Biological material

One hundred rabbit stomachs were used in the study. The rabbits were meat producing breeds (California, New Zealand, Chinchilla and crosses of these) and were 8 to 9 weeks old, males and females, weighting between 2 and 2.3 kg. The rabbits were sacrificed using the technique of desensitation by breaking the neck and bleeding through a cut in the jugular vein, following the standard NOM-033-ZOO-1995 (humanitarian sacrifice of domestic and wild animals) in the meat workshop of FES-Cuautitlán of the Universidad Nacional Autónoma de México.

Perivisceral fat, spleen, remnant of the esophagus and the pyloric portion with the rest of the duodena were extracted and the stomach contents eliminated. The stomachs were turned inside out to expose the gastric mucus, rinsed with tap water and left to drip for 30 min. The stomachs were divided into three lots: 1) 20 stomachs were submerged in a saturated NaCl solution (37 g in 100 g water at 20 °C) for 5 min; 2) the surface of 20 stomachs was covered with NaCl (2 g NaCl g of stomach); 3) 60 stomachs were not treated with salt. The stomachs were insufflated for drying in a forced air oven at 45 % relative humidity and 35 °C. They were considered dry when they reached constant weight (48 h approximately), and they were ground to a particle size of 25 𝛍m.

Enzyme extracts

One hundred mL extract was made with 1 g of stomach in acidified water (with acetic acid, pH 4.0). Coagulating capacity was evaluated in two stages. First, the effect of NaCl in brine, surface saturation and no NaCl was evaluated, it was carried out after 2 and 10 d of storage to determine its effect on curdling strength and proteolytic activity. In the second stage, the combined effect of NaCl (0, 1, 2, 3, 4 and 5 %) and ethanol (0, 1, 2, 3, 4 and 5 %) on curdling strength and proteolytic activity. These extracts were evaluated after 4, 8, 12 and 16 d of storage. All the treatments of the two stages were triplicated.

Curdling strength

Curdling strength was quantified in 10 mL of enzyme extracted added to 100 mL milk and defined as volume of milk (L) per gram of stomach coagulated in 40 min at 35 °C (^{Chazarra et al., 2007}), calculated with the formula proposed by ^{Spreer et al. (1998)}:

where Fc is curdling strength, Vl is volume of milk, 100 is the dilution of 1 g of stomach in acidified water gauged to 100 mL, 2400 s are 40 min, Vc is the quantity of curd and t is curdling time.

Curdling time was the lapse between application of the enzyme extract and the moment in which the curd could sustain a straw vertically (^{Chazarra et al., 2007}). If the milk did not coagulate at 40 min, the extract was considered to have no effect. For these tests, milk was pasteurized at 63 °C for 30 min and 20 °Dornic acidity with 50 % CaCl (2 mL 10 L^-1 milk).

Determination of proteins

Protein concentration was determined with the colorimetric method of ^{Bradford (1976)} at 595 nm in a spectrophotometer (Genesis 10 UV-VIS, THERMO™) and a standard curve of ovalbumin (2 mg mL^‒1 0.9 % NaCl) at concentrations between 0 and 30 𝛍g mL^-1. The results were expressed in 𝛍g protein mL^-1 stomach extract.

Proteolytic activity

Enzyme activity was determined with the methodology of ^{Corzo et al. (2012)}: 500 𝛍L of substrate (1 % casein in 0.1 M monobasic and dibasic sodium phosphate buffer solution, pH 6) with 50 𝛍L of enzyme extract were shaken for 30 s and kept 30 min at 35 °C. After 20 min, 1 mL 5 % trichloroacetic acid was added to stop the reaction. Proteolytic activity was quantified by spectrophotometry at 280 nm in the supernatant obtained after centrifuging (30 min at 3000 rpm, 4 °C). The activity was expressed as units of enzyme activity (UEA) (increase in absorbance of 0.001 per mg of protein per min). This analysis was performed in triplicate.

Experimental design and statistical analysis

The experimental design was complete randomized blocks, and data about the effect of NaCl on curdling strength and proteolytic activity were analyzed with ANOVA. The treatments were: condition without salt, with saturated brine, and surface saturation. The effect of blocking were days of storage. When differences were found, the Tukey test, p≤0.05 (^{Steel et al., 1997}) was used, with SAS 9.0.

For the combined effect of NaCl and ethanol the experimental design was completely randomized with a 6×6×4 factorial array: six levels of NaCl (0, 1, 2, 3, 4 and 5 %), six levels of ethanol (0, 1, 2, 3, 4 and 5 %) and four storage times (4, 8, 12 and 16 d). When significant differences were found, the Tukey test was applied, p≤0.05 (^{Steel et al., 1997}).

