The contribution of certain components of leucocytes and milk to the viscosity and precipitate which develop in the Whiteside mastitis test was studied to obtain information on the mechanism of the reaction. The viscosity obtained in the Whiteside test reaction was considerably less than that in the California mastitis test reaction and the Whiteside test was found to be more dependent on precipitation than increased viscosity.

Leucocyte nuclei were mainly responsible for the formation of the precipitate in the Whiteside reaction and CaCl2 dispersed the precipitate formed by the leucocyte nuclei into small clumps. Leucocyte protein and fibrinogen increased the amount of precipitate formed by the leucocyte nuclei. Fat globules also increased the amount of precipitate, probably by being trapped in the precipitate.

The addition of non-gelable or gelable nuclei resulted in the formation of similar amounts of precipitate in the modified Whiteside reaction. Furthermore, storage of milk for 5 days did not decrease the amount of precipitate formed, indicating that the DNA-protein complex of the leucocyte nuclei need not be present in the native form to contribute to the precipitate in this reaction.

Casein, whole αs-casein and β-casein were incubated for 3 and 14 h with crystalline rennin, at pH 6·60 and 36 °C, both in phosphate buffer and in milk dialysate. Products obtained from both systems, comprising 30–83% calciumsensitive (Cas) components, gave similar patterns on starch gel electrophoresis. Whole casein and whole αs-casein were not so soluble in milk dialysate as in phosphate buffer. No significant differences in composition were observed between the Cas and the calcium-insensitive (Ca1) products from the same source.

The αs1-component of the Cas product from rennin-treated whole αs-casein had faster gel mobility in comparison to the αs1-component in the Cas product from untreated whole αs-casein. Also, αs1-casein yielded one faster-moving degradation product, while αs2,3,4 appeared unaltered after 14h. The Cas product of rennintreated β-casein also had faster mobility than untreated β-casein and yielded one faster degradation product and several minor ones of slower mobility. Arginine was the only N-terminal amino acid found in the Cas product of both rennin-treated and untreated αs - and β-caseins. The arginine content increased from 3·48 and 4·98 moles/105g to 5·12 and 6·38 moles/105g in the Cas products from rennin-treated β-and αs-caseins, respectively.

An experimental ultra-high-temperature milk sterilizer, of 1140 1/h capacity and capable of operation as a plate-type indirect heating plant or as a steaminto-milk direct heating plant, has been installed for comparisons of the milk produced by the direct and indirect processes. The sterilizer and its ancillary equipment are described, together with methods of plant sterilization and milk processing. Time-temperature profiles of the plant are given for both modes of operation.

(1) The rate of release of fatty acids from milk by porcine pancreatic lipase was studied as a model system for the development of lipase taint. The rate was low initially but then increased logarithmically and later declined. Addition of milk which had been subjected to the action of lipase and then boiled to inactivate the enzyme enhanced fatty acid release. An extract containing monoglycerides from lipolysed milk had a similar action whereas an extract of free fatty acids had an effect mainly in the early stages, increasing the initial rate. Addition of Ca2+, which accelerated lipolysis of fresh milk, enhanced the action of fatty acids and depressed that of monoglycerides.

(2) In the early stages of lipolysis there was a preferential release of short-chain fatty acids and a high proportion of these passed into the aqueous phase. These effects were more pronounced at low temperature and at acid to neutral pH. At alkaline pH a greater proportion of the short-chain acids was present in the aqueous phase but the total release of these acids was less.

(3) Fresh milks differed in their response to ‘aeration’ and storage at 4°C. In those in which taint was induced there was a considerable release of fatty acids in response to aeration, and a further release and also a redistribution between the aqueous and fat phases during storage. No relationship was apparent between the amount of fatty acids released and the initial fatty acid content.

(4) The findings are discussed in relation to the development of lipase taint in commercial milks.

