For the preparation of the minimally purified coagulants from adult rabbit stomachs, protocol described by Lambert [17] was adopted. After thawing, the stomachs were opened and the gastric contents immediately removed including the excess perivisceral fat, and the rest of the esophagus, then washed with potable water in both external and internal superficies. For the enzyme extraction, two ways for salt applications were tested: directly on stomach superficies (SD) and immersed in a saturated saline solution (SS) (37%, m/v) for 5 min, and a third treatment without salt application (NS). After, all treatments were dried for 48 hours at 35˚C in an oven with continuous airflow, and dried tissues were cut into pieces of 5 × 20 mm approximately.

For the liquid coagulant preparation, five grams of dry tissues were immersed in a NaCl solution (12%, m/v) (1:10 ratio) and boric acid (1%, v/v), and stirred for 30 min.

A methodology of response superficies (MRS) was used to test the influence of the pH and time of enzyme activation. Two levels of both variables (maximum and minimum) were assayed, and the experimental design shown in Table 1,

t (h) time in hours; MCA (U/mL) milk clotting activity.

aimed to find the better condition for enzyme activation. Milk clotting activity was the response variable, following before methods described. Tissues of rabbit abomasum in NaCl solution (12%) were adjusted to pH with HCl (1N) and stay for the time of experimental design incubated at 37˚C in a closed oven. Then, the pH was raised to 5.6 with NaOH solution (1N), filtered on C41 filter paper (UNIFIL), and the liquid collected in amber glass bottles and stored at 5˚C.

The milk clotting activity was assayed using caseinate solutions as substrate. Ten mL of caseinate solutions, with sodium azide and CaCl₂, were put into glass tube. Then, 200 µL of coagulant were put and shaking on 1 min with stirred. Time of first coagulum appeared over glass tube was registered and MCA was calculated as followed equation:

MCA = ( 2400 ∗ V ) / ( t ∗ E ) (1)

Where:

MCA is the milk clotting activity; V is volume of milk (mL), t is the time of coagulation (min); E is the volume of coagulant (mL).

Calcium caseinate from bovine milk was used as a substrate for proteolytic activity assay following the procedure described by Ahmed [18] . Fresh whole milk was defatted by centrifugation at 2800 g for 10 min and 25˚C. Caseinate was obtained by isoelectric precipitation with milk acidification to pH 4.3, using an HCl solution (6N). The acidity milk was heated to 37˚C for 30 min and centrifuged at 2800 g for 10 min. The supernatant was discarded and the precipitated was washed three times with distilled water. Then the precipitated was rehydrated in distilled water (12% w/v) and the pH was adjusted to 6.5 using NaOH solution (1N), and finally left to stand for three hours at 4˚C to be subsequently frozen and dehydrated by lyophilization.

The substrate (1% w/v) was prepared from calcium caseinate in a buffer phosphate solution (pH 6.5) and stabilized at 30˚C of temperature in a water bath. The test started with the addition of RC in a ratio of 0.5 mL for 10 mL of the substrate. Samples were collected after the 5, 30, and 60 min of incubations and immersed in water at 95˚C, for the enzymatic inactivation. This assay included CC as control treatment following the same procedure described in the section before using de volume according to the manufacturer’s specifications.

Hydrolysis of casein with RC and CC was monitored using SDS-PAGE following the method described by Sams [19] with minor modifications. A volume of 75 µL of the digested substrate was collected and mixed with 25 µL of sample buffer (4× Laemmli, Biorad), and heated to 95˚C, for 10 minutes. Electrophoresis was developed in stacking and resolving polyacrylamide/bisacrylamide gels (5%, and 14%, respectively) in a Mini PROTEAN cell (Bio-Rad). After the electrophoretic run, the gels were stained with Coomassie Blue, with shaking for 1 h and decolorized with ethanol (30%), acetic acid (7.5%) and trichloroacetic acid (5%) for 12 hours. To compare molecular weights, a standard containing a mixture of 12 recombinant proteins with molecular weights between 2 kDa and 250 kDa was included in electrophorese gel. Gels’ images were analyzed using image density software (ImageJ).

