# Exploring New Fruit- and Vegetable-Derived Rennet for Cheese Making
## 3 Results
### Esterase and Protease Activities  
Before analyzing the coagulation capabilities of milk, the extracts were examined for their esterase and protease activities. To define the reference values for these activities, powdered calf rennet solubilized in water at 175 IMCU/mL was used. The vegetable and fruit rennet were obtained as reported in the Materials and Methods section, while the milky fig sap was used fresh and diluted 10 times in water. At first, the pH value of all the extracts was measured. The pH values were all in the range of 5.0-6.2 (Table 1), in line with the pH range of 5.0 and 6.5, which is the range values observed in curds. The pH value impacts the firmness and the water-holding capacity of the cheese [23]. When in vitro esterase activity was measured (Table 1), it was observed that the oyster mushroom extract showed an activity (0.016 U/mL) similar to that exhibited by calf rennet (0.016 U/mL), followed by the pineapple extract, which, in turn, appeared to be more active than fig milky sap. The least active extract was papaya, which showed only 25% of the esterase activity with respect to calf rennet (0.004 U/mL; Table 1).  
An assessment of the protease activity of the presumed vegetable rennet was also carried out. Indeed, all the vegetable matrices used in this study were chosen for the presence of known protease activity. In fact, cardosins A and B are present in cardoon and artichoke, fiction in fig latex, papain in papaya, bromelin in pineapple, and several proteases in oyster mushroom [24]. To evaluate the protease activity, using BSA as a substrate, the level of hydrolysis of BSA was considered. A difference in the hydrolysis pattern is the parameter that allows the activity to be measured and reveals the effectiveness of the different types of proteases in the extracts based on the activities recorded for calf rennet.  
The SDS-PAGE analysis showing the protease activity of the plant and fruit extracts is reported in Figure 1. Also reported in Table 1, this analysis revealed that calf rennet hydrolyzes approximately 46% of the BSA, while artichoke, thistle, and oyster mushroom more than 98%. A protease activity of approximately 50% is shown for the fig milky sap extract, while low hydrolysis rates were recorded for pineapple and papaya (28 and 18%, respectively). There are various products obtained from the BSA hydrolysis. With regard to calf rennet, there is a residual polypeptide of approximately 35.0 kDa; a similar polypeptide is also present for the artichoke plus other peptides that is approximately 20.0 kDa. The cardoon extract showed polypeptides that are approximately 20.0, 25.0, and 30.0 kDa, while the pineapple and papaya extracts allowed for short peptides of approximately 20.0 kDa to be released. A polypeptide of approximately 45.0 kDa was released by fig milky sap and oyster mushroom. The latter further showed polypeptides at approximately 60.0, 30.0, 25.0, and 20.0 kDa.  
Figure 1: The 12.5% SDS-PAGE analysis. To evaluate the level of proteolytic activity of the vegetable and fruit rennet, 5 ug of BSA was used for each sample. (1) Molecular weight markers; (2) control, only 5 ug BSA; (3) 5 ug BSA + 2 \(\mu\)L calf rennet; (4) 5 ug BSA+ 5 \(\mu\)L artichoke; (5) 5 ug BSA+ 5 \(\mu\)L cardoon; (6) 5 ug BSA+ 5 \(\mu\)L pineapple; (7) 5 ug BSA+ 5 \(\mu\)L papaya; (8) 5 \(\mu\)g BSA+ 5 \(\mu\)L fig milky sap; (9) 5 ug BSA+ 5 \(\mu\)L oyster mushroom.