Eight strains of the Lactobacillus acidophilus complex were subjected, as concentrated cultures in skim milk, to different freezing treatments (rapidly in liquid nitrogen and more slowly in cold air at −30 °C), then stored at −80 and −30°C for 9 months. Survival was determined by plate counting with and without bile salts or sodium chloride at the highest tolerated concentration for each strain, to distinguish the undamaged population from the total population. Fermentative activity was measured as total lactic acid production by thawed cultures under standard conditions. Higher survival rates and greater activity were always obtained by storing cultures at −80 °C, but most strains stored at −30°C also survived well. Analysis of variance revealed that the viability of the frozen cultures was affected more by storage temperature than by cooling rate. Selective media were unable to distinguish the active population from the total surviving population. The correlation between values for activity and survival with selective media was poor.

Cathepsin D is an indigenous aspartic proteinase in bovine milk. By competitive enzyme-linked immunosorbent assay the amount of immunoreactive cathepsin D and procathepsin D in bovine skim milk was estimated to be 0·4 μg/ml. Immunoreactive cathepsin D purified from whey consisted of a small fraction of mature cathepsin D, but the major form was the proenzyme procathepsin D. A preparation of bovine milk procathepsin D was, like mature cathepsin D, able to degrade purified αs1-, αs2-, β- and κ-casein and α-lactalbumin, while β-lactoglobulin was resistant to cleavage. The cleavage sites in these proteins were determined and compared with those of chymosin. Cathepsin D was capable of generating the αs1-I, β-I, β-II and β-III fragments originally described from the action of chymosin on the respective caseins, and these fragments were subjected to further proteolysis. Cathepsin D was also able to liberate the caseinomacropeptide from purified κ-casein, and to coagulate bovine skim milk. This demonstrated that milk contains an indigenous coagulation enzyme present mainly in the whey fraction.

Fresh rennet-coagulated cottage cheese whey was vacuum concentrated to 400 g total solids kg−1, and part of this evaporated whey was acidified to pH 4·6 to prepare whey protein concentrate. Both products were used separately to replace non-fat dried milk in yogurt. Diacetyl concentration increased on fortification with whey protein concentrate, and acetaldehyde increased with evaporated whey. However, the use of Bifidobacterium bifidum as a supplementary starter culture in addition to Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus reduced the concentration of diacetyl and acetaldehyde. Incorporation of whey solids stimulated the growth of Str. thermophilus and Bifid. bifidum in yogurt but the count of Lb. bulgaricus was reduced when Bifid. bifidum was incorporated. Examination of the organoleptic properties of the yogurts showed that both forms of whey solids were satisfactory replacements for non-fat dried milk. Fortification by whey protein concentrate improved the textural properties. Supplementation by Bifid. bifidum had only a marginal effect on the flavour of the product.

Plain yogurts (set type) fermented by three different starter cultures were subjected to different heat treatments (20 s at 72, 80 or 100 ·C, or 30 min at 60, 70, 80 or 100 ·C). DNA was extracted from each yogurt and analysed by agarose gel electrophoresis, by species-specific hybridization and by primer-specific polymerase chain reaction (PCR). Obvious degradation of DNA could be observed after heating at 80 ·C for 30 min and at 100 ·C for 20 s and 30 min. Identification of Lactobacillus delbrueckii using a species-specific hybridization fragment could be carried out in yogurt treated at 100 ·C for 20 s or for 30 min at lower temperatures. After treatment for 30 min at 100 ·C identification by hybridization was no longer possible. However, under the same conditions identification of starter microorganisms was still possible by primer-specific PCR, and this was demonstrated as an example for Streptococcus thermophilus.

A tributyrin esterase was purified from Lactococcus lactis subsp. cremoris E8 using FPLC chromatography. This was the major esterase activity observed in strain E8 and was associated with a single protein with a subunit molecular mass of 29 kDa and a holoenzyme of molecular mass 109 kDa. The enzyme was active against tributyrin and p-nitrophenyl butyrate. The N-terminal sequence of the enzyme was determined. The enzyme had a pH optimum in the neutral range, was stable on freezing at −20 °C, and had a half life of 1 h at 50 °C.

