An examination was made of the effect of varying the protein, lactose and total serum ion content of milk on the rate of production of heat-induced acidity (as measured by pH decrease), on heat stability (as measured by coagulation time), on the relationship between coagulation time and initial pH of milk (as measured by coagulation time-pH curves) and on the interrelationship of these parameters. In addition, the effect on these parameters and their interrelationship of varying the composition and volume of the headspace atmosphere in contact with the milk was investigated. Explanations are proposed for the observed effects on heat stability of varying the composition of milk and the heating conditions, with special reference to the influence of heat-induced acidity.

Indirectly heated ultra-high-temperature (UHT) processed milk was prepared with initially high, medium, and low dissolved O2 contents of 8·9, 3·6 and 1·0 ppm respectively, aseptically bottled, and tested at intervals during storage at room temperature for 150 d. Flavour acceptability increased to a maximum after a few days, but declined slowly after about 6 d; the increase was associated with less off-flavour described as ‘cabbagey’, and the decrease with more ‘stale’ off-flavour descriptions. Milks with higher initial O2 contents were preferred up to 8–13 d, but thereafter acceptability was independent of initial O2 content. Sulphydryl group (–SH) contents rapidly decreased and O2 levels correspondingly declined in the first few days as the flavour improved. Loss of –SH was lower with lower initial O2 contents, and moderate –SH content remained in low O2 samples for several weeks. Ferricyanide reducing (FR) values did not satisfactorily measure stale flavour development. They were initially high and decreased during the first 13 d at rates dependent on O2 content. After 20 d the FR values began to rise in high O2 samples, but continued to decline slowly in low O2 samples up to 90d although stale flavour was increasing.

High initial O2 contents resulted in rapid depletion of ascorbic acid and folic acid during storage. Losses of vitamin B12 were small, but were higher with high O2 contents than with low.

The beneficial effect of O2 on flavour, therefore, appears to be so slight and confined to such a short period in the early life of the milk as to be completely outweighed by the adverse nutritional effects.

Reduction of the level of colloidal calcium phosphate (CCP) progressively increased the heat stability of milk at pH values <~7·0 and increased the pH of maximum stability. Removal of 40% CCP also stabilized the system at the pH of minimum stability, but removal of ≥60% CCP rendered milk very unstable at pH values >7·2, an effect not offset by a 4-fold increase in κ-casein concentration. Doubling CCP had a slight destabilizing effect in the pH range 6·5–7·5.

Addition of β-lactoglobulin to serum protein-free casein micelles had a marked destabilizing effect at pH values > ~6·8, but increased stability in the pH range 6·4–6·8. β-Lactoglobulin had a similar and more apparent effect on the heat stability of Na caseinate dissolved in milk diffusate.

It is suggested that rather than being a stabilizing factor responsible for the maximum in the heat stability-pH curve, the true effect of β-lactoglobulin is to shift the curve to more acid pH values (reason unknown) and to sensitize the caseinate system to heat-induced Ca phosphate precipitation at pH values > ~7·0. Low stability at ~pH 7·0 introduces an apparent maximum in the heat stability-pH curve at ~pH 6·8, but this has no independent existence. At pH values >7·2, increased protein charge more than off-sets the influence of heat-precipitated CCP and stability again increases in micellar but not in soluble casein systems.

The formation of protein polymers in ultra-high-temperature (UHT)-treated milk during storage at various temperatures was examined by gel filtration. The extent of polymer formation was found to depend on both storage time and temperature. After some months of storage at the higher temperatures of 30 and 37°C, the extent of polymerization of the caseins and whey proteins due to reactions of the Maillard type was several times greater than the heat-induced changes resulting from the UHT processing itself. After storage for 6 months the following proportions of milk proteins were found to exist in the form of covalently bound polymers: 50% at 37°C, 40% at 30°C, 26% at 20°C and 21% at 4°C. In addition, further amounts of polymer were formed by disulphide bonding, the contribution of such polymers diminishing gradually in a temperature-dependent manner during storage due to continuing polymerization reactions. It appeared that αs1-casein may be preferentially involved in these polymerizations with β-casein reacting at a somewhat slower rate. Polymerization and associated reactions modifying molecular charge led to the expected alterations in electrophoretic mobility and a loss of definition in the bands due to the various protein components. Measurements of proteolytic breakdown indicated only very small increases in trichloroacetic acidsoluble fragments, suggesting that proteolysis was of minor significance.

A simple test procedure was used to predict the most suitable mixture of Noury rennet (Mucor pusillus Lindt proteinase) and swine pepsin for Cheddar cheesemaking. Duplicate cheeses were made with 6 Noury rennet–swine pepsin mixtures and with each enzyme alone, with calf rennet as the control. There were slight differences in the course of the cheese-making process and in the yields of the cheeses. Sensory evaluation showed that the most acceptable cheeses were those made by coagulant mixtures closely similar to the predicted mixture and these were as acceptable as cheeses made with calf rennet. The survival rates of different groups of bacteria were similar in all cheeses. There were differences in the products of proteolysis in the various cheeses throughout ripening, particularly with respect to products derived from αs1-casein. The Noury rennet was characterized for the proportion of milk clotting activity associated with each of 4 components and its stability in solution was determined.