The amounts of total lipid and fatty acids consumed, leaving the stomach and excreted in the faeces were examined in 4 sheep fitted with rumen and re-entrant duodenal cannulas. Diets of high (HM1) and low (CM1) roughage content were given at 0·9 times maintenance and the low-roughage diet was also given at 1·7 and 2·3 times maintenance. With all the rations more fatty acid left the abomasum than was consumed in the food, the difference being greater on ration CM1 than on ration HM1 and increasing irregularly with the amount of the low-roughage ration fed. Of the fatty acid entering the duodenum in the chyme, 72–89% was digested in the intestine. About 80% of the increase in fatty acids in the stomach was stearic acid and most of the remainder was palmitic acid. Almost all the polyunsaturated C18 acids ingested in the food were hydrogenated in the stomach, and the amounts of oleic acid were also greatly reduced, although more oleic acid entered the duodenum in the chyme with ration CM1 than with ration HM1. The possible origins of the increase in fatty acids in the stomach are discussed.

The metabolism, of pyruvate by resting whole-cell suspensions of Group N streptococci was studied over the pH range 4·0–7·0, in the presence and absence of oleic acid. In the absence of oleic acid pyruvate was utilized maximally at pH 4·5 with the formation of acetate (volatile acid), acetoin + diacetyl and carbon dioxide. The formation of acetate took precedence over the formation of acetoin + diacetyl. In the presenoe of oleic acid the utilization of pyruvate was maximal at pH 6·5 and completely inhibited at pH 4·5. The only products detected at pH 6·5 were acetoin + diacetyl and carbon dioxide. This effect of oleic acid on the metabolism of pyruvate was also obtained after treating the cells with acetone. The mechanism of action of oleic acid on cells of Group N streptococci and its possible influence on the formation of flavour compounds in cultured dairy products is discussed.

The variation of sporicidal efficiency with processing temperature was determined for an experimental ultra-high-temperature (UHT) milk sterilizer operating alternatively as an indirect or as a direct heater. Whole milk was inoculated with large numbers of spores of Bacillus subtilis 786 and Bacillus stearothermophilus TH24, and the proportion of spores surviving the sterilizing process was calculated from dilution and colony counts on the untreated and treated milk. The results for B. subtilis spores were unreliable, and the dilution count results for B. stearothermophilus spores were influenced by the inhibitory effect of the UHT processed milk. The results for the colony counts of B. stearothermophilus spores were preferred as a basis for the comparison of the direct and indirect processes. Over the range of processing temperatures 137–145 °C it was found that the sterilizing temperature had to be 3–4 degC higher with direct heating than with indirect heating to give equal spore destructions.

Absorbance measurements at the 1·93 μm water band on methanol extracts from butter, cheese and dried milk enabled moisture contents to be determined with coefficients of variation of 1·0, 1·1 and 2·0%, respectively. Butter, cheese and cheese curd analyses could be completed within a few minutes, but a 2-h standing period was required for dried milk to overcome difficulties associated with lactose precipitation.

The inactivation of lactoperoxidase (LP) by cultures of Group N streptococci was originally reported by Kiermeier & Kayser (1960). However, Pickering, Oram & Reiter (1962) found that incubation of Streptococcus cremoris 803 with LP did not completely inactivate the enzyme, but only modified its activity with certain substrates. This observation was subsequently confirmed by Kiermeier & Lechner (1966, 1967). In the present investigation, several strains of Group N streptococci were found to modify LP in such a way as to cause total or partial loss of enzymic activity with guaiacol. This loss of LP activity was found to be a consequence of the production of hydrogen peroxide by these organisms.

The bacteriological results of Franklin, Underwood, Perkin & Burton (1970) are analysed to show that colony counts of Bacillus stearothermophilus spores in UHT-treated milk are influenced by the inhibitory action of the milk, so that the sporicidal effects of the UHT process as calculated from the results are too high. Only the elimination of the inhibitory factor will allow true sporicidal effects to be determined.