During chill storage, cholesterol oxidation in feta cheese produced from bovine butteroil bleached at high temperature was compared with cholesterol oxidation in feta cheese produced from non-bleached butteroil. The bleaching was performed at two different temperatures, 265 and 280 °C, and the oil was bleached for 2·4, 3·8 or 4·3 min; a reference feta cheese was produced without bleaching. All cheeses were stored in brine at 4 °C, and cholesterol oxidation was measured during a storage period of 5 months. For the first 11 weeks of storage, the concentration of cholesterol oxides was comparable for the bleached feta cheeses, but on longer storage the concentration of oxysterols was highest in feta cheeses produced from the butteroil bleached at 280 °C. The bleaching temperature rather than the bleaching time affected cholesterol oxidation, which was minimal in the non-bleached reference cheese throughout the storage period compared with the bleached feta cheeses. 7-Ketocholesterol was found to be the dominant oxysterol in the feta cheeses at the end of the storage, comprising ∼ 50% of the total cholesterol oxides. In feta cheeses based on butteroil bleached at 265 °C, the concentration of 7-ketocholesterol ranged from 3·7 to 4·9 µg/g lipid at the end of the storage period, and in feta cheese based on butteroil bleached at 280 °C the concentration was 10·4–13·1 µg/g lipid. In the reference feta cheese the concentration of 7-ketocholesterol was 1·2 /µg/g lipid. There was no difference in yellowness, measured by tristimulus colorimetry as the Hunter b characteristic, of the feta cheeses bleached at 265 and 280 °C, and a small scale bleaching experiment with butteroil showed that it was possible to secure complete bleaching at temperatures down to 220 °C. We suggest that bleaching of butteroil for feta production should be performed at temperatures as low as possible in order to prevent cholesterol oxidation.

Polyclonal antibodies raised against partly purified aminopeptidase were specific to cell-free extracts of Lactobacillus casei subsp. pseudoplantarum UL 137, and did not cross react with any other proteins in Cheddar cheese, at least during the first week of maturation. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed in order to quantify cell-free extracts in the cheese curd, and was also used to determine the efficiency of encapsulation of cell-free extracts in liposomes. This method was very sensitive and exhibited a detection limit of ∼10 μg total protein/g cheese and ∼1 μg total protein/ml liposome suspension. The efficiency of encapsulation of cell-free extracts into liposomes was ∼55–60%. The retention of liposome-encapsulated cell-free extracts was ∼14 times that of non-encapsulated extracts.

Some media available for the isolation and enumeration of starter cultures employed for the manufacture of cheese, yogurt and bio-yogurt were examined. Reddy's medium or a modification of Elliker's medium was found to be most satisfactory for Lactococcus spp., while trypticase phytone yeast (TPY) agar with a mixture of antibiotics proved suitable for the discrete enumeration of Bifidobacterium spp. The inclusion of Prussian blue (PB) in reinforced clostridial medium or tryptone proteose peptone yeast extract (TPPY) agar gave excellent differential counts for the starter bacteria in yogurt even when the culture was imbalanced, while TPPY (PB) agar allowed the visible separation of all four of the organisms that might be found in a typical bio-yogurt, namely Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, a, Bifidobacterium sp. and Lb. acidophilus. It was noted that variation among different strains of any given species could change the expected reactions, so for quality control purposes the suggested media may need to be modified to cope with the specific cultures in use.

In a dairy spread (800 g lipid/kg, 10 g salt/kg) based on 750 g milk fat/kg and 250 g rapeseed oil/kg fat in 15 g extruded catering packaging, there was a more significant accumulation of cholesterol oxidation products than in butter (minimum 800 g lipid/kg, 12 g salt/kg) in 10 g extruded catering packaging when stored at 4 or at 20 °C. There was a lag phase of 7 weeks in cholesterol oxidation in dairy spread stored at 4 °C, while no lag phase was observed for storage at 20 °C. Total concentrations of oxysterols were, however, very similar for dairy spread stored at 4 and 20 °C after 13 weeks storage (∼ 12 μg/g milk lipid); storage at – 18 °C almost prevented cholesterol oxidation (∼ 4 μg/g milk lipid). For butter, cholesterol oxidation was less pronounced at 4 °C (< 3 μg/g milk lipid) than at – 18 °C (∼ 4 μg/g milk lipid) and 20 °C (∼ 7 μg/g milk lipid). 7-Ketocholesterol was the dominant oxidation product, with 1·3 and 5·7 μg/g milk lipid in butter and dairy spread respectively after 13 weeks storage at 4 °C.