The reduced availability and the increasing prices of calf rennet, coupled to the growing global demand of cheese has led, worldwide, to explore alternative clotting enzymes, capable to replace traditional rennet, during the cheesemaking. In addition, religious factors and others related to the vegetarianism of some consumers, have led to alternative rennet substitutes. Nowadays, several plant-derived milk-clotting enzymes are available for cheesemaking technology. Many efforts have also been made to compare their effects on rheological and sensory properties of cheese to those arising from animal rennet. However, vegetable clotting enzymes are still partially suitable for cheesemaking, due to excessive proteolytic activity, which contribute to bitter flavor development. This review provides a literature overview of the most used vegetable clotting enzymes in cheese technology, classified according to their protease class. Finally, clotting and proteolytic activities are discussed in relation to their application on the different cheesemaking products.

A total of 26 lactic acid bacteria isolates from both Italian and Brazilian cheeses were tested for their use in cheesemaking. Isolates were screened for salt tolerance, exopolysaccharide and diacetyl production, lipolytic, acidifying, and proteolytic activities. In addition, the aminopeptidase (Pep N and Pep X) activities, were evaluated. Most of the strains demonstrated salt tolerance to 6% of NaCl, while only two L. delbruekii (P14, P38), one L. rhamnosus (P50) and one L. plantarum (Q3C4) were able to grow in the presence of 10% (w/v) of NaCl. Except for 2 L. plantarum (Q1C6 and Q3C4), all strains showed low or medium acidifying activity and good proteolytic features. Furthermore, lipolytic activity was revealed in none of the strains, while the production of EPS and diacetyl was widespread and variable among the tested strains. Finally, regarding aminopeptidase activities, 1 L. delbrueckii (P10), 1 L. rhamnosus (P50), and 1 L. lactis (Q5C6) were considered as the better performing, showing high values of both Pep N and Pep X. Based on data presented here, the aforementioned strains could be suggested as promising adjunct cultures in cheesemaking.

In the present study, a kiwifruit aqueous extract was developed and used as a coagulant enzyme in cheesemaking. In detail, polyacrylamide gel electrophoresis (SDS-PAGE) was used to investigate the presence of actinidin, the kiwifruit enzyme involved in κ-casein hydrolysis, in different tissues (pulp, peel, and whole fruit) of ripe and unripe kiwifruits. Data revealed the presence of the enzyme both in the peel and in the pulp of the fruit. Although the aqueous extract obtained from the kiwifruit peel was able to hydrolyze semi-skimmed milk, it did not break down κ-casein. The aqueous extract obtained from the pulp showed a hydrolytic activity toward both κ-casein and semi-skimmed milk. The values for milk-clotting and proteolytic activity of the kiwifruit pulp extract were evaluated at different temperatures and pH parameters in order to obtain a high value of the MCA/PA ratio; we found that a temperature of 40 °C in combination with a pH value of 5.5 allowed us to obtain the best performance. In addition, the data revealed a higher hydrolytic activity of the enzymatic preparation from ripe kiwifruits than that from unripe ones, suggesting the use of the extract from pulp of ripe kiwifruits in the laboratory-scale cheesemaking. The data showed that 3% (v/v) of the ripe kiwifruit pulp extract determined a curd yield of 20.27%, comparable to chymosin yield. In conclusion, the extraction procedure for kiwifruit aqueous extract proposed in the present study was shown to be a fast, cheap, chemical-free, and ecofriendly technology as a plant coagulant for cheese manufacturing.

In recent years, there has been a significant shift in consumer preferences towards healthier and more diversified food options, with a particular emphasis on products derived from vegetable sources. This evolving trend is driven by an increased awareness of the link between diet and well-being, as well as a growing desire for sustainable and ethically sourced food choices. Consumers are becoming more health-conscious, with a heightened awareness of the impact of their dietary choices on overall well-being. The emphasis on plant-based diets, rich in fruits and vegetables, is supported by numerous studies linking diets to reduced risks of chronic diseases, to improved cardiovascular health, and to enhanced longevity. The modern consumer is increasingly seeking variety and novelty in their food choices, moving beyond traditional products. Vegetable-based products offer a diverse array of flavors, textures, and culinary experiences, appealing to consumers looking to experiment with their senses. The availability of innovative plant-based alternatives to traditional animal products, such as plant-based substitutes, has resulted in significant changes also in the dairy industry. In detail, the dairy market needs the search for animal rennet substitutes, because of the growth of vegetarian’s market niche as well as the request for Kosher and Halal foods. The role of milk-clotting enzymes is crucial in cheesemaking; among them, animal rennet is the most ancient milk - clotting enzyme and still the most widely used biocatalyst in cheesemaking procedures. The clotting properties of animal rennet are due to chymosin, an aspartic protease extracted from the abomasum of new-born ruminants with high specificity for cleaving k-casein Phe105-Met106 bond. However, the changed perception of the consumer, coupled to a diminishing supply of animal rennet, are responsible of the high demand for alternative milk-clotting enzymes. Several new vegetable sources of milk clotting enzymes have been investigated in the last twenty years and the development of new plants derived milk-clotting enzymes is now in progress. Plant enzymes are generally extracted by aqueous maceration of different plant organs, such as flowers, seeds, roots or leaves, followed by several different homogenization procedures. One innovative milk-clotting enzyme is Actinidin (EC 3.4.22.14), a cysteine protease extracted from kiwifruit (Actinidia deliciosa), that represents an interesting alternative to chymosin for milk coagulation. Previous studies demonstrated that the actinidin forms milk clots under the typical conditions used in cheese manufacturing. Nevertheless, plant proteases are characterized by a high proteolytic activity that generates bitter flavours in the final product. To reduce the bitter taste caused by the strong proteolytic activity, strains of Lactic Acid Bacteria (LAB) with high aminopeptidase activity are promising. Specific aminopeptidases (like Pep X and Pep N) are able to hydrolyze the bitter peptides formed during cheese ripening. Keywords: Milk-clotting enzymes, cheesemaking, vegetable coagulant, actinidin, lactic acid bacteria, aminopeptidase activity.