Fresh whole milk was supplied by the Tambo (cow milking area) of the Federal University of Santa Maria/RS, Brazil. Defatted dry matters (DFDM), protein, fat, lactose, mineral residue, and density were determined using the milk analyzer (MASTER MINI, Higmed, Tatuapé, São Paulo, SP). The pH was measured using a pH meter (Digimed DM20 SPLabor, Presidente Prudente, SP, Brazil), and acidity through titration with 0.1N NaOH, with phenolphthalein as an indicator.

For making of Prato type cheese coagulated by RC and CC, method described by Baptista [21] was followed, with minor modifications. The fresh milk was pasteurized (65˚C/30min) and cooled to 37˚C, then pH was adjusted to 6.5 with a lactic acid solution (1N). The mesophilic culture (Lactococcus lactis ssp. Lactis and L. lactis ssp. Cremoris, Rica Nata, Piracicaba, SP), were added (1%, v/v). Afterward, calcium chloride solution (CaCl₂, 50%) (2 ppm w/v) was added and, finally, the RC (3.6 mL·L⁻¹) or CC coagulant (0.9 mL·L⁻¹), with agitation for a maximum of 2 min, and left to rest for 45 min. Subsequently, the gel was cut with a knife to determine the clotting point. After 5 min of having been cut, the curd was treated, starting with gentle agitation for 5 min, which was repeated for 15 min with rest intervals of 3 min. The temperature was raised to 42˚C and the agitation and rest sequence was repeated for another 15 min. The whey was separated by draining, and the mass was placed in specific cylindrical molds. The cheeses were pressed applying at 10 times their weight for one hour, followed by turning and applying a pressure equivalent to 15 times their weight for another 12 hours. Pieces of cheese were salted in 20% saline solution for 3 hours and dried in the refrigerator at 8˚C for 3 hours. Finally, the pieces were vacuum-packed and stored in a refrigerator at 8˚C for sixty days.

To quantify the dry mass in samples of cheese and whey, 5 g of the sample was dried in an oven at 105˚C until constant weight [22] (method 925.23; AOAC, 2007). Protein was estimated by means of the Kjeldahl method (N * 6.38; method 954.01; [22] AOAC, 2007). Fat was determined by treating the sample with sulfuric acid and isoamyl alcohol [23] (IAL, 2008). Ashes were quantified by gravimetry after heating until the complete incineration of 2 g of the moisture-free sample in a muffle furnace at 550˚C [22] (method 935.42; AOAC, 2007). Acidity was determined by titrating the sample with an alkaline sodium hydroxide solution (0.1 N) (Brazil, 2018). The pH was estimated with a digital pH meter calibrated at 25˚C (Digimed DM20 SP Labor; Presidente Prudente, SP, Brazil) mixing 10 g of cheese with 40 mL of distilled water. All analyses were performed in triplicate.

The texture profile was performed according to Alves [24] (2017), with modifications. To cut the samples cylinders molds (20 mm in diameter and 20 mm in high) were used, and then the pieces were packed in plastic film, and then placed in a plastic bag, and kept in a polystyrene box with ice (5˚C ± 1˚C) for 1 h, in order to stabilize the temperature before the test. Texture profile parameters were analyzed with a TA-XT2 (Stable Micro System, Haslemere, United Kingdom) texture equipment using a 36 mm diameter aluminum probe (PB36). Three measurements were made for each treatment replica. The recorded parameters were hardness (N), cohesiveness, adhesiveness (J), elasticity, gumminess (N), and chewability. Data was collected by Texture Expert Software for Windows – 1.2 (Stable Micro Systems).

For 60 days, pH and acidity (mg lactic acid 100 g⁻¹) were registered following method described in the proximate composition of the cheeses (2.5.3).

The Proteolysis was described through the ripening extension index (REI) and ripening depth index (RDI) parameters that were determined by the Kjeldahl method, which allowed obtaining the amount of water-soluble nitrogen at a pH of 4.6 (SN-pH 4.6), and the soluble nitrogen in trichloroacetic acid (SN-TCA), and total nitrogen (TN), starting from a standard solution [25] .

The ripening extension index (REI) is obtained by Equation (3), below:

REI ( % ) = SN-pH 4.6 × 100 TN (3)

where:

SN-pH 4.6 = water-soluble nitrogen content at pH 4.6;

TN = the total nitrogen.