Stage 1: Effect of NaCl on curdling strength and proteolytic activity

Significant differences in curdling strength were found among the extracts from stomachs with and without NaCl. The extract without NaCl did not coagulate milk at any ofthe storage times. The extract from stomachs submerged in brine coagulated milk as did that from stomachs saturated with NaCl (Table 1). This result suggests that NaCl affected coagulating activity, as ^{Whitaker (1994)} indicated. This author states that NaCl concentrations above 6 % can affect enzyme activity, partially or totally inhibiting the process. ^{Sánchez and Burgos (1997)} reported that NaCl concentrations below 2 % increase protein solubility and unfolding and increases enzyme activity.

Table 1: Effect of NaCl on curdling strength (10³) and proteolytic activity of coagulant from rabbit stomach. 

♦ S E: No effect. UEA: Units of Enzyme Activity (increase of 0.001 in absorbance per mg protein per minute). ^†L of milk coagulated by g stomach. Treatments with different letters in a row are significantly different (p≤0.05).

Curdling strength and units of enzyme activity (UEA) were significantly different among treatments. Curdling strength was not affected by storage time. In the extracts without NaCl, UEA was greater (Table 1), but the milk did not coagulate. The reason for this may have been that the NaCl modified the conformation of the protein space and the active site. Moreover, the diminishing effect of the NaCl on proteolytic activity of the treatments is explained by the fact that salt promotes dissociation of calcium and phosphates of casein micelles, affecting the colloidal state of the milk and producing electrostatic interactions with the charged amino acids. Although, NaCl at concentrations below 5.8 % (1 M) seems to stabilize the original protein folding (^{Chazarra et al., 2007}), the method of applying the salt can affect activity, since high concentrations of NaCl delay proteolysis (^{Calvo et al., 2007}).

^{Moschopoulou et al. (2007)} pointed out that air-dried milk coagulants obtained from lamb stomachs maintain more activity for a longer time than those made from fresh or salted stomachs. In contrast, rennet from lambs lose their coagulating activity after four months of storage when salt is not applied (^{Moschopoulou, 2011}). Busamante et al. (2000) reported that the amount of salt added in making rennet from lambs does not its affect coagulating activity or its stability.

Stage 2. Effect of the coagulating extracts from rabbit stomachs supplemented with NaCl and ethanol on curdling strength and enzyme activity

Coagulating strength was not modified by NaCl. However, there were significant differences (p≤0.05) when ethanol was combined with NaCl (Table 2). Average curdling strength (g rabbit stomach L^-1 milk) (Table 3) was higher 4 (1:17 640), 8 (1:24 480), 12 (1:14 520) and 16 (1:31 710) than that obtained by ^{Florez et al. (2006)} between days 4 and 16 (around 1:1000 for rennet from goat kid) and by ^{Chazarra et al. (2007)} with 0, 34, 51 and 102 mN NaCl in extracts from thistle flowers (1:387 to 1:422 g^-1 thistle). This result may be attributed to the low NaCl concentrations used to prepare the extracts, indicating the importance of this factor since natural pepsin and chymosin without salt lose up to 26 % of their curdling strength after 6 months of storage (^{Kozelkova et al., 2012}). According to ^{Anifantakis and Green (1980)} coagulating activity was higher with coagulant enzymes extracted with 6 % NaCl.

Table 2: Combined effect of NaCl and ethanol on curdling strength (L milk coagulated g^-1 stomach) (10³). 

♦ S E: No effect; EE: standard error of the mean; treatments with different letters in a column are statistically significant (p≤0.05).

Table 3: Combined effect of NaCl and ethanol on proteolytic activity (UEA×10³). 

♦ Treatments with different letter in a column are statistically different (p≤0.05).

UEA observed in the treatment that contained 0 % NaCl and 0 % ethanol was significantly different (p≤0.05) from the treatments with 1 to 5 % NaCl at all levels of ethanol (Table 3). The increase in proteolytic activity observed when 2 to 5 % concentrations of NaCl were added may be due to improvement in solubility of the enzymes by ionic force, as reported by ^{Ahmed et al. (2009)}, that enzyme proteolytic activity is potentiated when using solutions of up to 5 % NaCl. According to ^{Irigoyen et al. (2001)}, chymosin proteolytic activity in rennet from calves may be affected by the NaCl concentration by modifying its specificity.