A gas-liquid chromatographic method is described for the simultaneous estimation of the carbohydrates attached to the glycopeptides released by the action of rennin on whole milk. The method can also be used for κ-casein. Certain of the findings may be applicable to other glycoproteins or glycopeptides. In particular: (a) the conditions for methanolysis of the carbohydrates from the glycopeptides must be chosen carefully to avoid decomposition of N-acetyl neuraminic acid; (b) it is not possible to obtain a satisfactory calibration for N-acetyl neuraminic acid using the free acid as a standard.

The rates of denaturation by heat of α-lactalbumin and β-lactoglobulin in skim-milk were measured by an immunodiffusion method over a wide range of temperatures. Both reactions showed an unusual temperature dependence.

The denaturation of α-lactalbumin is a first-order reaction; between 90 and 155 °C the kinetic constant k1 in s−1 is given by the equation

where T is the temperature in °K.

The denaturation of β-actoglobulin in skim-milk is second order with respect to time, and the kinetic constant k2 in 1g–1 s–1 is given by 2 equations, valid for different temperature ranges. Between 68 and 90 °C,

Between 90 and 135 °C

Results obtained by the immunodiffusion method agreed well with those found by salt fractionation of the milk proteins. The denaturation rates decreased when a specific reagent for sulphydryl groups was added, suggesting that such groups are involved in the denaturation of both proteins.

The nutritional deficiencies of co-precipitate as a growth medium for a strain of Lactobacillus acidophilus have been studied. Acid production by this organism was insignificant in a medium composed of 4% low-calcium co-precipitate and 5% lactose.

Additions of the amino acids phenylalanine, tyrosine, valine, cysteine and threonine as well as of nicotinic acid, manganese, magnesium and citric acid all increased acid production.

When only half the milk solids of skim-milk powder was replaced by the coprecipitate, fortification with manganese only was required.

Study of the dissociation of high-molecular-weight aggregates of preparations of αs1-, β-, κ-, and para-κ-casein by the detergent, sodium dodecyl sulphate (SDS), showed that there are differences in the aggregation properties of the individual caseins. Binding of detergent led first to the dissociation of casein aggregates and then to further interaction with the casein molecules. The amounts of detergent required to give the minimum sized protein-detergent aggregate when expressed as mg/mg casein were similar for κ-, para-κ- and αs1-casein but much less for β-casein. However, expressed as mole/mole the requirement for κ- and αs1-casein was similar but was twice that found for para-κ- and β-casein. The maximum amount of SDS bound was about twice that required for complete dissociation of the aggregates for κ-, para-κ- and αs1-casein but was 13 times greater for β-casein.

Complete dissociation of κ-casein aggregates by SDS alone was not possible due to the presence of aggregates formed by disulphide linkages. These aggregates, which consisted of 3±1 protein molecules, accounted for about one-third of the κ-casein in the preparations examined.

Bovine serum lipoproteins were fractionated by precipitation with dextran sulphate followed by ultracentrifugation and were examined by paper electrophoresis and by disk electophoresis in acrylamide gels. Gels containing concentration gradients of both sucrose and acrylamide gave better resolution of lipoproteins than did gels of uniform composition or gels with a concentration gradient of sucrose and a uniform concentration of acrylamide.

The densities of the lipoprotein classes isolated (listed in order of increasing mobility on electrophoresis in acrylamide gels) were as follows:

I, d < 1·019; II, 1·039 <d<1·050;

III, 1·019 <d<1·039; IVa and IVb, d > 1·050.

Lipoproteins of classes I, II and III were precipitated by dextran sulphate. On paper electrophoresis, lipoproteins of class II had β mobility and lipoproteins of classes IVa and IVb had α mobility. Lipoproteins of class I remained at the origin.

The lipoprotein classes of bovine sera resembled those of sheep and goat sera in that class IV predominated. In contrast, the sera of several non-ruminant species showed a predominance of lipoprotein classes of low electrophoretic mobility.

