II Processing of Dairy Products
Enzymes Applications for the Dairy Industry
Antonio Trani, Pasqua Loizzo, Angela Cassone and Michele Faccia
Department of Soil, Plant and Food Sciences, University of Bari, Bari, Italy
Milk contains a number of indigenous enzymes that play an important role in processing and quality of milk for direct consumption and cheese. Besides them, exogenous enzymes can be added for particular goals, varying from milk coagulation, to cheese processing and ripening, enhancing of shelf life and safety, and cleaning and treating dairy wastewaters. This chapter discusses only the use of exogenous enzymes, with particular emphasis to innovative applications. To this aim, applications have been classified into three main groups: use in cheese making, applications for shelf- life extension, and applications for functional and environmental purposes.
Use in Cheese Making
Proteases as Milk Coagulants
Milk coagulants belong to four main categories: animal rennet, plant-derived coagulants, microbial coagulants, and genetically engineered chymosin. Animal rennet contains the two main enzymes, chymosin and pepsin, in a ratio that depends on the age of the animals when slaughtered. Due to different specificity, the ratio between the two enzymes deeply influences proteolysis during cheese ripening (Santoro and Faccia, 1998; Irigoyen et al., 2002; Faccia et al., 2003). Calf chymosin is expressed in two major forms (A and B), and its technological properties have been extensively studied (Jacob et al., 2011). Lamb chymosin has similar coagulation properties as calf, but shows a slightly higher temperature dependency (Rogelj et al., 2001). Little information is available on kid and buffalo rennet, which are coagulants of local interest—both have maximum milk clotting activity at 30°C, and the former is more heat-stable than calf rennet (Mohanty et al., 2003; Kumar et al., 2006).
Besides liquid rennet, which is the most used type, paste preparations are also relevant, in particular for traditional ovine cheeses. Poor standardization and poor hygienic quality have long been the major limits of this type of rennet, especially when prepared at artisanal level. Efforts have been carried out to improve quality, including a filter sterilization process and the addition of probiotics (Calvo et al., 2007; Santillo et al., 2007; Santillo and Albenzio, 2008).
Advances in Dairy Products, First Edition.
Edited by Francesco Conto, Matteo A. Del Nobile, Michele Faccia, Angelo V. Zambrini, and Amalia Conte. © 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.
Plant-derived coagulants commonly derive from aqueous maceration of parts of plants such as leafs, flowers, and branches. The proteases that have a coagulant effect are a characteristic of the type of plant, such as ficin from fig tree (Ficus carica), bromelain from pineapple (Ananas sativa), cinarase and cardosin from cardoon (Cynara cardunculus), and papain from paw paw (Carica papaya). In general, they present disproportionate proteolytic activity with respect to milk-clotting action, and their use is associated to artisanal dairy practices or ethical concerns. Information about cheeses obtained with these coagulants is now available, thanks to the increasing interest towards traditional and lacto-vegetarian foods.
Mostly, information regards European cheeses from goat's and ewe's milk such as Cacioricotta from Italy, Serra da Estrela and Serpa from Portugal, La Serena, Torta del Casar, Los Ibores and Flor de Guia from Spain (Macedo et al., 1997; Roa et al., 1999; Sanjuan et al., 2002; Faccia et al., 2012). The study of new potential plant-derived coagulants is in continuous process (Katsaros et al., 2010).
Microbial coagulants are low-cost substitutes of rennet, since they are easily produced by fermentation. The main sources are fungi, and even though more than 100 fungal sources have been reported, the most used species are Rhizomucor miehei, Rhizomucor pusillus and Cryphonectria parasitica. Their technological properties are different from those of rennet: in particular, they have higher resistance to heat and stronger proteolytic activity during cheese making and ripening. A major concern of excessive or not well-balanced proteolysis is the development of bitter taste, which is related to intense degradation of p-casein (Jacob et al., 2011). Even though treatments for decreasing thermostability are currently applied by the producers, microbial coagulants are mostly employed for making fresh and short-ripened cheeses. Genetically engineered chymosin is produced with recombinant DNA technology. A number of microorganisms are currently used for the production of this enzyme, such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Kluyveromyces lactis, and others. Recombinant Bos taurus chymosin is by far the most produced genetically engineered clotting enzyme, but methods for obtaining different types of recombinant chymosin are also available (Kappeler et al., 2006; van Roijen et al., 2008). Despite of effectiveness in milk coagulation and well-balanced proteolysis in cheese ripening, use of recombinant rennet presents some ethical concerns in countries that have sensitivity to GMO-free policies.