J . Sci. FoodAgric. 1984,35, 165-172

The Effect of Dietary-induced Changes in Milk Urea Levels on the Heat Stability of Milk

WilliamBanks, John L. Clapperton, D. Donald Muir, Anne K. Powell and A. W. Maurice Sweetsur

The Hannah Research Institute, Ayr, Scotland HA6 5HL

(Manuscript received 8 April 1983)

Two experiments were carried out to investigate the relationship between milk urea content and the heat stability of the protein in the skim milk. In experiment 1, four cows were offered a diet of grass silage with different amounts of hay and a protein concentrate. Although there were individual differences between the cows in the relationship between coagulation time and milk pH, there was a significant correlation between milk urea content and the maximum coagulation time. In experiment 2, two groups of five cows were given a basal diet of hay with a supplement of either barley or soya bean meal. These diets were exchanged weekly over a 3 week period. A significant correlation between milk urea content and maximum coagulation time was observed, and a close relationship between milk urea content and the mean urea content of the blood found. It is concluded that, although milk urea content is not the sole determinant of coagulation time, there is considerable potential for manipulating the urea content of milk in order to increase the heat stability.

1. Introduction

There is general agreement that urea, which occurs naturally as a minor component of milk, has a significant effect on its heat stability.'-3 The addition to, or removal from, milk of urea3 causes marked changes in heat stability, but there is, also, a highly significant statistical correlation between seasonally-observed variations in heat stability and urea content^.^,^ Taken in conjunction, these two observations support the validity of the statistical relationship in the biological sense. Nevertheless, because virtually all the components of milk show considerable seasonal variations in content, direct proof that the naturally-occurring changes in heat stability reflect those in urea content has not been provided. The object of the work reported here was to remove any remaining ambiguity by using dietary manipulation of milk composition to relate urea content to heat stability. This type of dietary manipulation may also have some commercial significance, in that a growing proportion of skim milk powder is exported for subsequent recombination and use as a liquid product.

Urea-induced effects on heat-stability can be maintained during the manufacturing sequence for dried milk production.6 The effect is largely lost if the powder is used for the preparation of evaporated milk,' but even in this case, addition of stabilisers such as aldehydes or sugars' again emphasises the effect of urea.'. lo Alternatively, and perhaps of more industrial interest, high-temperature forewarming also renders the heat stability of concentrated milk susceptible to the effects of urea addition."

The main pathways for nitrogen digestion and metabolism in the ruminant are now fairly well elucidated,'* and it is clear that the relative availability of energy and protein in the diet of the cow will determine blood, and hence milk, urea levels.'*. l3 The work described here makes use of these established facts to examine the relation between the heat stability of milk and the naturally-occurring urea content.

165

166 W. Banks et ~ l .

2. Experimental 2.1. Feeding experiments

Two different experimental approaches were used in this work. In experiment 1, four Ayrshire cows which had calved, on average, 7 weeks before the start of the experiment were selected on the basis of yield and calving date, and were fed a basal diet of silage (D value=64.6) which was supplemented by addition of hay (D value=65.3) and a protein concentrate. The four treatments used were those described in detail by Retter and CastleI4 (Table 1) but only one group of cows was considered. The four treatments were carried out for 4 weeks each; milk was sampled in the final week of each experimental period. The cows were allocated at random to the treatment sequences in a 4x4 balanced Latin Square design format.

In experiment 2, there were two groups of five Ayrshire cows, on a basal diet of silage with either a barley-based concentrate or a soya meal supplement (Table 1). The animals in each group were fed the required diet for a period of 1 week, at the end of which a composite milk sample was taken from all five cows and then the diet was abruptly changed. The sequence for Group E was: control-treatment-control and for Group F the order was reversed: treatment-control-treatment.

Table 1. The food intake of the animals

Food intake (kg DM day-')

Extracted soyabean

Group Silage Hay Cubes Barley meal

- - - Experiment 1 A 7.0 3.7 - - B 8.8 - 2.2

C 7.5 1.8 2.1 D 6.4 3.6 2.2

- - - -

- 2.6 - - Experiment 2 E 2.6

'?Ahout 11 kg DMiday- '-not measured because periods were only 1 week long

- - - F

2.2. Milk analyses The fat, crude protein, lactose, casein, non-protein nitrogen and total solids content of the milks were measured by the techniques detailed in Muir et al. l5 The f?-lactoglobulin content was estimated by a method devised by J. C. D. White and reported by Muir and Sweetsur.'

After removal of fat by low-speed centrifuging,' the mineral partition in the skim-milks was characterised by the methods described by Holt and Muir,16 except that the partition of soluble and colloidal material was achieved by ultrafiltration at 25°C using a laboratory scale apparatus with a flat membrane (PM30 membrane in a TCF-10 ultrafiltration unit from Amicon Ltd, High Wycombe, England).