The ripening depth index (RDI) was determined by Equation (4), below:

RDI ( % ) = SN-TCA × 100 TN (4)

where:

SN-TCA = soluble nitrogen content in trichloroacetic acid (SN-TCA);

TN = is the total nitrogen.

Whole bovine milk presented contents in the percentage of defatted solids of 8.79 ± 0.13, protein 3.24 ± 0.05, lactose 4.82 ± 0.07, and mineral residues 0.72 ± 0.01. Density (g/mL) was 1.032 ± 0.001, cryoscopy point (˚H) −0.550 ± 0.01 and pH was 6.94 ± 0.01. In general, the results obtained were within the values required by Brazilian legislation [29] , except for the fat content, which was 2.56 ±

0.08, below the recommended. This fat value could be associated with the factor known as depression in fat in milk, which means that for the period of milk collection, factors such as diet or seasonality influenced the decrease in the production of this compound in milk [30] .

Initially, a rapid coagulation test was necessary for determinate de MCA due to high pH (6.94) of raw milk and was confirmed the needing of adjust the pH of milk to 6.5, like this was demonstrated in the Section 3.2. For attempt the same coagulation time, the volume of RC was 4 times the volume than the CC used to coagulate the milk within 45 minutes. During coagulation, the principal solid constituents of milk remain occluded in the proteinic matrix formed, a product of casein digestion, except for soluble proteins. Factors such as pH and temperature of milk influence the aggregation stage of protein fractions since κ-casein was hydrolyzed by enzymatic action [14] . Data from physical chemical analyzes of cheeses are shown in Table 2. Cheese composition generally did not show differences between treatments coagulated with RC and CC. The parameters considered as protein, fat, ash, and total solids obtained similar values for both treatments.

These results demonstrate that despite the possible presence of pepsin as the main enzyme present in the coagulant of the stomach of adult rabbits, RC can be

used as a coagulant for cheese formulation without loss of components due to probability excessive hydrolysis.

Another indicator of excessive hydrolysis can be reflected in the total solids on the whey [31] . In this study, the values found for total solids in the whey did not show differences between treatments.

These results express the conformation of a matrix with a greater capacity for retaining solids reflected in the coagulated treatment with RC. In contrast, when the bovine rennet made up mainly of pepsin used for cheese production, it can present a high proteolytic activity that can be reflected in a low yield of the product [31] . Alihanoğlu [16] found higher fat content in fresh cheeses (White Cheese) coagulated with gastric extract of young rabbits with a few weeks of age in comparison with coagulated treatments with renin bovine and camel renin.

During gel formation, fat, as well as other milk compounds, remain immersed in the protein structure derived from enzymatic hydrolysis on the casein mycelium, therefore, the retention of fat, minerals, water, and other elements may vary depending on the type of clot formed and is reflected in product characteristics and manufacturing performance [6] . Garg & Johri [32] consider that pepsin has some technological limitations to be used as a coagulant in cheese making, highlighting an excessive loss of fat during processing. The salt content was shown to be higher in the CC (p < 0.05), a response that could be directly associated with the moisture content, which also showed higher levels for the same treatment.

Cheese structure is highly dependent to casein and the way that it is affected by enzymatic action [6] , and the effects of hydrolysis can be observed through several aspects such as proximate composition. The protein content was similar in both treatments (p > 0.05), varying between 25.31% and 24.35% for cheese coagulated with RC and CC, respectively. Moatsou [33] tested artisanal rennet from goats and lambs abomasum, finding no differences compared between commercial rennet on the principal compounds as total solids, ash, sodium chloride (NaCl), fat, and total protein. [24] Alves et al. (2013) compared the coagulant capacity of enzymes produced from the Termomucor indicae fungus with a commercial coagulant, the latter is similar to that used as a control in the present study. These authors determined that the hydrolytic action of the enzymes produced by the fungus was alike to that of commercial chymosin.

The yield of cheese expressed as the final weight of the product in relation to the volume of milk used for their preparation can be considered an indicator to evaluate the effectiveness of the coagulant in the hydrolysis of the protein [34] . Excessive proteolytic activity of the enzyme on the casein matrix can result in low yield, which can be observed in the increase of the solids content during whey draining [1] . In our finding was clear that the action of RC was like the commercial coagulant CC, with no differences in total yield for both treatments (p > 0.05). For both treatments no differences were detected in total solids of whey and water activity (Aw), reinforcing the idea of similar action of RC and CC.