An examination of the spectra of some volatile carbonyl semicarbazones indicated that only compounds containing vicinal carbonyl groups formed semicarbazones which absorbed at 270 nm. This observation was used for the quantitative estimation of diacetyl. The presence of both acetaldehyde and acetoin did not interfere with the estimation of diacetyl by this method.

The dissociation of casein aggregates by the detergent sodium dodecyl sulphate (SDS) gave rise to difference spectra and these spectra were characteristic for each of the different types of casein. Increase in absorption by the chromophore groups, tyrosine and tryptophan, when αs1- and β-casein aggregates were dissociated indicated binding of the detergent at regions of the molecule containing these residues. A decrease in absorption when κ-casein was dissociated indicated that the tyrosine and tryptophan residues were not in the region of the molecule to which the detergent was bound and that in the κ-casein aggregate these residues were in a more hydrophobic environment. Peaks on the difference spectra were obtained at 280 and 288 nm for αs1-casein and 284 and 291 nm for β-casein and troughs at 278 and 286 nm for κ-casein. The difference spectrum reached a maximum value when the αsl- and β-casein aggregates were dissociated and the further binding of SDS did not alter this value. The large negative change in the difference spectrum of κ-casein did not occur until after most of the aggregates were dissociated and did not reach a maximum until binding with SDS was complete. The value obtained for ΔOD was found to be temperature-dependent for β-casein-SDS interaction, but not for αs1- and κ-casein. Changes in spectra were also observed when αs1- and κ-casein interacted to form aggregates. The data obtained confirmed the importance of hydrophobic binding in casein aggregate formation and indicated the possible involvement of tyrosine and tryptophan residues in this binding.

Four methods of estimating the cell content of bulk milk were studied, correlated statistically and their degree of reproducibility estimated. The relative merits of the 4 methods are discussed, and the need for defining the object of testing before selecting a test is stressed. If designed for use in a programme for mastitis control, or the control of milk quality based on cell content, each country must choose a screening test or a precise cell-counting technique, depending on the availability of laboratory resources, extension advisory services and other economic factors.

Screening tests studied were the California Mastitis Test (CMT) and the Brabant Mastitis Test (BMR), the former showing a correlation coefficient of 0·817 against the Electronic Cell Count (ECC) method in 320 samples. The more objective BMR correlated well with the ECC method, showing a correlation coefficient of 0·987 for 406 samples. Direct microscopic counts (DMC) were correlated with a centrifugal method and with a chemical method of preparing samples for electronic cell counting. For 430 samples in each case the correlation coefficients obtained were 0·930 and 0·966 respectively. The 2 ECC techniques were compared using 430 samples, and a very close relationship was found (r=0·988).

For regular examination of large numbers of milk samples using the electronic cell counter we consider the chemical method of sample preparation more suitable than the centrifugal method. Differences in the reproducibility and accuracy of both tests are negligible, but the former procedure is quicker, more economical in terms of cost, and more adaptable to a national testing programme.

Carboxypeptidase A (CPA, mol. wt = 34600) and 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride, mol. wt = 270) were shown to react with the 3 casein subunits, αs1-, β- and κ-caseins, which constitute the micelle. Virtually all of the C-terminal amino acids were released by CPA from these subunits without disrupting the micellar structure.

It was verified that the rate of dissociation of the micelle into low molecular weight complexes is considerably slower than the rates at which these reagents act on micellar components. It is concluded that dansyl chloride, CPA, and probably also rennin, whose molecular weight is similar to that of CPA, are able to penetrate to the interior of the micelles. This was confirmed in a quite independent way with a non-reacting agent, myoglobin (mol. wt = 17000).

It is postulated that the casein micelle has a sponge-like structure in which the 3 different subunits are distributed fairly uniformly.

Micellar fractions obtained by differential centrifugation from skim-milk and colostrum were examined for their content of αs1-, β-, κ- and para-κ-caseins by the use of CPA as described by Ribadeau Dumas (1968). The results confirm that there is a real difference in κ-casein content of the micelles, according to their rate of sedimentation.