# Exploring New Fruit- and Vegetable-Derived Rennet for Cheese Making
## 4 Discussion  
Herein, a comparative study among calf rennet and vegetable/fruit extracts used as rennet in cheese making was carried out. Some artisanal cheeses were prepared with aritchoke and cardoon [5], and it is known that the protease ficin, present in milky sap of figs, has clotting activity [4]. In this context, the putative clotting activities of the other three vegetables, namely papaya, pineapple, and the mushroom _Pleurotus ostreatus_, which are rich in proteases, were explored. In addition to the clotting activity, the esterase activity in these vegetable extracts was investigated since the hydrolysis of esters is critical in the development of cheese flavor. Protease and esterase activities were detected in vitro for all the vegetables and fruits. Different classes of proteases were revealed, which can act diversely. Moreover, aritchoke, cardoon, and _Pleurotus ostreatus_ showed high rates of BSA hydrolysis, being around 98-99%, whereas fig milky sap and calf rennet showed rates at about 50%, pineapple at 28%, and papaya at 18% (Figure 1, Table 1). The esterase activity was comparable for all the vegetable extracts with the calf rennet esterase activity. The papaya extract was the only one showing a low esterase activity (Table 1). The milk-clotting activity of vegetable and fruit rennet was also evaluated since this parameter is linked to the ability to obtain curd. As reported in Table 1, all the extracts used showed milk coagulation activity, but only the milky fig sap was comparable to calf rennet; all the others had coagulant activities approximately 10 times lower than calf rennet. In particular, it was verified that by using a time interval similar to that of calf rennet, a lower curd yield was obtained, but by increasing the incubation time, a good curd yield was obtained. Larger quantities of plant and fruit extracts were not used to avoid them altering the final flavor. The yield of the experimental cheeses, obtained starting from 500 mL of milk, was approximately 70 g of the curd with calf rennet, artichoke, thistle, papaya, and milky sap, while it was about 50 g with pineapple and mushrooms (Table 1). In terms of the chemical composition of the experimental cheeses, after 30 days of maturation, the moisture level was 10 to 30% higher than the calf rennet value (Table 2), whereas the protein content was comparable in all samples analyzed. When the free fatty acids (FFAs) produced at the end of maturation were analyzed, important differences were observed between the cheeses produced with the different vegetable and fruit rennet. The data acquired underline an enrichment of the nutritional value of these products, which could positively impact the health of consumers. Although FFA profiling has long been achieved through gas chromatographic analysis after derivatization, the coupling of UHPLC with mass spectrometry [26] could be considered valuable, as it exploits the acidity of the carboxyl hydrogen atom for correct ionization in negative ion mode. In fact, in recent years, this latter approach has become very popular [27]. The content of FFAs in cheeses depends on several factors that contribute to fluctuations. The first is obviously related to the milk used, not only with regard to the animal species but also the animal feed, feed seasonality, breed, lactation stage, etc. [28]. Apart from that, it should be considered that the FFA profile in cheeses could differ from that of the milk used for production. In fact, cheese production technology and ripening times are pivotal in determining their relative amount in the final product. Although the content of SFAs found in the present work is lower if compared to the literature data, the ratio with MUFA total amount, expressed in percentage, was in accordance with previous data regarding the FFA fluctuations due to different ripening times [29]. Moreover, the hypothesis that the relative content could also be due to the plant matrix used as milk-clotting agents should not be discarded.