar and amino acids (protein content). Reducing sugar and amino acids are responsible for the Malliard reaction that gives desirable brown color and flavor of foods [28].