Margaret E. Gregory

The effect of heat on the vitamin B6 of milk. 1. Microbiological tests.

1. Four test organisms were used for the microbiological assay of vitamin B 6 . The test organisms and the forms of vitamin B 6 active for them were: Saccharomyces carlsbergensis 4228 (pyridoxine, pyridoxamine and pyridoxal), Streptococcus faecalis R ATCC8043 and Str. faecium O51 (pyridoxamine and pyridoxal), and Lactobacillus casei ATCC7469 (pyridoxal). 2. By simultaneous assay of the test material with the four organisms the amounts of pyridoxine, pyridoxamine and pyridoxal present were determined. The differential assay technique has been improved by the use of Str. faecium , which is more specific for measuring total pyridoxamine plus pyridoxal, instead of Str. faecalis . 3. The optimum conditions for measuring vitamin B 6 in milk were studied and the differential assay technique was applied in an investigation of the effect of heat on the vitamin B 6 activity of milk. 4. It was found that pyridoxal accounted for about 80% of the vitamin B 6 activity of raw milk, the other 20% being due to pyridoxamine. 5. In the manufacture of evaporated milk there was a marked fall in the pyridoxal and a slight increase in the pyridoxamine content. The overall loss was some 60%. 6. On storage of evaporated and of sterilized liquid milk at room temperature there was a further loss of vitamin B 6 activity. Most of the residual activity in stored evaporated milk appeared to be due to a substance other than pyridoxal or pyridoxamine, possibly pyridoxine.

578. The composition of the milk of the blue whale

The detailed composition of samples of milk from three blue whales (Balaenoptera sibbaldi) is reported in terms of total solids, fat, solids-not-fat, total protein, casein, soluble proteins, non-protein nitrogen, lactose, chloride, ash, calcium, phosphorus, sodium, potassium, magnesium, vitamin A, thiamine, biotin, riboflavin, nicotinic acid, vitamin B6, pantothenic acid and vitamin B12.The composition of the milk of the whale is compared with that of other species.

THE COMPOSITION OF RABBIT'S MILK.

As part of a project on hand-rearing baby rabbits, it was necessary to know the composition of rabbit’s milk so that a suitable milk-substitute diet could be prepared. The gross chemical composition of rabbit’s milk has been studied in some detail by Folin, Denis & Minot (1919), Bergman & Turner (1937) and Neumeister & Krause (1956), but little is known about its vitamin content. We have therefore analysed samples of milk taken from New Zealand White and Chinchilla rabbits at different stages in the lactation cycle and report here their chemical composition and vitamin content. EXPERIMENTAL Milk samples Samples of milk were obtained from one Chinchilla and seven New Zealand White rabbits. Litters were removed from the lactating does on the evening before the sample was taken. The following morning each doe was given 2.5 i.u. oxytocin by injection into the ear vein and milk was expressed by gently massaging the mammary tissue. Milk flow usually began about 3 min after the injection and continued for about 15 min. Up to the 20th day of lactation volumes of 1-35 ml per doe were obtained, the average being about 20 ml; only very small quantities of milk could be expressed from does in later stages of lactation. Samples of colostrum were taken within 24 h of the birth of the litter. For full chemical and vitamin analysis of some of the samples, the milks from two rabbits at similar stages of lactation were bulked. In this way two mixed samples representing the 1st and 3rd weeks of lactation were obtained. Other samples were analysed individually. A portion of each sample (usually 5 ml) was set aside for chemical analysis. The remainder was weighed and centrifuged and the cream layer was removed, weighed and used for the measurement of vitamin A. The fat content of the cream, and thus of the whole milk, was determined. The water-soluble vitamins were measured microbiologically in the skim milk, but the results were recalculated in terms of whole milk.

The effect of heat on the vitamin B 6 of milk: II. A comparison of biological and microbiological tests of evaporated milk

1. For chicks and rats pyridoxine, pyridoxal and pyridoxamine were equally active in terms of the free bases when given separately from the diet. 2. Under our experimental conditions pyridoxine mixed with the chick diet was stable, but 20% of pyridoxamine, and a variable amount of pyridoxal was lost. 3. The vitamin B 6 activities measured with Saccharomyces carlsbergensis , chicks and rats respectively and expressed as μg. pyridoxine/g. freeze-dried milk were: raw milk 3·4, 3·2 and 4·9; evaporated milk 1·0, 2·1 and 2·7; stored evaporated milk 0·6, 1·4 and 2·0. For the chicks the milks were mixed with the diets; they were given separately to the rats. 4. The microbiological and biological results for raw milk agreed within the limits of experimental error. For the processed milks the differences between biological and microbiological tests were statistically significant. 5. All three methods of assay showed a 45–70% loss of vitamin B 6 activity on processing and a further loss of 30% of the remainder after storage for 6 months at room temperature. We are indebted to Mr J. Rothwell, Department of Dairying, University of Reading, for preparing the evaporated milk and to Dr B. Record, Ministry of Supply, Microbiological Research Establishment, Porton, for freeze-drying the milk. We should like to thank Dr S. K. Kon for his interest in this work.

