The evolution of antioxidative properties of protein-derived peptides of Mexican Panela goat and cow milk cheese during its shelf life

ABSTRACT In the current study, Mexican Panela cheeses manufactured from cow or goat milk were analyzed in terms of sensory characteristics, peptide concentration, proteolysis and antioxidant activity using the DPPH radical (1, 1-diphenyl-2-picrylhydrazyl) assay during its shelf life at 1, 7, 14, 21, and 28 days. There was no significant difference in sensory rating owing to the influence of the cheese based on a cow or goat milk, with high levels of like seen in both varieties of cheese (from 6 to 7 on average on the hedonic scale of 1 to 9). On the other hand, all cheeses, showed antioxidant activity, which was stronger in goat milk cheeses, which had a DPPH radical inhibition in the range from 11.59 to 83.11%. Additionally, substantial correlations were identified between the studied variables, suggesting that the antioxidant activity is related to the peptides contained in the water-soluble peptides extract by 92%.


Introduction
Milk and its derivatives have been classified throughout history as foods with high nutritional value and that also have certain beneficial effects on consumer health due to the presence of various bioactive components such as proteins (immunoglobulins, peptides), lipids (fatty acids), carbohydrates (oligosaccharides), hormones, vitamins, growth factors, and lactic acid bacteria with specific function and metabolism (Bhat & Bhat, 2014).Milk proteins may have latent bioactive qualities, the potential activity of which is revealed during proteolytic digestion when amino acids are released.The bioactive peptides have been defined as specific fragments of proteins that have a positive influence on physiological and metabolic functions or on the conditions of the organism and can have beneficial effects on health, among the most studied being antimicrobial, antihypertensive, antithrombotic, immunomodulatory, anticytotoxic and antioxidant (Baba et al., 2021;Giromini et al., 2018).These peptides have 2-50 amino acid residues (Mushtaq et al., 2015).Currently, dairy products, particularly cheeses, have piqued scientists' interest as a source of bioactive compounds, mostly peptides, particularly those with antioxidant potential (Kariyawasam et al., 2019).Said compounds might provide protection through a variety of ways, including hydrogen or electron transfer, reduction-inhibitiondeactivation of free radicals or their precursors and/or producers, or involvement in oxidative damage repair processes (Guangqing et al., 2018).
According to the Codex Alimentarius (2011), cheese is a solid or semi-solid product, ripened or fresh, in which the whey protein/casein ratio does not exceed that of milk, and is obtained by coagulation (total or partial) of the milk using enzymatic or other suitable coagulating agents, with a partial draining of the whey.In the other hand, the Mexicans cheeses are based on European cheese-making procedures, but the manufacturing in México have been modified over the years for various environmental and cultural influences (such as the addition or substitution of typical ingredients and the preference for unripened cheeses) to produce cheeses with unique sensory and technological characteristics (Van Hekken & Farkye, 2003).Panela cheese is a fresh Mexican cheese, not fermented, that is one of the three main types of cheese consumed in México.It is produced by the action of rennet (and/or specific enzymes), which produces a soft substance known as curd, which is pressed and drained to finish separating the whey (Jiménez-Guzmán et al., 2009).
The composition of milk impacts its nutritional quality and qualities in the creation of food items; goat's milk has great nutritional contents that only human breast milk surpasses.The proteins in cow and goat milk differ significantly in composition (Bidot-Fernández, 2017) and biological properties, such as high digestibility and thus amino acids absorbed more efficiently than those cow milk.In addition, goat is a main supplier of milk for rural regions and its importance intensifying due to allergy problem effect of cow milk especially among infants and for its functional effects especially antioxidants (Ahmed et al., 2015).As a result, the goal of this experiment is to assess the proteolytic activity and the synthesis of antioxidant bioactive peptides in Mexican Panela cheese prepared from goat or cow milk during the course of its shelf life (28 days).

Production of Panela cheese
The cheeses were made in three batches, separately for cow's (Boss taurus) and goat's (Capra aegagrus hircus) milk.Where: the milk was pasteurized at 65°C for 30 minutes before cooled to 37°C for the addition of CaCl 2 (ILM) (0.2 g/L) and the rennet (ILM) force 1:15000 L, and it was let to rest for 30 minutes at 37°C.After that, a cut of the curd (2 cm 3 ), was formed, and it was left to rest for 5 minutes.Following that, the curd was agitated for 10 minutes at 37°C before being moved to a drain to continue adding NaCl (ILM) (10 g/L of starting milk).An agitated-homogenized, to subsequently carry out a molding, a turning was carried out twice every 30 minutes, and it was essential to rest it for 12 hours at 5°C before packing.

