Cheddar cheese amino acids

The level of proteolysis in cheese varies from limited (e.g. Mozzarella) through moderate (e.g. Cheddar and Gouda) to very extensive (e.g. Blue cheeses). The products of proteolysis range from very large polypeptides, only a little smaller than the parent caseins, to amino acids which may, in turn, be catabolized to a very diverse range of sapid compounds, including amines, acids and sulphur compounds. 

Figure 10.24 Concentration of individual amino acids in 60-day-old Cheddar cheese, made with a single-strain starter Lactococcus lactis ssp. cremoris AM2, G11/C25 or HP (from...

During cheese ripening, proteases associated with starter culture organisms are released into cheese after cell lysis (Law et al. 1974). The proteolytic activity associated with lysed lactic streptococci is necessary for proper flavor development in Cheddar and other cheese varieties. The role of streptococcal proteases and peptidases appears to be production of flavor compound precursors such as methionine and other amino acids, rather than direct production of flavor compounds (Law et al. 1976A). Additional discussion of cheese ripening is presented in Chapter 12. 

Bitter peptides have been identified in hydrolyzates of casein (12,13), cheese (13a,b), and soy bean (14,15,15a). The bitter taste has been related to the hydrophobic amino acid content (16-20) and to chain length. Ney and Retzlaff (21) established a formula relating the bitterness of peptides to their amino acid composition and chain length. Too large a proportion of hydro-phobic amino acids gives rise to bitterness yet above a certain molecular weight, bitterness is not perceptible even when there are hydrophobic amino acids (21). Peptides that were responsible for bitterness in Cheddar cheese were rich in Pro, which occurred predominantly in the penultimate position (21a). 

Many enzymatic assays have also been developed for the analysis of proteolytic products. Total amino acids in Cheddar cheese were determined by Puchades et al. (1990) using the L-amino acid oxidase enzyme. Glutamic acid has been quantified by flow injection analysis using glutamate dehydrogenase (Puchades et al., 1989) and using the Boehringer-Mannheim kit (McSweeney et al., 1993). 

FIGURE 5.4 GC-FID chromatogram of Cheddar cheese extract showing the amino acid profile. Norvaline was used as the internal standard (IS). 

Free amino acids are further catabolized into several volatile flavor compounds. However, the pathways involved are not fully known. A detailed summary of the various studies on the role of the catabolism of amino acids in cheese flavor development was published by Curtin and McSweeney (2004). Two major pathways have been suggested (1) aminotransferase or lyase activity and (2) deamination or decarboxylation. Aminotransferase activity results in the formation of a-ketoacids and glutamic acid. The a-ketoacids are further degraded to flavor compounds such as hydroxy acids, aldehydes, and carboxylic acids. a-Ketoacids from methionine, branched-chain amino acids (leucine, isoleucine, and valine), or aromatic amino acids (phenylalanine, tyrosine, and tryptophan) serve as the precursors to volatile flavor compounds (Yvon and Rijnen, 2001). Volatile sulfur compounds are primarily formed from methionine. Methanethiol, which at low concentrations, contributes to the characteristic flavor of Cheddar cheese, is formed from the catabolism of methionine (Curtin and McSweeney, 2004 Weimer et al., 1999). Furthermore, bacterial lyases also metabolize methionine to a-ketobutyrate, methanethiol, and ammonia (Tanaka et al., 1985). On catabolism by aminotransferase, aromatic amino acids yield volatile flavor compounds such as benzalde-hyde, phenylacetate, phenylethanol, phenyllactate, etc. Deamination reactions also result in a-ketoacids and ammonia, which add to the flavor of... 

Several of the smaller volatile compounds formed from the catabolism of products of primary proteolysis (e.g., amino acids) can be determined by GC. The development of capillary columns and interfacing GC with MS has noticeably increased the sensitivity of this analysis. Over 200 volatile compounds have been identified in Cheddar cheese. A list of several of these compounds can be found elsewhere (Fox et ah, 2004a Singh et ah, 2003). The instrumental techniques available for the characterization of cheese aroma were also discussed recently (Le Quere, 2004 Singh et al., 2003). 

Lemieux, L., Puchades, R., and Simard, R. E. (1990). Free amino acids in Cheddar cheese Comparison of quantification methods.. Food Sci. 55,1552-1554. 

Figure 11.7. Effect of fat content on the levels of pH 4.6-soluble N (A) and free amino acids (B) in Cheddar cheeses aged for 30 ( ), 90 (O), 180 (A), or 225 (A) days (drawn from data of Fenelon and Guinee, 1999 Guinee et al., 2000a).

Jarrett, W.D., Aston, J.W., Dulley, J.R. 1982. A simple method for estimating free amino acids in Cheddar cheese. Aus. J. Dairy Technol. 37, 55-58. 

Lactate in cheese may be oxidized to acetate. Pediococci produce 1 mol of acetate and 1 mol of CO2 and consume 1 mol of O2 per mole of lactate utilized (Thomas et al, 1985). The concentration of lactate in cheese far exceeds that required for optimal oxidation, and lactate is not oxidized until all sugars have been exhausted. The oxidation of lactate to acetate in cheese depends on the NSLAB population and on the availability of O2, which is determined by the size of the block and the oxygen permeability of the packaging material (Thomas, 1987). Acetate, which may also be produced by starter bacteria from lactose (Thomas et al., 1979) or citrate or from amino acids by starter bacteria and lactobacilli (Nakae and Elliott, 1965), is usually present at fairly high concentrations in Cheddar cheese and is considered to contribute to cheese flavor, although high concentrations may cause off-flavors (see Aston and Dulley, 1982). Thus, the oxidation of lactate to acetate probably contributes to Cheddar cheese flavor. 

Significant concentrations of amino acids, the final products of proteolysis, occur in all cheeses that have been investigated (see McSweeney and Fox, 1993). Relative to the level of water-soluble nitrogen, Cheddar contains a low concentration of amino acids (see Fig. 19, Section VC2). The principal amino acids in Cheddar are Glu, Leu, Arg, Lys, Phe, and Ser (Wilkinson, 1992) (Fig. 15). Parmesan contains a very high concentration of amino... 

FIG. 15. Free amino acids in Cheddar cheese made using different starters and ripened at lO C for 42 days (Wilkinson, 1992). 

Amino acids in Cheddar cheese are deemed to be perceived if their concentration in the water extract is greater than their threshold concentration. Asterisks indicate degree of taste sensation. 

The small peptides in cheese can be fractionated by various forms of chromatography, e.g., gel permeation, ion-exchange, and especially RP-HPLC. Using these techniques, more than 200 peptides have been demonstrated in Cheddar cheese, many of which have been isolated and identified (see Section IVE). Free amino acids are usually quantified by ion-exchange HPLC with post-column derivitization using ninhydrin or by separation of fluorescent amino acid derivatives by RP-HPLC. 

FIG. 18. Concentration of total free amino acids (Cd-ninhydrin assay) in Cheddar cheese as a function of age (from O Shea, 1993). 

Hickey, M. W., van Leeuwen, H., Hiliier, A. J., and Jago, G. R. (1983b). Amino acid accumulation in Cheddar cheese manufactured from normal and ultrafiltered milk. Aust. J. Dairy Technol. 38, 110-113. 

Williams, A. G., Noble, J., Tammam, J., Lloyd, D., Banks, J. M. (2002). Factors affecting the activity of enzymes involved in peptide and amino acid catahohsm in non-starter lactic acid bacteria isolated from Cheddar cheese. International Dairy Journal, 12, 841-852. 

Table 10.33. Amino acid sequences of some small peptides from Cheddar cheese...

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