Preparation of black cumin extract: Superior quality of black cumin (Nigella sativa) was brought from the local market of Barishal district of Bangladesh. The fresh black cumin seeds were cleaned and washed using water and air-dried using a fan. Then the seeds were blended (IKA T25 digital Ultra-Turrax, Germany) with the addition of 9:1 (water: black cumin) ratio distilled water and filtered through Whatman® Grade 1 filter paper. The filtrate was frozen in a blast freezer at –20 ºC for 30 min and dried in a freeze dryer at –46 ºC. The extract from black cumin seed were shaded from light and stored at 41 ºC prior to use. Food grade BHA was used as a synthetic antioxidant and compared against the natural antioxidant extract. The five treatment groups were control (T0), 0.1% BHA (T1), 0.1%, 0.2% and 0.3% black cumin extract added beef patties as (T2), (T3) and (T4), respectively.
Sample collection and preparation: Beef was procured from the local market just after slaughtered and brought immediately to the “Animal Products and By-products Technology Laboratory” at Patuakhali Science and Technology University, Barishal-8210, Bangladesh. Then beef sample was deboned, trimmed of separable fat and connective tissue and chilled in a refrigerator at 4 ± 1 ºC for about 6 h and frozen at –18 ºC for further use. After that beef was cut into small pieces after partial thawing for 6 h at 4 ± 1 ºC. Then beef was doubled minced (10 mm plate followed by 8 mm plate) with a meat mincer (Tallers Ramon Model P-22, Barcelona). This ground beef was used in patties formulation.
Beef patties preparation: Beef patties (control) were formulated using 70% beef, 3% refined wheat flour, 5% condiment mix, 7% refined vegetable oil (soybean oil), 1.2% refined salt, 1.3% dry spice mix, 12% ice flakes, 0.5% sodium tripolyphosphate and 150 ppm sodium nitrite. Minced beef along with all other ingredients were mixed thoroughly in a bowl chopper (Seydelmann K20 Ras, Germany) to prepare emulsion for each treatment. 2 kg beef patties were formulated for each treatment. In treated formulation 0.1% BHA, 0.1%, 0.2% and 0.3% black cumin extract was added respectively according to weight basis. About 75 g of the emulsion was molded in a Petri dish (15 mm height and 75 mm diameter) to form patties and was cooked in a preheated electric oven (EO-60NK-3, SHARP Electric Oven, Bangladesh) at 180 ± 5 ºC for 15 min. Furthermore, patties were turned and allowed to cook for 10 min more till internal temperature reached about 75 ± 2 ºC recorded by a digital probe thermometer (TLC 700, ebro, Germany). Then they were allowed to cool at room temperature. The patties were aerobically packaged in a zippered bag after cooling to room temperature and stored at 4 ± 1 ºC for 15 days and analyzed at 0, 5th, 10th, and 15th day of storage for sensory attributes, pH, free fatty acids (FFA), peroxide value (POV) and thiobarbituric acid reactive substances (TBARS). Each treatment had three replications.
Sensory evaluation: Sensory qualities of beef patties were judged by a trained 6-member panel at 0 day and repeated at 5th, 10th and 15th days of storage. The different state of flavor, color, tenderness, juiciness and overall acceptability was measured in a 5-point scale. Sensory scores were 5 for excellent, 4 for very good, 3 for good, 2 for fair and 1 for poor. The judge's panel was selected among departmental staff and postgraduate students and trained according to the AMSA guidelines. Panelists have to present in an orientation program to become familiarized with the sensory quality scale. Sensory evaluation of patties was carried out in individual booths under controlled conditions of light, temperature and humidity. All samples were served in petri dishes.
pH measurement: The pH values of cooked beef patties were measured using a benchtop digital pH meter (CON60; Trans- Wiggens) from homogenate. A mixture of 5 g blended beef patties and 10 ml distilled water used for homogenate preparation.
Biochemical analysis: The biochemical properties as like as Free Fatty Acid (FFA), Peroxide Value (POV), Thiobarbituric Acid value (TBARS) were analyzed to know the biochemical quality of beef patties. FFA value, Peroxide value (POV) and Thiobarbituric Acid value (TBARS) was measured according to Rukunudin et al.; Sallam et al. and Schmedes et al. methods respectively.
Statistical analysis: The projected research experiment was a factorial experiment with two factors A (Treatments) and B (Days of Intervals), where all analyses were run in triplicate. Data were statistically analyzed using SAS Statistical Discovery software, NC, USA. Tukey's HSD test was done to determine the significance of differences among treatments' means.