# Exploring New Fruit- and Vegetable-Derived Rennet for Cheese Making
###### Abstract  
Cheese production is an ancient practice to preserve a perishable food, such as milk, for a long time. The first step of cheese processing involves the addition of rennet, which contains the enzymes necessary for the hydrolysis and coagulation of the caseins present in milk. Typically, animal-derived rennet, such as calf rennet containing chymosin, are used as source of enzymes for cheese processing. Alternatively, microbial chymosin or recombinant chymosin is used. However, recently, plant-derived rennet such as the ones derived from thistle and bitter orange flowers and from aritchoke (_Cymara cardunculus_ var. _scolymuts_) have also been demonstrated to be valid sources of enzymes for cheese processing. Therefore, herein, different plant and fruit extracts were tested and compared for their ability to coagulate milk caesines. In particular, beyond aritchoke and cartoon (_Cymana cardunculus_) extracts, those from pineapple (_Annaas comoss_ (L) [PERSON]), papaya (_Caria parqua L._), common fig (_Ficus carica_ L.) milky sap, and oyster mushroom (_Pleirotus ostravatus_ (Jacq, ex Fr.) [PERSON]) were investigated for their proteolytic, esterase, and milk-clotting activities. The extracts were then exploited as vegetable and fruit rennet for the experimental production of cheeses, which were examined, after 30 days of maturation, for their moisture, fat, protein, and free fatty acid (FFA) content. Interestingly, the aritchoke, cardoon, and thistle mushroom extracts showed high proteolytic activity compared to calf rennet, while the level of esterase activity appeared to be similar for all the extracts. The papaya extract showed the lowest proteolytic and esterase activity. Although the pH, moisture, fat, and protein contents were very similar to those of cheese made with calf rennet, the medium- and long-chain FFAs broadly differed among produced cheeses, with variations in the lipid quality indices. fruit- and plant-derived rennet; cheese making; milk-clotting activity; fatty acids +
Footnote †: journal: _Sciencedence_  
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fact, natural rennet is a mixture of different enzymes, such as chymosin or chymosin-like proteases (coagulation enzymes), lipase, esterase, lactase, and catalase [1]. Milk coagulation is characterized by the selective hydrolysis of \(\kappa\)-caseins, which leads to casein solidification and curd formation [2]. The main clotting enzyme used is chymosin, derived from calf rennet. The growing demand for calf rennet has led to the development of alternative rennet, among which the most used are those containing enzymes from _Rhizomucor miehei_, _Rhizomucor pusillus_, and _Aspergillus oryzae_. Alternatively, recombinant calf chymosin produced in _Escherichia coli_, _Bacillus subtilis_, and _Lactococcus lactis_ is also used [1; 2]. Some ancient artisanal cheeses produced in Italy, Spain, and Portugal use vegetable rennet derived from artichoke and its wild form, namely cardoon (_Cynara carduculus_) [3]. Furthermore, in some areas of the Mediterranean, the milky sap from fig (_Ficus carica_) is also exploited [1]. The development of alternative rennet, and in particular those of vegetable origin, has become not only a commercial but also an ethical requirement, considering dietary styles, such as vegetarian or vegan ones, which do not involve the consumption of foods of animal origin, or all those who do not consume these products for religious reasons. In general, the clotting activity of plant extracts is slower than that obtained from animal rennet, but plant-derived clotting enzymes are characterized by a high proteolytic activity against milk caseins, which generates peptides able to affect the cheese texture and contribute to the development of a bitter flavor [2]. During cheese production, one of the first steps is the solubilization of calcium phosphate by decreasing the pH of the milk. Calcium phosphate helps to stabilize casein micelles.