A comparison has been made of a number of different elution conditions for the separation by ion-exchange chromatography of the glycopeptides released by the action of rennin on whole milk. It was found that the most satisfactory separation was achieved using an NaCl gradient (0·10–0·18m) in 0·049 m-KH2PO4+0·001 m-Na2HPO4 at a constant pH of 5·1.

A relationship between temperature and sterilizing efficiency of heat treatment at constant time is described and checked experimentally using spores of Bacillus coagulans 604 in a pilot ultra-high-temperature plant. The relation is:

where N0 and N are initial and final concentrations of spores, T the temperature (°C), z the temperature coefficient (°C, z=10/logQ10) and A a dimensionless constant. A special effect of the injection of spores into live steam is observed. The sterilizing efficiency curve can be used in determining the heating temperature for a desired sterilizing efficiency and in predicting the influence of temperature fluctuation on sterilizing efficiency.

Data are presented which show that the use of glucono-δ-lactone as a chemical acidulant in cheese manufacture results in a much more rapid decrease in pH than that occurring in biologically acidified cheese. One of the consequences of the excessive early acid development was a very marked increase in the level of proteolysis during cheese-manufacturing operations. Consequently, chemically acidified cheese, as manufactured by the technique of Mabbitt, Chapman & Berridge (1955) (expt 17), does not appear to be a suitable ‘reference cheese’, especially for studies in which proteolysis during ripening is under investigation. The technique of Mabbitt et al. Avas modified to simulate the pH development pattern of starter cheese more closely; the level of proteolysis in curd made by the modified method was approximately equal to that in starter curd.

An attempt was made to link the propensity to develop bitterness of cheese made with fast acid-producing cultures with excessive early proteolysis in such cheese. The results show that fast- and slow-culture strains differed significantly in their acid-producing capability only after the cooking stage and consequently such cheeses differed little with respect to early proteolysis. Further, cheeses made with 0·5, 1·0, 2·0 or 4·0% of ML8 culture which differed considerably in rate of acid development and consequently in the level of early proteolysis did not develop bitterness, even when 4·0% starter was used.

It is concluded that gross proteolysis and bitterness are not correlated.

Bovine, ovine and caprine pepsin and lamb, piglet and kid chymosins (rennins) were isolated by basically the same procedure of selective pH precipitation, DEAE-cellulose chromatography in phosphate buffer, pH 6·5 and Sephadex G-100 chromatography. The homogeneity of the preparations was assessed by (a) re-cycling on Sephadex G-100, (b) chromatography on DEAE-cellulose using piperazine buffer, pH 5·3 and (c) ability to inactivate ribonuclease.

A method is outlined for accelerating ripening in Swedish semihard cheese by increasing the number of lactic starter bacteria present in the cheese without impairing characteristic texture and flavour. In addition to the normal starter inoculum, suitable lactic starter bacteria whose lactic-acid-producing activity had been greatly reduced by previous sublethal heat treatment, were added to the cheesemilk. When suspensions of streptococci and lactobacilli cultivated at a constant pH were heated at 59 and 69°C respectively acid production was retarded by 5–10 h, which was found to be sufficient for the cheese-making. Proteolysis was lowered only 10–30% by these heating temperatures. Bacterial cell suspensions, prepared by the methods outlined and added to the cheesemilk, were incorporated in cheese curd to extents depending on the amount added and the type of starter. The number in the final cheese could be increased to a maximum of 4–5 times that of control cheese. No adverse effect of the extra starter bacteria on pH, fat content and water content of cheeses 24-h old was observed. Proteolysis, measured as the increase in trichloroacetic acid and phosphotungstic acid (PTA)-soluble N, increased with increasing number of cells in the cheese. Organoleptic judgements showed a positive correlation (r = 0·81) between taste and PTA-soluble N, which in turn was influenced by the number of cells in the final cheeses.

Yeast extract was fractionated on Sephadex G-25 into 7 fractions. The fraction most stimulatory to the growth of Streptococcus lactis C10 contained over 70% of the amino N present in yeast extract and consisted of a wide variety of free amino acids and a small amount of peptide material. Examination of possible replacement factors for this fraction revealed that the amino-acid material present was largely responsible for the stimulation of Str. lactis C10. Purine and pyrimidine bases and inorganic constituents also contributed to the stimulation. In addition, yeast extract contained a component which decomposed H2O2, a metabolite which accumulates in the growth medium under aerobic conditions and inhibits growth. The nature of the stimulation was studied by isolating slow and fast acid-producing colonies of Str. lactis C10. It appeared that yeast extract and other amino-acid supplements prevented an observed inhibition of the growth of the slow variants below pH 6·0, apparently by satisfying a nutritional deficiency caused by a drop in pH.

