**3.5 Proteolytic activity**

The proteolysis of each of the FB was determined in triplicate based on the reaction of the free primary amines (NH3) with O-phthaldialdehyde (OPA) and b-mercaptoethanol, according to the Church method [26]. The OPA reagent was prepared as follows: 25 mL of 100 mM sodium tetraborate, 2.5 mL of 20% sodium dodecyl sulfate (SDS), 40 mg of OPA in 1 mL of methanol, 100 mL of b-mercaptoethanol, and capacity to 50 mL with tridestilated water. For the readings, 100 mL of each sample was taken and mixed with 2 mL of the OPA reagent by inversion of the

quartz cell, with 2 minutes of incubation at room temperature and inside the equipment to avoid exposure to light; the absorbance in a spectrophotometer (Genesys 10S UV-Visible, Thermo, USA) at a wavelength of 340 nm was read. The degree of proteolysis was determined as the difference between the proteolytic activity in the treatments (beverages fermented with probiotics) and the control samples (fermented beverages without probiotics).

#### **3.6 Total peptide concentration**

The concentration of the peptides contained in each of the FB was determined in triplicate using the Bradford method [27]. This is based on the reaction of the proteins with the bright blue dye of Comassie G-250, to form a colorful compound that absorbs strongly at 595 nm. For which, a calibration curve was made using eight bovine serum albumin (BSA) standards; at concentrations of 0.1–0.01 mg/mL, the standards were prepared using 0.15 M saline solution. The absorbance reading was performed in the spectrophotometer (Genesys 10S UV-Visible, Thermo, USA), and a calibration curve was made. A linear regression was made from the given curve, obtaining the following equation:

$$\mathbf{Y} = \mathbf{0}.\mathbf{312}\mathbf{3X} - \mathbf{0}.\mathbf{1007}\mathbf{R}^2 = \mathbf{0}.\mathbf{9977} \tag{2}$$

Based on the equation, the peptide concentration of each one of the filtrates during its shelf life could be determined from the given absorbance reading.

#### **3.7 Antioxidant activity**

This activity was evaluated by means of the spectrophotometric technique described by Pritchard [28], which determines antioxidant activities with the DPPH radical (1,1-diphenyl-2-picrylhydrazyl) in the presence of an antioxidant substance (in this case the content of FB), measuring the inactivation potential of said radical in aqueous medium. For this, we started from an initial concentration of free radical at 0.1 mM DPPH in ethanol, respectively, diluting 1500, 1000, and 750 μL plus 500 μL of the FB adjusting to a volume of 2 mL with water HPLC grade, which generated three concentrations of the radical (0.075, 0.05 and 0.0375 mM). Water HPLC grade dissolved in DPPH was used as control, according to the concentration used. Subsequently, the samples were subjected to centrifugation at 9470 g (Spectrafuge 16 M, Labnet, USA) for 2 minutes, and the absorbance at 517 nm was measured in the spectrophotometer (Genesys 10S UV-Visible, Thermo, USA). The percentages of inhibition were calculated by the following equation:

$$2\% \dot{m} \dot{n} \dot{n} \dot{c} \dot{n} \dot{\alpha} = \frac{A^{\text{control}} - A^{\text{extracto}}}{A^{\text{control}}} \propto 100\tag{3}$$

#### **3.8 Statistical analysis**

The analysis will be carried out using the SAS statistical package [29], in which an analysis of variance was carried out with the GLM procedure; considering a block design (three lots), treatments were used as qualifying variables and as variables of response (proteolysis, peptide concentration, and antioxidant activity). Considering the following model:

$$\text{yijkl} = \mu + \pi i + Dj + \{\pi D \} \text{ij} + \beta k + \theta \{\vec{ij}\} + \varepsilon \vec{v} \vec{j}kl \tag{4}$$

**23**

the milk of cow.

