**2.3 Estimation of levels of oxymyoglobin Mb (Fe2+) and metamyoglobin MetMb (Fe3+) of fresh meat emulsions**

The estimation of the levels of oxymyoglobin Mb (Fe2+) and metamyoglobin MetMb (Fe3+) in fresh meat emulsions was based on the method of Ning et al. [21] and Carlez et al. [22] with some modifications. Specifically, 2 g of the sample was homogenized with 20 ml of Na/K phosphate buffer at a concentration of 0.04 mol/L in a 50-ml Falcon flask in an Ultraturrax homogenizer for 20 s at 10,800 rpm. The samples were then centrifuged at 7000 rpm for 5 min after a total of 1 h in an ice basin, with the addition of aluminum foil externally to prevent oxidation. Immediately after filtration with whatman #1 filter type, the samples were supplemented with 25 ml of the same buffer. The absorbance of the samples at the following wavelengths was then measured: 525, 545, 565, and 572 nm, in a spectrophotometer (UV-1800, Shimadzu Co., Kyoto, Japan). The percentages of oxymyoglobin and metamyoglobin are calculated from the following equations [25]:

$$\text{MkO}\_2\text{\%} = \left( \text{O.882} \ast R\_1 - 1.2\text{\%}7 \ast R\_2 + \text{O.809} \ast R\_3 - 0.361 \right) \ast 100 \tag{1}$$

*Incorporation of Trigonella Foenum-Graecum Seed Powder in Nitrite-Free Meat Emulsion… DOI: http://dx.doi.org/10.5772/intechopen.104759*

$$\text{MetMb} \text{@} = \left(-2.541 \ast R\_1 + 0.777 \ast R\_2 + 0.800 \ast R\_3 + 1.098\right) \ast 100 \tag{2}$$

where factors *R*1, *R*2, and *R*3 are the absorption ratios and are calculated as follows:

$$R\_1 = \frac{A572}{A525}, R\_2 = \frac{A565}{A525}, R\_3 = \frac{A545}{A525}. \tag{3}$$

### **2.4 Deffating**

The defatting of T. foenum-graecum seeds was carried out based on the method of lipid extraction [26]. In this method, for the defatting of raw seeds, a Soxhlet apparatus was used utilizing diethylether as solvent. More specifically, a certain amount of T. foenum-graecum seed powder was weighed and placed in a cellulose extraction cartridge. The cartridge was plugged with cotton wool and then placed in the Soxhlet chamber, which was fitted to a pretared distillation flask containing dielthylether and two to three glass regulators. After extraction for 2 h in 50–60°C, the cartridge allowed to cool, and then, the cartridges were placed in an oven at 105°C for 12 h, following cooling in a desiccator and weighing. Placing in the oven for specific time, cooling in a desiccator and weighing was repeated until the difference between two consecutive weights was smaller than 2 mg.

### **2.5 Determination of color parameters**

Instrumental color was conducted by taking a direct reading of raw meat emulsions with a colorimeter (Konica Minolta CR-400, USA). Standard observer was 2° (Closely matches CIE 1931 Standard Observer [(x2λ, yλ, zλ)]); 8-mm diameter circular aperture and d/0 (D65,Diffuse illumination/0 < ° viewing angle) illuminate were used. The CIE-*L*\*, *a*\*, and *b*\* parameters were evaluated according to the methodology proposed by the American Meat Science Association (AMSA 2012). The calibration of colorimeter performed using a white plate, *L*\* = 97.58, *a*\* = 0.03, and *b*\* = 1.08. For the color analysis of raw meat emulsions, 10 g of each sample was placed on a 9-cm diameter petri dish at 1-cm thickness. The average of nine readings was reported. All tests were performed at room temperature on the second day after manufacture and then 5 and 7 days after manufacturing.

#### **2.6 Statistical analysis**

All statistical analyses were performed with SPSS (Version 23, IBM, USA). The homogeneity of variances was tested with the Levene test in the case of non-normal data with SPSS. The normality of the residuals was tested using UNIVARIATE of SPSS, and consideration was given to the Shapiro–Wilk test for normality. Once it was determined that the assumptions of the analysis of variance (ANOVA) were met for these data, the GLM procedure of SAS with a fixed effect of treatment and a random effect of replication was used for the statistical determination of all variables. Tukey's test *(P* < 0.05*)* was used to determine the differences between the treatment means. Least squares means were separated using a single-degree-of-freedom estimate statement to determine the difference between meaningful comparisons, which included Starch + NaNO2 versus Dtfg (3% inclusion). Differences were considered statistically different at *P <* 0.05*.* This experiment was completed in its entirety three times.
