Contribution of Lactococcus lactis Cell Envelope Proteinase Specificity to Peptide Accumulation and Bitterness in Reduced-Fat Cheddar Cheese

Abstract

Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the Lactococcus lactis extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of Lactococcus lactis as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of ␣ S1 -casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.Proteolysis and its secondary reactions play a major role in the maturation of Cheddar and many other bacterium-ripened cheese varieties (16). Proteolysis in Cheddar cheese is a complex process that involves endogenous milk enzymes, coagulant, and microbial proteinases and peptidases. Hydrolysis of intact casein (CN) is catalyzed almost exclusively by the added coagulant and endogenous milk enzymes, while proteinases and peptidases from Lactococcus lactis starter bacteria and adventitious (nonstarter) lactic acid bacteria are responsible for the production of water-soluble peptides and free amino acids (17). The contribution of individual enzymes in the cheese matrix to this process will also be influenced by specificity, relative activity, stability in the cheese matrix, and in the case of intracellular enzymes, access to appropriate substrates.In many bacterium-ripened cheeses, the L. lactis cell envelope-associated proteinase (lactocepin, EC 3.4.21.96) is the most important microbial enzyme for the conversion of largemolecular-weight (water-insoluble) peptides produced by coagulant or plasmin into the small water-soluble peptides needed for flavor development (10,17,35). Lactocepin is a 180-to 190-kDa membrane-anchored enzyme that belongs to the subtilisin family of serine proteases. Although lactocepins exhibit more than 98% amino acid sequence identity, purified enzymes may be differentiated by their relative affinity and specificity for individual CNs (23). Genetic studies showed that most differences in lactocepin specificity could be traced to amino acid substitutions in the enzyme substrate-binding regions, and this ...

Manufacture and biochemical characteristics during ripening of Cheddar cheese with variable NaCl and equal moisture content

“…Day 1 concentrations of the N-terminal peptide of primary chymosin cleavage of α S1 -CN, α S1 -CN(f1-23) (AN) appeared considerably lower than initial CEP-derived peptides, which in combination with the rapid accumulation of α S1 -I-CN confirms the importance of α S1 -CN(f1-23) conversion by lactococcal CEP in the early phase production of amino acid N in cheese . α S1 -CN(f1-9/13) (E/I) accumulated extensively during early ripening as shown previously (Broadbent et al 2002) followed, soon after, by marked accumulations of additional starter CEP-derived α S1 -CN N-terminal peptides, including α S1 -CN(f1-14/16/17) (J/Y/U) (Fig. 3, 30 days panel).…”

“…It is noted, however, that co-elution with particularly α S2 -CN(f148-207) caused an overestimation of the net increase in the concentration of β-CN(f193-209) during ripening at all salt levels (Table 5). Broadbent et al (2002) reported a slight increase during 6 months ripening of reduced-fat Cheddar cheese. This important, bitter peptide was formed initially in all cheeses, yet at a markedly higher rate with decreasing salt content (P<0.001), which largely ascribes aforementioned effect of salt on β-CN degradation to chymosin activity and agrees with earlier observations on variable S/M levels in Cheddar (Kelly et al 1996).…”

Occurrence of sensory defects in semi-hard ewe’s raw milk cheeses

“…Bitterness has been considered as the major cheese flavour defect for some matured cheese varieties, and in some cases, as for Cheddar cheese, it can become a serious economic concern (Engel et al 2001). Some authors reported that amino acids and peptides originated by hydrolysis of caseins are responsible for bitter flavour in ripened cheeses (Broadbent et al 2002;Engel et al 2001;McSweeney 2007). However, in this study only 4% of the cheese samples showed bitter flavour (Table 2).…”

“…Spanish sheep milk cheese production increased by 28% between 2002(FAO 2014. Ewes are mainly milked for the manufacture of typical dairy products, in particular cheeses, which often have a regional or local connotation of origin and quality (Boyazoglu and Morand-Fehr 2001).…”

Peptidase activity of selected heat-treated Lactobacillus strains

“…Specyficzność CEPs odgrywa zasadniczą rolę w wytwarzaniu gorzkich peptydów [4,20]. Peptydazy kultur bakterii kwasu mlekowego (LAB -lactic acid bacteria), takie jak: PepN, PepX, PepO2, PepO3 biorą udział w degradacji gorzkich peptydów i są odpowiedzialne za jakość sensoryczną produktów mlecznych [5, 6,26,18].…”