1030 kg/m3

*Comparision of Antioxidant Activity of Cow and Goat Milk During Fermentation…*

where yijkl = response variable measured over time; μ = general average; ti = fixed effect of the i-th treatment; Dj = effect of the j-th day (0, 7, 14, 21, 28); (tD) ij = fixed effect of the interaction between the i-th treatment and the j-th day; βk = random effect of the k-th block; Ɵ (ij) = random effect of the j-th experimental unit, nested in the i-th treatment; eijkl = random error distributed in normal form with zero mean and

A principal component analysis was also performed using the PRINCOMP procedure, and it was determined as response variables (proteolysis, peptide concentration, and antioxidant activity) within which its correlations will be determined.

The time elapsed after the pasteurization of the cow's milk until it reached a pH of 4.5 for the beverages inoculated with *Lactobacillus acidophilus* was 16 hours, while for the controls the necessary time was 27 hours. On the other hand, in goat's milk, the beverages inoculated with *Lactobacillus acidophilus* needed a time of 11 hours, whereas the controls 19 hours. In both types of milk for the controls, a longer time lapse is observed to reach the ideal pH; this because the fermentation of the milk in these treatments was carried out by the thermoduric microorganisms, which tolerate the thermal treatments applied to the milk. In the pasteurization process, it has also been observed that as the incubation temperature of the milk increases, there is

A physicochemical analysis was carried out in triplicate in cow and goat milk, as shown in **Table 1**. Between each parameter analyzed by the type of milk, a significant difference (p ≤ 0.05) occurred, because milk differs in its composition depending on the species where it comes from. For cow's milk, the average percentage of total solids that it must contain is 12.7 [31], fat 4.2, protein 3.3, lactose 4.7, and nonfat solids 8.8%, while in goat's milk, its fat content should be 4.5, protein 2.9–4.60, lactose 4.1, nonfat solids 8.9%, and total solids from 11.70 to 15.21%; however, all these values depend on several factors such as the breed of the animal, its age, the period of lactation, and feeding, among others [32]. For cow milk analyzed, the percentage of protein and total solids that was obtained is within the reported parameters, although a smaller amount was registered in the parameter corresponding to fat and a slight increase in the percentage of lactose and nonfat solids. On the other hand, in goat's milk the percentages of total solids and protein are within the established ranges; there was a slight increase in both fat and lactose and a lower percentage in nonfat solids. However the percentage of protein in goat's milk is within the parameters reported for a good quality milk compared to the percentage obtained in cow's milk that present a significant difference (p ≤ 0.05), surpassing

Regarding the physical properties, at 20°C the density of the milk is approximately

the freezing point, this is relatively constant and is between −0.510 and −0.560°C due to the natural fluctuations of the composition of the milk [32], the freezing point recorded in the sample of cow's milk was −0.580°C, so it may be that an balance in

density, while a lower density was registered in goat's milk (1027.5 kg/m3

, but this depends on its composition [8]. Cow's milk showed an optimum

). Based on

evidence of greater microbial development of thermoduric species [30].

*DOI: http://dx.doi.org/10.5772/intechopen.88212*

variance; and eij = N (0, s2).

**4. Results and discussion**

**4.2 Physicochemical analysis**

**4.1 Treatment of fermented beverages**

*Comparision of Antioxidant Activity of Cow and Goat Milk During Fermentation… DOI: http://dx.doi.org/10.5772/intechopen.88212*

where yijkl = response variable measured over time; μ = general average; ti = fixed effect of the i-th treatment; Dj = effect of the j-th day (0, 7, 14, 21, 28); (tD) ij = fixed effect of the interaction between the i-th treatment and the j-th day; βk = random effect of the k-th block; Ɵ (ij) = random effect of the j-th experimental unit, nested in the i-th treatment; eijkl = random error distributed in normal form with zero mean and variance; and eij = N (0, s2).

A principal component analysis was also performed using the PRINCOMP procedure, and it was determined as response variables (proteolysis, peptide concentration, and antioxidant activity) within which its correlations will be determined.