Sensory evaluation
The sample evaluation was conducted according by Hekmat and McMahoh (1992), with 80 untrained judges, were instructed to rinse before tasting each sample.The judges indicated the most and least preference samples and to evaluate flavor, texture and acidity of the product using a hedonic scale of 1 to 9 (I dislike it very much to I like it very much) and evaluated two cheeses' samples by a code with four-digit random numbers.Each sample was served at temperature constant (4°C).Evaluation was conducted at room temperature (25°C).

Determination of titratable acidity
It was measured in accordance with the Official Mexican Norm NOM-155-SCFI-2012(2012), at 1, 7, 14, 21, and 28 days (4°C), using 5 ml of sample, 10 ml of distilled water and 0.5 ml of phenolphthalein, with a triple titration with 0.1N NaOH.The titratable acidity was calculated using the following Equation 1: Where: V = Milliliters of 0.1N NaOH solution, spent in the titration; M = Molarity of the NaOH solution; S = Volume of the sample in mL; and 90 = Lactic acid equivalent.

Preparation of water-soluble peptides extract (WPE)
The cheeses samples were treated at 1, 7, 14, 21, and 28 days (4°C), as described by Donkor et al. (2007), with some modifications, in which 5 mL of each sample, were taken and mixed with 10 mL of 0.75% trichloroacetic acid (TCA), passing the mixture through filter paper (Whatman No. 1 of 150 mm), obtaining the water-soluble peptides extract (WPE), which were frozen (-20°C) until analysis (the proteolytic activity, the total peptide concentration and the antioxidant activity).

Proteolytic activity
This activity was determined in triplicate based on the reaction of the free primary amines (-NH 2 ) with o-phthaldialdehyde (OPA) and b-mercaptoethanol, described by Church et al. (1983).The OPA reagent was prepared and diluting to a final volume of 50 mL with triple distilled water and by the following reagents: 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 and 100 μL of b-mercaptoethanol.
For the readings, 0.5 mL of each sample was taken, and mixed with 1 mL of the OPA reagent by flip of the quartz cell.After 2 minutes of incubation at room temperature and inside the equipment to avoid exposure to light, the absorbance was measured in a spectrophotometer (Genesys 10S UV-Visible, Thermo, USA) at 340 nm.

Peptide concentration
The peptide concentrations in each WPE were measured in triplicate using the technique by Bradford (1976).This is based on the proteins reacting with the brilliant blue dye Coomassie G-250, resulting in a colorful product that absorbs significantly at 595 nm.They were taken 200 µL of WPE and 2 mL of Bradford's reagent and incubated at room temperature for 5 minutes without exposure to light.Each sample was measured in triplicate.A calibration curve was created using five Bovine Serum Albumin (BSA) standards produced in 0.15 M saline solution at concentrations of 0.01, 0.02, 0.04, 0.05, and 0.1 mg/mL.A calibration curve was created after taking an absorbance measurement with a spectrophotometer (Genesys 10S UV-Visible, Thermo, USA).From the provided curve, linear regression was performed, based on the Equation 2.

Antioxidant activity
The antioxidant activity was measured in triplicate using the technique of Pritchard et al. (2010), which determines antioxidant activities with the DPPH radical (1,1-diphenyl-2-picrylhydrazyl) in the presence of an antioxidant (in this case, the content of WPE) by measuring the radical's inactivation potential in an aqueous medium.With an initial free radical concentration of 0.1 mM DPPH in ethanol, subsequently was diluted with 800 or 1200 µL plus 750 µL of the WPE to complete a volume of 2 mL with tri-distilled water (450 or 50 µL), yielding two concentrations of the radical C1 = 0.04 mM and C2 = 0.06 Mm, respectively.Tri-distilled water dissolved in DPPH served as the controls (at the same concentrations).Following that, the samples were centrifuged at 9470 × g (Spectrafuge 16 M, Labnet, USA) for 2 minutes, and the absorbance at 517 nm, was measured in a spectrophotometer (Genesys 10S UV-Visible, Thermo, USA).The percentages of inhibition were calculated by the Equation 3.
Where: A = Absorbance at 517 nm.

Statistical analysis
The analysis was carried out using the SAS statistical package ( 2006), in which an analysis of variance was carried out with the GLM (General Linear model) procedure, considering a block design (three lots), treatments were used as qualifying variables, and as variables of response (proteolysis, peptide concentration and antioxidant activity).Differences between the treatment means were compared at the 1% level of significance using Tukey´s test.Furthermore, a Principal Component Analysis (PCA) was performed using the PRINCOMP procedure, and it was determined as response variables at day 1 of shelf life (proteolysis, peptide concentration, and antioxidant activity with a concentration of 0.06 mM of the DPPH reagent) within which Pearson correlations will be determined.

