Development of a plant-based milk-clotting enzyme and a debittering microbial culture for the formulation of halal-certified healthy cheese
Authors:   , Nicosia Fabrizio Domenico 
Published: August 2024

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.