alpha-Keto acids. 3

Although it is not fully clear, the rate-limiting step in amino acid catabolism appears to be the action of aminotransferases on amino acids. Hence, there has been attention paid to accelerating this step by increasing in cheese the concentration of the co-substrate for aminotransferase action, alpha-ketoglutarate.

alpha-Ketoglutarate may be produced from glutamic acid by the action of glutamate dehydrogenase (essentially a reversal of aminotransferase action) or, in citrate-positive lactococci, by the citrate-oxalate pathway. Another possible pathway involving citrate metabolism, the citrate-isocitrate pathway, does not appear to be operational in lactic acid bacteria.

A number of authors (e.g., Yvon et al., 1998; Banks et al., 2001; Shakeel-Ur-Rehman and Fox, 2002) have added alpha-ketoglutarate to cheese and seen its effect on the products of amino acid catabolism. For example, Banks et al. (2001) added alpha-ketoglutarate to Cheddar curd at salting and saw a statistically significant increase in the concentration of some volatile flavour compounds derived from amino acid catabolism (see table).

Another strategy (e.g., Rijnen et al., 2000) involves cloning the gene for glutamate dehydrogenase into a strain of Lactococcus; likewise, these authors also found an effect on the production of volatile flavour compounds in model cheeses.

Further reading

Banks, J.M., Yvon, M., Gripon, J.C., de la Fuente, M.A., Brechany, E.Y., Williams, A.G. and Muir, D.D. (2001). Enhancement of amino acid catabolism in Cheddar cheese using a-ketoglutarate: amino acid degradation in relation to volatile compounds and aroma character. Int. Dairy J. 11, 215-243

Curtin, Á.C. and P.L.H. McSweeney (2004). Catabolism of amino acids in cheese during ripening. In Cheese: Chemistry, Physics and Microbiology, Volume 1, General Aspects, 3^rd edition, P.F. Fox, P.L.H. McSweeney, T.M. Cogan and T.P. Guinee (eds), Elsevier Applied Science, Amsterdam. pp. 436-454.

Rijnen, L., Courtin, P., Gripon, J-C. and Yvon, M. (2000). Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds. Appl. Environ. Microbiol. 66, 1354-1359

Shakeel Ur-Rehman and Fox, P.F. (2002). Effect of added a-ketoglutaratic acid, pyruvic acid or pyridoxal phosphate on proteolysis and quality of Cheddar cheese. Food Chem. 76, 21-26.

alpha-Keto acids. 2

In addition to conversion to hydroxyacids by the action of 2-hydroxyacid dehydrogenases, alpha-keto acids can be decarboxylated to the corresponding aldehyde. In the example of the degradation of tryosine, its alpha-keto acid (p-hydroxy phenylpyruvate) is converted to phydroxy phenylethanal, which in turn can be oxidised to the corresponding alcohol (p-hydroxy phenylethanol) or reduced to the corresponding carboxylic acid (p-hydroxy phenylethanoic acid). alpha-Keto acids can also undergo a series of chemical degradations leading, in the example of tyrosine to products such as p-hydroxy benzaldehyde.

alpha-Keto acids. 1

The alpha-keto acids produced by aminotransferase activity are relatively unstable and do not accumulate in cheese but are rather degraded via a range of pathways. Taking tyrosine as an example, its alpha-keto acid (p-hydroxy phenylpyruvate) can be degraded by 2-hydroxyacid dehydrogenases to the corresponding hydroxy acid (p-hydroxy phenyl lactate). Other pathways of degradation of alpha-keto acids include decarboxylations and chemical degradations forming other volatile flavour compounds, which will be discussed in future posts.

Aminotransferases

The catabolism of amino acids to a wide range of volatile flavour compounds is amongst the most important series of reactions in the development of cheese flavour. The key enzymes in the degradation of free amino acids appear to be aminotransferases (ATases) from lactic acid bacteria. ATases are intracellular enzymes whose physiological role is in the interconversion of amino acids. These enzymes require pyridoxal-5'-phosphate (PLP) as a co-factor and catalyse the transfer of the amino group of a donor amino acid (leucine in the example below) to an acceptor molecule, usually alpha-ketoglutarate, forming a product alpha-keto acid corresponding to the donor amino acid (alpha-ketoisocaproate in this example) and glutamic acid. The catalytic mechanism of ATases involves two steps: firstly, the amino group of the donor amino acid is transferred to PLP to yield the product alpha-keto acid and enzyme-bound pyridoxamine-5'-phosphate. Secondly, the amino group is transferred from pyridoxamine-5'-phosphate to the acceptor alpha-keto acid, thus regenerating PLP. The alpha-keto acids formed by ATase action are unstable and degrade to a wide range of compounds via enzymatic and/or chemical pathways.