Physicochemical analysis of raw milk
The physicochemical study was performed on cow's and goat's milk (Table 1), and the density parameters, non-fatty solids, and proteins percentages are within the cited parameters, according to NOM-155-SCF1-2012 (2012).Furthermore, differences (p < .01)were found across all the characteristics assessed based on the milk type.The findings are consistent with those of Ocampo et al. (2016), who investigated the physicochemical profiles of cow's and goat's milk and found that goat's milk had less proteins (3.01 ± 0.29%), but lactose was found to be higher in cow's (4.5 ± 0.01%) and goat´s milk (4.2 ± 0.01%), as in the current study.

Sensory evaluation
The data obtained from the sensory evaluation are reported in Figure 1, which demonstrates that the panelists assessed the cheeses with the highest frequency between 6 and 7 (on a scale of 1 to 9).Additionally, they are indicating that there were no differences in satisfaction (p > .01) in the sensory evaluation of both types of cheeses.Coinciding with the study of Rasheed et al. (2016), for cottage cheese, was observed a high overall acceptability of goat's milk cheese and cow's milk cheese (µ = 8.6 and µ = 8.4, respectively in a 9 point Hedonic scale) with no significant differences (p > .05).

Determination of titratable acidity
Titratable acidity was measured and represented in Dornic degrees from day 1 to day 28.The results are given in Table 2, where there is a consistent and substantial increase in the acidity of both kinds of cheese over their shelf life (p < .01).Similarly, it was feasible to confirm that the change in acidity, while consistent, was substantially different for each kind of cheese studied (p < .01),with higher values in goat's milk cheeses.On the other hand, Van Hekken and Farkye ( 2003), mention that four commercial brands of Mexican fresh cheese with cow´s milk, reporting a variable pH ranging from 5.84 ± 0.06 to 6.33 ± 0.06, which, when translated to Dornic grades, is within the range reported in our study until day 14 (28.33 ± 5.77).

Proteolytic activity
The assessment of proteolytic activity revealed significant differences (p < .01) between cow's milk and goat's milk cheeses, with the goat's milk cheese beginning with greater proteolytic activity and rising of the life on shelf (Table 3).It can also be seen that the proteolytic activity in both kinds of cheese increases continuously as the shelf-life time elapses (p < .01),implying that the interaction of the shelf-life impact with the kind of cheese was also significant (p < .01).It should also be mentioned that the statistical interaction between milk types and monitoring days was significant (p < .01),that is, a reciprocal relationship between both effects studied.Additionally, results that are mainly due to the differences between the composition of both types of milk, where especially in proteins it is qualitative and quantitative type (Ahmed et al., 2015).In the other hand, comparing the results to other similar studies in Italian cheese, Masotti et al.The results were expressed as a mean value with standard deviation.A,B Different literals indicate significant differences (p < .01) between parameters by type of milk.(2017), discovered that there was wide variability among the different cheese samples analyzed for the proteolysis and volatile compound profile variables, both of which are closely related to the odor and flavor attributes of these cheeses and the proteolysis was low compared with other cheeses of its type and significant differences were observed too in this variable by effect of monitoring day.In contrast, Rodríguez-Hernández and Chávez-Martínez ( 2018), used the same spectrophotometric technique to show that the proteolytic activity in goat milk yogurt enriched with probiotics ranged from 1.6 to greater than 90%, observing that with the addition of probiotics, the percentages of proteolysis were elevated.However, because the current study is based on a non-fermented fresh cheese, low values of proteolysis are observed in comparison to a fermented dairy product.

Peptide concentration
The peptide concentration analysis findings are provided in Table 4, and they reveal that there are significant differences (p < .01) between the two varieties of cheese, with goat's milk cheese having the highest peptide concentration.Which was to be expected since initially the concentration of protein was higher in goat's milk than cow's milk (Table 1).There was also a variation attributable to the influence of monitoring day (p < .01),with the highest peptide concentration reported on day 14.It should also be mentioned that the interaction between milk kinds and monitoring days was significant (p < .01),that is, a reciprocal relationship between both effects studied.

