Solubility of carbon dioxide in renneted casein matrices: effect of pH, salt, temperature, partial pressure, and moisture to protein ratio

The solubility of carbon dioxide (CO2) in the moisture and protein components of cheese matrices and the influence of changing pH, salt and temperature levels remains unclear. In this study, model casein matrices were prepared, by renneting of micellar casein concentrate (MCC), with modulation of salt and pH levels by adding salt and glucono delta-lactone, respectively, to the MCC solutions prior to renneting. Different moisture-to-protein levels were achieved by freeze-drying, incubation of samples at different relative humidities, or by applying varying pressures during gel manufacture. The CO2 solubility of samples decreased linearly with both increasing temperature and salt-in-moisture content, whereas solubility of CO2 increased with increasing pH. A non-linear relationship was observed between CO2 solubility and the moisture-to-protein ratio of experimental samples. Overall, such knowledge may be applied to improve the quality and consistency of eye-type cheese, and in particular to avoid development of undesirable slits and cracks

Solubility of carbon dioxide in renneted casein matrices: Effect of pH, salt, temperature, partial pressure, and moisture to protein ratio

peer-reviewedThe solubility of carbon dioxide (CO2) in the moisture and protein components of cheese matrices and the influence of changing pH, salt and temperature levels remains unclear. In this study, model casein matrices were prepared, by renneting of micellar casein concentrate (MCC), with modulation of salt and pH levels by adding salt and glucono delta-lactone, respectively, to the MCC solutions prior to renneting. Different moisture-to-protein levels were achieved by freeze-drying, incubation of samples at different relative humidities, or by applying varying pressures during gel manufacture. The CO2 solubility of samples decreased linearly with both increasing temperature and salt-in-moisture content, whereas solubility of CO2 increased with increasing pH. A non-linear relationship was observed between CO2 solubility and the moisture-to-protein ratio of experimental samples. Overall, such knowledge may be applied to improve the quality and consistency of eye-type cheese, and in particular to avoid development of undesirable slits and cracks

Plasmid-Encoded Diacetyl (Acetoin) Reductase in Leuconostoc pseudomesenteroides

A plasmid-borne diacetyl (acetoin) reductase (butA) from Leuconostoc pseudomesenteroides CHCC2114 was sequenced and cloned. Nucleotide sequence analysis revealed an open reading frame encoding a protein of 257 amino acids which had high identity at the amino acid level to diacetyl (acetoin) reductases reported previously. Downstream of the butA gene of L. pseudomesenteroides, but coding in the opposite orientation, a putative DNA recombinase was identified. A two-step PCR approach was used to construct FPR02, a butA mutant of the wild-type strain, CHCC2114. FPR02 had significantly reduced diacetyl (acetoin) reductase activity with NADH as coenzyme, but not with NADPH as coenzyme, suggesting the presence of another diacetyl (acetoin)-reducing activity in L. pseudomesenteroides. Plasmid-curing experiments demonstrated that the butA gene is carried on a 20-kb plasmid in L. pseudomesenteroides

HdcB, a novel enzyme catalysing maturation of pyruvoyl-dependent histidine decarboxylase

Pyruvoyl-dependent histidine decarboxylases are produced as proenzymes that mature by cleavage followed by formation of the pyruvoyl prosthetic group. The histidine decarboxylation pathway of Streptococcus thermophilus CHCC1524 that consists of the pyruvoyl-dependent histidine decarboxylase HdcA and the histidine/histamine exchanger HdcP was functionally expressed in Lactococcus lactis. The operon encoding the pathway contains in addition to the hdcA and hdcP genes a third gene hdcB. Expression of different combinations of the genes in L. lactis and Escherichia coli followed by analysis of the protein products demonstrated the involvement of HdcB in the cleavage of the HdcA proenzyme. The HdcA proenzyme and HdcB protein were purified to homogeneity and cleavage and activation of the histidine decarboxylase activity was demonstrated in vitro. Substoichiometric amounts of HdcB were required to cleave HdcA showing that HdcB functions as an enzyme. In agreement, expression levels of HdcB in the cells were low relative to those of HdcA. The turnover number of HdcB in vitro was extremely low (0.05 min(-1)) which was due to a very slow association/dissociation of the enzyme/substrate complex. In fact, HdcB was shown to co-purify both with the HdcA S82A mutant that mimics the proenzyme and with the mature HdcA complex

Comparison of the Hydrolysis of Bovine κ-Casein by Camel and Bovine Chymosin: A Kinetic and Specificity Study

Bovine chymosin constitutes a traditional ingredient for enzymatic milk coagulation in cheese making, providing a strong clotting capacity and low general proteolytic activity. Recently, these properties were surpassed by camel chymosin, but the mechanistic difference behind their action is not yet clear. We used capillary electrophoresis and reversed-phase liquid chromatography-mass spectrometry to compare the first site of hydrolysis of camel and bovine chymosin on bovine κ-casein (CN) and to determine the kinetic parameters of this reaction (pH 6.5; 32 °C). The enzymes showed identical specificities, cleaving the <i>Phe105</i>–<i>Met106</i> bond of κ-CN to produce <i>para</i>-κ-CN and caseinomacropeptide. Initial formation rates of both products validated Michaelis–Menten modeling of the kinetic properties of both enzymes. Camel chymosin bound κ-CN with ∼30% lower affinity (<i>K</i>_M) and exhibited a 60% higher turnover rate (<i>k</i>_cat), resulting in ∼15% higher catalytic efficiency (<i>k</i>_cat/<i>K</i>_M) as compared to bovine chymosin. A local, less dense negatively charged cluster on the surface of camel chymosin may weaken electrostatic binding to the <i>His</i>–<i>Pro</i> cluster of κ-CN to simultaneously impart reduced substrate affinity and accelerated enzyme–substrate dissociation as compared to bovine chymosin