Partly purified extracellular aminopeptidase from Brevibacterium linens was used to accelerate Cheddar cheese ripening. It was found that although the aminopeptidase was unstable in acidic buffer, it was highly stable in Cheddar cheese; negligible amounts of the enzyme activity were lost during 3 months' maturation. A better score for flavour in sensory analysis of enzyme-treated cheese was obtained by the combination of the aminopeptidase and a commercially available metalloproteinase (Neutrase) than by the metalloproteinase alone.

The cheesemaking properties of recombinant chymosin from Kluyveromyces lactis (Gist-Brocades Chymosin 610) were Compared with those of standard rennet in parallel trials with the same milk and mixed culture starter. For each pair of cheeses the cheesemaking characteristics, mass balance results, composition of the cheeses at 6 weeks and maturation rates were similar. A taste panel was not able to differentiate between the cheeses at 3, 6 or 12 months.

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 °C and pH 7·0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3·5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5·40±0·05 and a mol. wt of 40200±2100. It is heat-stable having D-values at 74 and 140 °C of 1·6 and 1·0 min respectively; 40 and 70% of the original activity remained after HTST (74 °C/17 s) and ultra high temperature (140°C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.

A commercial neutral proteinase was encapsulated in liposomes (multilamellar) and incorporated into cheese curd via the cheese milk. The milk proteins were protected from proteinase attack during curd manufacture. A significant proportion of the liposome-entrapped enzyme was retained in the curd and its subsequent release into the cheese matrix was inferred from an increased rate of β-casein breakdown.

Intracellular cell-free enzyme extracts (CFE) of cheese starter bacteria significantly enhanced the flavour-accelerating effect of commercial neutral proteinase without increasing the gross proteolysis in Cheddar cheese. Peptide and amino acid N were released more rapidly in CFE-treated cheeses, compared with those treated with neutral proteinase or untreated controls.

Optimum conditions have been determined for the use of Hide Powder Azure (HPA) to estimate extracellular proteinases produced by psychrotrophic Gram-negative bacteria. This assay is quicker, more precise and 3·2 times more sensitive than assays of soluble N released from casein. It provides a simple alternative method for monitoring proteinase purification schemes. With the modification described it is now possible to determine proteinase activities directly in milk. In its simplest form the HPA assay is sufficiently sensitive to detect proteinases produced by as few as 1·5 x 106 c.f.u. ml-1 psychrobtrophs without the necessity for any sample processing.

The development of typical flavour in Cheddar cheese was accelerated by the addition of bacterial neutral proteinase to the curds, but not by the addition of fungal acid proteinase which produced a bitter defect even at low concentrations. An optimum level of neutral proteinase addition was established at which general proteolysis was increased by about 20% in 2 months and treated cheeses of this age had flavour intensities typical of 4-month-old untreated cheeses. Enzyme-treated cheeses were softer-bodied and more brittle than untreated cheeses of the same age.

The effect of proteolytic, psychrotrophic strains of Pseudomonas fluorescens, Ps. putida and Acinetobacter spp. on cheese-making with stored milk has been investigated. Ps. fluorescens and Ps. putida growing for 72 h in raw milk at 7·5 °C to levels of approx. 107 colony-forming units/ml caused a low degree of β- and к-casein breakdown detectable by gel electrophoresis, but this was insufficient to affect N losses in whey or cheese yields. Variations in cheese-making times with pasteurized milks were not attributable to the counts of psychrotrophs in the corresponding raw milks. The water-soluble and TCA-soluble N fractions of maturing cheeses were unaffected by psychrotroph counts in raw milks, but small differences in levels of casein fractions of cheeses made from milks stored for 72 h were detected by quantitative polyacrylamide gel electrophoresis. The incidence of casein breakdown in raw milk and subsequently in cheese were not necessarily related. None of the cheeses developed off flavour related to excessive protein breakdown but many became lipolytically rancid, despite the selection of strains with low lipolytic activity on a diagnostic medium. It is concluded that the numbers of psychrotrophic bacteria likely to occur in stored raw milk under commercial conditions are unlikely to cause significant changes in the yields or quality of Cheddar cheese through their proteolytic activity.

Strains of coryneform bacteria which produce methanethiol from L-methionine in laboratory culture have been isolated from Cheddar cheese and raw milk Many Gram-negative raw milk isolates including Pseudomonas fluorescens, Ps. putida, Ps. fragi, Ps. aureofaciens, Acinetobacter, Aeromonas and Serratia marcescens also produced methanethiol. Representative strains of coryneforms and pseudomonads produced methanethiol only from free L-methionine; glycyl-L-methionine and D-methionine were not degraded. The aerobic nature of the methanethiolproducing bacteria and the instability and alkaline pH-dependence of their thiol producing enzymes explained their failure to increase methanethiol concentrations in experimental cheese, even when large numbers (5 × 107/g milled curd) of pregrown cells were added. Other commonly occurring cheese bacteria such as starter streptococci, micrococci or lactobacilli either did not produce methanethiol or produced only traces. It is concluded that the methanethiol normally present in Cheddar cheese is unlikely to be the product of bacteria or their enzymes.