Antioxidant activity
The results demonstrate that there was a difference (p < .01),as indicated in Table 5, between the varieties of cheese made from cow's and goat's milk.As can be shown, the goat's milk-based cheese had higher antioxidant activity, with DPPH radical inhibition percentages reaching up to 80%.There was also a variation in the influence of the monitoring day (p < .01)and the two concentrations of DPPH reagent employed (p < .01).Milk and its derivatives are foods with antioxidant activity owing to the presence of several bioactive components such as bioactive peptides (Giromini et al., 2018).On the other hand, in antioxidant techniques, antioxidant chemicals and radicals can respond differently depending on the method's reaction system (Ames et al., 1993), therefore two concentrations of the DPPH radical were evaluated to guarantee that there were no changes in this variable, as determined.In the study of Alyaqoubi et al. (2014), inhibition percentages of the DPPH radical were found in goat milk, ranging from 55.29 to 64.77%, indicating that higher values were reached in this work.In a more recent study, Kariyawasam et al. (2019) detected antioxidant activity in cottage cheese, observing a constant increase in this bioactivity as its shelf life The results were expressed as a mean value with standard deviation.A,B Different literals in the same row indicate significant differences in the samples (p < .01)due to the effect of milk type.a,b,c,d Different literals in the same column indicate significant differences in the samples (p < .01)due to the effect of monitoring day.The results were expressed as a mean value with standard deviation.A,B Different literals in the same row indicate significant differences in the samples (p < .01)due to the effect of milk type.a,b,c,d Different literals in the same column indicate significant differences in the samples (p < .01)due to the effect of monitoring day.The results were expressed as a mean value with standard deviation.A,B Different literals in the same row indicate significant differences in the samples (p < .01)due to the effect of milk type.a,b,c,d Different literals in the same column indicate significant differences in the samples (p < .01)due to the effect of monitoring day.
increased, and in turn, percentages of approximately 15 to 45% DPPH radical inhibition were detected in the watersoluble extracts of the cheeses analyzed, a trend also observed in the current work.

Principal component analysis
According to the PCA in the Figure 2, it is observed that the types of cheeses (made with cow's milk and the other with goat's milk), presented a very different behavior from each other for the three variables included in this analysis (proteolysis, peptide concentration, and antioxidant activity).Additionally, to the results of the Pearson correlation analysis (Table 6), there are high correlations (all greater than 92%) and direct proportional with high significance (p < .01),relationships between the variables of proteolytic activity, antioxidant, and peptide concentration, implying that the antioxidant activity determined in the water-soluble extracts is largely due to a source of peptide origin present in them.This was predicted given that several peptide sequences originating from milk proteins have been shown to have antioxidant activity (Baba et al., 2021), which has been found in various dairy products such as cheeses (Kariyawasam et al., 2019), and specifically with fresh cheese, the biological activity is mostly related to the peptides of milk and by rennet action during cheese manufacture (Hernández-Galán et al., 2017).

Conclusion
All the cheeses analyzed presented antioxidant activity, with higher percentages observed in goat's milk cheeses.In the evaluation of proteolytic activity, peptide concentration and antioxidant activity, significant differences were presented by effect of type of cheese (cow's or goat's milk) and by monitoring day effect (1, 7, 14, 21 and 28 day), that is, they suffered variations on the shelf life.Likewise, high correlations were observed between the variables analyzed, which suggests that the antioxidant activity in 92% is derived from molecules of peptide origin in these cheeses.Additionally, it The results were expressed as a mean value with standard deviation.can be said that greater antioxidant activity was observed in goat's milk cheese and no significant differences were observed in sensory evaluation, so it represents an option to consume these types of cheeses with greater functional properties and that maintain sensory quality.

Figure 1 .
Figure 1.Sensory acceptance of attributes (flavor, texture and acidity) in Panela cheeses made from cow's milk (black) and goat's milk (gray).With standard error bars.There are no significant differences because of cheese type between attributes (p > .01).
A, B, Different literals indicate significant differences in the samples (p < .01)due to the effect of milk type.a, b, c, d, Different literals in the same column indicate significant differences in the samples (p < .01)due to the effect of monitoring day.1,2Different numbers in the same row indicate significant differences in the samples (p < .01)due to the effect of DPPH reagent concentration.

Figure 2 .
Figure 2. Loading plots from the Principal Component Analysis (PCA) carried out on all variables at day 1 of shelf life (proteolysis, peptide concentration, and antioxidant activity).1: cheeses with cow's milk and 2: cheeses with goat´s milk.Prin: Principal Component.Figure obtained statistical package Statistical Analysis System (SAS, 2016).

Table 3 .
Proteolytic activity (%) in water-soluble peptides extract of Panela cheese made with cow´s and goat´s milk.

Table 4 .
Peptide concentration (mg/mL) in water-soluble peptides extract of Panela cheese made with cow´s and goat´s milk.

Table 6 .
Pearson correlation coefficients in water-soluble peptides extract of Panela cheese made with cow´s and goat´s milk.