The urea levels in both the milk and blood of individual cows were measured by the method of Fawcett and Scott.2, l7

2.3. Measurement of heat stability The coagulation time-pH profiles of each milk were measured by the subjective methods of Sweetsur and White."

3. Results 3.1. Experiment 1 3.1.1. The effect of diet on the nitrogen distribution of milk The effect of diet on the partition of milk proteins in experiment 1 is shown in Table 2. The diet had no significant effect on either the total concentration of protein in the milk, or on the relative

Diet and heat stability of milk 167

Table 2. The effect of diet on the nitrogen distribution of milk

Nitrogen distribution

Crude protein Casein" plactoglobulin" Urea Diet (g kg-7 (%) (%) (g W ' )

A 30.9 78.5 8.9 0.493 B 29.6 79.9 9.1 0.359 C 30.7 76.9 10.2 0.514 D 30.9 77.9 9.4 0.503 1.s.d.' 1.10 0.98 1.79 0.100

Expressed as a percentage of total nitrogen. ' I.s.d.=least significant difference at a significance Pi0.05

proportions of whey protein and casein. This result agrees with the more general observation of Retter and Castle14 who found that, on this particular feeding regime, the only significant effect of diet on gross composition was that the milk fat concentration was slightly elevated under treatment B where the fat content was 38.2 g kg-' compared to a mean value of 35.2 g kg-' for the other three diets. However, the dietary manipulation did have a significant effect on the urea concentrations of

r d

pH of milk

Figure 1. Coagulation time-pH (CT-pH) profiles for animals on dietary treatment C. a , cow 22; b, cow 45; c, cow 135; d, cow 136.

168

Table 3. The effect of dietary-induced changes on heat stability of milk

W. Banks el al.

Heat stability

Coagulation Maximum Minimum time coagulation time coagulation time

Diet (min) (min) (min)

A 11.4 22.6 6.3 B 8.6 15.4 6.6 C 21.0 24.1 9.3 D 13.9 23.2 7.7

the milk, although the effects were comparatively small. In treatment B, the urea level was significantly lower than all the other diets.

It is also worth noting that there were significant differences in Plactoglobulin content between animals. Although the average Plactoglobulin content (as a percentage of total nitrogen) for cows 22,45 and136was 10.1% (s.e.=0.25) thecomparablemeanvalueforcow 135was7.3% (s.e.=0.62). When the whey proteins were isolated and separated by polyacrylamide gel electr~phoresis'~ it was found that the three animals of similar nature were of the AB /3-lactoglobulin genotype while in cow 135, the Plactoglobulin genotype was BB. As will be discussed later, this had a significant effect on the heat stability characteristics of [he individual cow's milk.

u - 0 0 4

*: 10

0 b

0 /

5 E

I

._ x 0

d

t t 1 I I I I I I I I I I

3b0 I 400 500 600 I 360 I 400 500 600

Urea (mg kg-')

Figure 2. Individual cow responses to changes in diet. a, cow 22 (r=0.978*); b, cow 45 (r=0.801"'): c, cow 135 (r=0.988'); d, cow 136 (r=0.990**).

Diet and heat stability of milk 169

3.1.2. The effect of diet on the heat stability of milk There were distinct differences between both individual animals and the dietary treatments when the heat stabilities were examined. Three of the four cows had coagulation-time (CT)-pH profiles with marked maxima and minima around the natural pH of the milk, but the remaining animal (cow 135) had an almost inflectionless CT-pH profile. The CT-pH profiles for all four animals on dietary treatment C are shown in Figure 1. Cows 22, 45 and 136 exhibited typical type CT-pH profiles, but cow 135 had a profile approaching the type B character described by Rosez1 for milks with low levels of Plactoglobulin.

Three indices of heat stability were then considered: (a) the coagulation time at the natural pH of the milk; (b) the maximum coagulation time in the pH range 6.66.9; and (c) the minimum coagulation time in the pH range 6.8-7.1. As shown in Figure 1, two of the points can be coincident. The effect of diet on the indices of heat stability are shown in Table 3. In all three cases, the optimum heat stability occurred when cows were fed diet C, and the relative order of stability coincided with the urea level in the milk (Table 2).

3.1.3. The relation between urea level and heat stability The correlation between urea level and maximum CT was then considered, and the individual animal regressions are shown in Figure 2. For each animal a clear relation between urea and maximum CTwas found (although in the case of cow 45 the regression was not statistically significant with only 4 data points) and the regression accounted for between 64% and 98% of the observed variance in CT. The differences between individual animals were probably partly caused by variations in nitrogen partition and mineral equilibria. However, in this experiment no measurements of salt balance in the milks were carried out.

3.2. Experiment 2 3.2.1. Changes in milk composition induced by dietary manipulation Experiment 1 considered individual cow responses to changes in diet, but experiment 2 was designed to evaluate similar dietary manipulations in groups of five cows, i.e. in a more practical situation. The detailed analysis of milks from cows in groups E and F on control and test diets are shown in Tables 4 and 5. In these short-term experiments, the significance of small changes in gross composition cannot be readily evaluated. However, it is clear that, as in experiment 1, changes in fat, protein and lactose contents of the milks were small. There were also small changes in the mineral balance but these were not of practical significance.