An effect of enzymatic activity on the casein can be seen in the physical structure of the cheeses, which can be assessed through the texture profile. Table 3 shows the results of the evaluation of the texture profile in the RC and CC cheeses. When analyzing hardness, differences between the samples were evidenced, being higher (p < 0.05) in the CC with values of 34.37 ± 4.37 N, a result that shows the composition of a more rigid structure compared to RC. This result, probably, could be associated with a higher intensity of proteolysis on the casein matrix and fat retention in the treatment with RC.

The gummy and chewable parameters were also lower (p < 0.05) on the RC cheese. These parameters reflect that this treatment has a less compact and less firm structure, and less effort is required to generate the deformation that is necessary for chewing. The result also confirms that the impact of the hydrolysis product of coagulation with RC is relatively greater on casein when compared to CC, giving a more fragile structure in cheese.

Alihanoğlu et al. [16] compared cheeses coagulated with bovine chymosin, camel’s chymosin, and gastric extract of young rabbits showing a texture profile where parameters such as hardness, gumminess, and chewability were superior in treatments when coagulated with RC and camel chymosin and lower in cheeses which were coagulated with bovine chymosin. Although these authors did not specify the age of the rabbits, it is likely that there is still residual enzyme chymosin, acting in a similar way to bovine chymosin.

The pH of the cheeses was monitoring during for 60 days of storage, showed changes due to the ripening process (Figure 5). It is important to highlight the need to adjust the pH of the milk to 6.5 in the initial preparation stage, since the

RC cheese coagulated with rabbits gastric extract; TC Cheese coagulated with commercial chymosin; CV coefficient of variation. Different lower case letters indicate significant differences by the Tukey test (5%).

milk had a pH of 6.9, and almost zero coagulant activity of the RC at pH above 6.9 was demonstrated. After two days of storage, the pH decreased for both RC and CC varied between 6.1 and 6.4, respectively, showing slight differences (p < 0.05). This comportment is probably associated with the residual of coagulant or the action of added starter cultures, which act on milk sugar, transforming it into lactic acid [14] .

On day 30, there was a decrease in pH compared to the first period, thus showing differences between treatments as well. At the end of day 60, the pH remained the same in both treatments, RC and CC, maintaining differences between both treatments. The stability of the pH value can be explained by the decrease in the transformation of lactose into lactic acid.

In a study carried out by Alihanoglu [16] with the application of a gastric extract of young rabbits in the formulation of fresh cheese (White cheese), there was a more intense pH decrease, obtaining a cheese with a pH of 4.57 at 60 days of ripening in the treatment elaborated from RC. Alves [24] obtained pH values of 5.3 in the first 5 days after the manufacture of Prato cheese when they evaluated the coagulant capacity of the extract produced by fermentation of the fungus Thermomucor indicae. The pH values on the 60th day of ripening were within the values found by Batista et al. [35] in the evaluation of commercial-branded Prato cheeses.

During the ripening of cheese occur degradation process on protein, lipids, and carbohydrates, the main components of curd, because of starter cultures added, as well as residues of the coagulant used. The product results in the generation of other substances such as medium-chain peptides and free amino acids, as well as such as short-chain fatty acids and phospholipids, and lactic acid, derived from the biochemical processes known as proteolysis, lipolysis, and glycolysis, respectively [36] .

The ripening extension index (REI) is represented by SN-pH 4.6 fraction and indicates the percentage of soluble nitrogen when the sample is precipitated at pH 4.6. During the early stages of ripening, much of the released nitrogen comes from digestion by the action of the residual coagulant [36] . In this study, the REI was similar between treatments and times of collect the samples, the percentage of soluble nitrogen at pH 4.6 for the first 30 days was between 17.8% and 18.4% for cheese coagulated with RC and CC, respectively (Figure 6(a)). The RDI represents the percentage of nitrogen soluble in trichloroacetic acid (TCA), also related to total nitrogen, and it refers to the nitrogen released by the action of lactic acid bacteria. Likewise, no differences (p > 0.05) were found in both samples for the REI and RDI (Figure 6(b)).