In this chapter, the use of microbial non-coagulant proteases, microbial lipases, and microbial transglutaminase in the cheese making procedure is discussed. Microbial proteases and lipases have been used for over 30 years to accelerate cheese ripening and consequently to enhance the cheese flavor development by increasing proteolysis and lipolysis level in a shorter time. They are commercially produced by bacteria and fungi species. Transglutaminase is a relative new enzyme, which catalyzes the cross-linking of peptide bonds and helps to improve the cheese texture and to increase the cheese yield. Today, cheeses from almost all cheese categories are produced using these enzymes.
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It is commonly accepted that the most important exogenous enzymes in cheese industry are the milk-clotting enzymes. The rennet enzymes as well as the milk coagulants, i.e. enzymes from non-ruminant animals, plants, microorganisms or biotechnologically invented, belong to aspartic group of proteases (EC 3.4.23). Although microbial coagulants are aspartic proteases they have special molecular characteristics and specificity (Moschopoulou, 2017). The exogenous non-coagulant enzymes used in cheese making for various purposes are mainly proteases and lipases and in some cases transglutaminase. Production, characterization and use of exogenous microbial enzymes in dairy processing have been extensively reviewed (Neelakantan et al., 1999; Jooyandeh et al., 2009; El-Hofi et al., 2011; Feijoo –Siota et al., 2014; Garcia et al., 2016; Romeih & Walker 2017).
Proteases are generally classified according to their optimum pH range as:
- 1.
Acidic,
- 2.
Neutral or
- 3.
Alkaline proteases
and according to their catalytic mechanism of action, as
- 1.
Serine proteases (EC 3.4.21),
- 2.
Serine carboxyl proteases (EC 3.4.16),
- 3.
Cysteine proteases (EC 3.4.22),
- 4.
Metallo proteases I (EC 3.4.24) or metallo carboxyl proteases (EC 3.4.17).
They are also classified according to their specificities for amino acid sequences to be cleaved (Garcia et al., 2016). Microbial proteases are bacterial or fungal, having different molecular characteristics and mechanism of action. Bacterial neutral proteases may exhibit activity at the pH range 6.5-8.5, and at 10-70oC (optimum temperature at about 55oC) and are characterized by their high affinity for hydrophobic amino acids pairs. Some of the neutral proteases belong to the metallo-protease type and require divalent metal ions for their activity, while those belonging to serine proteases are not affected by chelating agents. Bacterial alkaline proteases present activity at high pH values e.g. pH 9.0, have broad substrate specificity and show optimal temperature at about 60oC. Fungi produce a big variety of proteolytic enzymes. For example, Aspergillus oryzae produces acid, neutral and alkaline proteases, which are active at a wide pH range, i.e. pH 4-11 and show broad substrate specificity. Fungal acid proteases are mostly used in milk clotting, while fungal neutral proteases and metallo-proteases combined with bacterial proteases are used to reduce bitterness in cheeses. They hydrolyze hydrophobic amino acid bonds, are active at pH 7.0 and are inhibited by chelating agents (Rao et al., 1998; Tavano, 2017). Production of microbial enzymes is made either by submerged fermentation or by solid-state fermentation. Bacterial commercial proteases are mostly produced by submerged fermentation, whereas fungal proteases are produced by solid-state fermentation (Aguilar et al., 2008).