The acid phosphatase activity levels in a number of Greek cheeses and in Cheddar cheeses were found to be unaffected by storage for up to 18 months and 12 months respectively. In Cheddar cheese, starter organisms made an insignificant contribution to this activity. Studies of acid phosphatase prepared from Streptococcus cremoris-lactis NCDO 762 starter cultures showed that the enzyme was of high molecular weight and largely particle-bound. The pH of optimum activity was 5·2 and the enzyme was inhibited by F−, Al3+, a number of heavy metals, oxidizing agents and sulphydryl-modifying reagents. Kinetic measurements at pH 5·2 gave a Km value for p-nitrophenyl phosphate of 1·2 mM. Orthophosphate, pyrophosphate and isoelectrically precipitated casein behaved as competitive inhibitors to the hydrolysis of p-nitrophenyl phosphate with K1 values of 1·2 mM, 1·0 mM and 1·1 mM respectively. In spite of this binding to the enzyme, casein provided a very poor substrate for the starter acid phosphatase. The properties of acid phosphatase present in Cheddar cheese made with Str. cremoris NCDO 924 starter were consistent with the enzyme being exclusively of milk origin and small differences between this and the acid phosphatase previously isolated from bovine milk were attributable to the binding of peptides produced during the cheese maturation to the enzyme molecules. It was concluded that in cheese, phosphatase action was due largely to the enzyme of milk origin, with that provided by the starter being of minor importance.

The effects of 3 different thickening agents on the microstructure of yoghurt were examined by scanning and transmission electron microscopy. Unsupplemented yoghurt consisted of casein micelles fused together and linked in long chains; they seldom appeared in clusters. The addition of 0·5% gelatin caused no observable change in the yoghurt structure. Carrageenan (0·4%) formed a markedly fibrillar microstructure which connected large clusters of casein micelles. The fibres were thin and long and no free terminations were observed. Pregelatinized waxy maize starch (2%) appeared under scanning electron microscopy in the form of short fibres and sheets. The fibres frequently had free terminations and only some of them were connected with small clusters of casein micelles. Transmission-electron microscopy did not distinguish between pregelatinized starch and carrageenan additives.

In ripening and mature Cheddar cheese, 2 distinct types of birefringent microscopic crystalline inclusions are found. One of these is an irregularly shaped large aggregate of needle-like crystals (LAC). The histochemical demonstration of Ca in the LAC together with their total absence from cheese made with δ-gluconic acid lactone (GAL) instead of starter bacteria suggests that they are composed of Ca lactate.

The other type of inclusion is a smaller aggregate of crystals (SAC). They are more numerous than the LAC and are concentrated in areas which probably correspond to the lines of curd particle fusion. The SAC appear in cheese immediately after pressing and increase in number until the cheese is about 1 month old. They can be detected in unsalted, milled curd after 1 d and are present in large numbers in cheese made with GAL. These observations together with histochemical evidence that they may contain phosphate groups suggest that the SAC are probably composed of Ca phosphate. Their location in spaces between the fat and casein phases of the cheese indicates that they crystallize from pockets of residual whey. Each SAC is composed of a large number of very long (maximum length 4·5 μm), thin (maximum diam. 25 nm) crystals each of which is covered by a thin layer of electron-dense material tentatively identified as casein.

Citrate utilization and diacetyl, acetoin and acetaldehyde production by 2 strains each of Leuconostoc cremoris and Streptococcus diacetilactis in milk were studied. With the leuconostoc bacteria no growth and little citrate utilization occurred unless a stimulant (yeast extract) was present, when complete utilization of citrate without concomitant production of diacetyl or acetoin was obtained. The addition of Mn2+ stimulated growth and citrate utilization in the presence of yeast extract. Addition of citric acid after 65-h growth resulted in diacetyl and acetoin production. Destruction of diacetyl and acetoin occurred when the citric acid level fell to c. 1000 and 600 μg/g in the case of Leuc. cremoris FR8-1 and CAF1, respectively. Only strain FR8-1 produced acetaldehyde. In contrast, Str. diacetilactis produced diacetyl, acetoin and acetaldehyde concomitant with citrate utilization.

Several peptidase activities in the 3 species of Group N streptococci were partly separated by gel filtration on Sephadex G-200. The peptidases identified were a general aminopeptidase of wide specificity, a tripeptidase, a proline iminopeptidase (prolyl-β-naphthylamidase), a proline iminodipeptidase and an aminopeptidase-P. The effects of temperature and pH on the stability of the enzyme activities, and the influence of the type of N source used in the growth medium on the elution pattern of the enzymes were examined.

Buffalo αs-casein (α-sb) was found to be heterogeneous; on starch gelurea electrophoresis 4 electrophoretic components were detected.

The αsb-fraction was isolated by urea and alcohol fractionation. A preparation containing only the 2 main components was obtained by chromatography on DEAE-cellulose. Its amino acid pattern was different from that of cow αs1-casein. The fraction was also free of carbohydrate, cystine and cysteine.

Buffalo αsb-casein was less stable than cow αs-casein in solutions containing Ca, and the maximum stability of the αs-/κ-casein complex occurred at a lower Ca concentration.