# Exploring New Fruit- and Vegetable-Derived Rennet for Cheese Making
###### Abstract
When chymosin or a clotting enzyme are added to milk, the specific hydrolysis at the Phe\({}_{105}\)-Met\({}_{106}\) of \(\kappa\)-casein leads to the exposure of its hydrophobic surface, facilitating the aggregation of casein and the formation of clots [2]. Microbial rennet-like coagulants have properties like those of calf rennet; specifically, they are aspartic proteases (EC 3.4.23) able to hydrolyze bovine \(\kappa\)-casein at the Phe\({}_{105}\)-Met\({}_{106}\) bond, like calf rennet. Among plant proteolytic enzymes that show clotting activity are ficin, a cysteine endopeptidase, from the milky sap of _Ficus carica_ (EC 3.4.22.3) [4] and papain from the latex _Carica papaya_ (EC 3.4.22.2), a cysteine protease. The latter hydrolyzes proteins with broad specificity for peptide bonds, while preferring an amino acid bearing a large hydrophobic side chain at the P2 position and not accepting Val at P1 [4]. Furthermore, aspartic proteases from cardoon and artichoke show specific activity for the Phe\({}_{105}\)-Met\({}_{106}\) bond of bovine \(\kappa\)-casein [5]. In particular, the main proteases are cardosin A and cardosin B. The first enzyme is involved in the coagulation process, it has an activity like that of chymosin, which is responsible for the hydrolysis of \(\kappa\)-casein in milk, while cardosin B has an activity similar to that of pepsin and is responsible for specific proteolysis during cheese ripening [6]. It has been reported that the high proteolytic activity of plant-derived rennet causes lower cheese yields, bitter flavors, and texture defects when they are used for bovine milk, if the amounts of extract used are exceeded [4; 7; 8]. In some cases, plant extracts, such as those from cardoon and fig milky sap, have been used for making sheep and goat cheeses. It has been reported that the type of milk (bovine, sheep, or goat) and the milk composition influence the yield and quality of the cheese [4]. A study on an Italian artisanal cheese, obtained from raw cow's milk with an aqueous extract of _C. carduculus_ flowers, demonstrated that the curd forms slowly and that, after 30-45 days of maturation, the aroma of the cheese is intense with the scent of thistle and wild grass, while the flavor is slightly bitter [9]. Furthermore, it has been observed that cheese, obtained by using raw milk, contains lactic acid bacteria (_Lactococcus lactis_ and _Leuconstoc mesenteroides_) up to two weeks of ripening, indicating that enzymes derived from _C. carduculus_ will not have a relevant effect on cheese ripening and in the release of volatile compounds that contribute to the cheese flavor [10]. Herein, artichoke, cardoon, fig latex, and papaya extracts were further investigated for valuable rennet for producing cheese, while other sources, such as pineapple (_Ananascomosus_ (L.) [PERSON]) and oyster mushroom (_Pleurotus ostreatus_ (Jacq. ex Fr.) [PERSON]), were of interest, and their clotting ability, as well as their milk coagulation activity, were innovatively analyzed and compared to that of calf rennet. Therefore, some physical parameters of the curds obtained from pasteurized whole milk were measured. In addition, the lipase/esterase activity of the extracts was measured since this activity influences the cheese flavor. The free fatty acid profile was further investigated, focusing on medium- and long-chain compounds, especially essential \(\omega\)6- and \(\omega\)-3 fatty acids (FAs), which can positively affect lipid quality indices and the nutritional value of FA-containing food products.