Table 4. Changes in milk composition induced by short-term dietary manipulation (Group E)

Dietary treatment

Control Test Control

Gross composition (g litre-') Fat 53.8

Lactose 45.0 CP (NX6.38) 33.4

Mineral composition (mM litre-') Calcium Total 33.0

Soluble 9.9

Magnesium Total 4.9 Soluble 3.0

Phosphate Total 19.8 Soluble 9.2

Citrate Total 12.5 Soluble 10.7

47.0 33.9 44.7

33.2 8.7

4.7 2.7

23.2 9.8

10.7 9.2

48.1 32.5 44.4

31.7 9.4

4.6 3.0

20.0 10.7

11.5 9.6

12

170 W. Banks r f al.

Table 5. Changes in milk composition induced by short-term dietary manipulation (Group F)

Dietary treatment

Test Control Test

Gross composition (g litre-') Fat CP (Nx6.38) Lactose

Mineral composition (mM litre Calcium Total

Soluble Magnesium Total

Phosphate Total

Citrate Total

Soluble

Soluble

Soluble

41.0 35.2 43.2

- 7 31.5 8.8 4.5 2.7

21.0 10.4

10.1 9.1

49.0 32.2 44.8

33.2 8.5 4.7 2.6

20.9 9.4

11.6 9.8

43.1 34.2 43.6

31.2 9.1

4.7 2.8

20.9 10.4

11.0 9.5

3.2.2. Changes in urea level and maximum heat stability The relative changes in urea concentration and maximum heat stability for the groups of cows in experiment 2 are shown in Figure 3. Although the experimental periods lasted only 7 days, there was a very clear step in both urea level and maximum CT when the diet was changed. Irrespective of the order of dietary treatment, when the test diet was fed, milk urea levels were elevated and a corresponding increase in maximum CT was observed. It is therefore apparent that the urea level in milk can be reliably changed over a very short time.

Control Treatment Control Treatment Control

Group E Group F Treatment

Figure 3. The response of urea levels in milk and heat stability to short-term changes in diet. 0, maximum coagulation time at 140°C (min). B, urea concentration (mg kg-I).

171 Diet and heat stability of milk

60( 0

0 J 0

I I 300 600

Blood urea (mg kg-’)

Figure 4. Blood urea and milk urea in 10 individual cows given silage plus either barley (*), or soya bean meal (0).

The slope of the regression between CT maximum and urea was within the range reported for creamery milk samples by Holt eta^,^ which suggests that these experiments on small groups of cows are relevant to bulk milk as a whole.

3.2.3. The relation between milk urea level and blood urea level Four samples of blood were collected from the jugular vein at 2-hourly intervals from 09.00 to 15.00 hours from each of the cows in experiment 2 on the last day of each experimental period, and equal amounts of plasma were pooled for analysis. Corresponding samples of milk were also taken at the morning and evening milkings, and these were combined in proportion to yield. The relation between blood urea concentration and that in the milk is shown in Figure 4; the urea levels are highly correlated (P<O.OOl, r=0.979). These results are very similar to those quoted by Thomas” but the correlation coefficient is much higher than that described by Journet et al.I3

4. Discussion

The results presented in this paper clearly demonstrate a causal relation between the diet of the cow and the heat stability of the milk. It is evident from the data presented for both experiments 1 and 2 that if milk urea levels change, a consequential change in heat stability occurs. The effects of dietary-induced changes are most highly correlated with the maximum coagulation time. Although the maximum CT does not always coincide with the CT at the natural pH of the milk, this fact does not detract from the potential value of the work, since small changes in pH can be readily induced in milk by addition of stabilising salts permitted with the Dried Milk regulation^.'^

In addition, it is worth noting that the techniques advocated for increasing the urea content of milk, i.e. supplementation of a basal silage diet with a protein concentrate, have beneficial effects on dry matter intake of the cow, with subsequent improvements in milk yield. Nevertheless, under some conditions a slight depression of fat content of milk was although in this work the effect was absent or small.

172 W. Banks et al.

Notwithstanding the consequential effects on yield of fat, this work demonstrates that there is considerable potential for increasing the heat stability of milk by manipulating the diet to enhance urea concentration in milk. As the relative proportion of milk going to manufacture of sterilised milk products increases, the heat stability of milk will assume more practical importance and the methods advocated may find more extensive application.

The results also show that urea is only one determinant of the heat stability of individual cow milk, and further work will be required to elucidate the others.

Acknowledgements

Mrs S. J. Golightly, Mrs N. West and Mrs M. McGhee are thanked for their expert technical assistance. Dr M. E. Castle and Dr W. Steele are also thanked for their enthusiastic co-operation in providing milk and blood samples from experimental animals.

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