2.1. Research design
The study was done 2x3x3 factorial design comprising white lupine bean from 2 soaking conditions, 3 roasting temperatures, and blended with 3 categories of supplementation of wheat flour. All white lupine bean was prepared in duplicate/triplicate following a randomized completely block design (RCBD). Using this design the effect of blending ratios and processing were the interaction of factors on proximate compositions, minerals content, and sensory evaluation of bread was investigated. Upper and lower levels of variables were selected based on the different composite to wheat flours (13, 14).
2.2. Study area
This study was carried out for breadmaking was done at Debre Markos University where the whole apparatuses and chemicals available there, to get a good product.
2.3. Wheat flour preparation
Wheat grain sample was purchased from the local market and the sample was graded, sorted, and cleaned manually to remove foreign matters, immature and damaged. They cleaned 15 kg of whole wheat grain was milled (Retsch, Cyclon Miller-Twister) and sieved to pass a 60µm sieve. The flour samples were packed in a sack and stored at room temperature.
2.4. White lupine bean preparation
A white lupine bean sample was purchased from the local market and the sample was graded, sorted, and cleaned manually to remove foreign matters, immature and damaged. They cleaned 5kg of the sample was roasted at 120,130 and 140 ºC by using an electric oven and lasted for about 12 minutes. Then it was allowed to cool for about 10minute at room temperature and then soaked in a bucket of tap water (1:10, lupine: tap water) for 10 and 15days, after each 8hours changed the water. The hull of beans was taken off by pressing on the bean with hands finger, then washed with tap water, and dried at 60ºC by using an electric oven. They dried, milled with (Retsch, Cyclon Miller-Twister) grinder and sieved to pass a 60µm sieve, stored in sealable polyethylene bags in plastic, and stored at room temperature before analysis and making baked food (15).
2.5. Flour blending
Wheat and processed white lupine flour were blended in the percentage of 95:5, 90:10, and 85:15, and 100% of wheat flour as control with each sample weighed out into different places using a weighing balance. The blends were homogenized manually.
2.6. Breadmaking
The bread samples were produced in batches by mixing and kneading manually each of the above flour blends with the ingredients using a stainless-steel bowel. Ingredients used for making control bread samples were included 500gm of wheat flour with 4gm of salt, 4gm of yeast, and 300ml of water. The supplemented bread was prepared using white lupine flour at 5, 10, and 15%. After thorough kneading in each case, the dough was allowed to ferment and develop for 15min before being knocked back and then molded into a cylindrical shape. After molding in each case, the dough was then placed in a well-oiled baking pan where it was proofed for 40minutes at room temperature before it was baked in a cabinet oven pre-heated. All different bread formulas were baked at 250°C for 10-15min then cooled and stored at room temperature for chemical analysis after organoleptic evaluation.
2.7. Proximate analysis
The American Association of Cereal Chemists (16) procedure was used to determine the proximate compositions (crude protein, crude fat, crude fiber, and total ash) of the bread samples made from wheat-lupine flour. Protein concentration was determined by Kjeldahl method by Digester, Automatic Distillation and Titration System (VELP- SCIENTIFICA, UDK-119), ash was quantified by incineration of the samples in a muffle furnace, total fat was extracted with petroleum ether using a Soxhlet apparatus (LOBA CHME PVT.LTD), and crude fiber was quantified by the enzyme-gravimetric method. All analyses were carried out thrice.
2.8. Mineral analysis
For mineral determination, the American Association of Cereal Chemists (16) procedure was used to determine minerals content. Calcium, zinc, and iron were determined using a UV-VIS spectrophotometer.
2.9. Anti-nutritional factor analysis (total alkaloids)
The alkaloid content was determined gravimetrically by the method of (17). A sample (0.5g) was dispersed in 10mL of 10% acetic acid solution in ethanol. The dispersed solution was well shaken and then allowed to stand for about 4hrs before it was filtered. The filtrate was evaporated to one-quarter of its original volume. Concentrated NH4OH was added dropwise to precipitate the alkaloids. The precipitate was filtered into pre-weight filter paper and washed with 1% NH4OH solution. The precipitate in the filter paper was dried in the oven at 60 ºC for 30 min and reweight until it attains constant mass. Alkaloid was expressed in percentage.
2.10. Sensory evaluation
Sensory evaluation of white lupine bread taste, color, flavor, appearance, and overall acceptance as the method described by using a 5-points hedonic scale method (18). Bread samples were evaluated organoleptically by a panel of 20 panelists. On the day of evaluation, bread was placed on small plates and labeled with three digital random codes. Panelists were provided with distilled water and unsalted crackers to clean their palates between samples. The bread samples were presented in random order and panelists were asked to rate their assessment of taste, color flavor, appearance, and overall acceptance on a 5-point hedonic scale (ranging from 5 = like very much to 1 = disliked very much). The raw scores were assembled and statistically analyzed using appropriate software.
2.11. Statistical analysis
All experiments were replicated at least thrice. All data were statistically analyzed by three-way ANOVA (Analysis of Variance) using SPSS version 25 and expressed as mean values ± standard deviations. Duncan’s multiple range tests were used to determine significant differences. Differences were regarded as significant at 95% (p < 0.05).