Study of Enterobacteriaceae throughout the manufacturing and ripening of hard goats' cheese.

The evolution of the counts and the species of Enterobacteriaceae as well as some physico-chemical parameters (pH, aw and NaCl and moisture contents) during manufacturing and ripening of a hard Spanish goats' cheese of the Armada-Sobado variety were studied. Enterobacteriaceae (mean log counts 4.45 g-1 in milk) increased 0.71-2.18 log units in curd and afterwards decreased until they disappeared after 2-4 weeks of ripening. This premature disappearance seems to be due to the decrease in aw values and in moisture contents. However, the low pH values, reached from the beginning of the ripening process, could also contribute to this phenomenon. The most abundant species in milk was Serratia liquefaciens (57.5% of isolates), followed by Morganella morganii (27.5%), Hafnia alvei (5%), Klebsiella oxytoca (5%) and Yersinia enterocolitica (5%). Yersinia enterocolitica was not subsequently isolated from either curd or in cheese. Hafnia alvei numbers increased in curd and in 1-week-old cheese where this micro-organism was the most abundant (47.5% and 75% of the isolates respectively). Escherichia coli, which was not isolated from milk, curd or 1-week-old cheese, was the predominant organism in 2-week-old cheese (57.8% of isolates). This confirms the finding of other authors who have shown that it is one of the most resistant species in ripening cheeses.

Kinetics and thermodynamics of diacetyl reduction with NADPH by alpha-dicarbonyl reductase from pigeon liver.

alpha-dicarbonyl reductase from pigeon liver catalyzes diacetyl reduction with NADPH via an ordered Bi-Bi mechanism in which the coenzyme is the leading substrate, as deduced from the inhibition pattern by products and by acetone. The activation energy of the reaction has been calculated as 16.6 kcal/mol, delta H and delta F as 15.6 and 15.3 kcal/mol, respectively, and delta S as 1 cal/mol per k. Kinetic constants obtained for substrates (KmNADPH = 15 microM, KsNADPH = 10 microM; Kmdiacetyl = 0.5 mM, Ksdiacetyl = 0.35 mM) and products (KiNADP 50 microM; Kiacetoin = 100 mM) are about 10 times lower than those reported for this enzyme in the reduction of diacetyl with NADH. This confirms that NADPH is its physiological coenzyme.

Further purification and characterization of diacetyl reductase from pigeon liver.

Electrophoretically pure preparation of an enzyme from pigeon liver which is classified in the I.U.B. Enzyme List as a specific diacetyl reductase (Acetoin: NAD oxidoreductase, EC 1.1.1.5) have been obtained. This enzyme has been characterized as an alpha-dicarbonyl reductase (L(+)-alpha-hydroxicarbonyl: NAD(P) oxidoreductase, EC 1.1.1...) similar to the one recently discovered from beef liver by the authors' group. Although differences in mol wt among these two reductases had been reported, both of them are oligomers of 25,000-26,000 dalton subunits.

Kinetics of alpha-dicarbonyls reduction by L-glycol dehydrogenase from hen muscle.

Michaelis constants of L-glycol dehydrogenase from hen muscle (isozyme of pI 7.2) for the alpha-dicarbonyls tested (glyoxal, 2,3-pentanedione, methylglyoxal, and diacetyl) range from 35 microM for pentanedione to 0.41 mM for glyoxal. The enzyme shows a high affinity for NADPH, Km (2.2-3.1 microM), and Ks (1.2-1.9 microM) being so much lower than its tissue concentration that L-glycol dehydrogenase has to operate in vivo saturated with the coenzyme; this condition is very unfavorable to play a role in regulating the equilibrium oxidized/reduced forms of the pyridine nucleotides, as it has been proposed for some similar enzymes. Convergence of the double reciprocal plots and the pattern of inhibition by products and by acetone, a substrate analog, demonstrate that glyoxal reduction--and most likely that of diacetyl--proceeds via an ordered Bi-Bi mechanism in which NADPH is fixed before the addition of the carbonyl; the reduction of methylglyoxal and 2,3-pentanedione could follow the same model, but our experimental results are also consistent with that of Theorell-Chance.