The effect of ultra-high-temperature heat treatment on the content of thiamine, vitamin B 6 and vitamin B 12 of milk

Results are given for the effect of different ultra-high-temperature milk sterilizing processes on the thiamine, vitamin B 6 and vitamin B 12 contents of milk. For all the processes examined, the loss of thiamine is negligible, and the losses of vitamins B 6 and B 12 lie between zero and 35%. Indirect-heating processes appear to cause more loss of vitamin B 12 than of vitamin B 6 , but the reverse is true of directheating processes.

The effect of hydrogen peroxide on the nutritive value of milk

Treatment of milk with 0·05% (w/v) H 2 O 2 for 8 h at 24°C had no significant effect on the concentrations of seven of the B-complex vitamins or of the fatsoluble vitamins A and E or of carotene. Rat tests showed that the nutritive value of the milk proteins was slightly reduced. This finding was confirmed by microbiological tests with Streptococcus zymogenes , which also showed that the loss was probably connected with that of methionine. The concentration of H 2 O 2 used was effective in controlling bacterial growth during the incubation period, even in a sample of milk with a high initial bacterial count.

COPROPHAGY AND VITAMIN B12 IN THE RAT.

The importance of coprophagy in nutritional studies has long been recognized; already in the early days of vitamins numerous workers demonstrated the presence of B vitamins in the excreta of birds and mammals (cf. Kon, 1945; Elvehjem, 1948; Mickelsen, 1956) and began keeping experimental rats on screens to prevent their consuming their droppings. Rats kept in this way have been used for assays of members of the vitamin B complex and have shown characteristic signs of deficiency in the absence of any one of them. However, additional measures have usually been necessary to cause rats to exhibit deficiency of biotin, folic acid or vitamin B,, . These vitamins are needed in only small amounts by the rat and are synthesized in appreciable quantity by many micro-organisms. It has been generally assumed that sufficient biotin and folic acid were synthesized and absorbed from the caecum to supply the animals’ requirements, for the appropriate deficiency could be induced when synthesis was depressed by the inclusion in the diet of sulphonamide drugs (cf. Mickelsen, 1956). The picture with vitamin B,, was less clear; the absorptive mechanism for this vitamin is more complicated and it seemed unlikely that absorption occurred from the caecum. However, it was considered that the difficulty in inducing deficiency was due to storage of the vitamin in the tissues, and it was found that the onset of deficiency could be hastened on increasing the metabolic rate by inclusion of desiccated thyroid in the diet (Ershoff, 1947). It now seems probable that coprophagy was the main reason for these difficulties in inducing certain vitamin deficiencies, and it is noteworthy that du Vigneaud, Chandler, Moyer & Keppel(1939) pointed out already 25 years ago that rats can practise coprophagy even when kept on screens. The full extent of this practice was not really appreciated until Barnes, Fiala, McGehee & Brown (1957) designed an effective cup for collecting faeces (described in the rest of this paper simply as ‘cup’) which they used to show that rats kept on screens normally ingest about half their daily output of faeces. Later, using the same technique, they were able to induce deficiencies of biotin (Barnes, Kwong & Fiala, 1959), folic acid (Barnes, Fiala & Kwong, 1963) and vitamin B,, (Barnes & Fiala, 1958) in rats given appropriate deficient diets and without recourse to other expedients. Subsequently, Morgan (1960a, b) showed that the condition of refection

The occurrence of pyridoxamine phosphate in milk

The presence of pyridoxamine phosphate in extracts of freeze-dried raw and evaporated milks has been demonstrated by separation and identification of the vitamin B 6 active compounds by chromatography and electrophoresis on paper. Its presence in the milk extracts is the cause of the higher values for vitamin B 6 activity measured with Streptococcus faecalis as compared with those obtained with Str. faecium or Saccharomyces carlsbergensis . This is because Str. faecalis can utilize the phosphate for growth as readily as free pyridoxamine, whereas Str. faecium and S. carlsbergensis cannot. The mild acid treatment used for extracting the vitamin B 6 active compounds from the milk samples for microbiological assay was found to be insufficient to hydrolyse the pyridoxamine phosphate. Further treatment of the acid extracts with intestinal phosphatase released the pyridoxamine from its phosphate and increased the vitamin B 6 activity measured with S. carlsbergensis and Str.faecium so that the total vitamin B 6 activities of the freeze-dried raw and evaporated milks measured microbiologiclly, were then in agreement with the values found in previous tests with chicks and rats. Pyridoxamine phosphate could only be detected in small amounts in a sample of fresh milk. The possibility that more of it was formed during the processing and storage of the freeze-dried samples is discussed.